Staining and Examining Medullary Rays

filled with starch, which appears dark blue. This is best seen in stems cut in autumn. Examine the medullary rays closely in the thinnest part of the section, under the highest power, and note the small triangular intercellular spaces, which take a horizontal course along the medullary rays, and are therefore cut here transversely. In close connection with these 2. The xylem-parenchyma, the cells of which also contain starch, and are thus easily recognized : note that cells of the parenchyma more or less completely surround 3. The vessels, the walls of which are stained yellow, and present those characters already observed in radial sections. The interspaces are filled by 4. Masses of xylem-fibres, which appear as before. VIII. Cut tangential sections of the phloem of a similar stem : treat as before, and observe 1. The form and arrangement of the medullary rays as in the xylem, but the walls of the cells are thinner, and not lignified : intercellular spaces may be 106 PRACTICAL BOTANY noted here also, similar to those above described as occurring in the medullary rays of the wood. The cells contain copious protoplasm. 2. Phloem-parenchyma, the cells of which differ in their cell contents a. Some contain crystals. b. Others have copious protoplasmic contents. Both forms will be seen to have been derived by division from original elongated cells with pointed ends, since they are arranged in groups of this form. Compare the form of the cambium cell (IX.). 3. Sieve-tubes answering to the description given for radial sections. The sieves are oblique, the form of the successive segments oblong. The sieves are callous (see p. 115), and are easily recognized in sections stained with iodine or eosin. 4. Bast-fibres as before in radial sections. IX. Cut tangential sections through the cambium of the stem of Elm : treat with dilute potash or "eau de javelle," and mount in glycerine. Examine first with a low power, and note that the general arrangement is similar to that already seen in tangential sections through the mature tissues, also that the form of the cells, in each part of the cambium-zone, is like or similar to the average form of the elements of the mature portion of wood or bast, which borders on it in a radial direction. Thus the cambium is differentiated into 1. Cambium, of medullary rays, which appears to consist of roundish cells, resembling cells of the mature medullary rays in form. 2. Cambium, from which all the other tissues are derived, the cells of which have a prismatic form. ELM STEM 107 To gain a clear idea of the process of secondary thickening, the actual form of the cambium-cells and their arrangement must be recognized : as stated above (p. 7), it is necessary, in order to fully realize the form of a cell as a solid body, to cut sections in three directions at right angles to one another : the cambium-cells - /' FIG. have now been seen in transverse, radial, and tangential sections and the results are represented diagrammatically in Fig. 9, A, B, C, which are based upon results of Sanio's investigations of Pinus, but the main points are the same for Dicotyledons. Fig. 9, A, shows diagrammatically four radial rows of cambiumcells (1, 2, 3, 4,), as seen in transverse section : of these row (2) 108 PRACTICAL BOTANY. is a medullary ray. Note in row (3) the single initial cell (i), oblong in transverse section, and the shorter diameter placed radially ; according to Sanio's law of cambial division there is only one such initial cell in each radial row : from this successive segments (w w) which go to form wood have been cut off on the inner side, others (b, b, b] which go to form bast on the outer side : each is represented in the diagram as dividing into two by a periclinal wall ; this is typically the case in Pinus, but the divi- sion is not so regular in Dicotyledons. In row (1) is represented a segment (w') recently cut off from the initial cell on the side next the wood, in which this division has not yet taken place ; in row (4) there is a similar undivided segment (6'), which, after division, will go to form bast. Fig. 9, B, shows diagrammatically the arrangement of the cells of one of these rows (3), as seen in radial section : the length of the cells is much greater than their width, and the ends are square : w i is, as before, the initial cell of the row : w, pairs of cells formative of wood : &, 6, 6, pairs of cells formative of bast : m. r. is a medullary ray put in so as to show the relative position and form of the cells. Fig. 9, C, represents the appearance of the cambium cells, i, i, i, in tangential section : they are obliquely pointed, and their A width