to . . ..... !......,.. . ~ ~ I ( , · - ~ - •. ..._j _ - ' - ' -___ I PRACTICAL PLANT ANATOMY --, oJ;J;),~ PRACTICAL PLANT ANATOMY AN ELEMENT ARY COURSE FOR STUDENTS BY COMYNS J. A. BERKELEY, B.Sc. Lecturer in the Department of Biology, Chelsea Polytechnic WITH A FOREWORD BY B. BARNES, D.Sc., PH.D., F.L.S. UNIVERSITY OF LONDON PRESS, LTn. 10 & II WARWICK LANE, LONDON, E.C.4 1 934 Printed in Great Britain for the UNIVERSITY OF LONDON PRESS, LTD., by HAZELL, WATSON AND VINEY, LTD., London and Ayl e sbury . PREFACE Tms book is written with the purpose of assisting students in what usually proves to be the most difficult part of their botanical practical work during the intermediate stage. It is believed that a s _ tudent who works conscientiously through the course will acquire some appreciable dexterity in using the microscope for studying botanical material and gain a good basic knowledge of the vegetative ana- tomy of angiosperms. Much of the subject-matter consists of directions and hints that have been found valuable in _ personal and teaching experience. The remainder is a set of instruc- tions in relation to exercises the range of which should give sufficient experience to students studying for examina- tions up to and including the intermediate science of the University of London. The book is divided for convenience of reference into numbered sections, each of which covers one practical method, a group of closely allied ones or a set of exercises of similar character. Each working method is described just prior to the exercise in which it is first used and reference is made to the description, by the number of the section containing it, in closely following exercises requir- ing its use. A section number is frequently used in exer- cise instructions in order to obviate the necessity of repeating a long description of a process. Three factors have been considered in choosing the sequence of the exercises : difficulty of manipulation of material, difficulty of understanding the prepared object 5 6 PREFACE and the theoretical relationships of the exercises the one to the other. As far as possible the first two have been given most weight, so that difficulty may march hand in hand with increasing dexterity. On the other hand, for the benefit of students who are forced to work with little or no guidance, the difficulty sequence has been broken with- out hesitation when it has been felt that the theoretical implications of an easier exercise will be best grasped if - one of greater difficulty is undertaken first. Students will find that their progress is at first slow; much has to be learned before a result is attained. Many will feel the impulse to get on somehow and to hurry through those lines of the book which do not seem to lead to the immediate end. Others will feel more comfortable working in a way other than what is advised. Let those who would hurry glance ahead through the book. They will find less and less instructions are given as the course proceeds. More and more knowledge is assumed, and if the preceding pages have been thoro · ughly assimilated, progress will be faster and faster. No claim is made that the methods suggested in this book are per- fect, but they are without doubt ones which give good results and students will be well advised to follow them closely until they are at least moderately skilled. Then perhaps they may modify them to their own taste for the better. COMYNS ] . A. BERKELEY. CHELSEA POLYTECHNIC, LONDON, S.W.3. September 1934. CONTENTS CHAPTER I THE COMPOUND MICROSCOPE AND THE METHOD OF USING IT PAGE I. THE PRINCIPLE OF THE COMPOUND MICROSCOPE 13 2. THE PARTS OF THE COMPOUND MICROSCOPE . 13 3. PREPARATION OF OBJECTS FOR EXAMINATION 17 4. METHOD OF WORK WITH THE MICROSCOPE 19 5. COMMON FAULTS AND THEIR REMEDIES 23 6. ESSENTIAL RULES FOR MICROSCOPY 24 7. EXERCISES IN SIMPLE MOUNTS 25 CHAPTER II CUTTING AND MOUNTING SIMPLE SECTIONS- MICRO-CHEMICAL REACTIONS 8. THE RAZOR g. SECTION CUTTING 10. EXERCISES IN SIMPLE SECTIONS II. MICRO-CHEMICAL REACTIONS 12. EXERCISES IN MICRO-ANALYSIS 13. CELL-WALL DRAWING • CHAPTER III CUTTING, MOUNTING AND DRAWING STEM SECTIONS 14. TYPES OF SECTION 15. TISSUE STUDIES . 16. COLOURATION OF SECTIONS • 17. HOW TO STUDY STEM SECTIONS 18. LONGITUDINAL RADIAL SECTIONS . 19. DRAWING SECONDARY TISSUES 20. STRUCTURES IN WOODY STEMS 2I. EXERCISES IN STEM STRUCTURE 7 28 30 32 33 36 40 41 43 48 53 59 61 63 67 8 22. BUNDLING • CONTENTS CHAPTER IV OVERCOMING DIFFICULTIES 23. HARDENING IN SPIRIT 24. EMBEDDING 25. EXERCISES IN ROOT STRUCTURE • 26. EXERCISES IN LEAF STRUCTURE, ETC. APPENDIX I PAGE 75 76 78 85 87 INDEX OF MATERIAL AND TABLE OF SOURCES gr APPENDIX II METHODS OF PREPARATION AND PRESERVATION OF MATERIAL • IOO APPENDIX III STAINS AND REAGENTS APPENDIX IV STUDENTS' REQUIREMENTS • GENERAL INDEX . IIO III LIST OF ILLUSTRATIONS FIG. PAGE I. PRINCIPLE OF THE COMPOUND MICROSCOPE 12 2. PARTS OF THE COMPOUND MICROSCOPE 14 3. SECTIONAL VIEW OF AN EYEPIECE 15 4. SECTIONAL VIEW OF A LOW POWER OBJECTIVE 15 5. SECTIONAL VIEW OF A HIGH POWER OBJECTIVE 16 6. METHOD OF PUTTING ON A COVERSLIP 18 7. METHOD OF HONING THE RAZOR 29 8. METHOD OF HOLDING MATERIAL FOR CUTTING TRANS- VERSE SECTIONS 31 g. THE PLANES OF THE THREE TYPES OF SECTION 41 IO. PARENCHYMA CELLS FROM ECBALLIUM STEM . 44 II. COLLENCHYMA CELLS FROM ECBALLIUM STEM AS SEEN IN TRANSVERSE SECTION 12. TWO STAGES IN DRAWING SCLERENCHYMA FIBRES AS SEEN IN A TRANSVERSE SECTION OF ECBALLIUM 44 STEM 45 13. PHLOEM AS SEEN IN TRANSVERSE SECTION OF ECBAL - LIUM STEM 45 14. STAGES IN DRAWING THE XYLEM OF ECBALLIUM STEM AS SEEN IN TRANSVERSE SECTION 46 ' g IO LIST OF ILLUSTRATIONS FIG. 15. HAND LENS DRAWING OF TRANSVERSE SECTION OF SUN- FLOWER STEM . 16. ZONE DRAWING OF TRANSVERSE SECTION OF SUN- FLOWER STEM . 17. METHOD OF PLANNING A HIGH POWER DRAWING. PART OF TRANSVERSE SECTION OF SUNFLOWER STEM 18. METHOD OF HOLDING MATERIAL FOR CUTTING LONGITUDINAL RADIAL SECTIONS 19. STAGES IN DRAWING SECONDARY XYLEM. TRANS- VERSE SECTION OF SUNFLOWER STEM PAGE 53 56 57 60 62 FOREWORD BOTANY cannot be studied profitably unless the plant itself is investigated, and it is therefore essential that the student shall acquire some facility in a technique which, while presenting no real difficulties, demands care and attention to detail. An attempt has been made in this book to help the student to build up a satisfactory knowledge of botanical technique, by providing a series of exercises and practical hints, all of which have been tested by the author during his considerable experience in teaching students of diverse types. Every exercise is well within the power of anyone of ordinary capacity and application. The book will be of assistance to students working under guidance, and to those who, for one reason or another, have to find their own way into the delights of practical botany. The student who really masters the exercises will gain, not only a valuable introduction _ to the study of plant structure, but also that measure of confidence which will help him to direct his own work and to build up his own methods. Those who use this book can best show their appreciation by making it a foundation for a thorou~h study of the plants. · B. BARNES, D.Sc., Ph.D. (Lond.), F.L.S. CHELSEA POLYTECHNIC, September 1934. Head, Department of Biology. II Eye ----Eyepiece ---- PrimaPy real image ----- Objective -----·· Oiject Final virtual ima e FIG . r.-Pr i nciple of the Compound Microscope. 12 CHAPTER I THE COMPOUND MICROSCOPE AND THE METHOD OF USING IT I. The Principle of the Compound Microscope WHEN light coming from an object passes through a suit- able lens correctly placed (Fig. r, objective) an image larger than the object will be formed on the far side of the lens. This image could be received on a screen and is therefore called a real image. If another suitable lens (eyepiece) is correctly situated between this image and the eye of an observer, another image will appear as if placed farther away than the objective and of much greater size. There is thus formed by the eyepiece a second image which since it cannot be received on a screen is called a virtual image. The objective forms a real" primary" magnified image of the object and the eyepiece forms a" secondary" and further magnified virtual image of the primary one. 2. The Parts of the Compound Microscope Fig. 2 indicates the parts of a modern compound micro- scope which may be considered in four groups. The Supporting Parts The supporting base or foot carries the arm or limb · by means of a transverse joint so that the microscope may be inclined out of the vertical. The arm supports the body tube on a rack and pinion and also the stage upon which objects to be examined are placed. The body tube bears 13 14 PRACTICAL PLANT ANATOMY at its lower end the nosepiece into which are screwed the objectives. It can be lengthened by means of a draw -.J.-----Draw tube - 4 - - - t - - - - - - - - - - - - - Coarse ac(;uslment Double nosep1ece \ . - - - ' . - - - - - - - - - - - F i a l m1f'l'0f' L - - ~ ' - . - - - - - - ~ - - Concave m1rl'or -&----root FIG. 2.-Parts of the Compound Microscope. tube which carries the eyepiece. Under the stage is a substage. The Lenses The eyepieces or oculars most commonly used are numbered r, 2, 3 or 4 and magnify respectively 5, 6, 8 and ro diameters. An eyepiece (Fig. 3) is a tube carrying an upper or " eye " lens and a lower or " field " lens and containing THE METHOD OF USING THE MICROSCOPE r5 a diaphragm at the focal point of the eye lens. The eyepiece may be fitted with a pointer or with cross wires for locating objects of special interest. The objectives, which are tubes each fitted with a system of lenses, FIG . 3.-Sectional Vie w of an Eyepiece. are screwed into a rotating and dust- proof nosepiece so that either may be brought into position for use. The low power objective (Fig. 4) has the larger external lens, and the one most commonly used is marked thus : tt i in. (English), r6 mm. (American and some Continental makes). These figures indicate the focal length of the system of le~ses and not the working distance, i.e. not the dis- tance between the outer lens and the object when in focus, which is only about 7 mm. and thus less than the focal length. FIG . 4. - S ec - ti on a l V i ew o f a Low Po wer Ob- j e cti v e . The low power objective usually magnifies the object about ro times, but the primary image formed by it within the eyepiece is again magnified by the eyepiece lenses forming an e nlarg e d secondary virtual image (see Fig. r). Thus using a No. 2 eyepiece, x 6 and a two-third objective, we get a total magnification of ro x 6 = 60 dia!Ileters. Modern objectives are corrected for a definite length of body tube, usually 6 or 7 ins. (r6o mm. or r8o mm.). To obtain this it may be necessary to pull out the draw tube a little, hence the latter · should be graduated in inches or millimetres. Further extension of the draw tube increases the magnification of the primary image formed by the objective but does not increase the direct magnification of r6 PRACTICAL PLANT ANATOMY the object, and so does not give any further details of structure. The high power objective (Fig. 5) has the smaller external lens and is marked ¼ in. or 4 mm., the working distance being about o.6 mm. The primary magnification is about 40 diameters, so that with a No. z eyepiece, x 6 and 160 mm. tube, the total magnification is 40 X 6 = 240 diameters. The Illuminating Parts FIG. 5. - Sec- tionalView of an High Power Ob- jective. The two mirrors, one plane and the other concave, are mounted back to back in a frame on a universal joint which allows them to be so placed that either can catch light from any direction and reflect it through the aperture in the stage. The mirror must be so arranged that its centre is on the optical axis of the microscope in order to act efficiently. If the mirror is swung out so that its centre is not on the optical axis, oblique illumination results and shadows are cast upon the object. This must be avoided. The flat mirror reflects parallel rays of light and is used with strong light or with a condenser. This is a system of lenses which concentrates the light coming from the mirror. It is not necessary to use it with low powers, but it can be used to advantage with i in. objectives. The concave mirror also concentrates the light and so is used when a bright illumination is required, and the condenser is not in use. The silbstage and fittings. Below the stage is a substage which contains an iris diaphragm, the aperture of which may be altered by rotating a collar. By modifying the THE METHOD OF USING THE MICROSCOPE 17 size of the aperture the amount of light passing to the objective may be modified and the clearness or definition of the image improved. The substage also carries the condenser when one is fitted and is then provided with some form of adjustment for focussing the condenser on to the object. The Focussing Adjustments The coarse adjustment is actuated by a pair of large milled heads which rotate a spiral pinion whose teeth engage on a rack attached to the body tube. Turning the milled head so that its upper edge moves towards the observer raises the body tube. The fine adjustment is actuated either by a single milled head with a vertical axis, or by one or two lateral milled heads with a horizontal axis. Objects to be examined under objectives up to and in- cluding ¼ in. should always be found and focussed with the coarse adjustment. The fine adjustment is intended only for critically focussing parts of an object already in view, and the milled head should be turned backwards and forwards only, not right round. 3. Preparation of Objects for Examination Objects are usually examined by transmitted light, and in order that light may pass through them they must be very thin. Consequently, except in the case of powders, fibres or very small objects, thin slices or sections have to be prepared and rendered translucent. The smallest quantity of material which will show what it is desired to see should always be used . An object to be examined is mounted on a glass slide 2 18 PRACTICAL PLANT ANATOMY 3 ins. by r in. and is covered with a thin sheet of glass called a coverslip. Coverslips i in. square, No. 2 thickness, are suitable for elementary plant anatomy. A liquid called the mounting medium is nearly always Comz~~ ""- __ Wale" =.~:-c-:-·,._ t---Skd e , FIG. 6.-Method of Putting on a Coverslip. included in the space surrounding the object between the slide and the coverslip . . EXERCISE r.-To MAKE A SIMPLE MOUNT OF A POWDER SUCH AS POTATO STARCH r. Clean both slide and coverslip thoroughly. Dust or dirt on either may be mistaken for parts of the object and in any case prevent distinct vision. 2. Place a very small quantity of the starch-about enough to cover a pin's head-in the centre of the slide. 3. Catch a small drop of water from the tap on the end of a glass rod and mix the starch in this so that all the grains are thoroughly wetted and distributed. . 4. Hold the coverslip vertically with its edges in contact with finger and thumb of left hand ; bring the lower edge down on to the left-hand end of the slide (Fig. 6) ; move the cover- slip to the right until it touches the water drop: then, support- ing its upper edge with a needle, allow it to fold down gently on to the wa~er drop. In this way the inclusion of air bubbles, which prevent distinct vision, is avoided. 5. The coverslip should now adhere sufficiently firmly to the slide, so that if the latter is held at a slope the former will not THE METHOD OF USING THE MICROSCOPE 19 move. If, when this test is applied, the coverslip shifts, too much mounting fluid has been used, and a little should be drawn away by applying the edge of a piece of blotting-paper to the edge of the coverslip. If the mounting fluid does not fill the underside of the coverslip, touch the slide and edge of coverslip with a drop of water on a glass rod ; capillarity will cause water to run from the rod to fill the space. 6. Take great care that liquids do not get on top of the coverslip or under the slide. If they do so they may be trans- ferred either to the objective, which is then rendered useless, or to the stage, in which case moving the preparation becomes difficult and damage is done to the microscope. 4. Method of Work with the Microscope EXERCISE Ia.-SETTING UP THE INSTRUMENT Position.-Place the microscope on the bench opposite the left * shoulder with the rear edge of the stage parallel with the front edge of the bench. · Tilt the arm on the foot a little so that by nodding the head the left eye* comes naturally into position above the eyepiece. Illumination. (A) Without a Condenser See that the low power objective (larger external lens sur- face) is in position ; that the swinging rod of th _ e mirrors is central and that the iris diaphragm is open. Hold.the mirrors between the fingers and thumbs of both hands and move them on the universal joint (not the swinging joint) until the field of view appears brilliantly illuminated. (B) With a Condenser Proceed as in (A), being sure that the flat mirror is reflecting the light in this instance. Then, after obtaining a fair light, * A left-handed person should read right for left. 20 PRACTICAL PLANT ANATOMY place on the stage the object to be examined and focus it with the low power. Move the condenser up and down until the image of the light source appears accurately focussed on the preparation. Then slightly lower the condenser till the image of the source of light just fades out of view. Once the microscope is set up it should not be moved bodily nor should the light source be shifted till work is concluded. EXERCISE rb. - EXAMINATION OF THE OBJECT Place the preparation to be examined over the aperture in the stage with the coverslip uppermost. Secure it by means of the clips. Always examine with the low power first. Focussing the Low Power.-Look at the side of the micro- scope and rack the objective down with the coarse adjustment until the ~xternal lens surface and its reflection seen in the coverslip nearly meet. Apply the right eye* to the eyepiece and turn the coarse adjustment upwards and towards yourself until the object comes into view. Now use the left finger and thumb and turn the fine adjustment backwards and forwards to study the details and depths of the object. It is essential to acquire the habit of keeping both eyes open. Selection of Part for Higher Magnification.