Introduction

Richard M. Holaian fclOJLOGr A COURSE OF PRACTICAL INSTRUCTION IN BOTANY. A COUESE OF PKACTICAL INSTRUCTION IN BOTANY BY F. 0. BOWER, M.A, F.L.S., LECTURER ON BOTANY AT Till NORMAL SCHOOL OF SCIENCE, SOUTH KENSINGTON AND SYDNEY H. VINES, M.A., D.Sc, F.L.S., FELLOW AND LECTURER OF CHRIST'S COLLEGE, CAMBRIDGE, AND READER I{J ,BOTANX jy, THE UNIVERSITY. WITH A PREFACE BY W. T. THISELTON DYER, MA., C.M.G., F.R.S., F.L.S., ASSISTANT-DIRECTOR OF THE ROYAL GARDENS, KEW. PART I. PHANEROGAM^PTERIDOPHYTA. MACMILLAN AND CO. 1885. Tlie Eight of Translation and Reproduction is Eeserved. BIOLOGY LIBRARY RICHARD CLAY AND SONS, PRINTER^, . BREAD STREET HIpL, T^PON, E.G., IN MEMORIAM PREFACE. A FEW words may be said to explain the origin of the work of which the present portion is a first instalment. In 1873 I was invited by the Science and Art Department to conduct a course of instruction in what is now the Normal School of Science at South Ken- sington. It was a condition of the undertaking that the instruction should be carried on continuously from day to day and throughout the working hours of each My day. friend Mr. Lawson, late Professor of Botany at Oxford, was so good as to give me his assistance. We had the use of Professor Huxley's convenient and well-appointed laboratory, and we determined to attempt a course of instruction which should embrace the lead- ing morphological facts of every important type in the vegetable kingdom. We, in fact, resolved to adopt exactly the same plan of work as Professor Huxley in his own teaching had found convenient for the animal side of morphology. At this time, as far as I am aware, no previous at- tempt had been made in this country to give an extended 981914 VI PREFACE. course of botanical instruction of this kind. Professor Lawson and myself found our own difficulties scarcely less considerable than those of the students. The interest, however, which the novelty of the new method of work excited in the class soon became very obvious. The enthusiasm of the more skilful students at once stimulated and assisted us, and at the conclusion of the course we found that there was scarcely anything of importance in the rather comprehensive range which had been attempted which the students had not been able to study, examine, and draw for themselves. This course was an experiment. It was repeated at irregular intervals during the next few years. It gradually took a more systematic shape, and with the appointment of Mr. Bower as . Lecturer on Botany in the Normal School, it is likely, I think, to settle down into a permanent system of instruction. I had always hoped to put together the results of the experience in teaching methods acquired at South Kensington in the form of a handbook, which should save teachers who wished to follow our example from much of the trouble and difficulty which I, and those who, at different times, have taught in this way, have had to face. But, in the meanwhile, I had been drawn off to administrative duties which have left a steadily diminishing leisure for purely scientific work. For- tunately, my friend Mr. Bower was willing and with far greater competence to take up the task which I was unable to perform, and to him are entirely due the PREFACE. Vll laboratory instructions for studying the different types selected. Dr. Vines Las very kindly supplied the chapters on methods and on the morphology of the cells. But besides this he has at every step given the assistance of his own extensive experience in practical teaching. It had been our intention to preface the directions for the study of each type with a short account, in language fairly intelligible to the general reader, of its salient morphological facts. This would have represented the brief lecture with which the work of each day began in the course as originally organised. To carry out this intention would have postponed the publication of the teaching directions already prepared by Mr. Bower, and, in justice to him, it has seemed the best course to issue what is already finished without delay. It is intended to follow the present part with another, which will comprise the remaining types of the vegetable kingdom. Should the book be found as useful to students as it is hoped may be the case, I look forward to seeing the original scheme upon which it was planned still carried out in a future edition. ROYAL GARDENS, KEW, December 1884. W. T. THISELTON DYER. TABLE OF CONTENTS. INTRODUCTORY CHAPTERS. I. MET BODS AND REAGENTS. PAGE A. Making Preparations 1 B Micro-chemical Reagents 17 II. STRUCTURE AND PROPERTIES OP THE CELL. A. General Structure 24 B. Micro-chemistry of the Cell 29 C. Micro-physics of the Cell 37 CONTENTS. PRACTICAL DIRECTIONS FOR THE STUDY OF TYPES. PHANEROGAMS. I. ANGIOSPERMS. t VEGETATIVE ORGANS. A. DICOTYLEDONS. PAGE EMBRYO AND GERMINATION 44 STEM HERBACEOUS TYPE. * Mature 45 ** Young 62 Apical Bud 64 Node 67 STEM ARBOREOUS TYPE 68 STEM AQUATIC TYPE. ....... 