corresponds to that shown in : m. r., as before, the medullary rays. Fig. 9, D, shows the form of a single isolated cambium-cell as a solid body, drawn to the same scale as the other figures : if such a cell be cut transversely, radially, or tangentially, it would give the appearance presented by the initial cells (i) in Figures A, B, and C. Taking cells of this form as a starting-point, the several tissues above described are derived from them in the following way : Phloem. (i.) a. Sieve-tubes, sion, and by lateral distenconversion of the oblique walls into sieve-plates. b. Parenchyma, by division of the cells by transverse septa. ELM STEM ' 100 (ii.) Xylem. c. Fibres (sclerenchyma), by elongation and interweaving of cells, the width of the cells at the same time being relatively reduced : the ends of the cells slide past one another as the cells elongate. a. Vessels, by lateral distension, and absorption of cell- contents, and of the oblique walls. b. Parenchyma, by division of the cells by transverse septa. c. Fibres, by elongation and inter- weaving of the cells, while the width of the individual cells is relatively reduced. Observe intermediate stages between cambium-cells and these several mature tissues : this may best be done in sections cut from stems in early summer. X. To investigate the nature of the crystals, several times observed in the parenchyma of the stem of the Elm, cut tangential sections of the phloem or of the cortical tissue, mount in water, and having found one or more crystals (i.) Run some iodine solution under the cover-slip : the crystal is not stained. (ii.) Acetic acid : it is not attacked. (iii.) Dilute nitric acid : it is more or less completely dissolved. These reactions, coupled with what is known from the analysis of ash, point to the conclusion that these are crystals of calcium oxalate. 110 PRACTICAL BOTANY STEM -AQUATIC TYPE Note the cylindrical smooth stein of the Mares-tail (Hippuris vulgaris), bearing whorls of simple leaves. Cut the stem transversely, and note the central vascular cylinder, which is easily seen with the naked eye, and the broad band of cortex with large intercellular lacunae. For the microscopic work fresh material may be used : if the material has been kept in alcohol, the sections should, after being cut, be allowed to swell in water before mounting. I. Cut transverse sections of an internode of the stem of Hippuris vulgaris : mount in glycerine and examine with a low power. Observe A 1. well-marked epidermis with cuticle. Here and there are to be seen radiating scale-hairs. These occur especially in the axils of the leaves. 2. Cortical parenchyma : a broad band consisting of thin-walled, chlorophyll-containing cells, with large intercellular spaces. A 3. well-marked bundle-sheath, with the usual characters, which immediately surrounds 4. The central vascular cylinder. This is com- posed of A a. basis of thin- walled parenchyma, in which are distributed HIPPURIS STEM 111 I. In the centra] part vessels of the xylem with lignified walls : c. Towards the periphery elements with the characters of soft bast ; the sieve structure is in this case doubtful. II. Cut thick transverse sections of nodes ; treat with potash, mount in glycerine ; and observe, with a low power, that the distribution of tissues is in the main the same as in the internode, but 1. The large intercellular spaces are divided by horizontal diaphragms, consisting of single layers of cells. 2. Branch-bundles leave the central cylinder, and pass horizontally outwards to the bases of the leaves. III. Take a terminal bud of Hippuris : remove from it the largest external leaves, and then dissect off the inner and smaller leaves with needles in a drop of water on a glass slide : in the centre of the bud will be found the elongated conical and colourless apical cone. Cover with a cover-slip, and examine it under a low power : the smooth cylindrical apical cone will be well seen, the inner tissues of it being marked by a reticulum of dark lines : these are the intercellular spaces filled with air. Note especially the leaves, which appear as rounded outgrowths laterally on the axis : the larger ones are seated lower down the axis, and they are successively smaller as the apex is approached. IV. Cut median longitudinal sections of the apical bud of Hippuris, so as to pass through the elongated apical cone ; treat with potash, or with " eau de javelle," and mount in dilute glycerine. Examine first with a low power, and observe 112 PRACTICAL BOTANY 1. The axis, which is wide below, but tapers upwards to the rather elongated apical cone (punctum vegetationis). The axis is composed of the several tissues already noticed. Note especially in the lower part of the section a. The rectangular intercellular spaces, divided transversely by diaphragms at the nodes. b. The axile vascular cylinder, which may be followed far up into the apical cone, and which gives out lateral branches to the leaves. 