-Unclip the slide and shift t it until a suitable part of the preparation appears in the centre of the field of view. Clip the slide in this position. For a powder the part chosen should show typical particles fairly well separated. In a section it should be a representative area which is thin. Changing Objectives.-Rack the tube up till both objec- tives are well above the level of the stage. Turn the double nosepiece till the high power "clicks" into position. * Left for left-handed person. t Note that the actual movement of the object is apparently reversed when viewed through the microscope. THE METHOD OF USING THE MICROSCOPE 21 Focussing the High Power.-Using the coarse adjustment and looking at the objective sideways, bring it down until its external lens surface almost touches the coverslip. * Now rack upwards with the eye at the eyepiece: very little movement of the coarse adjustment is necessary. If, after half a complete tum of the coarse adjustment the object is not in view, it has been missed: the objective has been carried up too far or the object is not directly underneath. Commence the whole focussing process again. Never attempt to focus down- wards with the eye at the eyepiece. The object would be missed again and the objective ultimately forced on to the - coverslip and both broken. Having found the object under the high power, study details and depths with the fine adjust- ment. The starch grains resemble oyster shells. They are flattened and mostly oval or triangular with rounded angles, the hilum being nearer to the narrow end and the laminations eccentric. Compound grains with two or three grainlets and semi- compound grains are fairly common. EXERCISE Ic.-DRAWING Place the paper by the side of the microscope opposite the right shoulder. Use the left eye t for the microscope and th e right eye to view the paper, so that either object or drawing may be seen by transferring attention from one to the other, but without moving the head. Use the left hand on the fine adjustment. Thus, in order to avoid strain, which leads * Microscopes are made so that after an object has been focussed under the low power, it is only necessary to turn the nosepiece and change the objecth '. es for the object to appear ill; approximate focus under the high power. In laboratories where numerous microscopes are in use, however, interchange of objectives-, repairs and ;eplacements tend to derange their original adjustment. The above precaution is necessary when the worker does not know f9r _ certain that the adjust- · ment of the particular instrument he is using is correct. t For a left-handed person the positions are reversed, the microscope having been set up opposite the right shoulder. 22 PRACTICAL PLANT ANATOMY to poor work, one eye is used for preliminary examination and the other for viewing the object during drawing. Type of Drawing Required It should be the aim of the student to portray on paper the features of the object in as clear · a manner as possible. For this purpose line drawings to a large scale should be used. Attempts at shading usually result in indefinite- ness and for this reason should be a voided. Do not use coloured pencils. All parts of objects shown in drawings should be " labelled." Their names should be written at the side of the drawing, each name being joined to its · appropriate part by a clear straight line. EXERCISE Id.-MICROCHEMICAL TEST FOR STARCH Refocus the low power on the starch. Place a drop of iodine solution on the left-hand side of the slide so that it just touches the edge of the coverslip. Then, while watching the starch, touch the right-hand side of the coverslip with a piece of blotting-paper. Water will be drawn out, iodine will run under the coverslip and will tum the starch blue. The blue substance is iodide of starch and the process just performed is called "irrigating " with iodine. Now irrigate the iodide of starch with caustic soda: it will be seen to swell, gelatinise and lose its colour. EXERCISE 2.-AIR BUBBLES Put a drop of water on the slide and allow a coverslip to drop horizontally on to it. Note the bubbles of air that are included. Focus one of the small bubbles and see that its surface appears as a black ring caused by the refraction of the light at the air-water surface. THE METHOD OF USING THE MICROSCOPE 23 5. Common Faults, their Causes and Remedies in Sequence as they may appear r. INSUFFICIENT LIGHT. May be due to: High power in position · by mistake . Change objectives. Swinging rod of mirrors not central . . Centre it. Flat mirror being used with too weak a light or hollow mirror with condenser Change mirror. Too far from or too near light source Diaphragm shut . Adj'ust distance. 2. SPECKS IN FIELD OF VIEW : Dirt on objective or eyepiece If specks rotate . If they do not Open it. Rotate eyepiece body tube. Clean eyepiece. Clean objective.* 3. THE OBJECT CANNOT BE FOCUSSED CLEARLY: m Coverslip not perfectly pol- Remove coverslip, re- ished polish and replace. Exterior of objective dirty* Polish with a dry duster. · If no improvement, dirt may be soluble in water . If still no improvement, dirt may be cariada balsam Polish with a damp duster and dry off. Polish with ; a duster moistened with xylol and dry off. * The objectives must on no account be ta,k~n to pieces; only the external surface of the lens is to be :i:ioU§h~d, 24 PRACTICAL PLANT ANATOMY 4. OBJECTS APPEAR TO BE LATERALLY ILLUMINATED AND OFTEN APPEAR SIL VERY : Swinging rod of mirrors not central . Centre it. 5. INSUFFICIENT LIGHT (see · No. r) : Preparation too thick . Prepare a new and thinner section. 6. Essential Rules for Microscopy r. The microscope must be straight on bench opposite left shoulder. (For left-handed people, right shoulder.) 2. Always see that the low power is in position first. 3. Do not move microscope or the light source after you have arranged the lighting. 4. Always use the low power first. 5. Always focus upwards with coarse adjustment for both low and high power objectives. 6. Never twist the fine adjustment right round, but only turn it backwards and forwards. 7. Keep both eyes open and use right eye for microscope during preliminary examination and left while drawing. (Vice versa for left-handed people.) 8. Select under the low power a portion of the object suitable for higher ~agnification. g. Make and keep drawings of all objects examined. In the case of large objects, such as sections of stems, always first make a large outline drawing of the object, naming the parts, and then make a detailed drawing of representative parts, using the high power. ro. Keep coverslip, objectives and stage dry and clean. rr. Neither eyepieces nor objectives may be taken to pieces except by a qualified optician; only the external surfaces of the o~ter lenses may be cleaned. THE METHOD OF USING THE MICROSCOPE 25 rz. Always leave the microscope in a clean and dry condition after use. Failing to do ' this spoils the work of the next student as well as damaging the microscope and objectives. 7. Additional Exercises in Making Simple Mounts The following material, except where otherwise stated, may be mounted and drawn as that in Exercise r, p. r8 et seq. EXERCISE 3.-WHEAT STARCH The grains are flat circular discs, with a central hilum and concentric laminations, which are very difficult to see until the grains are slightly swollen with caustic soda. There are very numerous minute starch grains in wheaten flour. EXERCISE 4.-PEA OR BEAN STARCH The grains are kidney shaped or elliptical, with a long central hilum and concentric lamination. Very often a crack appears in the region of the hilum from which clefts radiate. The crack is due to the unequal distribution of water in the central and peripheral parts of the grains so that they contract un- equally on drying and swell unequally on soaking. EXERCISE 5.-OATMEAL STARCH The grains are large, compound and more or less spherical ; they show a network marking due to the outline of the grainlets into which they very readily break up. EXERCISE 6.-FLOOR SWEEPINGS Many foreign objects frequently · seen in microscopical preparations may be found · in sweepings. Fibres of:- Cotton, which . appear as flattened twisted tubes with rounded ends. 26 PRACTICAL PLANT ANATOMY Linen, which have pointed ends, never twist and are seldom flattened. Silk, which appear as solid rods with a more or less smooth surface. Wool, which appear as solid rods, but have an outer coating of overlapping scales. Pine wood, which have characteristic button-shaped marks on their sides. Mineral particles of various shapes and colours, all recognisable by their angular faces. Tiny particles of dust which are ~oo small for their shape to be seen but which, on account of the fact that they _ are suspended in a fluid, are " wobbling" in Brownian movement. EXERCISE 7.-SCALE OF ONION BULB Tear an onion scale obliquely and from the skin of the inside of the scale, which will project from one of the torn edges, cut · out with a pair of scissors a piece about I cm. square. Mount this in water. Examine under the low power and draw a few of the close- fitting cells, showing the cell wall and the nucleus of each. Irrigate witR iodine; focus under the high power and draw one cell and label to show : The wall and its attachments to surrounding cells. The cytoplasm. The nucleus suspended by cytoplasmic strands. The vacuole. EXERCISE 8.-LEAF OF Elodea Canadensis Mount a: single leaf of Elodea, upper side down, in a drop of the warm water (about 27° C.) in which the plant has been kept for half an hour. Find one of the cells at the edge of the leaf that projects as a t _ ooth. Examine with the high power THE METHOD OF USING THE MICROSCOPE 27 and find the nucleus. Draw the projecting cell and label the drawing to show the cell wall, cytoplasm and nucleus. Find and draw stages in the fission (division) of the chloro- plastids. Find the elongated cells in the midrib of the leaf. Focus into them with the high power. Notice the rotation of the chloroplastids, which are carried round in the moving cyto- plasm .. Observe that the direction of movement differs even in adjacent cells and in each cell there is a central neutral zone without movement. Draw two adjacent cells and indicate direction of movement by arrows. EXERCISE 9.-FILAMENTS OF LIVING Spirogyra Mount a few threads of Spirogyra in water. Examine under the low and the high power of the microscope and draw one cell as seen under the high power. Irrigate with iodine and look for grains of iodide of starch round the pyrenoids. EXERCISE IO.-CELLS OF ROSE FRUIT Remove a piece of skin from a ripe rose fruit. Scrape some of the cells from the inner layers of the skin into dilute glycer- ine on a slide. Observe and draw a few selected cells with chromoplastids. CHAPTER II CUTTING AND MOUNTING SIMPLE SECTIONS- MICROCHEMiCAL REACTIONS IN order to study the internal construction of plant organs very thin slices or sections are cut with a razor and viewed under the microscope by transmitted light. 8. The Razor The kind of razor most suitable for section cutting is one sold specially for the purpose. It is of the old "cut- throat" type, but differs in three essentials from the one used by the barber. It is a much weightier instrument, a quality which assists its passage through hard materials; one side of the blade only is hollow ground, the other being flat so that the blade travels straight ; it is made of fairly soft steel so that when the edge is spoiled in use a new edge may be readily ground on it. Sharpening the Razor The razor should be sharpened every time it is used, so that the edge never gets even slightly dull. For this purpose a fairly fine hone or oilstone and a strop are necessary. EXERCISE II.-CARRY OUT THE PROCESS: I. Wet the st.one with dilute glycerine or oil* and set it lengthways along the right-hand edge of the bench. * One or the other must always be used on one particular- stone. 28 SIMPLE SECTIONS-MICRO-ANALYSIS 29 2. Open the razor so that the handle continues the line of the blade. 3. Lay the flat side of the blade flat on the stone at its far end so that the length of the razor makes an angle of about 45° FIG. 7.-Method of Honing the Razor. with the stone and the heel of the edge points towards you. (Fig. 7, a.) 4. Pull the blade along to the near end of the stone, at the same time pressing the blade very gently* with the fingers of the left hand to keep it flat on the stone. 5. Using the back of the blade a_s a fulcrum, roll the razor over so that the hollow side comes to lie flat on ~he stone; pivot the razor at the heel so that it again lies at 45° to the stone and the heel points away from you (Fig. 7, b). 6. Push the blade to the far end of the stone, keeping it flat * Heavy pressure will lead to a wire edge which is detrimental to section cutting. 30 PRACTICAL PLANT ANATOMY as before by gentle, though this time slightly heavier, pressure from the fingers of the left hand. 7. Roll the razor on its back and readjust into position as in 3 above, and then repeat 3 to 7 as often as is necessary.* 8. Finish off_ the edge by a similar application on the strop, except that in this instance the back of the razor travels first. On the Stone. Razor flat. Heel of edge leading. Do not press hard. Roll razor on its back. Holding the Razor for Cutting On the Strop. Keep strop stretched tight. Razor flat. Back leading. Press moderately hard. Roll razor on its back. Bend the handle of the razor on its pivot so that it takes on an angle of 90° with the blade and so that the edge of the blade is directed towards you and the main mass of the handle away. Grasp the razor with the tips of the index and middle fingers and thumb on the metal to the left of the handle and the third finger on the slight hook which continues the blade to _ the right of the handle. 9. Cutting, Selecting and Mounting a Section For first attempts use a piece of fresh potato tuber about r x r x 5 ems. in dimensions. Keep the material thoroughly wet with water. Any drying of the material will lead to air bubbles in the section, which are to be avoided. Holding the Material for Cutting The material should be held between the index finger and thumb of the left hand, with the finger as straight as * The edge should be sharp enough to shave hairs from the back of the hand or wrist when it is fit for using in section cutting. SIMPLE SECTIONS-MICRO-ANALYSIS 3r possible, the material perfectly vertical and projecting a few mm. above the level of the finger, and the thumb as far as possible below (Fig. 8). EXERCISE IZ.-CUTTING Holding the razor and the material as explained above, flood both with water. Then lay the flat of the blade flat on the FIG. 8.-Method of Holding Material for Cutting Transverse Sections. index finger of the left hand (Fig. 8) with the heel of the edge touching the side of the material. Draw the razor from left to right with a long slicing action : using the elbow and shoulder joints but keeping the wrist stiff. Do not use the razor as a chisel. A slab of material should be removed to expose a _ flat face. This operation is called "trimming off the materiai." Do not alter the grasp of the material. Now lay the razor flat on the index finger again, but this time have the edge of the blade covering about half of the trimmed surface of the material (Fig. 8). , 32 PRACTICAL PLANT ANATOMY Repeat the slicing action, pressing the flat of the razor on to the index finger to steady it and thus remove a very thin slice or section of the material. · Obtain a number of sections in this way. EXERCISE rza.-SELECTION OF SECTION FOR THINNESS When a fair number of sections have accumulated on the razor, slide them off into · a shallow dish of water. Stir the liquid and while it is still in motion look for a shadow on the bottom of the dish that is more obvious than the section which casts it. Then find this section. EXERCISE rzb.-MOUNTING THE SECTION Carefully transfer the selected section by means of a section lifter or camel-hair brush to the middle of a perfectly clean · slide. Put on a drop of dilute glycerine and a coverslip as in Exercise I, p. 18. EXERCISE rzc.-DRAWING Make a clean-cut line drawing of the section as seen under the low power to show the shape and relationships of the cells and the distribution and shape of the starch grains. Also draw one grain as seen under the high power. 10. Additional Exercises in Cutting Simple Sections Cut and select sections of the following material in the way described in Exercise rz, p. 31 et seq., but if the material has been preserved in spirit use zs¾ spirit instead of water for flooding the razor and the material while the sections are being cut, and for immersion of the sections during selection. EXERCISE: 13.-ORCHID (Phajus grandiflora) OR POTATO TUBER, OR IRIS RHIZOME SUITABLY PRESERVED Mount selected sections in water. Observe the rod-shaped leucoplastids often clustered round SIMPLE SECTIONS-MICRO-ANALYSIS 33 the nucleus in the outer cortical cells. In the inner cells look for stages in the formation of large starch grains, each fully formed grain having a large rod-shaped leucoplastid attached to its broad end. Irrigate with iodine. Protoplasmic structures go yellow to brown ; starch goes blue. Draw cells containing leucoplastids only and others showing stages in the formation of starch grains. EXERCISE r4.