82 SIEVE TUBES 84 LATICIFEROUS TISSUES 87 LEAF PETIOLE 90 LAMINA Bifacial Type 91 Centric Type 97 ROOT 99 Apex of the Hoot 102 B. MONOCOTYLEDONS. EMBRYO AND GERMINATION STEM HERBACEOUS TYPE STEM ARBOREOUS TYPE LEAF ROOT Apex of Root . 104 107 112 114 118 120 tt REPRODUCTIVE ORGANS. DEVELOPMENT OF THE FLOWER STAMEN CARPEL AND OVULES FERTILISATION DEVELOPMENT OF THE EMBRYO. i. Dicotyledon ii. Monocotyledon .122 125 129 131 132 134 CONTENTS. PHANEROGAMS (continued). II. GYMNOSPERMS. t VEGETATIVE ORGANS. EXTERNAL CHARACTERS HISTOLOGY OF THE STEM LEAF ROOT tt REPRODUCTIVE ORGANS RIPE SEED AND GERMINATION XL PAGE 136 ... 138 .149 .151 154 160 PTERIDOPHYTA. A. LYCOPODIN.E. I. SELAGINELLA. SPOROPHORE OOPEORE II. LYCOPODIUM. SPOROPHORE 162 , 173 175 13. FILICINE.E. ASPIDIUM. MATURE SPOROPHORE. EXTERNAL CHARACTERS 186 ANATOMICAL CHARACTERS TO BE OBSERVED WITH THE NAKED EYE 188 MICROSCOPIC OBSERVATIONS. Stem 190 Root ....... 198 Leaf . . . ."..'. ; 201 Sporangia .... 202 OOPHORE 204 YOUNG SPOROPHORE 210 C. EQUISETINE^l. EQUISETUM. SPOROPHORE Sporangia OOPHORE. 212 224 .... 226 PKACTICAL BOTANY. i. METHODS AND REAGENTS. A. Making Preparations. Preservation of Material. In many cases it is possible, and even preferable, to use fresh material, but it is often convenient to keep it for a time ; the best liquid for this purpose is ordinary methylated alcohol, in such quantity as to completely cover the material. It must be remembered that this will extract the green colouring matter (chlorophyll) from the material immersed in it, as well as resin and other substances. Hardening. It is not necessary, for the general study of the histology of plants, to harden them, for the tissues are usually sufficiently firm to admit of their being cut satisfactorily. In the case of exclusively parenchymatous tissues, especially those of cellular plants, it is necessary to harden them somewhat, and for this purpose dilute alcohol (50 per cent.) may be used. B 2 PRACTICAL BOTANY. When it is desired to study the structure of the protoplasm and of the nucleus, special methods must be employed for hardening them, or rather, for fixing them as nearly as possible in the condition in which they were during life. For this purpose the fluids mentioned below must be used. Care must be taken that the objects are of small size, that the quantity of hardening fluid is very large relatively to the bulk of the object, and that the fluid has ready access to all parts of it. The following are the best fluids for this purpose : 1. Absolute alcohol. 2. Picric acid (saturated solution in water). 3. Chromic acid (O'l 0'5 per cent, solution in water). 4. Osmic acid ('1 - 1 per cent, solution in water). These reagents can only be applied to fresh material. The following is a useful method for preparing sea-weeds : to a quantity of saturated solution of picric acid in sea-water add three or four times its volume of sea-water, and treat the tissue with it for | hr. 2 hrs. : then treat successively with 30, 50, 70, and 90 per cent, alcohol. When absolute alcohol is used, the object may be kept in it for an indefinite period. Such treatment generally makes the object brittle ; this may be reme- died when the object is to be mounted in glycerine by placing it, for at least twenty-four hours before it is to be cut, in a mixture of glycerine and absolute alcohol in equal parts, leaving it exposed to the air so that the alcohol may gradually evaporate. The glycerine slowly saturates the object and restores its consistency. This METHODS. 3 can of course only be done when the sections are to be mounted in glycerine. When picric or chromic acid is used, the object should be immersed in it for several hours ; the length of time varies with different material, and, in the case of chromic acid, with the strength of the solution used, from a few minutes to twenty-four hours. The objects must then be washed with dilute alcohol (50 per cent.), and then placed in stronger alcohol (70 per cent.), and finally in absolute alcohol (or 90 per cent.), which must be changed so long as any colour is still extracted from the objects. They may be preserved in this for future use. When osmic acid is used, the fixing effect is produced much more rapidly; in the case of simple structures, such as unicellular or filamentous Algae a few minutes (5 15) generally suffices; in the case of more complex structures, such as ovules, sporangia, growing-points, &c., the object may be left in the acid till it looks black on the exterior : it must be then well washed with dilute alcohol (50 per cent.), and left in it for \some time, and be then removed to 70 per cent. The sections are best mounted in dilute glycerine. In some cases osmic acid produces an