2. The leaves, diminishing in size towards the apex. Note the scale-hairs about the bases of the leaves. Put on a high power, and examine the apical cone. Note 1. The dermatogen, a continuous layer of cells, which covers the apical cone externally. Trace it backwards from the apex : it will be seen to give rise to the epidermis. 2. The periblem, consisting of 4-5 layers of cells, Avhich may be traced backwards, and be thus shown to give rise to the cortex. A 3. central cylinder of plerome, which is continu- ous with, and gives rise to, the vascular cylinder (compare the diagram, Fig. 5, p. 80). Note that the leaves originate from the outgrowth of the dermatogen and periblem, the plerome taking no part in their formation. Also that the vascular system of the stem is already developed at a higher point on We the axis than that of any of the leaves. have thus an instance of cauline vascular bundles, that is such as are proper to the stem, as distinguished from com- mon vascular bundles, which terminate at their upper extremities in the leaves. SIEVE-TUBES 113 SIEVE-TUBES i. Gucurbita Though the sieve-tubes of the Sunflower are fairly large, the soft bast does not occur in large masses. In the Vegetable Marrow, however, the sieve-tubes are of extraordinary size, and occur in large numbers: this stem is thus excellently fitted for the study of the sieve-tubes of the type usually found in herbaceous stems. The material should be hardened in alcohol. I. Cut transverse sections of the stem of the Vegetable Marrow, stain with eosin, and mount in water or glyce- rine. The general arrangement of tissues in this stem differs in several important points from that in the Sunflower, and, indeed, from that in most herbaceous Dicotyledons. Thus it will be seen on examination under a low power that 1. There occurs at a short distance below the epidermis a thickwalled band of sclerenchyma with lignified walls (yellow, with chlor-zinc-iodine, or acidulated aniline sulphate). This is quite distinct from the vascular bundles. 2. The vascular bundles are always separate and distinct : though an interfascicular cambium is formed in old stems, no secondary vascular tissue is derived from it. 3. The structure of the individual bundle is abnormal, there I 114 PEACTICAL BOTANY being in each bundle a central mass of xylem with the phloem masses lying the one on the central, the other on the peripheral side of it : this type of bundle is termed bi-COllateral. Between the xylem and the peripheral phloem mass is the cambium layer. The structure is fundamentally the same in both phloem masses : either will therefore serve for the study of the sieve- tubes. In the soft bast, which resembles that of Helianfhus but has larger constituents, observe (i.) The transverse, circular, punctate sieve-plates, having the same appearance as in Helianthus, and easily recognized by their contents being stained with eosin. (ii.) The companion-cells, appearing as though cut off from the side of a sieve-tube by a curved wall. Cambiform cells. (iii.) Treat some sections with chlor-zinc-iodine : all the walls of the soft bast turn blue (cellulose), but the sieve-plates appear yellow or brown. II. Cut longitudinal sections through the soft bast : either radial or tangential sections will do. Mount some in iodine solution. The transverse sieve-plates will be brought into prominence by the deep yellowish brown staining of the mass of substance, which surrounds them : this may consist of A a. callus-mass, which surrounds, and often com- pletely invests the sieve-plate : the size of the callusmass is variable according to season, age, &c., being greatest in autumn, and in old sieve-tubes. b. Protoplasm, which is usually collected in close contact with the sieve-plate (or with the callus if present), and more especially on its upper side, SIEVE-TUBES 115 Note, I. the oblong form of the segments composing the sieve-tubes. ii. The companion-cells, short with granular protoplasm, and nucleus, iii. Cambiform cells of similar form to the segments of the sieve-tubes. Other sections should be stained with eosin, then washed, and mounted in glycerine. The sieve-tubes will be readily seen, as their contents will be stained deeply. III. (a) Mount some sections in water, and having found a sieve-plate with callus, run some dilute potash under the coverslip. The callus-mass swells ; the protoplasm also swells : the section