-Pellionia pulchra STEM Cut sections as nearly as possible at right-angles to the length of the stem. Mount in water . . Examine closely under the high power and observe the small chloroplastids in the outer cortical cells and those in the inner cells, each developing a starch grain. In the cells nearest the centre the fully formed starch grains appear to have a green cap (actually the chloroplastid) at the broad end. Irrigate with iodine. · Draw stages in the formation of starch grains. IeI . Micro chemical Reactions Many structures seen under the microscope may be recognised by their shape, but in order to be perfectly certain as to the material of which they are composed,, ch e mical tests are performed upon them which give definite changes with particular substances. Thus, if a colourless oval granule thought to be starch is treated with iodine and a blue to black colour results, the assumption becomes definite knowledge. This principal microchemical test may be followed by a confirmatory test. When the blue iodide of starch is treated with caustic soda or caustic potash, swelling and loss of colour confirm the presence of starch. Sometimes two reagents 3 34 PRACTICAL PLANT ANATOMY are used one after the other to produce the primary result. Sometimes one section is used for the primary test and a second section has to be treated differently for confirma- tion. TABLE OF PRINCIPAL SIMPLE MICROCHEMICAL TESTS In the table given below, the method of application and the reagent are followed by the change in colour or other Method of application. r. Mount in ... .. Examine (quickly in the case of preserved material) for .. S ect ion will also show . . ...... . which may enclose or ... . ....... . or ........... . 2. Heat (r) ..... . 3. Remount ( 2 ) in 4. Mount in ..... 5. Soak sections of fresh material in ......... . 6. Mount in ...... . 7 . Mountin 8. Mount in 9 . Soak in ...... . ro . Mountin - , Reagent. water. dilute acetic acid .. 75% sulphuric acid absolute alcohol .... ferric chloride solu- tion ........... . mixture of iodine and 1o% ammonia .. tincture of alkanet . go% alcohol ..... . Positive result. Crystalline cell con - tents Crystals dissolve ... Crystals do not dis- solve .. .. ..... . . Crystals dissolve .. . Crystals do not dis- solve . .... . .... . Crystals dissolve and r eprecipitate as small needles .... Precipitate in cells . Blue-black or green colour ......... . Bright red colour .. Red or pink coloura - tion ........... . Material dissolves .. . Indicating. { Inulin } Calciumcarbon- (2) ate Calcium oxalate Cell walls (ro) Liquid contents only (5, 6, rg) Homogeneous solid contents (8) Contents not homo- geneous ( 2 10 1 ) Inulin ( ) . . . . . . . . . . . . . . . . (3) Calcium carbonate. . .... . ......... . (4) Calcium oxalate. Sugar or Inulin (rg) Tannin (7) Tannin confirmed. Resin (g) or Oil (15) Resin confirmed. iodine . . . . . . . . . . . . Blue to black . . • . . Starch (II) or Yellow to brown . . . Protein (1 2 and 13) Lignified wall (14) Cuticularised wall (15) Suberised wall (15) SIMPLE SECTIONS-MICRO-ANALYSIS 35 Method of application. 11. Remount (10) in 12. Cover with .... Add ......... . 13. Mount in ..... . q. Soak in ...... . Drain off excess and add .... Drain off. excess and mount in. 15. Mount in 16. Irrigate (15) with Examine quickly 17. Soak in Drain off and mount in . ... 18. Mount in ..... . 19. Cover with ... . and heat ... . Reagent. caustic soda solution strong nitric acid and warm* strong ammonia (Xan lioproteic test) Millon's reagent and heat .......... . phloroglucin, 3 mins. strong hydrochloric acid, glycerine •........ sudan III ........ . ether . iodine, 3 mins. 75% sulphuric acid chlor-zinc iodine ... (Schultze's reagent) Fehling's solution 20. Cover with . . . . dilute hydrochloric acid. Heat. Neutralise with . Cover with . .. . and heat ... . 2r. Cover with ... . Add ......... . Warm ....... . strong caustic soda. Fehling's solution 10% so luti on of a.-oaphthol. few drops of strong sulphuric acid . Positive result. Loss of colour,'and gelatinisation ... Orange yellow ..... Brick reel ....... . Red to purple .... Slowly red ...... . Red drops dissolve . Blue and swelling .• Brown, hut no swell- ing even after some hours . ... . Blue-violet ....... . Red precipitate .... No red precipitate .. Red precipitate .... Deep violet. cl • Indicating. Starch confirmed. Protein confirmed (13) Protein confirmed . Lignified walls con firmed (possibly also suberised walls) (15) Suberised or cuticu larised wall con firmed (17) Fat or Oil in the ce ll (16) Fat or Oil confirmed. Cellulose wall (18) Cuticularised wall confirmed. Cellulose wall con- firmed. Gluccse, Fructose or Maltose. • . . . . . . . . . • . . • (20) Sucrose or Inulin ('21) Inulin confirmed . ' ~ * Care must be taken that ' the fumes of this acid do not come into contact with the microscope. change which indicates the presence of a particular sub- stance. Numbers following the indicated substance refer to the: 36 PRACTICAL PLANT ANATOMY number of the confirmatory test which should be applied. Numbers following the method of application indicate that the same section from the test so numbered should be used again. Otherwise a new section should be used for each test. In all cases the reagent should be applied to the section on the slide and it should be covered with a coverslip before it is examined under the microscope. 12. _ Exercises in Simple Micro-analysis Cut and select sections of the following material in the way described in Exercise 12, pp. 31 and 32, but if the material has been preserved in spirit, use 25 % spirit in- stead of water for flooding the razor and keeping the material wet, or, if the material has been preserved in the dry condition, cut it with a dry razor. Having obtained positive results from the tests enumer- ated in the exercises be sure to apply the confirmatory tests indicated in the table, pp. 34 and 35. Record the methods used, make notes on the results obtained and deductions made in a table similar to that given in No. rr. EXERCISE 15.-FRESH APPLE, PEAR OR GRAPE Treat fairly thick sections as _ directed in the table, Nos. 1, 5, 19 and 17. After 19, if the section is gently washed and mounted in water or glycerine the granules of copper oxide will be seen in the cells which contained sugar. EXERCISE 16.-FRESH PARSNIP Treat sections as directed in the table, Nos. 1, 5 and 19. Note the results. SIMPLE SECTIONS-MICRO-ANALYSIS 37 EXERCISE 17.-ARTICHOKE (Helianthus tuberosus) TUBER, FRESH Treat sections as directed in table, Nos. I, 5, 19, 20, 21. Note the i"esults. EXERCISE 18.-ARTICHOKE (Helianthus tuberosus) TUBER, PRESERVED _ Treat sections as directed in table, Nos. I, 2, 21. Note the results. Also mount a section in glycerine and make a drawing under the low power to show distribution of the sphrerocrystals of inulin in the cells. EXERCISE 19.-PEA OR BEAN SEED, PRESERVED IN SPIRIT Remove the seed coat and cut sections at right-angles to the flat face of the cotyledon. Treat as directed in table, Nos. I, 10, I I , 12, 13. Note the results. Also mount a thin section in dilute glycerine and observe the mass of small rounded protein granules in which the starch grains are embedded. EXERCISE 20.-ALMOND, DRY Mount a section in water, warm and observe the drops of oil in and around the section. Treat other sections according to the table, Nos. IO, 12, 13, 15 and 16. Note the results. EXERCISE 21.-CASTOR OIL (Ricinus communis) SEED, DRY Break the brittle seed coat from one end only of the seed. Cut sections of the endosperm with a dry razor and _ treat as in Exercise 20. Note the results. Also dissolve out the oil from a number of sections with absolute alcohol in a watch glass. Mount one section in dilute glycerine and observe the sharply defined crystalloid and globoid in each aleurone grain, .v 38 PRACTICAL PLANT ANATOMY the outer layer of the grain having swollen up and become transparent. Mount another section in glacial acetic acid and observe the crystalloids swell up and disappear while the globoids appear alone for a time and then dissolve. EXERCISE 22.-BRAZIL NUT OR WALNUT, DRY Remove the outer shell and cu _ t sections of the food reservoir. Treat as in Exercise 21, p. 37. Note results. The aleurone grains have crystalloids but no globoids. EXERCISE 23.-SUNFLOWER (Helianthus annuus) FRUIT, DRY Treat as in Exercise 21, p. 37. Note results. The aleurone grains have no crystalloids. EXERCISE 24.-ANNUAL LUPIN (Lupinus hirsutus) SEED, DRY Cut a seed across with a sharp penknife, and so expose a flat face. Make the usual section-cutting actions, with a dry razor, applying firm pressure but not force to the dry seed, and a roll of tissue will be removed. Transfer this to a drop of water on a slide. The roll will uncurl and become flat. Proceed in the same examination according to Note results. to prepare other sections for e, Nos. ro, 12, 13, 17 and 18. Examine, also, ~ a section mounted in dilute glycerine and observe the thick cell walls; the intercellular spaces (black, • owing to the optical effect of the included air) ; and the simple tablet aleurone grains. ExERCIS~ 25.-Tetragonia crystallina or Myriophyllum STEM Cut sections of the stem as nearly as possible at right-angles to the length. Mount in water, draw cells to show the cluster crystals and SIMPLE SECTIONS-MICRO-ANALYSIS 39 treat sections . according to the table, Nos. 1, 2, 3 and 4. Note result~. EXERCISE 26.-HYACINTH (Hyacinthus orientalis) BuLB, SCAPE OR L , EA VES Cut a bulb or a piece of scape vertically down the middle and then cut sections parallel with the surface so exposed, or hold four or five pieces - of hyacinth leaf in a bundle and cut sections parallel with the length of the leaf. Treat as in Exercise 25, p. 38. Note results. Draw cells containing raphides. EXERCISE 27.-BELLADONNA (Atropa Belladonna) RooT Cut sections at right-angles to length. Treat as in Exercise 25, p. 38. Note results. Find cells showing innumerable minute crystals ( crystal sand). EXERCISE 28.-Kleinia hastata OR LIME (Tilia) STEM Cut sections of the outer tissues parallel with the length of the stem. Draw cells containing single tablet crystals and treat as in Exercise ~5, p. 38. Note the results. EXERCISE 29.-Sedum spectabile STEM Cut sections at right-angles to the length. Treat as in the table, Nos. 1, IO, 14, 15, 17 and 18. Note results. EXERCISE EXERCISE EXERCISE This space is left blank for notes on exercises other than those given above. L- 40 PRACTICAL PLANT ANATOMY 13. Conventional Methods of Cell Wall Drawing In order to avoid obscurities and complications in drawings the student is advised to follow certain conven- tions in drawing cell walls. (a) A cellulose wall unless it is very thick should be represented as one line, the thickness of the · wall being ignored. (b) A very thick cellulose wall such as occurs in collen- chyma should be represented by a breadth of paper between two suitably spaced lines. In effect the cavities of the contiguous cells are delineated. (c) . A lignified wall should be represented in a similar manner, but distinguished from a thick cellulose wall by a median line in the position of the middle lamella of the wall which is usually visible in the section. EXERCISE 30.-ELDER (Sambuc u s ni gr a) STEM, YOU N G Cut a ver y, thin section as nearly as possible at right-angles to the lengtli of the stem. Treat with phloroglucin and hydrochloric acid (Microchemical Table, p. 34, test 14), focus under the low power and draw: Three or four non-coloured cells with thin ce1lulose walls from the middle of the stem. A few of the cells with thick cellulose walls that are to be found in patches just under the surface. A few of the "red," lignified elements. CHAPTER III CUTTING, MOUNTING AND DRAWING STEM SECTIONS 14. Types of Section SECTIONS of stems may be cut with advantage in three planes (Fig. g). (a) Transverse sections, in the plane at right-angles to the length of the stem. · (b) Longitudinal radial sections, parallel with the length of the stem, with one d f h · f Transverse e ge O t e section orm- section ing a radius of the trans- verse section. (c) Longitudinal tan- gential sections, parallel with the length of the stem and at right- angles to (b), but not ~-----Longitudmal · radial seclton ~~~_..,q...;;..i ;,, Long,tudmal ~ tantJenbal sec!,on ~ i ~ ~ '.1i, 1· passing through the centre. Transverse sections FIG. 9.-To illustrate the Planes of the Three Types of Section. are of value in studying the general distribution of the tissue elements in relation to one another. Longitudinal sections must be made for the purpose of studying the individual element and the distribution and construction of such structures as medullary rays. Nearly every element in a stem is so constituted that certain of its walls are in the transverse and certain in the longitudinal planes. Since the thinnest section has some 41 42 PRACTICAL PLANT ANATOMY thickness, unless it is in one or other of these planes it will not appear distinct under the microscope. In truly transverse sections the longitudinal walls of the elements stand vertical on the slide, and hence the part of the wall that is not in focus at the moment will be masked by the part that is. Thus provided a section be perfectly transverse it may be fairly thick and yet be clearly seen. On the contrary, in a section which is even slightly oblique, the longitudinal walls stand sloping on the slide. As a consequence, no matter at what level the microscope is focussed, parts that are in focus will be blurred by those which are not. Truth of direction of sections depends primarily on the accuracy with which the material is trimmed and subse- quently on ~aintaining the trimmed surface true. This latter can best be accomplished, when transverse sections of cylindrical objects such as stems or roots are to be cut, by rolling the material between the finger and thumb a quarter of a complete turn after each section is removed. Comparison of Transverse and Oblique Sections EXERCISE 31.-SEDUM (Sedum sp e ctabile) STEM Trim off the end of a piece of Sedum stem at right-angles to the length (Exercise 12, p. 31) and cut a number of perfectly transverse sections. Float the sections off the razor into a dish of 25 % spirit. Deliberately trim off the end of the stem to form an angle of about 6o 0 with . the length and then cut sections parallel with this sloping surface. Float the sections off into a second dish of 25 % spirit. From the two batches select two sections which appear equally thin (Exercise 12a, p. 32). Transfer both to the middle of one slide and treat with phloroglucin and hydro- CUTTING AND DRAWING STEM SECTIONS · 43 chloric acid (No. II, test 14), and mount in dilute glycerine under one coverslip. Examine under the low power and note : (a) That the oblique · section appears blurred in comparison with the transverse one. (b) That as the focus of the microscope is altered the oblique section appears to shift laterally while the truly transverse one does not. Examine under the high power and note : (a) That cell wall edges (and perhaps the surfaces of a few end walls) only are visible in the transverse section. (b) That the sloping side walls can be seen in the oblique section. This knowledge should be applied in selecting every trans- verse section for examination. I5, Tissue Studies EXERCISE 32.-MARROW (Cucurbita Pepo ) STEM Cut a number of truly transverse sections of Cucurbita stem. Select one for thinness (Exercise 12a, p. 32) and truth ~f direction (Exercise 31, p. 42) . Treat with phloroglucin and hydrochloric acid and mount in dilute glycerine. Examine under the low power. A ring of red elements just within the edge of the section are sclerenchyma fibres. Groups of red elements deeper in are xylem vessels. Find some rounded thin-walled cells between the sclerenchyma and the xylem. They are parenchyma (Fig. ro). Note that although the generai appearance of the cells is circular, where contact between them occurs the walls are flat. The only part of the wall that is really curved is that which bounds an intercellular space. Draw a few cells, using the single line to indicate the thin cellulose wall. Label the drawing. Find some thick-walled colourless collenchyma (Fig. II ) just inside the surface. Focus under the high power. Draw 44 PRACTICAL PLANT ANATOMY by outlining the cell cavities and allowing paper surface between to represent the thick cellulose walls. Small angular cavities · are intercellular spaces. Label the drawing. Wall flattened - - - between two cells '\ - - - - Cell cavity FIG. ro . - Parenchyma Cells from Ecballium Stem. X 275 approx . focus the sclerenchyma under the high power (Fig. 1 2 ). The walls are thick and have running through them a central layer of different optical properties to t he main mass of the wall. This layer shows as a line in transverse sections and is called the middle lamella . Draw the middle lamella first very lightly (Fig. 12, a ). (In 0 \..J ~ 0 V-L,mlt of cellcav1ty ) Q~ ov llntercellulal' space 0 0 O "'c-/-Th,clmess of wall )tfoo 0..., \\ Diameter of 1 ~ cell cavtty FrG. r r. - Collenchyma Cells from E cballium Stem as seen in Transverse Sections. X 275 appro x . places where it is not obvious it may be assumed to run through the middle of the wall.) Then outline the cell cavity (Fig. 12, b), taking care to get the correct proportion between the CUTTING AND DRAWING STEM SECTIONS 45 diameter of the cell cavity and wall thickness. Label the drawing. Find the phloem (Fig. 13) just outside · the xylem. _ The A Oiame!er of cav,fy FIG. 12.-Two Stages in drawing Sclerenchyma Fibres as seen in a Transverse Section of Ee ballium Stem. X 275 approx. walls are of cellulose and relatively thin, hence they are to be represented as single lines. There are no intercellular spaces, so the elements are polygons whose corners meet. In this instance the sieve tubes are large, companion cells small and phloem parenchyma cells of intermediate size. In drawing commence with one polygonal element. Then - Phloem parenchyma Compamon Cell Sieve tube FIG. 13.-Phloem as seen in a Transverse Sectiop of Ecballium Stem. X 275 approx. draw the walls of the contiguous elements that project from its corners. Complete the elements so partly drawn and repeat · the process. Label the drawing. 46 PRACTICAL PLANT ANATOMY The xylem consists of xylem vessels and other elements (Fig. 14). The latter may have lignified walls or cellulose ones. They may be tracheids or lignified parenchyma cells or non- lignified parenchyma. All the elements are more or less · polygonal. To draw: Sketch in lightly the large xylem vessels (Fig. 14, a). Draw out from them, in the correct positions, lines for the A 0 0 B C FIG. r4.-Stages in Drawing the Xylem of Ecballium Stem as seen in Transverse Section. X 275 approx. radiating walls of the non-lignified contiguous elements or for the middle lamella of lignified ones (Fig. 14, b). Flatten off the faces of the outside of the xylem vessels , by drawing chords between the inner ends of the radiating walls of the contiguous elements. Complete the latter and continue as for phloem (Fig. 14, c). Then draw in the line representing the inner surface of the wall of all lignified elements, taking care that the true propor- tions are maintained (Fig. 14, d). Label the drawing. CUTTING AND DRAWING STEM SECTIONS 47 EXERCISE 33.