excessive blackening of the cells, which can be removed by treatment with chlorine-water. It is advisable in cases in which the cell-walls tend to swell up excessively (as in many Algas) to use solu- tions of picric, chromic, and osmic acids, to which an equal volume of absolute alcohol has been added. Of these hardening reagents the most serviceable are absolute alcohol, or 90 per cent, alcohol, and picric acid. B2 4 PEACTICAL BOTANY. A Cutting Sections. sharp razor is the best cutting instrument. Care must be taken to keep the object and the razor wet during the process of cutting, in order to avoid the entrance of air into the tissue, and to prevent adhesion of the section to the razor. When fresh material is cut, water or very dilute alcohol may be used for this purpose, but if material which has been hardened is cut, it is advisable to use alcohol of the same strength as that in which the material has been preserved. When a successive series of sections of an object is required, a microtome may be used. Imbedding. The objects are frequently so large that they may be held in the hand whilst they are being cut. If they are too small for this it is convenient to imbed them in some substance. The simplest method is to fix the object into a slit in a piece of pith. Elder-pith is the best. When the sections are to be made with a microtome, it is more convenient to imbed in some easily fusible substance ; by this means also the position of the object is less likely to be distorted in the process of cutting. Many mixtures of waxy and fatty substances are used for this purpose, of which the following is perhaps the best : Solid paraffin (melting-point about 58 C.) : 2 parts. Yaselin : 1 part. These must be melted together and well stirred. The resulting substance is sufficiently transparent to enable the exact position of the object to be ascertained ; it is easy to cut, and it is readily soluble in carbolic acid and turpentine. The relative proportions of paraffin and METHODS. 5 vaselin may be varied somewhat to suit the object ; a softer mixture is produced by increasing the proportion of vaselin. For soft objects cacao-butter, which has the advantage of being soluble in ether or chloroform, is useful. The method of imbedding is to make a cavity in a piece of the substance sufficiently large to contain the object, which must have been previously washed with alcohol to remove all traces of water from its surface ; a small quantity of the substance is then melted and poured into the cavity so as to surround and cover the object. When it is cold it may be cut. Another method of imbedding is to moisten the object in water, and then suspend it by means of a pin attached to a thread in some white of egg, which has been previously well shaken up, and then strained through muslin. The white of egg should be in an evaporating dish. The object should be left thus sus- pended for some hours, so that the white of egg may come into close contact with all parts of it. Heat is then applied by means of a water-bath, and the white of egg coagulates. The part surrounding the object is now cut out and hardened in alcohol for some days. This method is useful for making sections of buds and flowers. It is important to keep the imbedded objects wet with alcohol during the process of cutting, in order to prevent the drying-up of the object, and its consequent contraction away from the substance in which it is imbedded. A third method of imbedding is very useful when it is desired to obtain sections of very small objects, such 6 PRACTICAL BOTANY. as spores, pollen-grains, &c. This is effected by means A of gum. thick layer of strong clean gum is laid on the flat surface of a piece of pith ; this is allowed to become nearly dry ; and then the pollen grains or spores are dusted on to it these are then covered with an; other thick layer of gum, and the whole is allowed to dry. Sections are now made of the dried gum, and, on their being placed in water, the gum is dissolved, and the sections of the pollen-grains or spores are set free. Staining. It is often useful to stain sections in order to bring out certain points in their structure, which are difficult to observe under ordinary circum- A stances. great number of colouring matters have been used for this purpose, among which may be men- tioned as the most useful: Hsematoxylin, Carmine, Cochineal, Gold Chloride, various preparations of Aniline, such as Safranin, Nigrosin, Fuchsin, Methyl- green, Eosin, and Methyl-violet. Staining is best performed by placing a few drops of the staining-fluid in a watch-glass and immersing the sections in it. The exact strength of the fluid, and the time of exposure of the sections to its action varies in each case, and must be ascertained by preliminary trials. As a rule, when differentiated staining is desired, the best results are obtained by using a dilute solution, and by exposing the sections for a long time to its action. A Haematoxylin. number of preparations of this colouring-matter are in use ; of these the following are those generally employed for vegetable tissues. 