thus becomes more transparent, and the cellulose basis or true sieve becomes more apparent, and its pores can be easily seen. (&) Treat another section in which a callus has been found with Eussow's callus -reagent (see Appendix A) : the callus stains a deep brown. (c) Treat another preparation with corallin-soda (see Appendix A), and mount in glycerine : the callus stains pink. (d) Stain another preparation with Hoffmann's blue in 50 per cent, alcohol for 24 hours, wash with water, and mount in glycerine : the callus is stained deeply. IV. Treat some fresh sections with iodine, then dry off the superfluous fluid with blotting-paper, and mount in a single drop of strong sulphuric acid. The cellulose walls and callus will swell; the protoplasm will contract. Look carefully over the protoplasmic contents of the sieve-tubes for the points where sieveplates have been ; ,here it will be found that fine strings of protoplasm, which passed through the sieve-plate, connect the protoplasmic masses on opposite sides of ihe I2 116 PRACTICAL BOTANY sieve with one another. By this reaction the continuity of protoplasm through the sieve is demonstrated. Another method by which similar results may be obtained is to add a small quantity of dry Hoffmann's blue to a little strong sulphuric acid in a watch-glass, and mix well. Treat the sections with this for a short time, wash with water, and mount in glycerine. It will be noted that the sieve-tubes of Cucurbita closely resemble those of Belwnthus, the sieve-plates being transverse and simple. This is the usual type of sieve-tube to be found in primary phloem, and gene- rally in herbaceous stems of Angiosperms. In the secondary phloem of ligneous stems a more com- plicated type of sieve-tube is frequently found. This will be studied now in the stem of the Lime. ii. Tilia (Lime). I. Cut radial sections of the phloem of a stem of Lime more than three years old. Stain with eosin, wash, and mount in glycerine ; examine them with a high power for sieve-tubes. The general arrangement of the phloem is similar to that in the Elm. The sieves, which will appear stained pink, occur on oblique walls facing the radial plane, and are therefore here seen in surface view. Note that they have a similar appearance to those above described, but here three or more sieve-plates occur on each oblique wall. II. Cut tangential sections of the same ; stain, and SIEVE-TUBES 117 mount as before. The oblique walls are here cut longi- tudinally ; the sieve-plates are often callous, especially in autumn. Note the form of the segments of sieve-tubes ; it is fundamentally the same as that of the cambium cell as seen in tangential section (compare Fig 9, C, D). 118 PRACTICAL BOTANY LATICIFEROUS TISSUES The material for the study of these tissues should be prepared by treatment with alcohol to coagulate the latex. Care should be taken to place the material in alcohol directly it is cut, or at least the cut surfaces should be wetted with alcohol so as to check the flow of latex from them. If the latex be allowed to escape, the laticiferous tissues are emptied, and are then much less easily traced than when they are full. The best method is perhaps to preserve the whole plant without injury in alcohol, in which case the latex will not be lost at all. Draw from a piece of the fresh stem of Euphorbia a drop of latex upon a slide : examine it quickly under the microscope, and observe the fluid is at first almost uniformly milky, but that in a short time a coagulum separates in irregular masses from the more transparent fluid. The coagulation is effected more completely and rapidly on addition of a drop of alcohol. i. Laticiferous Vessels. I. Cut tangential sections from the phloem of the root of the Dandelion (Taraxacum officinale), mount in potash and glycerine, and warm ; examine under a low power. The main constituents of the tissues are parenchy- LATICIFEROUS VESSELS 119 matous cells, with thin walls (phloem -parenchyma) : sieve-tubes are to be met with here and there. The whole mass of tissue is permeated by a ramifying, and profusely anastomosing network of laticiferous vessels. The communication of these tubes with one another is demonstrated by the continuity of their coagulated contents (latex), which appear brown and granular. The course of the vessels is mainly longitudinal, while lateral, horizontal branches frequently connect the parallel tubes. With a high power make out more accurately the course of a group of the vessels. By staining other sections with alkannin (Appendix A), or with solution of potassium bichromate, good preparations may be obtained. II. Cut transverse sections of the same; mount in glycerine, and