-MACERATED SUNFLOWER (Helianthus annuus) STEM Mix a very small drop of macerated sunflower stem with dilute glycerine on a slide and put on a coverslip. Find and draw the following isolated elements: A parenchyma cell. Some will appear almost round', but most are cubical. A sclerenchyma fibre, which may be recognised by its long narrow · shape, its sharp pointed ends and its very thick wall. A length of pitted vessel. Vessels usually break up into lengths during the process of maceration. These are recognised by their tubular shape and the numerous spots-pits-in their walls. A length of spiral vessel, which resembles a length of spiral spring. A length of annular vessel, which may be recognised by the separate rings of thickened wall. Sometimes maceration causes solution of the cellulose tube in which these are set and the rings become isolated. EXERCISE 34.-MARROW (Cucurbita Pepo) STEM Cut longitudinal tangential sections by shaving away one of the ridges of the stem. Treat some with phloroglucin and hydrochloric acid and mount in dilute glycerine. Mount others in magenta and cotton blue in lactophenol. * The sections from nearest the surface will show collenchyma, parenchyma or sclerenchyma. Those from deeper in that have passed through the vascular bundle ( seen as a whitish streak) will show phloem or xylem. Search for sclerenchyma and xylem in the phloroglucin preparation and make labelled high power drawings of a few elements of each. In the magenta and cotton blue preparations search for * See No. :r6, p. 48. 48 PRACTICAL PLANT ANATOMY regions which show dense blue transverse blobs. These are sieve plates. Draw, under the high power, one or two sieve tubes with their companion cells, and label the drawing. EXERCISE 35.-BOTTLE CORK Cut a very thin section of bottle cork. Soak in alcohol to remove the air and then mount in sudan III. Note that the walls gradually pick up the colour of the stain, orange yellow to red (Microchemical test for suberised walls). 16. Colouration of Sections In · order to make the various structures in a section more readily visible, and also to assist in distinguishing the kinds of elements, a section is often treq,ted in such a way as to colour some or all of them. TEMPORARY PREPARATIONS The microchemical reactions listed in No. r r may be used for this purpose. They yield results which are diagnostic of the facts to which they apply, but the preparations so made cannot be kept in a satisfactory condition for more than a few days. Hence such prepara- tions are called temporary preparations. DOUBLE STAINED AND PERMANENT PREPARATIONS Oth~r methods of treatment entail the staining or dyeing of the substance in the section, and in many instances different material in the same section may be stained contrasting colours. The result is a " double stained preparation." The colourings obtained by these methods ;lre seldom of accurate diagnostic value, but such preparations may usually, by suitable subsequent treat- ment, be made into "permanent preparations" which will keep for a period of years. CUTTING AND DRAWING STEM SECTIONS 49 (a) Magenta and Cotton Blue Method A very thin section is mounted in a solution of two dyes, magenta and cotton blue in lactophenol.* Almost immediately, protein, protoplasm and mucilage stain blue, lignified walls always, and suberised walls, sometimes, stain red slowly and cellulose walls stain blue ultimately. The section may be examined and drawn while still in the stain mixture, and this is advisable since staining im- proves as the sect.ion soaks. The section may be kept, in a horizontal position, mounted as it is in the mixture which has no tendency to dry, and in about 7 to 14 days' time staining will be at its best. The preparation may then be made permanent. (b) To Make a Magenta and Cotton Blue Preparation Permanent _ r. Remove the coverslip, wipe away as much stain as possible without touching the section or letting it dry. 2. Put a few drops of methylated spirit on one end of the slide; tip it backwards and forwards over the section and then off the slide into the sink. 3. Repeat this process a number of times, substituting absolute alcohol for methylated spirit in the last two washings. This should remove all traces of water. (Dehydrate.) 4. Give one final rinse with absolute alcohol and quickly cover the section with a drop of xylol. The section should become much more transparent as the oily liquid soaks into it and hence the process is called clearing. * Ann. Bot., XLVIII, No. CLXXXIX, Jan. 1934. 4 50 PRACTICAL PLANT ANATOMY 5. Put a drop of canada balsam on to the section and then cover it with a coverslip. 6. Stick a label on one end of the slide and on it write a brief description of the object mounted. 7. Keep the slide horizontal till the balsam has set .. (c) The Safranin and Light Green Method r. Soak a very thin section in a few drops of safranin on a slide for 5 minutes. If the stain, which is dissolved in 75 % spirit, tends to dry, add a little more. 2. Tip off the excess stain. 3. Put a few drops of methylated spirit on one end of the slide ; run . it backwards and forwards over the section and then tip it off into the sink. 4. Repeat 3, using absolute alcohol, till a quick glance at the section under the low power of the microscope shows that the red dye has been nearly removed from all except the lignifi.ed walls. 5. Give one final rinse with absolute alcohol and quickly cover the section with a drop of light green in clove oil and a coverslip. The light green dye will gradually stain the cellulose walls and the oil clears the section. The section may riow be drawn and will keep in this condition for a few days. It should not be kept too long in the light green in clove oil or the red will be masked by excessive green staining. (d) To Make a · safranin and Light Green Preparation Permanent r. Lift the coverslip. 2. Rinse off the oil of cloves with xylol. * · * Unless the oil of cloves is completely removed the preparation will fade. CUTTING AND DRAWING STEM SECTIONS 5r 3. Transfer the section with a section lifter or small brush to a clean slide. 4. Quickly put a drop of canada balsam on top of the section. 5. Put on a coverslip and keep the preparation hori - zontal till the balsam has set. FAULTS WHICH MAY APPEAR IN DOUBLE STAINED PREPARATIONS Method : Magen ta and cotton blue. Safranin and light green . Magenta and cotton blue, or safranin and light green. Mounting in balsam. Fault. Sufficient light can- not be made to penetrate a tem- porary mount. Differential staining does not occur. Safranin remains in non-lignified ele- ments. Safranin persists after repeated washing with abrn- lute alcohol. The removal of sa- franin from non- lignified elements is accompanied by its removal from lignified ones. Whole section in xylol or oil of cloves appears whitish to the naked eye and cel- lulose walls blue- black or green- black under the microscope. Patches of the sec- tion have the ap- pearance noted above. Air bubbles in sec- tion . Air bubbles in bal- sam. White patches in bal- sam. Cause. Section is too thick. Section not left long enough in stain. The method is not suitable for the particular material. Incomplete washing with · absolute . al- cohol. Cells contain tan - nin or mucilage. }section is too thick Section not left long enough in sa- franin. Water in section. (a) Incomplete dehydration, or (b) Breathing on section after dehydration . Slide was not dry. Section allowed to dry or to fl.oa t on top of one or other of the liquids used. Balsam stirred too vigorously. Slide not dry . Cure. Cut a thinner section. Rerooun t the section in the stain mix- ture. Treat another section in another method. Repeat the process. Ignore . Cut a thinner one . Commence staining again. Repeat washings with absolute alcohol and restain if necessary. Repeat dehydration and use clean dry slide;. Soak section for some hours in absolute alcohol or prepare a new one. Prick bubbles with a hot needle. Use another. 52 PRACTICAL PLANT ANATOMY (e) (!) This page is left blank for notes on other methods of staining which may be preferred to those given in th e prec e ding pages. CUTTING AND DRAWING STEM SECTIONS . 53 6. Stick a label on one end of the slide and on it write a brief description of the object mounted. I 7. How to Study Transverse Stem Sections For first attempts use pieces of Sunflower (Helianthus annuus) or Dahlia stem that have been selected to show the primary structure and have been preserved in 75% spirit. Keep the material immersed in 253/o spirit when it is not actually being cut, c!,nd while it is being cut keep both the material and the razor blade wet with the same liquid. In studying any structure always examine gross features first and work through to smaller and smaller detail. Hand Lens Study EXERCISE 36.-PREPARING A SLICE FOR HAND LENS STUDY Trim off the end of a piece of stem at right-angles to its length. ~ - - - Eptderm1s Codex Medullary My Vascular bundle ---r-:r----'~Pith : · Part shown 1n ':"""zone drawmq ; See Fig . 16. I FIG. 15.-Hand Lens Drawing of Transverse Section of Sunflower Stem. x ro approx. Cut a fairly thin slice right across the stem; transfer it to the middle of a slide, treat it with phloroglucin and hydro- 54 PRACTICAL PLANT ANATOMY chloric acid (No. II, test 14), and mount in dilute glycerine. Examine under a hand lens. It will be possible to see the vascular bundles, each showing "red" patches • of sclerenchyma and xylem · towards the outside and inside respectively. Drawings should show only so much as can be seen accurately. _ It will be found in the present instance that although '), the xylem vessels can be seen, their exact shape is not discernible, nor for that matter is it easy to decide the exact shape of the groups of xylem, though one can just do so in the case of the bundles as a · whole. · All that can be determined is: The shape of the section. The number, position, relative size and shape of the vascular bundles. The position of the cqrtex, pith and medullary rays. EXERCISE 36a.-'DRAWING UNDER THE HAND LENS Construct a labelled drawing of the transverse slice of the stem seen under a hand lens, indicating the facts e numerated above only. Draw to -such a scale (which · state) that all the above- mentioned parts may be accurately delineated, and label them all (Fig. 15). _ Microscope Study. Until students become expert in section cutting they are advised to aim to cut sections which include samples only of the entire structure and to avoid trying to obtain sections right across the organ. CUTTING AND DRAWING STEM SECTIONS 55 EXERCISE 3;',-CUTTING AND SELECTING A SECTION FOR STUDY UNDER THE MICROSCOPE Having trimmed off the material at right-angles to its length, lay the razor flooded with 25 % spirit on the trimmed surface so thatits edge is along a diameter. Cut with a slicing action and aim to remove a section which is a sector and includes one ..: eighth to one-quarter of the complete transverse section (Fig. 16). After cutting the first section rotate the material about one-quarter of a complete turn so that the razor enters it to cut the second section near where it emerged after cutting the first. Repeat _ the processes and so obtain a number of sections. If, d~ring sectioning, the end of the material becomes even slightly oblique, trim it off to renew the perfectly transverse surface. Select a section for ( a ) thinness (E x ercise 12a, p. 32). (b) truth of direction (Exercise 31, p. 42 ) . Treat with phloroglucin and hydrochloric acid and mount in dilute glycerine (No. II , test 14). Examine under the low power. The Zone Drawing It will be found that in a satisfacto_ry section, especially at its thin edges where the razor entered or came out of the material, every element may be seen. Many of them, however, are too small for their exact shape to be deter- mined and hence they cannot be drawn. On the other hand, in addition to what was visible under the hand lens one can now. see the lines of demarcation between the collenchyma just under the epidermis and the parenchyma within; the sclerenchyma _ and the phloem; the phl o em and the cambium; and the cambium and the xylem. EXERCISE 37a.-DRAWING UNDER THE Low POWER Construct a" zone drawing" in which the various groups · of tissue are drawn true to shape in outline, but in which no · 56 PRACTICAL PLANT ANATOMY individual element is shown (Fig. 16). The drawing should be · labelled and include sufficient of the section to allow the rest to be imagined, and should be to a stated scale of about FIG. 16 . -Zone Drawing of Transverse Section of Sunflower Stem. X 20 approx. x 50. When the drawing is completed its area should be indicated in the hand lens drawing (Fig. 15). High Power Drawing The hand lens and low power zone drawings show the distribution of the tissues : all that remains to be repre- sented is the shape and relationships of the various ele- ments. For this purpose it is best to draw a radial strip of the section as seen under the high power. This need be only two or three cells broad, and where any tissue is deep radially, much ofit may be omitted if the amount omitted is indicated in the drawing (Fig. 17). Students usually find great difficulty in deciding on what scale to make their drawings, and having done so in keep- CUTTING AND DRAWING STEM SECTIONS 57 ing to it. Drawings cannot be too large except for con- venience; students frequently make them too small for The DepJh Unit of Zone Drawing - } l One • Componton Cell ~ '§ f,.i II') {1 'X_ylemlw,ce ~ \ as deep as e Phloem Metaxjlem vesseJ t, :-(::) i.t Pf'o!orylem vessel ~ { Xylem parencf!Jlma c£ ~ FIG. 17.-Illustrating a method of planning a High Pow e r Drawing. Part of Transverse Section of Sunflower Stem. X no approx. accuracy. They are advised to follow some scheme for determining the scale and maintaining the proportions such as is given in Exercise 37b. 1 58 PRACTICAL PLANT ANATOMY For the drawing reproduced in Fig. r7 the companion cells were represented with an average diameter of z mm. The complete phloem region in the section had a radial depth equal to 15 companion cells= drawing depth of 30mm. The sclerenchyma region was found to be one and a half times as deep as the phloem and the xylem twice as deep, and so on. To avoid much repetition of drawing and to allow the complete sequence of tissues to be shown in one drawing it was decided to omit : ¾ of the sclerenchyma. ½ of the phloem. ½ of the xylem. In Fig. r7 the sieve tubes are on the average two and a half times as broad as the companion cells. They were drawn approximately 5 mm. in diameter. EXERCISE 37b.-DRAWING UNDER THE HIGH POWER Select under the low power the thinnest and clearest region of the section. Indicate this region in the z~ne . drawing. Focus under the high power. Find the smallest type of element-often the companion cells of the phloem. The exact shape can be seen and the scale of the high power drawing must be large enough to allow accurate representation and full labelling. Make a trial of the size necessary. Use the actual cell as a unit by which to estimate the extent of the various tissues in the section, and the drawing just made of it as a unit of paper measurement, and by this means plan out in light lines a large zone drawing of the area it is intended to draw under the high power. Then draw in the elements. CUTTING AND DRAWING STEM SECTIONS 59 To assure that the individual elements are drawn to scale, count the number of elements along a radial line through the zone and divide the corresponding region of the zone drawing similarl y . Also check the estimate of element size so made by comparing with the unit (com- panion cell). r8. Longitudinal Radial Sec_tions EXERCISE 38.-SUNFLOWER (H elianthus annuus) STEM P re paration of Surface for Cutting Select a piece o{ stem one to one and a half inches long, and with a sharp knife or razor cut it lengthways down the middle so that the cut divides two vascular bundles on opposite sides of the stem. Hold in g th e Mat e r ia l Grip one piece of the halved stem about midway along its length between the tips of the thumb and middle finger of the left hand, so that the curved face of the material is directed towards the palm of the hand. Support the material in a \. horizontal position by putting the tips of the index and third fingers under the rounded face (Fig. 18). C uttin g Lay the razor flat on the trimmed face of the material with its edge about 2 mm. from the end nearest the little finger (Fig. 18), and with a slicing action aim to remove a very thin section including all tissues from the surface to the centre of . the stem. After a number of such sections have been removed the vascular bundle will have been cut away from the end · few mm. Then commence cutting by laying the razor on · 60 PRACTICAL PLANT ANATOMY the material with its edge about 4 mm. from the end, and so on. Selection of Section and Mounting Select a section for thinness as in Exercise rza, mount it in water and examine under the low power. If it has not been cut approximately longitudinally the lumina of the vessels FIG. 18 . -Method of holding material for cutting Longitudinal Radial Sections . will appear as obvious holes in the section. If these do not show, the section is sufficiently truly longitudinal. Treat it with phloroglucin and hydrochloric acid and mount in dilute glycerine. Examine under the low power. A truly radial section will show annular, spiral and pitted vessels in that sequence from the inside outwards. <" 1o · CUTTING AND DRAWING STEM SECTIONS 6r Interpretation of Longitudinal Sections Owing to the fact that most of the elements have curved or faceted longitudinal walls, longitudinal sections are not as diagrammatic as transverse ones. Comparison of position of structures seen in the longitudinal section with the same relative position in transverse ones will often assist students in interpreting them. Drawing Zone drawings need not be constructed of longitudinal sections unless the distribution of the tissues differs from what would be expected from the appearance of the transverse section. That is not the case in the present instance; hence a high power drawing only is necessary. Sketch out the zones lightly, determining the proportions in the same way as when drawing the transverse section (Exer- cise 37b, p. 58). Use that drawing to help you. Then draw in the individual elements. Some of each . type should be drawn in surface view and others as they appear in optical section, as the section would appear were it thin enough. Label the drawing. Care should be taken that the ends of elements of each type are included, as these are characteristic in certain instances. 