1. Alum solution of Hrematoxylin. Dissolve 035 grammes of hsematoxylin in 10 c.c. of water, and add to it a few drops of a METHODS. 7 solution of alum consisting of 1 gramme of alum to 1 c.c. of water. 2. Kleinenberg's hsematoxylin. Saturate some 70 per cent, alcohol with calcium chloride ; let the mixture stand for twelve to twenty- four hours over alum, shaking occasionally ; add eight parts of 70 per cent, alcohol ; filter, and then add a solution of hsematoxylin in absolute alcohol until the liquid has a purple-blue colour; let it stand in a corked bottle exposed to sunlight for about a month ; it is then fit for use. The liquid is to be diluted as required with alum solution. 3. Expose a few crystals of hsematoxylin to the action of gaseous ammonia in a watch-glass under a bell-jar : then add water, and a good colouring fluid is obtained. The disadvantage of this is that it has to be freshly prepared every time it is required. The alum-solutions will stain all parts of the cell, including the cell-wall. Their especial uses are (a) to make the cell-walls more evident when they are naturally transparent and colourless ; (b) to stain the protoplasm, so as to make its intimate structure apparent ; (c) to stain the nucleus, so as to demonstrate its pre- sence and to show up its structure. The ammoniacal solution is especially adapted for differentiated staining. If a dilute solution be used, the first thing to become stained is the chromatin of the nucleus, then, after a time, the rest of the nucleus (achromatin), then the protoplasm. The cell- walls do not stain with this fluid, or only slightly. Kleinenberg's hsematoxylin stains in a few minutes, whereas the alum-solution is much slower in its action. Hsematoxylin may be used either for fresh material, or for sections which have been previously hardened with alcohol, or with picric or chromic acid. In the latter case the sections must be washed repeatedly in distilled water to remove every trace of the acid, which, if present, would interfere with the proper action of the hsematoxylin. If the section becomes too deeply stained, as sometimes happens when the alum-hasma- toxylin is used, the excess of colouring-matter may be removed by washing with watery solution of alum. 8 PRACTICAL BOTANY. Sections stained with alum or with Kleinenberg's hgematoxylin are to be mounted in Canada balsam (or Dammar). Those stained with the ammoniacal solution are to be mounted in glycerine. Carmine. The two best preparations of carmine are those of Beale and Thiersch : carmine possesses, however, but little differentiating power. 1. Beale's Carmine To prepare this 0'6 gramme of carmine is dissolved in 2 c.c. of boiling solution of ammonia ; the solution must then stand for an hour or so to cool, and to allow of the escape of the superfluous ammonia ; to the solution are added 60 c.c. of distilled water, 60 grammes of glycerine, and 15 grammes of absolute alcohol. The mixture must be allowed to stand for some time it is then to be filtered. ; 2. Thiersch's Carmine Four grammes of borax are dissolved in 56 c.c. of distilled water ; to this 1 gramme of carmine is added, and then twice its volume of absolute alcohol is added to the liquid. After nitration the liquid is ready for use. Carmine readily stains the protoplasm and the nucleus ; Thiersch's preparation is especially useful for bringing out the structure of the nucleus. It can very well be used for sections which have been previously treated with picric, chromic, and osmic acids. The time during which the section is to be exposed to its action varies very much, as is the case with hsematoxylin. The rule is in both cases, that the most satisfactory results are obtained by a prolonged immersion in a dilute solution. In case of overstaining, the section may be washed for a moment in water, to which a trace of ammonia has been added. Preparations stained with carmine are best mounted in glycerine. METHODS. 