examine with a low power. The laticiferous vessels appear circular in transverse section, and have brown contents : they are distributed in groups, which form more or less regular concentric rings round the central xylem. They may be recognized still more distinctly in sections stained with alkannin, or with potassium bichromate. Note in these sections the presence of spherecrystals of inulin : in the former section they will have been dissolved by the treatment with potash. Observe that they are formed quite irrespective of the cell-walls, which are often included in them. The development of the laticiferous vessels may be traced by cutting thin longitudinal sections through the cambium of the root of the Dandelion. By careful comparison of such sections it 120 PRACTICAL BOTANY will be found that they originate from a number of originally separate cells of the cambium, the cavities of which are thrown together by the partial or complete absorption of the walls. Such fusions may appear in the terminal or the lateral walls. Note on Timlin. From a fresh tuber of the Jerusalem Artichoke (Helianihus tiiberosus), or from fresh Dandelion roots cut sections in any direction, and mount them in a very small quantity of water : examine under a low power, and observe that the tissue is chiefly composed of cells with transparent contents. Irrigate the sections well with alcohol : a granular precipitate will appear in the fluid surrounding the sections, and a similar precipitate will also be seen within the cells, which may be so bulky as to make the whole section appear opaque. Now irrigate thoroughly with water : the precipitate will again dissolve. Cut sections from material which has been kept for some weeks, or better for some months, in alcohol : mount in glycerine, and observe the transparent tissue as before : here and there will be seen large circular patches of highly refractive substance, which are of such size and position as often to extend over a number of cells : these are the sphere-crystals of inulin, Treat with iodine solution : they are not appreciably coloured. Irrigate well with water : they are slowly dissolved, their solution being hastened by warming, and they show a radiate structure as solution progresses. Treat with potash : they are dissolved more quickly, without any coloration. A similar substance, hesperidin, is found in young Oranges : if these be treated for a long time with alcohol, the tissues will be found crowded with sphere-crystals, which have similar reactions to those of inulin, but on dissolving in potash the solution takes a yellow colour. ii. Laticiferous Cells. I. Cut tangential sections of the cortex of Euphorbia splendens (other species will do) just outside the vascular LATICIFEROUS CELLS 121 ring, and mount in water, or dilute glycerine : or stain with alkannin, and mount in glycerine. Examine with a low power. Eunning through the cortical parenchyma will be seen long tubes, with thick cellulose walls and granular contents. These are the laticiferous cells, which differ from the preceding in being developed, not by fusion of originally distinct cells, but by continued apical growth of single cells. Note cases of branching of these cells. Included in the granular contents are starch-grains of peculiar dumb-bell form. Treat sections with iodine solution, and observe the effect on these bodies. II. Cut transverse sections of the same stem, and note the distribution of the laticiferous cells ; they may be recognized by their walls, which are thicker than those of the surrounding tissues, and appear circular in section. III. Separate the whole cortex from a piece of the stem ; boil it in potash for about five minutes, and tease out the long laticiferous cells with needles ; mount, and observe with a low power. They appear as long cylindrical tubes, with thick walls. Observe occasional branching. They are usually broken at the ends, the length of the tubes being greater than that of the parts teased out. In longitudinal sections through the apical region of the stem of Euphorbia it may be shown by staining with haematoxylin that numerous nuclei are present near to the blind endings of the tubes. 122 PRACTICAL BOTANY LEAF i. The Common Bifacial type A. PETIOLE Observe that the mature leaf of the Sunflower consists of an upper, flat, expanded portion the lamina, and a lower, narrow stalk the petiole, by which it is inserted on the stem. Note the channelled upper surface of the petiole, and the broad insertion on the stem : in the angle between the petiole and the stem may usually be observed an axillary bud, or shoot. I. Cut transverse sections of the petiole, and mount in glycerine. The details of