19. Drawing Secondary Tissues EXERCISE 39.-SUNFLOWER (Helianthus annuus) STEM. ORIGIN OF lNTERFASCICULAR CAMBIUM A hand lens drawing need not be constructed, since the difference between this stem and that already drawn in Exercise 36a, p. 54, would not be obvious at so small a magpification. Cut a number of transverse sections. Select one (Exercises 12a, p. 32 and 3r, p. 42), and mount in magenta and cotton blue in lactophenol (No. r6, a). Construct a zone drawing to show position of interfascicular cambium and high power drawing of part of the cambium. Label the drawings. ✓ J 62 PRACTICAL PLANT ANATOMY EXERCISE 40.-SUNFLOWER (H elianthus ann _ uus ) STEM. SECONDARY THICKENING Cut a moderately thin but complete transverse slice and treat with phloroglucin and hydrochloric acid (No. II, test 14). Make a hand _ lens drawing to show the zones of epidermis, cortex, fibres, phloem and xylem. Cut transverse section, mount in magenta and cotton blue in lactophenol. Secondary xylem will stain red a little earlier than primary xylem. Make a zone drawing to show the A C D E regions and group your labels accord- ing to the origin of the tissues from : Apicai meristem. Fascicular cam- bium. Interfascicular cambium. Make a high power drawing to include samples of all tissues (Exercise 37b, p. 58). When drawing the secondary FIG. 19. - Stages in drawing Secondary tissues pursue the Xylem. Transverse Section of Sunflower Stem. x 275 approx. following method~ (Fig. 19). Sketch µi, lightly, parallel lines the correct distance apart for the rows of secondary elements (Fig. 19, a).. Sketch in, in · their correct positions · and proportions, the large xylem elements (Fi g. 19, b). Draw the tangential walls of the phloem and non-lignifi.ed xylem . elements and the middle lamellre of the tangential walls of the lignifi.ed elements (Fig. 19, c). · CUTTJNG AND DRAWING STEM SECTIONS 63 Complete the non-lignined elements and the middle lamellre of the lignified ones (Fig. 19, d). Draw in the inner limits of the lignified walls (Fig. 19, e). 20. Structures in Woody Stems EXERCISE 41.-LIME (Tilia) STEM, 3 TO 4 YEARS Orn. TRANSVERSE SECTION Treat in general as in No. 17 and refer to Exercise 40, ,/ p. 62, when making the high power drawing. Use the phloroglucin preparation for the hand lens drawing and show in it: Periderm. Narrow cortex. Radiating triangles of secondary phloem (red on account of the sclerenchyma present). Medullary rays (yellow triangles continued from their apices as lines ih the . xylem). Annual rings of wood. Pith. When cutting the transverse section be sure to include all tissues from pith to periderm, or if this proves too difficult, make two sections, one of the pith outwards and the other of the periderm inwards, and build up the zone drawing from both. In the zone drawing differentiate in addition to those features shown in the hand lens drawing: The medullary sheath and the primary xylem at the border of the pith. The open texture of the spring wood. The cambium. Sclerenchyma groups and sieve tube containing areas in - the phloem. · Parenchyma and collenchyma in the cortex..: -· ' Phellogen and phelleni in the periderm. Primary and secondary medullary rays. 64 PRACTICAL PLANT ANATOMY High Power Drawing: Use the phloroglucin preparaHon for the lignified elements and the preparation in magenta and cotton blue in lacto- phenol (No. 16, a) to study living elements. For the high power drawing select a strip whose midline is one of the primary medullary rays. Draw four parts only of the strip : The part including the periderm and cortex. A small region around the point where a medullary ray crosses the cambium. •.;_~ , A small part where spring and autumn wood ar~ in contact. A part of the pith and primary xylem. In the xylem region show : The large vessels. These have bordered pits on the side walls adjoining other vessels qr tracheids. The tracheids with cavities smaller than the vessels and bordered pits on the , side walls. The wood fibres, recognised by their small cavities, simple pits and the absence of contents. Xylem parenchyma cells which all have a small cavity containing cytoplasm and a nucleus. The medullary rays, consisting of rows of living oblong cells with half-bordered pits where they abut on a vessel. In the phloem show : Lignified bast fibres. Flattened parenchyma cells, sometimes containing starch or crystals of calcium oxalate. The large sieve tubes with small companion cells. The narrow secondary medullary rays, crossing all the zones radially. EXERCISE 42.-LIME (Tilia) STEM, 3 TO 4 YEARS OLD. J LONGITUDINAL RADIAL SECTION Treat as in No. 18. The development of the lime stem is often eccentric, so that CUTTING AND DRAWING STEM SECTIONS - 65 the pith is situated a little to one side of the centre of the stem. The arrangement of the tissues is radial around the pith, hence in this case the stem must be split through the centre of the pith (the organic centre) instead of through the true centre. A zone drawing should be constructed in which the limited depth of the medullary rays is indicated. EXERCISE 43.-LIME (Tilia) STEM, 3-4 YEARS OLD. LONGITUDINAL TANGENTIAL SECTION S e ctions required: Longitudinal tangential sections are cut longitudinally down the stem and at right-angles to the longitudinal radial section (Fig. 9, p. 4;r:). They are specially useful for studying the "end on" appearance of medullary rays, the edges of radial walls and the surface of tangential walls of the elements. The only parts of these sections that are of valu,e are the middle parts close to line A-Bin Fig. g. On either side of this line the medullary rays, etc., run obliquely through the section and are difficult , to see and understand. Hence when it is required to study the tangential appearance of material, sections must be cut at various depths corresponding to the distribution of the tissues. In this instance the construction of the secondary phloem and xy lem are of most importance. C uttin g th e S e ct i o n s Hold the material which has been used in the last exercise as directed in No. 18, but with the rounded face uppermost. Trim off just sufficient of the rounded surface to remove the periderm and the cortex. Then cut small sections from the surface of the exposed phloem. Later trim down to the xylem and again aim for small sections cut at right-angles to the medullary rays. 5 66 PRACTICAL PLANT ANATOMY Mounting Treat some sections with phloroglucin and hydrochloric acid and mount in dilute glycerine. Mount others in magenta and cotton blue in lactophenol. The latter will show the sieve plates and the contents of living cells stained blue. Drawing Construct two zone drawings ( 1) of the phloem region and (z) of the xylem region. Make high power drawings of representative parts of both. EXERCISE 44 . .:..._ELDER (Sambucits nigra) STEM GATHERED AT V END OF FIRST YEAR'S GROWTH. LENTICEL AND PERIDERM - Make a morphological drawing to show the distribution and shape of the lenticels to be seen as brown oval patches in the surface of the stem. Preparation of Material for Cutting Select a large lenticel near one end of the piece of stem and trim the material down till the lenticel just shows in the perfectly transverse surface. Cut away, at about 45°, the end of the stem on the opposi _ te side to the lenticel so that only about a quarter of the transverse surface with the lenticel in its middle remains. Cutting Hold the material with the oblique surface towards the razor hand. Lay the razor on the transverse surface with the edge about r mm. away from the lenticel and aim for a radial strip which includes the lenticel. By cutting soft outer tissues and hard inner ones at one and the same time, splitting of the secti0n is usually a voided. CUTTING AND DRAWING STEM SECTIONS 67 Mounting Treat some sections with phloroglucin and hydrochloric acid and mount in dilute glycerine, and mount others in magenta and cotton blue in lactophenol. Drawing Make a zone drawing to show the regions: Pith, primary xylem, secondary xylem, cambium, secondary phloem, cortex, periderm and len ticel. Make a high power drawing of the lenticel and part of the neighbouring cork, cork cambium, cortex, etc. 2 I. Additional Exercises in Stem Structure EXERCISE 45.-CURLED DocK (Rumex crispus) STEM Study as in Exercises 36 and 37, p. 53 et seq., and make an ~dditional mount in magenta and cotton blue in lactophenol. Especially observe the distribution of the sc;:lerenchyma and collenchyma, and the patches of chlorenchyma inside the epidermis. EXERCISE 46.-WHITE DEADNETTLE (Lamium album) STEM Study as in Exercises 36, 37 and 38, p. 53 et seq. Observe the four pillars of collenchyma at the angles; the four open vascular bundles and the hollow pith. EXERCISE 47.-MARROW (Cucurbita Pepo) OR WHITE BRYONY (Bryonia dioica) STEM Study as in Exercises 36, 37 and 38, p. 53 et , seq. In the zone drawing show: Epidermis with hairs. Cortex of collenchyma and parenchyma. Starch sheath. Pcricycle ring of sclerenchyma. ,t. 68 PRACTICAL PLANT ANATOMY Open bicollateral vascular bundles with: External phloem. Cambium. Xylem. Internal phloem. Pith in which there is often a cavity. EXERCISE 48.-DEADLY NIGHTSHADE (Atropa Belladona) STEM Study as in Exercises 36 and 37 and make an additional mount in magenta and cotton blue in lactophenol. Keep this last and make it permanent (No. 16, b). Note especially the small groups of medullary phloem just inside the ring of primary xylem elements which border the pith. EXERCISE 49.-MAIZE (Zea Mais) STEM In monocotyledon stems the vascular bundles are arranged in several concentric circles or scattered. They are closed, i.e. do not possess a cambium, and generally have a scleren- chymatous sheath. Study as in Exercises 36, 37 and 38, p. 53 e t s e q., and make additional preparations according to No. 16. In the zone drawings show : Epidermis. C t { Hypoderma of sclerenchyma. or ex Parenchyma. Scattered vascular bundles, of which the outer ones are small and have a thick sclerenchyma sheath, the inner ones are larger and have a thinner sheath. Under the high power draw the outer part of a radial strip from the cuticle to the inner limit of one of the outermost vascular bundles, and also one of the inner vascular bundles. CUTTING AND DRAWING STEM SECTIONS 69 Each bundle is a closed collateral one and contains: The phloem, consisting of sieve tubes and companion cells. The xylem, consisting of two large pitted vessels, several spiral and annular protoxylem vessels. The innermost protoxylem vessel becomes torn so that isolated rings lie in a cavity. The sclerenchyma sheath. It is advisable that students with limited time at their disposal postpone the remainder of this section till they have studied typical roots and leaves. EXERCISE 50.-LILY (Lilium Martagon ) STEM Repeat Exercise 49, but note that the sclerenchyma ring is here in the pericycle and has outside it a quantity of chlor- enchyma. EXERCISE 51.-CREEPING BUTTERCUP (Ranunculus repens) STEM Study as in Exercises 36 and 37, p. 53 e t s e q . , and make a double stained preparation according to No. 16, c and d. The vascular bundles are each almost enclosed in a scleren- chymatous sheath and the cambium in the bundles is poorly developed and inactive. The . phloem consists entirely of sieve tubes and companion cells. Thus the bundle strongly resembles in structure a mono- cotyledon bundle. EXERCISE 52.-JAPANESE ANEMONE ( Anemone japonica) STEM Study as in Exercises 36 and 37, p. 53 et seq., and make a double-stained permanent preparation a~cording - to 1-fo. 16, c and d. There are several circles of bundles and the bundles have a very poor cambial growth; thus we have a dic o tyled o n stem resembling a monocotyledon stem. 70 PRACTICAL PLANT ANATOMY EXERCISE 53.-ELM (Ulm us ) STEM, 1 TO z YEARS OLD Study as in Exercises 41, 42 and 43, p. 63 et seq. In the drawings show : Epidermis with hairs (trichomes) and stomata. The periderm (if present), consisting of cork and cork cambium formed in the layer under the epidermis. The cortical parenchyma, amongst which are a few large cells with thickened mucilaginous walls and here and there a crystal sac. The irregular ring of sclerenchyma (bast fibres). The phloem zone. The cambial zone. The secondary xylem, containing vessels, fibres and par- enchyma. The primary xylem bundles, forming wedges round the pith. The medullary rays. The pith or medulla, parenchyma cells with pitted walls and a few large mucilage cells. · EXERCISE 54.-ELDER (Sambucus nigra) STEM GATHERED IN EARLY SUMMER Treat as in Exercise 44, p. 66, and No. r6, c and d. Study the · origin of the cork cambium in the hypoderma and of the lenticels inside stomata. EXERCISE 55.-BLACK CURRANT (R ibes nigrum) STEM Study as in Exercise 44, p. 66, and No. 16, c and d, but omit lenticel structure. , Note the deep periderm including cork cambium and phelloderm, also the dead cortical and epidermal cells. CUTTING AND DRAWING STEM SECTIONS 71 EXERCISE 56.-APPLE (Pyrus Malus) STEM GATHERED IN EARLY SUMMER Study as in Exercise 44, p. 66, and No. 16, c and d, but omit lenticel structure. Note the origin of the cork cambium in the epidermis. EXERCISE 57.-DRAGON TREE (Dracama) STEM. MONO- COTYLEDON WITH SECONDARY THICKENING Make preparations according to No. II, test 14, and No. 16, c and d. In the zone drawing show: The central primary region with scattered primary bundles which are concentric and closed, the xylem enclosing the phloem. (Theground tissuecellsarenotinstraightrows.) The zone of secondary tissue in which the ground tissue parenchyma is lignifi.ed and in radial rows. The secondary bundles, like the primary, but elliptical or elongated radially. (The xylem consists chiefly of pitted tracheids . ) The cambium, or meristem zone, with new bundles developing. The secondary cortex of thin-walled parenchyma cells in radial rows. The primary cortex ~ith - small leaf-trace bundles and crystal sacs. (The cells are not in straight rows) The periderm, consisting of cork cells in radial rows and the phellogen. There may be the dead remains of the epidermis. Under the high power, draw representative parts of a radial strip (see Exercise 37b). EXERCISE 58.-BoG-BEAN (Menyanthes trifoliata) STEM. MARSH PLANT Study as in Exercises 36 and 37, p. 53 et seq., and make preparations according to No. 16, a, c and d. Note: The large air s:paces and leaf-tra.ce bundles in the c;orte:x:. 72 PRACTICAL PLANT ANATOMY The large vascular stele enclosed in an endodermis. The collateral vascular bundles with vestigial cambium, but well-developed xylem and phloem. EXERCISE 59.-RusH (Juncus) STEM. MARSH PLANT This is another example of a marsh plant. Its stem has a pith of stellate parenchyma and an outer zone of girders. Study as in Exercise 58, p. 71. EXERCISE 60.-MARE's TAIL (Hippuris vulgaris) STEM. SUBMERGED WATER PLANT Treat as in Exercise 58. When cutting, hold gently so as to avoid crushing the stem and lay the razor on the trimmed surface with its edge nearly touching the stele. · Aim to cut the stele ; the cortex will look after itself. In the zone drawing show: The epidermis. The broad cortex of thin-walled parenchyma with large intercellular spaces. The endodermis with thickened walls and lignified bands in the radial walls. The central rope-like vascular cylinder of: Xylem vessels enclosing a parenchymatous pith. Sometimes a vestjge of cambium. Phloem, which is a zone of small cells just external to the xylem. A pericycle, between endodermis and outermost sieve tubes. In the high power drawing of the radial strip omit most of the cortex. EXERCISE . 6ra.-WATER VIOLET (Hottonia palustris) STEM, SUBMERGED PART Treat as in Exercise 60. CUTTING AND DRAWING STEM SECTION.$ 73 EXERCISE 61b.-WATER VIOLET (Hottonia palustris) STEM, AERIAL PART Study as in Exercises 36 and 37, p. 53 et seq. Note : The strong tissue near the surface; the middle is hollow. The pericyclic zone of sclerenchyma just inside the endo- dermis. The ring of separate vascular. bundles with xylem vessels. Other exercises on water stems are given in Exercises 67 and 71, pp. 77, 79. EXERCISE 62.-DUTCHMAN's PIPE (Aristolochia sipho) STEM, y OUNG. A CLIMBING STEM Observe inside the endodermis a continuous zone of scler- enchyma constituting the pericycle. As secondary growth takes place this is burst open and the gaps are at first repaired by the formation of sclerotic cells. The inter-fascicular cambium forms parenchymatous medul- lary rays only, hence the primary bundles retain their indivi- duality. In older stems the seconcfary growth of the vascular bundles crushes the pith and breaks the sclerenchyma ring. EXERCISE 63.-LILY OF THE VALL~Y (Convalla1 ' ia 1najalis) RHIZOME Make preparations according to No. II, test 14, and No. 16, a, b, c and d. In the drawings indicate: ✓ The thick-walled epidermis. The broad cortex. The thickened endodermal cells. The narrow pericycle. The ring of collateral vascular bundles. The inner concentric vascular bundles. 74 PRACTICAL PLANT ANATOMY EXERCISE EXERCISE EXERCISE EXERCISE EXERCISE EXERCISE This space is left blank for notes qn, e:x;ercises in stem stru.ctu,;e other than those set out above, CHAPTER IV OVERCOMING DIFFICULTIES WITH SMALL, THIN OR SOFT OBJECTS 22. Bundling IN cutting thin material it is difficult to maintain the true direction of the sections. For transverse sections of sufficiently firm material, grasping a number of short lengths together in a bundle and cutting them simultane- ously often overcomes the difficulty, truth of direction being judged by the appearance of the cut end of the bundle rather than of the individuaf structure. Cylindrical material such as roots, stems or cylindrical leaves may with advantage be tied into bundles with a wrapping of cotton. EXERCISE 64.