9 3. Picro-carmine (or ammonium picro-carminate) is another useful preparation of carmine. It is prepared by adding a strong ammoniacal solution of carmine to a quantity of concentrated solution of picric acid in water, until a precipitate begins to be formed ; it is then evaporated to about one-fifth of its bulk filtered, and the filtrate is evaporated to dryness. The crystal- line residue is dissolved in water so as to make a 5 per cent solution, and this may be diluted as occasion requires. Another method (Gage) is to dissolve a quantity of picric acid in ] 00 parts of water, and an equal quantity of carmine in 50 parts of solution of ammonia ; these are then mixed, filtered, evaporated to dryness, and the residue dissolved in 100 parts of water. Picro-carmine is used especially for staining nuclei, the staining being more uniform than when carmine alone is used : it has this further advantage, that a prolonged exposure to it does not produce overstaining, as is the case with the other preparations of carmine. The objects should be previously kept for some time in absolute alcohol. If it is desired to retain the double staining which this reagent produces, the sections must be mounted at once in glycerine ; but if the carmine staining only is required, the sections must be washed in water, which will dissolve out the picric acid. When stained sections are mounted in glycerine, a small quantity of picro-carmine must be added to the glycerine in order to preserve the colours. The various preparations of carmine can be used as well for tissues which have been hardened in chromic, picric, or osmic acid, as for fresh tissues, but the former stain less readily. 4. Cochineal. The ordinary preparations of carmine frequently fail to give good results, especially when the tissue has been previously treated with chromic acid. Other preparations of the same colouring matter made directly from the cochineal insect have therefore been employed. A 1. Alcoholic Solution. quantity of finely powdered cochi- neal (best grey) is extracted for several days with 70 per cent, alcohol ; the liquid is filtered off and is ready for use. 2. Solution in water. Seven grammes of cochineal and an equal quantity of burnt alum are rubbed up together in a mortar until the whole is a fine powder : the powder is then added to 700 c.c. of 10 PRACTICAL BOTANY. distilled water ; the whole is then boiled, and evaporated to 400 c.c. : when it is cool a trace of carbolic acid is to be added, and then the liquid is passed two or three times through a filter. A dirty-red substance remains on the filter, and the filtrate is a clear fluid, thin layers of which appear red and thicker layers violet. This fluid will keep well for some months, but every now and again a trace of carbolic acid must be added to it, and it must be filtered. Both these preparations give good results, the differentiation being very marked. In using the alcoholic solution, the sections must be first soaked in 70 per cent, alcohol before they are placed in the staining liquid : it is also necessary, when sections are to be stained, to dilute the solution considerably with 70 per cent, alcohol. The watery solution acts very rapidly, staining fresh or alcohol material in a few minutes (3 5). The solution of cochineal in water stains especially the bastfibres of vascular bundles. In some cases the whole of the wood stains, but if the section be treated with dilute hydrochloric or sulphuric acid, the colour will be removed from all the cell-walls except those of the bast-fibres. Gold-chloride, in 0'5 per cent, solution in water, has been employed for staining Fungi. They must remain in it from one to six hours, and be mounted in dilute glycerine. A Aniline colouring-matters.; large number of these have been employed, only the more important are mentioned here ; they all stain rapidly. 1. Safranin. This is used in solution in absolute alcohol. It is especially adapted for staining sections which have been previously hardened with chromic or picric acid ; it is not quite so good for those which have been treated with osmic acid. The sections must be well washed in distilled water, and then placed in a small quantity (1 c.c.) of the saturated alcoholic solution mixed with an equal volume of distilled water ; they require to be left for several hours in the staining fluid. They must then METHODS. 11 be removed, and washed for a short time in alcohol ; then they must be placed in absolute alcohol and kept there until they appear transparent. The sections can now be mounted in dis- tilled water in order to see if the results are satisfactory, or, if they are to be preserved, they must be cleared by means of oil of cloves, and mounted in Canada balsam or Dammar. By this means very successful preparations of the structure of nuclei can be obtained. 2. Fuchsin. This is used in alcoholic solution. It is useful for bringing out the structure of thickened cell-walls. The sections must be previously treated with alcohol. It is also a good reagent for corky tissue. When a section is stained and is then washed with absolute alcohol, the coloration is removed from all parts excepting the corky tissue. A 3. Methyl-green. tolerably strong alcoholic solution of this is used. The sections of the object, which must have been previously kept in absolute alcohol, are to be treated with the staining-fluid for from 5 25 minutes, then quickly washed with distilled water, and mounted in glycerine. The nucleus stains of a green or bluish-green colour, the protoplasm remaining un- coloured. It is especially good for staining nuclei which are dividing. It has been used for staining chlorophyll-corpuscles, and is also useful in bringing out the nuclei and protoplasm in the cells of Fungi, which have been previously preserved in absolute alcohol and in glycerine. Strasburger recommends the following method for obtaining A preparations of nuclei : section of the fresh tissue is mounted in 1 per cent, acetic acid solution, to which a little methyl-green has been added ; the nuclei are fixed almost instantaneously. 4. Methyl-violet, This is used in concentrated alcoholic A solution. It is especially useful for staining bacteria. few drops of the solution are added to 15 20 c.c. of distilled water, and a drop or two of the mixture should then be placed on the bacteriamembrane (zooglcea), and be allowed to remain there for a short time until the membrane appears to be coloured : if the solution used is too strong, the substance between the bacteria will become stained. The colouring-matter is then washed off with distilled water, or better with a 10 per cent, solution of acetate of potash. The preparation may then either be allowed to dry in the air and 12 PKACTICAL BOTANY. be then mounted in Canada balsam, or it may be mounted in a 50 per cent, solution of potassium acetate in water. A useful preparation of methyl-violet is the following : Some of that substance is dissolved in strong sulphuric acid, forming a brownish-green solution : on the gradual addition of water the violet colour reappears. This is especially useful for sieve-tubes. If a section be treated with this fluid for a short time, and be then washed with water, it, will be found that the cell-walls have become swollen and transparent, that the protoplasm has become deeply stained, aod that the sieve-plates are very well brought out. Lignified tissues treated with this fluid assume a yellow colour, as they do when treated with aniline sulphate. 5. Hanstein's Aniline-violet. This is prepared by dissolving equal parts of fuchsin and methyl- violet in alcohol. It stains cellulose cell- walls of a faint violet colour, and lignified cell-walls red. It is especially useful for bringing out the different parts of the bast, since the bast-fibres stain red, whereas the sieve-tubes and the parenchyma scarcely stain at all. The protoplasm is stained pink ; amyloid substances, gums, and nuclei stain different shades of red, resins blue, and tannin brick-red. 6. Hoffmann's Blue. Used in solution in dilute alcohol slightly acidified with acetic acid : it is a useful reagent, inas- much as it stains the protoplasmic cell-contents and not the cellwall : it stains also the callus which closes the perforations of the sieve-plates during the winter in perennial plants. (Water blue is almost as good a reagent.) 7. Methylene blue. Used in solution in water : stains the cell-wall but not the protoplasm. To produce the differentiated staining mentioned in 6 and 7, the preparations must be washed in water after staining, and also before staining if the material has been previously kept in alcohol. 8. Alizarine. Many of these aniline-dyes will not stain the protoplasm of Fungi. Alizarine will do so at least in some cases. 9. Eosin. Used in strong alcoholic solution for demonstrating the structure of sieve-tubes, as it stains the protoplasm deeply : a solution in water may also be used. A 10. Corallin (rosolic acid). solution in 30 per cent, sodium carbonate colours lignified tissue, the callus of sieve-tubes, and starch grains pink. METHODS. 13 To the detailed instructions given above, the following general remarks may be added. All the abovementioned staining-fluids may be used for protoplasm and nuclei. The stain produced by aniline-colours is apt to fade, so that they are not to be recommended for preparations which are to be kept for a long time. The staining of haematoxylin also fades, but more slowly. In order to prevent fading, the preparations should be kept in the dark. Clearing the preparations. If it is not desired to observe the details of structure of the protoplasm or of the nucleus, the best clearing agent is a solution of potash, either in water or alcohol. The most generally useful is the 5 per cent, solution made by dissolving five grammes of solid caustic potash in 100 c.c. of distilled water. The alcoholic solution is made by adding strong alcohol (ordinary methylated alcohol will do) to a quantity of a concentrated solution in water until a precipitate begins to be formed. The mixture must then be well shaken, and allowed to stand and settle for twenty-four hours; the clear fluid is then poured off. For use a mixture of equal parts of this solution and of distilled water may be made. The clearing action of potash is due to the swelling of the cells and their contents, so that they become more transparent ; at the same time it dissolves many of the granules in the protoplasm, and saponifies the oil-drops. The swelling caused by the action of the solution in water is often too great, especially when it is desired to see the cell-walls distinctly; this difficulty may be got over by the use of the alcoholic solution. 