-BROOM (Genista prcecox) STEM. ASSIMILATING SWITCH STEM For cutting, hold a bunch of short lengths of stem together and aim to cut transverse sections of the central ones. Treat sections according to No. 16, a, b, c and d. In the drawings show : The epidermis with very thick cuticle and with stomata restricted to the grooves between the ridges of the stem. The cortex, with strands of sclerenchyma beneath the epidermis in the ridges, and assimilatory tissue ( chloren- chyma) occupying the sides of the riqges bordering on the grooves. · · The hairs in the grooves. The vascular tissue enclosing a · pith and consisting of primary xylem, secondary xylem, cambium and secondary phloem. 75 76 PRACTICAL PLANT ANATOMY EXERCISE 65.-OTHER SWITCH STEMS (a) Casuarina equisetifolia, (b) Broom (Cytis u s scoparius) , (c) Gorse (Ulex europceus), (d) Whortleberry (Vaccinium M yrtillus), ( e) Rush (J uncus). J EXERCISE 66.-FLAG (Iris) ROOT. TYPICAL MONOCOTYLEDON RooT STRUCTURE Select a number of fairly straight pieces of root. Cut them into lengths of about one inch. Hold them in a bundle and cut transverse sections. Treat selected sections according to · No. II, test 14 and No. r6, a and b. Construct suitable zone and high power drawings to show: The piliferous layer with remains of root hairs. Several outer layers of the cortex thickened and cuticu- larised ( exodermis). The parenchymatous inner cortex. The endodermal cells with inner and radial walls strongly thickened and with thin-walled passage cells placed here and there opposite the protoxylem strands. The pericycle between the protoxylem and endodermis. The xylem strand with external protoxylem. The phloem strand. The ground ( conjunctive) tissue consisting of central pith and the cells between the xylem and phloem strands ; this may be more or less thickened and lignified. 23. Hardening in Spirit When material is so soft that it gives in front of the razor and is difficult to cut for this reason, it should be soaked i:i:i strong spirit. The longer it soaks the harder it will become, and after a time sectioning will be possible. Even then both the material and the razor must be kept wet with a strong spirit during the process. OVERCOMING DIFFICULTIES 77 Objects thick enough should be held singly. · Thin objects which become sufficiently rigid may be bundled, otherwise they must be embedded in pith or carrot, No. 24. EXERCISE 67.-WATER STEMS The material given below is usually soft. Cut transverse sections of single lengths, bundles or pieces embedded accord- ing to the thickness and rigidity of the object. (a) Myriophyllum: Central stele as in Hippuris, but one circle of large spaces in the cortex. Cells with cluster crystals of calcium oxalate projecting into the spaces. (b) Elodea: Resembling Ceratophyllum (Exercise 7r, p. 79). (c) Potamogeton natans: The vascular bundles retain their individuality, but the xylem vessels have disappeared and are represented by spaces; small bundles of sclerenchyma may be seen in the cortex. EXERCISE 68.-BUTTERCUP (Ranunculus repens) RooT. V TYPICAL DICOTYLEDON ROOT This root is often rather soft : harden by soaking in spirit for a time and then cut by the bundling method. Treat selected sections according to No. rr, test r4 and No. r6, c. and d. In drawings show: The piliferous layer with root hairs. The cortex of parenchyma. The endodermis with cuticularised radial walls. The pericycle. The small number of xylem and phloem strands alter- nating with one another. The conjunctive tissue between the strands. , EXERCISE 69.-CRESS SEEDLINGS. ROOT HAIRS Shave off some of the surface tissue of the root with root hairs attached. Mount in magenta and cotton blue in 78 PRACTICAL PLANT ANATOMY lactophenol. Observe that the root hairs are unicellular and consist of a cellulose wall lined with cytoplasm containing a nucleus. There is a central vacuole containing sap. 24. Embedding in Carrot or Pith Sometimes neither of the methods described in N os. 22 and 23 leads to success. Resort must then be had to embedding. In this method the material is clasped between two pieces of carrot or the two halves of a piece of elder stem pith. The carrot or pith is cut at the same time as the material to which it gives support. Obviously, since the material is hidden in the embedding substance, it is impossible to judge whether truth of cutting direction is being maintained, unless the material is embedded with accurate reference to the lines of the carrot or pith. For . transverse sections the material should be embedded perfectly longitudinally, and for longitudinal ones truly transversely in the pith. EXERCISE 70.-PEA OR BEAN ROOTS, YOUNG. THREE REGIONS OF ROOT GROWTH Hold a piece of pith (4-5 ems. long), or a piece of carrot ( I x I x 5 ems.), vertical on the bench between the finger and thumb of the left hand. With a sharp razor ~r scalpel make a vertical cut down the pith or carrot for about half its length, pressing the finger and thumb together while the cut is being made. Insert, perfectly longitudinally between the two cut faces, a thin paint-brush handle or a matchstick, and squeeze the two parts on to it so as to make grooves down both of the cut surfaces. Into the channel so made insert the root, tip upper- most. Cut transverse sections of pith and root together. From OVERCOMING DIFFICULTIES 79 amongst the first few sections select a thin one and mount according to No. 16, a. Examine the apical meristem cells. Cut other sections and mount selected ones from various levels in the root and so determine the three regions of growth : The region of cell division just under the root cap where the cells have dense protoplasmic contents and large nuclei. The region of cell vacuolation just below, where the cells are elongating rapidly, and are seen to have large vacuoles. The region of differentiation where growth is approaching completion and the cells are taking on their final form as xylem vessels, sieve tubes, etc. Make a drawing of the characteristic appearance of cells in each of the first two regions, and zone and high power drawings of the completed primary structure as seen in the third. EXERCISE 71.-HORNWORT ( Ceratqphyllum) STEM. SUBMERGED WATER STEM Embed longitudinally in pith, cut transverse sections and make preparations according to No. 16, a and b only. In drawings show : The epidermis without cuticle or stomata. The cortex of parenchyma with a ring of air spaces and many tannin sacs. The true endodermis. The central vascular cylinder with the xyJem reduced to a central passage with thickened walls and sur- rounded by xylem parenchyma: there are no vessels. The phloem, containing a ring of well-marked sieve tubes. EXERCISE 72.-MAIZE (Zea . Mais) RADICLE. STRUCTURE OF ROOT APEX Cut off the apical 5 mm. of the radicle, harden in spirit, embed horizontally in pith and cut a complete series of longi- ✓ 80 PRACTICAL PLANT ANATOMY tudinal sections. Look through them all and select the most median section. If it is thick, mount in caustic soda, other- wise in glycerine, and draw- to show: The root cap. The periblem which gives rise to the cortex. · The plerome which forms the central cylinder. EXERCISE 73.-BEAN (Vicia Faba) RooT, 6 TO 7 INS. FROM APEX. EARLY SECONDARY THICKENING OF ROOT Treat as in Exercise 70, p. 78. Make additional preparations according to No. r6c. Construct drawings to illustrate: The origin of the cambium between the xylem and the phloem. The . appearance after some secondary growth has occurred, indicating: The primary xylem strands in the centre. The secondary xylem external to the primary xylem. The cambium region. The region of secondary phloem. The position of the primary phloem, which is indicated by the four groups of bast fibres, external to the second- ary phloem and alternating with the primary xylem strands. The cortex. EXERCISE 74.-PINE (Pinus sylvestris) RooT. WOODY RooT The pine root is included here, although not an Angiosperm, on account of the ease with which the primary xylem groups may be recognised. · Cut a transverse section, being sure to include the organic centre of the root where the · primary xylem is situated. Treat selected sections according to No. rr, test 14 and No. r6, a. In the drawings indicate : The primary xylem with resin ducts nearly encircled by the protoxylem elements. OVERCOMING DIFFICULTIES Sr Secondary xylem with resiri ducts. Cambium. Secondary phloem. Medullary rays. Pericycle with resin ducts. Periderm. · EXERCISE 75.-ELM (Ulmus) RooT. WooDY ANGIOSPERM ROOT Treat as in Exercise 74, p. 80, and make suitable drawings. EXERCISE 76.-CHERRY LAUREL (Prunus Laurocerasus) LEAF. J TYPICAL BIFACIAL LEAF From the miadle of the leaf cut out about 1·5 cm . of the - midrib 4 with about ·5 cm. breadth of lamina on either side of it . Insert this into the cleft made in a piece of pith ( Exercise 70, p. 78) so that the midrib is vertical in it. Hold the pith for cutting in such a way that the edge of the razor is parallel with the surface of the leaf, so that the razor may have the minimum distance to travel through the- material and the section cannot be thin at the side the razor enters and thick at the other. Cut transverse sections of pith and leaf together. Treat sections selected for thinness, etc., especially of the midrib, according to No. II, test 14 and No. 16, c and d. Treat sections selected for thinness, etc., especially of the mesophyll, according to No. 16, a, b, c and d. Construct a zone drawing to show the regions of the leaf and a high power drawing of a strip through the leaf in~luding one side of the midrib and a contiguous part of the lamina. In the midrib there is to be seen: The thick-walled epidermis. The collenchyma internal to both the upper and lower epidermis. 6 82 PRACTICAL PLANT ANATOMY The crescent of vascular tissue with parts in the following order, beginning from above: A group of sclerenchyma fibres. Xylem. Cambium. Phloem. Sclerenchyma. The bundle sheath consisting of several layers of parenchyma cells containing tannin. At the side of the midrib the section should show: The upper epidermis with cuticle but no stomata. The palisade layer of elongated mesophyll cells, each with many chloroplastids. The spongy layer of mesophyll with larger intercellular spaces and fewer chloroplastids. The lower epidermis. The stomata on lower side only. Small groups of xylem and phloem may be present in the lamina. They are the veins-vascular bundles cut across in various directions. EXERCISE 76a.-CHERRY LAUREL LEAF. LEAF SURFACES Roll a piece of leaf round a piece of pith and cut very thin surface sections from both faces of the leaf. Mount selected sections in glycerine and make high power drawings. In the upper surface section there should be visible: The tabular epidermal cells without chloroplastids and with no stomata among them. The ends of the palisade cells showing through the epidermis. The air spaces between the palisade cells. In the lower surface section one should see : The epidermal cells with stomata among them. The cells of the spongy parenchyma with large inter- cellular spaces between them. OVERCOMING DIFFICULTIES 83 EXERCISE 77.-CHERRY LAUREL PETIOLE Cut sections from the middle of the petiole and treat accord- ing to No. rr, test 14 and No. r6, a. The construction is very similar to that of the midrib of the leaf. EXERCISE 78.-HYACINTH (Hyacinthus orientalis) LEAF. J TYPICAL lSOBILAJ:ERAL LEAF The hyacinth leaf tends to be soft: harden, if necessary, by soaking in strong spirit, and while cutting keep the razor wet · with strong spirit. Sections may be obtained by embedding in pith as in Exercise 76, p. 8r, bµt owing to the softness of the material c:i;ushing usually occurs. Better results are obtained by a modification of the bundling method. Take one length of leaf which has been hardened and gently roll it up so that the veins run down the roll. Roll a second leaf round the first and repeat till a convenient sized roll is obtained. Then cut the roll transversely, taking care not to crush it with the left hand. Mounting to Avoid Crushing The section is even more delicate than the complete piece of leaf, and the weight of the coverslip may crush it unless precautions are taken. To avoid this lay two coverslips a little less than a coverslip breadth apart on the slide and mount the section between them, bridging the two supporting slips with the actual covering one. Mount the transverse section of the hyacinth leaf according to No. r6, a and construct drawings to show: The epidermis with cuticle and stomata on both surfaces. A well-marked respiratory cavity under each stoma. The mesophyll, consisting of about two layers of rounded cells containing chloroplastids and central, thin-walled, rounded parenchyma cells containing few or no chloro- plastids. (Irregular cavities may be formed by some of 84 PRACTICAL PLANT ANATOMY these cells breaking down. There are raphides in some of the cells.) The vascular bundles, consisting only of xylem and phloem, the former towards the morphologically upper side of the leaf; here and there small transverse bundles connecting the main parallel longitudinal ones. EXERCISE 79.-HYACINTH LEAF, FRESH. MOVEMENT OF Gu.ARD CELLS Cut the surface of the leaf with a scalpel and lift a small tongue of the surface tissue. Bend this back, and then with a quick pull strip off the tongue and immediately immerse the stripping in water. Part of this will appear as a thin trans- lucent skin. This is the epidermis. Cut off part of the epi- dermis and mount outside uppermost in water. Draw, under the high power, a stoma with the surrounding epidermal cells. Remount in 5 % salt solution and watch a stoma until the pore is closed and the cytoplasm just commences to shrink away from the walls at the ends of the guard cells. Draw the stoma in the closed condition. Rinse off the salt solution and remount in water. Note the reopening of the stomata. J EXERCISE 80.-HAKEA (Hakea aciculare) LEAF. TYPICAL CENTRIC LEAF This is a compound leaf with tough, almost cylindrical seg- ments. It illustrates the centric type of structure. Make a bundle of a number of leaf branches and cut as described in No. 22, p. 75. Treat selected sections according to No. rr, test r4 and No. r6, c and d. In zone and high power drawings show : The epidermis with thick cuticle, and stomata with de- pressed and over-arched guard cells distributed all round. Each stoma has a large respiratory cavity beneath it. OVERCOMING DIFFICULTIES 85 The well-developed palisade zone, consisting of two layers of cells supported by a series of tubular idioblasts · , slightly lignified and extending right to the epidermis. The central parenchyma, almost destitute of chlorophyll, and with scattered cells containing tannin. The collateral vascular bundles supported by bundles of sclerenchyma. EXERCISE 8r.-THE RuE (Ruta graveolens) LEAF Treat as in Exercise 76, p. Sr. Include in the high power drawing: The loose texture of the palisade layer. Groups of the outer palisade cells, each joining on to one inner cell which is often broadened to receive them ; this is a collecting cell. The spherical oil glands just inside the upper and lower epidermis, the cavity bounded by a layer of thin-walled cells surrounded by flattened cells with thicker walls. In the fresh leaf the cavity contains a globule of volatile oil soluble in alcohol. Outside each gland is a group of flattened epidermal cells. 25. Additional Exercises in Root Structure EXERCISE 82.-PEA ROOTS, GROWN IN SAWDUST AND PRESERVED IN SPIRIT. ROOT BRANCHING The roots are translucent. Draw under the hand lens to show young roots arising internally. EXERCISE 83.-SWEET FLAG (Acorus (:_alamus) RooT. ORIGIN OF ROOT J:3RANCHES Treat as - in Exercise . 7r; p. _ 79, and draw a section which has passed through a young lateral root. 86 PRACTICAL PLANT ANATOMY EXERCISE 84.-SUNFLOWER RADICLE, t TO ½ IN. LONG. SECOND TYPE OF ROOT APEX Treat as in Exercise 70, p. 78, and draw to show: The root cap. The meristem, which gives rise to the root cap and to the pilif erous layer. The periblem, which gives rise to the cortex and endoder- mis and in a median section is seen to form a single layer of initial cells just at the apex. The plerome, which gives rise to the central cylinder from the pericycle inwards and forms a group of initial cells at the apex. EXERCISE 85.-PEA OR BEAN RADICLE. FURTHER TYPE OF ROOT APEX In some dicotyledons with broad root apices the periblem and plerome are not distinct right up to the apex, but there exists a broad transverse zone of meristem under the root cap which gives rise to periblem and plerome lower down. Treat as in Exercise 70, p. 78. EXERCISE 86.-EPIPHYTIC ORCHID (Dendrobi u m) AERIAL ROOT These roots hang freely in the air and absorb _ rain or dew. The cortical cells contain chloroplastids and the root appears green when wetted. Treat as in No. rr, test 14 and No. r6, a, b, c and d. Draw to show: Several layers of non-living cells containing air, whose walls are strengthened by a :fibrous thickening. These cells constitute the velamen and occupy the position of the piliferous layer in other roots. The exodermis, a single layer of cells with thickened radial walls. OVERCOMING DIFFICULTIES 87 The general parenchymatous cortex. The endodermis with much-thickened walls and passage cells opposite the xylem strands. The pericycle and conjunctive tissue, often with thickened and lignified walls. Many alternating xylem and phloem strands embedded in the thick-walled ground tissue. 26. Additional Exercises in Leaf Structure XEROPHYTIC - STRUCTURE IN LEAVES EXERCISE 87.-HOLLY (flex aquifolium) LEAF Treat as in Exercise 76, p. 8r. Draw to show: The upper epidermis with no stomata and the outer wall differentiated into three layers, (r) Cuticle, (2) Cuticu- larised cellulose, (3) Cellulose. The hypoderma, a single layer of thick-walled supporting cells. The palisade mesoph y 11. The spongy mesophyll, with here and there cells containing crystals of calcium oxalate. The vascular bundles cut across in various directions, each enclosed in a sheath of parenchyma cells without intercellular spaces; the largest bundles with scleren- chyma, xylem and phloem; the smaller ones with xylem and phloem; and the smallest with one or two tracheids only, enclosed in a sheath. The lower epidermis with cuticle, small stomata and small respiratory ea vities. EXERCISE 88.