14 PRACTICAL BOTANY. After treatment with the aqueous solution, the sections should be washed in distilled water, and after treatment with the alcoholic solution in dilute alcohol; the sec- tions, in both cases, should be mounted in glycerine. Another method of clearing, which is especially re- commended for obtaining good preparations of growing points, is to treat sections with calcium chloride. The sections are placed on a slide in a drop of water, and are then covered with some dry powdered calcium chloride the slide is then warmed over the flame of ; a spirit-lamp until the water has nearly all evapo- rated; a drop or two of water is now placed on the sections, and they are to be mounted in glycerine. In the case of tissues, which have been hardened in alcohol, with or without treatment with other hardening agents, another method of clearing may be used. The sections, after staining, if that is desired, should be placed for a few minutes in absolute alcohol ; they should then be transferred to a watch-glass, containing either a mix- ture of turpentine and creosote (four parts of the former to one of the latter), or some oil of cloves ; sections which have been stained with aniline dyes are best cleared by cedar-wood oil ; they should be left in this for a short time, until they appear to be quite trans- parent, and should then be mounted in a drop of Canada balsam or Dammar. Mounting. For the observation of the coarser features of the histology of plants, it suffices to mount the sections in a drop of water, or, in certain cases, in a drop of alcohol. This is the only possible method when micro-chemical observations have to be made. Sections of objects which have been hardened, or otherwise METHODS. 15 specially prepared, and which it is desirable to preserve, should be mounted in glycerine, or in glycerinejelly, or in Canada balsam or Dammar. Glycerine and glycerine-jelly may be used for sections which have been prepared by any of the methods described above. Dilute glycerine should be used for this purpose, consisting of a mixture of pure glycerine with an equal bulk of water. The cases in which these media are especially suitable have been mentioned. Only those sections which have been treated with absolute alcohol, and either oil of cloves or the mixture of turpentine and creosote can be mounted in Canada balsam or Dammar. When preparations are mounted in glycerine-jelly, a trace of carbolic acid should be added in order to prevent the growth of Fungi. The sections should be previously soaked in glycerine so as to remove water or alcohol from them. In order to make the preparations mounted in glycerine quite permanent, the cover-slip should be fixed to the slide by applying a coating of gold-size or Brunswick black round its edge with a brush. Care should be taken that no glycerine is on the slide outside the vcover-slip; if any is there it should be removed by means of blotting-paper before applying the varnish. It is better to varnish Dammar preparations in this way also ; but it is not necessary for those in Canada balsam. Preparations of green parts of plants in glycerine lose their colour. These may be best put up in a drop of a strong solution of potassium acetate, or of alu- minium acetate. The cover-slip must be fastened down as above described. 16 PRACTICAL BOTANY. It is often desirable to observe objects in the living state for a considerable time under the microscope. This must be done in a moist chamber. A moist chamber A may be readily constructed as follows : piece of thick rough cardboard is cut to the size of the glass slide, and a circular hole is punched out of the middle of it of such a size as to be covered by a cover-slip. The piece of cardboard is then soaked in water (or boiled in water when pure cultures of Fungi are to be made), so as to A saturate it, and placed on the glass slide. drop of water (or solution as described below), is placed on the cover-slip, the object is immersed in it, and the cover- slip is then inverted over the hole in the piece of card- board. Thus the object is suspended in a drop of liquid on the under surface of the cover-slip. Any loss from the chamber by evaporation is prevented by occasion- ally wetting the cardboard on the slide. The liquid to be used will of course vary with the nature of the object to be observed. In the case of AlgaB, water may be used ; in the case of Fungi, decoc- tions of various organic substances (fruits, animal tissues, &c.), or a solution of sugar, according to the habit of the Fungus. For observing the germination of the spores of Mosses and Ferns, water will suffice ; but in the case of pollen-grains a solution of sugar is neces- sary (1 20 or even 30 per cent, the concentration being different for different plants) ; for observing the process of cell-division in the hairs on the stamens of Tradescantia, a 1 per cent, sugar solution may be used. REAGENTS. 