-RosEMARY (Rosmarinus officinalis) LEAF The stomata are restricted to grooves in the under surface of the leaf and are further protected by a feltwork of hairs, Tr~M fl.~ in Exercise 76, p. 8r, J J 88 PRACTICAL PLANT ANATOMY EXERCISE 89.-0LEANDER (N e rium oleander) LEAF The guard cells of the stomata project from the bottom of pits in the lower surface of the leaf. The pits contain hairs. There are several layers of hypodermal water storage tissue. Note also cluster crystals of calcium oxalate. Treat as in Exercise 76, · p. 81. EXERCISE 90.-THE INDIA-RUBBER PLANT (l!icus e lastica) LEAF The cuticle is thick. There is a three-layered epidermis for water storage, in which special large cells contain cystoliths. The guard cells of stomata are depressed. Embed pieces of the lamina in pith so that the parallel lateral veins are cut transversely. Treat as in Exercise 76, p. 81. EXERCISE gr.-CROWBERRY (Empetrum nigrum) LEAF The leaf is permanently ~olled. The lower surface lines the ea vity so formed and contains the stomata. Treat as in Exercise 76, p. 81. V -EXERCISE 92.-MARRAM GRASS (Psamma arundinacea) LEAF The leaf is rolled up when dry by the collapse of special thin-walled cells placed at the bottom of longitudinal grooves. The stomata are placed along the sides of the grooves. Treat as in Exercise 80, and No. 16, a and b. EXERCISE 93.-ALOE (Aloe arbor_ea) LEAF The cuticle is thick and the stomata are depre sed, The central colourless tissue is for • wa.ter storage. Treat as in Exercise 76, p. 81. Note.-This material seldom stains clearly. OVERCOMING DIFFICULTIES 89 A FLOATING AQUATIC LEAF EXERCISE 94.-WHITE WATERLILY (Nymphcea alba) LEAF Treat as in Exercise 76, p. 8r. Draw to show: The upper epidermis with cuticle and small stomata. The thick palisade layer of mesophyll. The spongy parenchyma with very large intercellular spaces. The lower epidermis without . cuticle or stomata. The vascular bundles. The branched internal supporting cells which are thick- walled, partly lignifi.ed and show deposits of calcium oxalate. Two LEAF-LIKE ORGANS EXERCISE 95.-BUTCHER's BROOM (Ruscus aculeatus) PHYLLOCLADE Embed in pith. Cut transverse sections and treat sections according to No. II, test r4 and No. r6, a, b, c and d. In the drawings show : Thick-walled epidermis with cuticle and stomata. The guard cells protected by a plate of cuticle projecting partly over the pore. The assimilating zone of spongy tissue with chloroplastids. The large thin-walled, clear, water-storing cells (aqueous tissue) in the centre. The vascular bundles in a row. EXERCISE 96.-PHYLLODE OF Acacia Cut transverse sections in pith and treat sections according to No. II, test r4 and No. r6, c and d. ~ EXE~q~~ : 97.-0THER LEAF fETIOLES Sunflower, Bean, Black currant. 90 PRACTICAL PLANT ANATOMY EXERCISE EXERCISE EXERCISE EXERCISE EXERCISE EXERCISE This space is left blank for notes on exercises in root and leaf structure other than those given above. APPENDIX I INDEX OF MATERIAL USED AND TABLE OF SOURCES THE table set out in the following pages -l~sts the mat~.rial required in the exercises. It may be used for tracing 'i:he number of the exercise in which a particular object is cut, but its main end is to supply information as to when and where supplies may be obtained. In the third column the figures I to 12 designate the months of the year, and the actual figures given against a particular plant organ indicates the months which experience has shown are most convenient for collection. In many instances it is possible to obtain what is required during other months, but material is not always in its best condition; gardeners are more ready to cut plants at one time than another, and heavy frosts may preclude digging during part of the winter. More - over there is-more probability of the collector being near some kinds of vegetation at certain times of the year and of recog- nising wild plants if they are in flower. The sources from which plants can be obtained are tabu - lated so that a glance will show what may be obtained from a particular place. The last two columns refer to Appendix II, in which will be found notes as to the ways in which material must be prepared for use and subsequently preserved till it is required. gr Material. .... 0 SOURCES OF ,d Q & .... 0 "' .... "' ::, O' -< - - - - - - - - - - - ~ - - - - - - - - - - - - - - - - - - - - Acacia, phylloclade Acorus Calamus, root Almond Aloe arborea, leaf . Anemone japonica, stem. Apple, fruit . ,, , stem, see Pyrus. Aristolochia sipho, stem, young Artichoke, tuber, fresh ,, ,, , preserved Atropa Belladonna, root. , stem. Bean, seed ,, , root, older ,, ,, , young. ,, , petiole . Belladonna, see Atropa. Black currant, see Ribes. Bog-bean, Buck-bean, see M enyanthes. Brazil nut . . . Broom, see Cytisus and Genista. ,, , Butcher's, see Ruscus. Bryonia dioica, stem Buttercup, see Ranunculus. Castor oil seed . . Casuarina equisetifolia, stem Ceratophyllum, stem Cherry laurel, see Prunus. Convallaria majalis, rhizome Cork, bottle . Cress seedlings Crowberry, see Empetrum. Cucurbita Pepo, stem Cytisus scoparius, stem Deadly nightshade, see Atropa. Deadnettle, see Lamium. 96 6-9 83 6-7 20 l-12 93 l-12 52 6-9 15 l-12 62 7-9 17 I0-3 18 ro-3 27 6-8 48 6-8 19 l-12 73 l-12 71 l-12 97 6-8 22 47 5-9 21 l-12 65 5-7 7.l 6-7 63 5-8 35 l-12 69 l-12 32 6-8 34 47 65 5-7 Dendrobium, aerial root . 86 7-9 Dock, see Rumex. Dracaena, stem 57 7-9 Dragon tree, see Dracaena. Dutchman's pipe, see Aris- tolochia. . . 92 + + + + + + + + + + + + + + + + + + \0 (J.) I I I I I I I I I I I 1 · I I I I 0 ..p.. I I I I I I I I I I I I I I I I 0 ..p.. I I I I I I I I I I I I I I I I + I I I I I I I I + I I I I I I 0 0 .I>, .I>, I I I I I I I 1 - 1 I I I I I I I I +I I I I I I I I I + I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I+ I I I I I I I I I I I I I I I I I I I I I I _ I I I I I + I I I I +II I I I I , I I I I I I I ~• 0 00 • 0 ..P..W-1>- Ul-1'-W -I>- I I I I I II II I 11 11 + 1 · 1 Bogs and Pond Sides. ~ II II II \ Wet Pl a c e s. t,:j I I I I I II I I I ::0 I Ditch e s. > I I I I I II II I II I I II r' I I I I I II I II I II I II ] Hedg e rows . I I I I I + + II I II I I I I J R o ad s id e$ a nd / or W as t e Pl a c e s. I I I I I I II II II II I I I Fi e ld s . I I I I I II I II II I I II J M e adow s . I I I I 1 · II II I II II II J S a nd Dun es. I I I I I II II I II II II J H e ath s . I I I I I II II I II I I II J Wood s . I I I I I II I II II II II ] Old Plant a ti o ns. I I I I I I I I I I +II I I I \ Or c h a rd s . I + II I I I + + I II II I I \ Kitch e n Gard e n. I I I I I I I I I + I + I II I I Gard e n . I 1+ +1 I I I I I I I I I I I I Grow from S ee d . I I I I I I I I I I I I I · I I I I Hou se hold Vacuum . I Prepu , tion '"d 000:i,. '"d @ • 000 • 0 • 00 • 00 ( L e tt e r s r e fer to @'d is: H 0 App e ndix II). U> ll>('D ~ ; _ ~ I ""'""'' lion !l) 0 0. C' . ::, 0 w -1=--!>--!>-W +,i:..+ >-<-!>- 1-<.j>. .j>. :.,J .j>. .j>. (Num e rals ref e r p~- to Appendix II) . 0. Material. Elder, see Sambuci,s. Elm, see Ulmus. Elodea, leaf ,, , stem Empetrum nigrwm, leaf Ficus elastica, leaf. Flag, see Iris. ,, , Sweet, see Acorits. Genista praeco:x, stem Gorse, see Ule:x. Hakea aciculare, leaf H elianthus annuu , s, fruit . ,, ,, , petiole ,, ,, ,radicle ,, ,, , stem, with interfasci- cular cam- bium , stem, prim- ary struc- ture. , stem, secon- dary struc- ture. Hippuris vulgaris, stem . Holly, see Ile:x. Hornwort, see Ceratophyllum. Hottonia, stem, submerged ,, ,, , aerial Hyacinth, bulb . ,, , leaf, preserved , ,, , fresh Ile:x aquif olium, leaf India-rubber plant, see Ficus. Iris, rhizome ,, , root J uncus, stern Kl t inia hast a ta, stem 8 4-10 - 67 4-10 - 91 4-8 - go 7-9 64 6 - 9 Bo 6 - 9 - 23 l-1 2 - 97 ' 6-9 - 84 l-12 - 39 6-7 - 40 9-10 - 60 6-9 - - 61a 61b 26 26 78 79 87 5-8 5-8 9-12 - 12-3 12 - 3 l - 12 - 13 II - 3 66 II-3 5 9 6 - 9 65 6-8 94 VI · .:: 0 ~ + + + + .... 0 SOUR CE S OF + + + + + + + + + '° v, I I I I I I I I I I I I I I I I p .... I + I I I I I I I I I p I. I I I p .... I I I I I I I I I I I I I I I I I I I I I I I I I I I I I + I I I I I I I I I ++ I I I I I I I ,. Pt-1 p ........ .... I I I I I I I I I I I I I I I I I I I I I +I . I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I + + II I I+ + I I I I I I I I I · 1 I I I I I I > P>PP p"IJ >,:j p p ... Ulv.l.l>,Ul Ul -I>- -I>- ] Bogs and Pond Sid es. a:: I I I I I I I I I I ~ I Wet Places. tTJ I I I I I I I I I I ::0 ~ I I I I I I I I++ I Ditches. r I I I I I I I I I I I Hedgerows . I I I I I I I I I I I Roadsides and/or Waste Places. I I I I I I I I I I \ Fields . I I I I I I I I I I \ Meadows. I I I I I I I I I I I Sand Dunes. I I I I I I I +II \ He aths. I I I I I I I I I I ' \ Woods. I I I I I I I I I I \ Old Plantations. I I I I I I I I I I \ Orchard s . I I I I I I . I I I I I Kitch e n Garden . + I+ I I + I I I , I I Gard e n. I +II I I I I I I \ G r ow from S eed . I I I I I I I I I I \ Hous e h old Vac uu m. I Prnpuation "d >,:j ~P>P p p PP> (Lett e rs refer to ~~~ p Appendix II). u, I)) Cl> ~~Er I Pre~ITation . ~ g.g. c-.::, 0 -I>- -!>--1>-W-!>- -I>- .I>,. .j>, Ul l\l (Numerals refer ? ~ t- to Appendix II). p. Material. Lamium albwm, stern Lilium Martagon,, stem . Lily of the valley, see Conval- laria. Lily, water, see Nymphaea. Lily, see Liliu , m. Lime, see Tilia. t .a § z Cl) "' . B Cl) ~ ~ - 46 50 llD -~ -~ t.l ~ Cl) 0 u .a .... u .8 ., ] "' -B ~ Q 0 .a :s ~ - - - - 3-9 7-8 Lupinus hirsutus, seed . 24 1-12 - Maize, see Zea . Mare's tail, see Hippuris. Marram grass, see Psamma. Marrow, see Cucurbita. Menyanthes, stem. Myriophyllum, stem N erium oleander, leaf Nymphaea alba, leaf Oleander, see Nerium . Onion bulb . . . Orchid, see Dendrobium or Phajus. Parsnip Pea, radicle , , , root, young ,, , ,, , long ,, , seed . . Pellionia pulchra, stem Phajus grandifiora, tuber Pinus, root Potato tuber ,, ,, , young Potomogeton natans, stem Prunus Laurocerasus, leaf ,, ,, , petiole Psamma arundinacea, leaf Pyrus M alus, fruit. , stem, gathered early summer Ranunculus repens, stem , root. 58 5 -9 67 5-8 89 l-1 2 94 6-9 7 1-1 2 16 ro-3 85 I - 1 2 70 I-1 2 82 l-1 2 19 1-1 2 14 13 74 9-ll 12 l-12 13 6-7 67 5-8 76 6-9 77 6-9 92 6-9 15 1-12 56 5.:...6 51 5-9 68 5-9 9~ - ..; 8 0 0 - - .... 0 "' "@ ..; -~ 8 ..; ci 0.. 0 CU U) !i'o ~ "' ~ 'O c:: ·"@ :a Cl) ·c: 1l ~ 0 t.l ~ ~ U) 0 - - - - - - - - + + + + + + + SOURCES OF g "§ '8 CU (!) '3 t.l ·B .A _g 0 0 ::i:: p'.1 - - - - + + + + 'O Q 0 p.. .... 0 "' ·c: CU ::, O' < - - + + + + '-l \0 --J I I I I I I I I I I I I ++ I I I I I I I · I I I I I I I I I I I ()() .,..,. I 111111111 I 11 1111111 I 1111+1111 I 11111111 1 I 111111111 I 111111111 I 111111111 I 1+1111111 I 111111111 I 1111111+1 I 111111111 + II I I II I I I I . 11111+111 I I I++ I I I I I I 11111+ 111 I 111111111 >rj >(')(')(')(')~>(')H () ..... Hoi,.oi,..i:..j>,..j>,. H.j>,..j>,. II II II I I I II II II I I I II I I II I I I II II II I I I II I II I I I I II I II I I 1 , 1 II I I II I I I II II II I I I II II II I I I II II II I I I II II II I I I I I II II I I I II I II I I I I +11111 I I+ I I+++ I I I I II I II I I I I 0 '< (")(")(") H>>()HH> > ()() .j>,. H\;l .i,. .j>,. .i,. H H Ul .i,. I+ I I I I Bogs and Pond Sides. ~ > >-l I I I I I I Wet Plac es. trj ::0 I Ditches. > I I I I I ~ I I I I I [ H e dg e rows. I I I I+ I Roadsides and/or Waste Places. I I I I+ I Fields. I I I I I j Meadows. I I I I I j Sand Dunes. I I I I I j Heaths. I I I I I I Woods. I I I I , I I Old Plantations. I I I · I I \ Orchards. I I I I I \ Kitchen Garden. I I I +I \ Garden. I I I I I I Grow from Seed. I I I I I Household Vacuum. Preparation "d "d.., ()() > () () (Letters refer to ~~ Appendix II). (/) ~ Cl>.., ... ~ Preservation ~ g. ~.::, Ul .j>,. \;l .,..,. (Numerals refer 0 ::, ~ to Appendix II). . ::, p,. Material. ... 0 SOURCES OF "' ... "' ::, O" < - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - Ribes nigrum, stem , petiole R~;e fruit . Ros ernarinus officinalis, leaf Rue, see Ruta. Ru mex crispus, st em . Rusc u s aculeatus, phyll oclade Rush, see Juncus. Ruta graveolens, l e af Sambucus nigra, stem, very young ,, , ear l y summer ,, , end of . first year S e dmn spectabile, stem Spirogyra Star ch, oatmeal ,, , pea ,, , potato ,, , whea t Sunflow e r, see H e li anthus . Sw ee ping s , floor T e trago ni a crystallina, stem Tilia, stem, 3 to 4 years old U lex europaeus, stern Ulmus, stem, r to 2 yea rs old . ,, , root . Vaccin i um myrtillus, stem Violet, water, see Hottonia. Water lily, see Nymphaea. Whortleberry, see Vaccinium. Zea Mais, stem ,, , radicle 55 8-9 97 6-9 10 9-10 - 88 6-9 45 6-8 95 6 - 9 8 1 6-9 30 4-5 54 5-6 44 9- 10 - 29 7 - 9 31 9 4-5 9-10 5 l-12 + 4 I-12 + l 3 l-12 l-12 + + 6 l-12 - 25 7-9 28 9-11 - 41 42 43 65 2-8 53 9-II - 75 9-11 - + + + + 65 4-8 - - - - - - - - - - 49 7-8 72 l-12 - 98 '° '° I I I I I I I I I I I I I I I I I I I I I I I I - I I I + ++ I I , . :;0 -l"--1"- I I I I I I I I I I I I I I I I I I I I I ++I 1 · I I I 1 I I I I + I I+ I ++I I I I I I I I I I I I I I I I I I I I I I I I , I () () () () -I"- -I"- 0\ 0\ I I I I I I I I I I I I I I I I I I I I I I I I I i' I I I I I I I I I I I I I+ I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I +I I I I I I I I I I I I I I I I I I I I I I I I I I I I I +I I I I I I I + I I I I I I I I I I I I I + I I I I I 1 . 1 ()() • •••• • ':r:i 0() 0\-1"- v> v> v> v> v> l -) -l"--1"- I I I I I I I I I J Bogs and Pond Sides. ls: ~ I Wet Places. t:r:I I I I I I I I I I ~ > I I I I I I I I I I Ditches . r- ++ I I I I+ I I I Hedgerows . I I I · I+ I I I I I Roadsides and/or Waste Places. I I I I+ I I I I J Fi e lds. I I I I I I I I I j Meadows. ·1 I I I I I I I I I Sand Dunes. I I I I I I I I I I He aths. I I I +I I I I I I Woods. I I I I I I I I I I Old Plantations. I I I I I I I I I I Orchards. I I I I I I I++ I K i tchen Garde9 . I I + +I +II I I Garqen. I I I I I I I I · I J Grow from Seeq,. I I I I I I I I I Househo l d Vacuum . Preparation "d;w ':r:i':r:i 0 • 0':r:i ()() () ()() (L etters refer to ~] ~ () Appendix II). I Presemtinn · ~ g.[ c-. t:, -l"-,IS, ,IS, -l"--1"- ,IS, 1-<,IS,,IS, (Numerals refer § "' 8, to Appendix II). . ~ 0. APPENDIX II METHODS OF PREPARATION AND PRESERVATION OF MATERIAL LETTERS and figures in heavy black type refer to the last two columns in Appendix I. A. Objects which are used in the exercises in the fresh or dry condition need no preparation other than what is part of the actual examination. B. Some seeds show their cell contents best if they are soaked before being preserved. Peas should be soaked in water for 24 hours before being fixed by the chroma-acetic method ( see I below). C. Radicles of seedlings and young roots may be obtained all the year round by germinating the seeds. For this purpose shallow earthenware saucers or boxes are best. They are filled with sawdust, washed sand or Sphagnum moss, and the previously soaked seeds are set so that the radicle points downwards. The seeds are kept just moist and warm. A few days suffices for the emergence of the radicles, which are allowed to grow to the required length and are then removed ahd preserved. D. Young potatoes may be dug early in the season or they may be obtained by keeping a mature potato in a dark and not too dry place. The mature tuber will shoot and, after a time, tiny tubers will be formed at the expense of the mature one. E. Maize plants may be grown readily from seed sown in the garden in the spring. It will be found that the common maize grains, such as are usually seen in chicken food, yield the hardiest and most successful plants. Much of the material used is derived from commonly grown plants, and where it is the intention to collect from the garden 100 APPENDIX IOI it is advisable to make sure that more than is required for ordinary garden use is planted. Whether plants are collected from a garden close to the laboratory or have to come some distance, it is absolutely essential to ensure that they suffer the minimum drying before they are preserved. F. In some instances it is important that an experienced botanist should select the part of the plant that is to be cut by the novice. This is true in relation to the sunflower stem. During the early summer the plant makes rapid growth and the stem commences to undergo secondary thickening even before the primary structure is fully completed. To the novice this condition is confusing. Later, when the rate of growth slows down, however, it is possible, by making trial sections, to select lengths in which the primary structure is fully completed, but in which the interfascicular cambium has not yet commenced to form. Later still much of the stem will have produced secondary growth. Similar precautions apply to the apple and elder stem that" are used to show the origin of the cork cambium. G. Most material is examined in the preserved condition and prior to preservation must be killed, fixed so that the cells and their contents retain as nearly as possible the condition in which they were during life, and hardened so that subsequent treatment may not alter their appearance. Hardening is also necessary so that objects that were soft may offer sufficient resistance to the razor. These three processes may usually be satisfactorily accomplished in anatomical - material by soaking in alcohol, which confers in addition certain other advantages. It displaces the air which is always present in the intercellular spaces of living material and so leads to sections that are not opaque by reason of air present in them. It also removes water and so paves the way for dehydration which is necessary before sections can be cleared in oily liquids such as xylol or oil of cloves. The objects to be fixed are cut up into pieces of suitab l e size, about r inch long in the case of stems. The pieces are roz PRACTICAL PLANT ANATOMY then put into a jar and covered with industrial spirit.* The jar is tightly closed with a bung through which the suction pipe of a filter pump projects, and by the action of the filter pump air is drawn out of the vessel. By this means the air in the intercellular spaces is rapidly extracted to allow the fixative to enter, and in due course the material sinks. When this has occurred the pump may be disconnected. The material is left in the industrial spirit for the remainder of 24 hours and is then transferred to 75 % spirit for storage.* I. For cells whose contents are to be examined, other fixing fluids usually yield better results. One of these is chromo- acetic acid . * Tiny objects, such as root tips, are cut off and dropped immediately into the fixing fluid; larger ones, such as young potatoes, should be cut up into small pieces first, and seeds such as peas, after having been soaked, should be stripped of their seed coats and split in halves before being immersed in the fluid. The vessel containing the fixing agent and the material is attached to the filter pump and air is withdrawn as in the alcohol method. The objects are left in the fixing agent for 24 hours after they have sunk and are then thor- oughly washed with water to remove the fixing agent. The best way to do this is to soak the material in warm water, changed every half-hour for, say, 12 hours. Subsequently, the water must be removed and the material hardened. This process must be gradual or shrinkage will result. Thus after washing is completed the material is transferred to ro¾ spirit for half an hour, then to 20% spirit for 1 hour, and then through successive grades of alcohol in a similar way up to 95 % , in which it is left for 12 hours. It may then be returned to 75% spirit for storage. r. Some of the objects to be examined must be obtained fresh every time they are required for use. Such material as * Fixing fluids must always be used in excess. The material to be fixed has a considerable effect on the fluid, and unless there is plenty of the latter it cannot act effectively. Storage fluids need be present in quantities only sufficient to cover the material. APPENDIX rose fruits show the facts for which they are examined only if they have been recently collected from the bush. If they are kept, they wither and the cell contents disintegrate; more- over they do not lend themselves to methods of preservation which will retain the characteristic parts. 2. There are a few objects, such as Spirogyra and Elodea leaves, which must for the best results be examined in the fresh condition, but which may be kept healthy in jars or tanks of water in the laboratory for some months. Precautions should be taken that the water is kept moderately fresh and is not exposed to direct sunlight. 3. The material that is the least trouble is that such as starches and some seeds which, after being purchased, are simply stored in bottles or boxes in the condition in which they are obtained. 4. Most stems and leaves are best stored in 75% spirit in airtight containers. It is very important that the stoppers of the vessels should be effective, or during a period of weeks evaporation will reduce the concentration of the spirit con- siderably and the material may go mouldy. 5. Certain stems and leaves, such as Elodea stem and Hyac inth leaves, are best preserved in strong spirit since they require much hardening. On the other hand, soaking for too long in this liquid will make them brittle. 6. Others are too hard for cutting even when they are collected. They should be stored in equal parts of spirit and glycerine. APPENDIX III STAINS, REAGENTS, ETC. The reagents marked * are frequently needed, the others should be available in the laboratory. IT is advisable that students test the effect of reagents and stains on known material such as is given in No. 12 prior to applying the tests to unknown material. ACETIC Acrn, glacial, is used as a mounting medium to bring out the parts of compound aleurone grains. . ACETIC Acrn, dilute. Glacial acetic acid Distilled water . I C.C. . 99 c.c. Dissolves calcium carbonate with the evolution of bubbles of carbon dioxide. *ALCOHOL, dilute spirit. A 25 % solution of industrial spirit in water may be conveniently kept in wash bottles of about 500 c.c. capacity. It is used for flooding the razor and the material during section cutting, for the immersion of the material while it i s not actually being cut, and for the immer- sion of sections while they are being selected. ALCOHOL, 75 % spirit. · A 75 % solution of industrial spirit in water is used for preservation of material which is to be cut. Stronger concentrations may be advantageously used for material that tends to be soft. Glycerin~ may be added when material is hard or becomes too brittle. *ALCOHOL, industrial spirit, 64° 0.P., 90% spirit. In- dustrial spirit is a satisfactory fixative for most plant organs that are to be examined anatomically. It may be used to precede absolute alcohol in the process of dehydration for the 104 APPENDIX ro5 economy of the latter fluid. It precipitates sugars and inulin, coagulates proteins and dissolves chlorophyll, other colouring matters, resins and some fixed oils. *ALCOHOL, absolute, must be used _ as a final dehydrating agent prior to transferring sections to xylol or oil of cloves. Its capacities for dissolving and precipitating are much the same as those of industrial spirit. ALKANET. A solution of commercial alkannin in absolute alcohol is prepared, diluted with an equal volume of distilled water and filtered. · The solution gives a pink to red colour with oils, resin and caoutchouc, but the action is often very slow. AMMONIA, strong, following nitric acid and warmth, gives a yellow colour (Xanthoproteic reaction) with protein. AMMONIA, ro¾, mixed with iodine solution gives a bright red colour with tannin. *CANADA BALSAM. Dissolve 25 grms. of 25 c.c. Canada balsam in Pure xylol. Filter and allow the filtrate to evaporate to the consistency of glycerine. Is used as a mounting medium for permanent preparations. Objects so mounted must be previously dehydrated with alcohol and cleared in xylol or clove oil. CHLOR-Zrnc-IomNE (ScHULTZE's REAGENT). Dissolve roo grms. 300 c.c. Dissolve 12 grms. Zinc in Pure hydrochloric acid and evapor- ate the solution to ' 150 c.c. (sp. gr. 1·8). Pqtassium iodide in as little water as possible and add 0·15 grm. Sublimed iodine crystals. Mix the two solutions. ro6 PRACTICAL PLANT ANATOMY This reagent causes starch grains to swell and turn blue, cellulose walls to turn blue to violet, lignified walls, suberised walls, protoplasm and protein to turn yellow to brown. CHROMIC Acrn is a constituent of many fixing reagents, e.g. chromo - acetic acid. CHROMO - ACETIC Acrn. Dissolve 0·3 grm. 100 C.C. . add 0·7 c.c. Chromic acid in Distilled water and Acetic acid. This is a suitable fixative for the material needed for Exercises 12, 13, 71, etc. CLOVE OIL is used as a clearing agent after dehydration with absolute alcohol. It is also a solvent for some stains, e.g. light green. When it is used as a solvent it serves two purposes simultaneously. COPPER SULPHATE is used in the preparation of Fehling's solution. COTTON BLUE stains protein, protoplasm and mucilage blue. See Stains in lactophenol, below. ETHER is a solvent for fat and oil. FEHLING's SOLUTION. Solution A Dissolve 69·28 grms. Copper sulphate in 1,000 c.c. Distilled water. Solution B Dissolve 346 grms. Rochelle salt (Sodium potassium tartra te) and 130 grms. Caustic soda in 1,000 c.c. . Distilled water. Keep the two solutions separately and just prior to use mix equal parts of · A and B. The mixture when heated with glucose, fructose or maltose g i ves a yellow to red or brown precipitate. APPENDIX 107 FERRIC CHLORIDE, neutral solution, produces a blue-black to green colour with tannin. GLYCERINE is a valuable temporary mounting medium because it has a high refractive index and does not evaporate. It may be used pure for this purpose. It is added to alcohol in equal proportions for the preservation of material that is hard or tends to become brittle. It is a constituent of lacto- phenol. *GLYCERINE, dilute. Glycerine. Water 50 c.c. . 50 c.c. Dilute glycerine is used more frequently than pure glycerine as a temporary mounting medium since it is less treacly to handle. *HYDROCHLORIC Acrn, strong, is used in conjunction with an alcoholic solution of phloroglucin as a test for lignification. HYDROCHLORIC Acrn, dilute. · Pure hydrochloric acid Water IO C.C. 80 c.c. Dilute hydrochloric acid may be used with heat to invert cane sugar into a mixture of glucose and sucrose. *IODINE. . Dissolve I grm. IOO C.C. add 0·3 grm. Potassium iodide in . Distilled water and Sublimed iodine. Shake at intervals till the latter is dissolved. Iodine gives a blue to black colour with starch, a brown colour with protein, protoplasmic structures, cuticularised walls and lignified walls, and a faint yellow with cellulose walls . . In combination with sulphuric acid a blue colour is produced in cellulose walls which at the same time swell. 108 PRACTICAL PLANT ANATOMY LACTOPHENOL. Mix roo c.c. 200 c.c. roo c.c. Add roo grms. Lactic acid. Pure glycerine. Distilled water. Phenol crystals and allow to dissolve. Lactophenol is a temporary mounting medium of high refractive index in which many stains are soluble. It may often be used in place of glycerine. *LIGHT GREEN. A saturated solution in oil of cloves is used for staining cellulose walls. As a counter-stain to safranin it is valuable. MAGENTA is used to stain lignifi.ed walls in the mixture of stains ( q.v.) in lactophenol. MILLON 1 S REAGENT. Stand a small beaker containing in a fume cupboard and add r c.c. Mercury 9 c.c. Cone. nitric acid. Close the fume cupboard and when the action is complete add ro c.c. Distilled water. When proteins are warmed in this reagent they turn brick red. NITRIC Acrn, strong, colours cuticularised walls and proteins yellow, and causes cellulose walls _ and lignifi.ed walls to swell. It is used with ammonia (q.v.) in the Xanthoproteic test for protein. PHENOL (CARBOLIC Acrn) is a c~mstituent of lactophenol, q.v. *PHLOROGLUCIN. A strong solution of the solid in indus- trial spirit is µsed with strong hydrochloric acid as a test for Jignifi.ca tion. · *SAFRANIN. Dissolve r grm. of IOO C.C. of add 50 c.c. of Safranin in 95 % spirit and Distilled water. APPENDIX Safranin is a useful stain for lignified walls. It also stains nuclei. ScHULTZE's REAGENT, see Chlor-zinc - iodine. SODA, caustic. A 5 % solution in water is commonly used. It causes iodide of starch to swell and lose its colour, saponifies fats and gives a reddish colour to cells containing tannin. SODIUM CHLORIDE. A. 5 % solution. *STAINS in lactophenol (magenta and cotton blue). Stock solutions of stains. r % cotton blue in lactophenol. r¾ magenta in lactophenol. Mixture for use. r % cotton blue in lactophenol r¾ magenta in lactophenol Lactophenol SUDAN III. 4 c.c. z c.c. . 50 c.c. Make a saturated solution of the solid in 70% absolute alcohol. After some days filter. Cuticularised walls, suberised walls, fat and oil, when mounted in the stain, gradually pick up the colour: the longer they are left in it the deeper their colour becomes. SULPHURIC Acrn, concentrated, dissolves cellulose and starch. *SULPHURIC Acrn, 75%, causes the conversion of calcium oxalate into calcium sulphate, the change of crystal shape indicating that the process is going on. It also - gives a blue colour with cellulose walls previously treated with iodine. *XYLOL is a solvent for canada balsam, and is also used as a clearing agent. APPENDIX IV STUDENTS' REQUIREMENTS · BOTANICAL razor. Scalpel. Pair of forceps. Camel-hair brush. Pocket lens. Duster. Microscope slides. Coverslips. Notebook, containing plain pages of large area with a hard surface, and an HB pencil. Microscope. · Light source. Small white porcelain dish. Wash bottle of 25 % spirit. Water supply above a sink or a wash bottle of water and a vessel for used liquids. Rack of stains and reagents marked * in Appendi x III. The other reagents listed in Appendix III should be available in the laboratory. IIO INDEX Adjustments of mi croscope , 17 Air bubbl es , 1 8, 22, 51 Air spaces, 38, 43, 71 , 72 Al e urone grains, 37, 38 Annual rings , 63 Apical mer i stem, 62, 79 Apparatus, 100 . Assimilating tissue, 75 Bast fibres, 64 Brownian movement, 26 Bund l e sheath, 82 Bundling, 75 Calcium carbonate, 34 ,, oxa l ate, 38, 39 Cambium, 55, 56, 57, So, 8 1 , 82 , co rk, 67, 70, 71 , fascicular, 62 ,, , interfascicular, 62, 73 ,, , ves tigial, 69, 72 Cel!, cavi t y, 44 ,, divi sion, 79 ,, , simple, 26 ,, vacuolation, 79 , , wal l , 26, 27 ,, ,, , drawing, 40 , , , , , tests for, 34, 35, 38, 39 Chlorenchyma, 67, 75 Chloroplastids, 26, 27, 33, 82, 83 Chromop l ast id s, 27 C le aring, 49 Collenchyma, 43, 47, 57, 63, 67, S r Companion cell, 45, 58, 64, 69 Gonfumatory test, 33, 34 Conjunctive tissue, 76, 77 Cork, 48, 67, 70 Cortex, 54, 56, 57, 63, 68, 71, 72, 73, 75, 76, 77 Coverslip, 1 8, 19 Crystalloid, 37, 38 Crystals, 38, 39 Cuticle, 57, 82, 84, 87, 88, 89 Cystolith, 88 Cytoplasm, 26, 27 Dehydration, 49 Differentiation, r eg ion of, 79 Dirt, 18, 23 Drawing, 21, 22, 24, 32 Endodermis, 57, 72, 76, 77, 79, 87 Epidermis, 55, 56 , 57, 67, 68, 70, 72, 73, 75, 79, 8 1, 82, 84, 87, 89 Exodermis, 76, 86 Fat, 35 Faults in u sing the microscope, 23 , , in double stained preparations, 51 Fib r es, 25, 45, 47, 64 ,, , bast, 64, So ,, , wood, 64 Fie ld of view , 1 9 ,, ,, ,, , spec k s in, 23 F i ss i on of chlo r op l ast id s, 27 Foca ll eng th, 15 Focussing, 20, 21, 23 Foreign objects in mounts , 25 Glo b oid, 37, 38 Glucose, 35 Growth , three regions of, 78 Gua rd cells, movemen t of , 84 Hairs, 56, 75, 77, 87, 88 Hand l ens st ud y, 53, 61, 62 Hardening in spirit, 76 Hilum , 2 1 Hone, 28 Hypoderrna, 68, 70, 87, 88 Idioblast, 85 Illumination - , 19, 23, 24 Image, 1 3, 1 5 . Intercellular spaces, 38, 43, 44, 45, 57 Inul in, 37 Irrigating, 22 L abe lling, 22, 44, 46 Laminations, 21 L e af, a quatic, 89 , , , bifacial, 81 , , , centric, 84 ,, , isob ila tera l , 83 ,, -like o rgan s, 89 ,, p etio l e, 83, 89 ,, · , ro ll ed, 88 , , surfaces, 82 , , - tr ace bundles, 71 L eaves, xerophytic structure in, 87 Lenticel, 66, 70 Leucoplastids, 32 Magnifica t ion , 15, 16 , accuracy of, 5 4 , high power, 56, 61, 64 , labelling of, 22, 44, 46 , low power, 55 . Maltose, 35 Materia l , co ll ec tion an d preparation of, 100 of elements in transverse section, 43-46 of l ongitudinal radial sect i ons, 61 of l ongitudina l tangentia l sections, 66 of secondary ti ss u es, 6 1 , 62 of transverse sections, 5 4-59 , sca l e of, 54, 56, 57 , select ion of part of sect i on for, 58 ,, , zone, 55, 61, 6z, 63 , 65, 66 Dust, 18 Embedding, 78 III ,, , list of, 91 Medullary ra ys, 54, 56, 63, 64, 65; 70, 73, S r , , sheath, 63 Mesophyll, 82, 83, 87, 89 Microchemical r eac tions, 22, 33 ,, , tabl e of, 34 ,, reag ents, pr e paration of, 104 Microscope, method of work with, 19-24 , p a rts of, 13 ,, , principle of, 13 Middlelamella, 44, 46, 62, 63 Mineral particles, 26 Mounting m e dium, 1 8 , 19 to avo id c rushing, 83 II2 INDEX Movement, apparent, of o bj ect, 20 Mucilage cells, 70 Nucleus, 26, 27 , 33,64 Oil, 37 , , g l ands, 85 Oilstope, 28 Palisade, 82, 85, 87, 89 Parenchyma, 43, 47, 55, 63 , li gnified, 46 , phl oem, 45 , s t e ll ate, 72 , , , xylem, 64 P eri blem, So, 86 Pericycle, 69, 73, 76, 77, 81, 86, 87 Periderm, 63, 66, 70, 71, 81 Ph e ll em, 63 Phellogen, 63 Phloem, internal, 68 ,, , medullary, 68 ,, , primary, 45, 47, 55, 58, 69, 71, 72, 76, 77, So, 82, 84, 8 7 ,, , secondary, 62, 63, 64, 65, 70, So, 81 Phy!loclade, 89 Phyllode, 89 Piliferous l ayer, 76 , 77, 86 Pith, 54, 56, 63, 64, 68, 70 Pits, 47, 64 Plerome, So, 86 Powders, 17, 20 P reparation, doubl e s t a in ed, 48-51 of objects, 17, 18, 32 ,, , permanent, 49, 50 ,, , temporary, 48 Protein gran ul es, 37, 38 Protop l asm, 33, 49 Pyreno id , 27 Radicle, 79, 86 Raphides, 39 Razor, 28-30 R eagen ts, 33, 3 4, 104 Resin, 34 ,, ducts, So Respiratory cavity, 83, 84, 87 Rhizome, 73 Root, add iti o n a l exercises in structure of, 85 , , , aerial, 8 6 apex, 79, 86 branchin g, 85 cap, S o, 86 h airs, 77 , r eg i on of growt h of, 78 , secondary thickening of, So , , , sections of, 75 , typical di coty l edon, 77 ,, , ,, monocotyledon, 76 ,, , woody , So, 81 Rules for microscopy, 24 Sclerenchyma :fibres, 43, 47, 54, SS, 58, 63, · 82, 85 Sclerotic cells, 73 Seconda ry thickening of r oots, So ,, ,, stems, 62, 71 , , tissues, drawing of, 61 Section c utting . , 30 , 31, 53 - 5, 59, 65, 66, 75, 76, 78 . Sections, comparison of tr ansverse and o bliqu e, 42 S e ctions, longitudinal rad i a l , 41, 59 ,, tang en tial, 4 1 , 65 , selection of for thinness, 32 ,, ,, truth of direction, 43, 60 , transv e r se, 41, 42, 53, 54, 75 , , , types of, 41 Sieve plates, 45, 48, 66 ,, tubes, 45, 57, 63, 64 Slide, 17 Sma ll and soft objects, 75 Sphrerocrystals, 37 Spongy tissue, 82, 89 Staining with magen_ta and cotton blue, 49 ' · ' ,, safranm and li ght green, 50 Starns, 104 Starch grains, 18, 21, 22, 25 ,, ,, , formation of, 33 Stem, aquatic, 72, 73, 77 ,, , ass i milating, 75, 76 , climbing, 73 ,, , marsh plant, 71, 72 ,, , monocotyledon with secondary thickening, 7 1 ,, , organic centre of, 65 , switch, 75, 76 ,, , structur e, additional exercises in, 67 ,, , primary, 53-61, 67-69, 71-73 ,, , secondary, 61-67, 70, 71, 73 ,, ,, , sections for, SS, 59, 65 , typica l herbaceous dicotyledon, 53 ,, , ,, . ,, monocotyledon, 68 ,, , woody dicotyledon, 63-67 ,, , water plant, 72, 73, 77 Stele, 84 Stomata, 75, 82, 83, 84, 87, 88 Stomata! mechanism, 84 Strain, avoidance of, 2 1 Strop, 28, 30 Sucrose, 35 Sugar, 34, 36 Tannin, 34 ,, sacs, 76 Tissues, drawing of, 43, 46 Tracheids, 64 Vacuole, 26 Vacuolation, region of, 79 Vascu l ar bundle, closed collateral, 69 , concentric, 71 , open bicollateral, 68 , , , collateral, 57 , primary, 7I, 73 Ve l :i'men, 86 , , secondary, 71 Walls, tests for, 34, 35 Water stora-ge cells, 88 Working distanc e, 15 Wood, autumn, 64 ,, , spring, 63, 64 Xerophytic l eaves, 87 Xylem, metaxylem, 57 , primary, 63, 64 , protoxylem, 57 , redu ced, 77 , secondary, 62, 64, 81 , vessels, 43, 46, 47, 54, 62 I . I Couverture / cover Faux-titre Page de titre Préface Contents List of illustrations Foreword Chapter I. The compound microscope and the method of using it Chapter II. Cutting and mounting simple sections - microchemical reactions Chapter III. Cutting, mounting and drawing stem sections Chapter IV. Overcoming difficulties with small, thin or soft objects Appendix I Appendix II Appendix III Appendix IV Index