17 B. Miero-ehemical Reagents. Besides the fluids which are used for hardening and staining the tissues, a considerable number are employed, which, on account of the characteristic effects produced by their action on cell-walls and cell- contents, may be regarded as chemical tests for the various substances which may be present. The follow- ing are the principal reagents which are used in this way : the mode of preparing them is also given, and some indication of their uses; but this latter subject is more fully treated in the next chapter. I. Acids. Sulphuric acid. This is used either concentrated or dilute (1 to 3 of water). It causes, in either case, the swelling-up of cellulose cell-walls, starch-grains, &c. when ; cellulose cell-walls which have been pre- viously saturated with solution of iodine are treated with sulphuric acid, they turn blue. Concentrated sulphuric acid dissolves cellulose and starch, but cuticularised cell-walls and the middle lamella of lignified cells resist its action. It is used with cane-sugar, as a test for proteids, and with aniline sulphate as a test for lignin. Nitric acid. It colours cuticularised cell-walls and proteids yellow ; it also causes swelling-up of cellulose and of lignified cell-walls. It is useful for dissolving the crystals of calcium oxalate which are frequently present in the cells. It is used with ammonia as a test for proteids (xanthoproteic reaction), and with potassium chlorate as a test for suberin, and as a macerating fluid. C 18 PRACTICAL BOTANY. Hydrochloric acid. Used, with aniline chloride phloroglucin, or carbolic acid, as a test for lignin. By itself it turns lignified cell-walls yellow ; when its action is prolonged, the cell-walls become violet, owing to the presence of various substances such as phloro- glucin, coniferin, and pyrocatechin. A Chromic acid. strong aqueous solution of this acid dissolves lignified and cellulose cell- walls ; cuticu- larised cell-walls resist its action ; but they become A very transparent, and may be easily overlooked. dilute solution brings out the stratification of cell-walls very clearly. Acetic acid. This is used as a dilute aqueous solution (1 per cent.). It dissolves crystals of calcium carbonate ; it causes swelling-up of cell-walls, starch- grains, &c. ; it brings out nuclei very clearly ; it is useful as a corrective after treatment of a preparation with potash. II. Alkalies. Potash. This may be used either in a dilute or a concentrated solution in water. The dilute solution is chiefly used for clearing preparations, as already de- scribed. It causes cell-walls, starch-grains, &c., to swell up very much, and it dissolves proteid crystalloids, and most aleurone-grains. It gives a reddish colour to cells in which tannin is present. It may be used as a macerating fluid ; when woody tissues are boiled in potash, the cells of the vascular bundles become more or less isolated, for the lignin of their walls undergoes solution. It dissolves inulin. The concentrated solution is used as a test for suberin. When sections of cork are boiled in strong REAGENTS. 19 potash, the suberin escapes in the form of yellow viscid drops ; when the sections are only slightly warmed in potash solution the cuticularised cell-walls assume a yellow colour. Potash is also used, together with copper sulphate as a test for proteids, and for various kinds of sugar. Ammonia.. The solution in water is often used instead of potash for clearing preparations, as its action is less intense. It is used, together with nitric acid, as a test for proteids, and with copper sulphate as a solvent for some forms of cellulose. III. Non-Metallic Elements. Iodine. This is .one of the most useful microchemical reagents. It is used in solution, in water, or alcohol, and in the chloride of zinc mixture. 1. Solution in water. Dissolve a small quantity of potassium iodide in the requisite quantity of water ; then dissolve iodine in it until the liquid has a dark sherry colour. This may also be prepared by diluting the liquor iodi of the pharma- copoeia. 2. Alcoholic solution. Dissolve iodine in alcohol until it has a dark sherry colour. This may also be prepared by diluting with alcohol the tinctura iodi of the pharmacopoeia. Iodine stains proteid substances brown, cellulose faintly yellow (as a rule, see next chapter), cuticularised and lignified cell-walls yellow, gum purple, starch blue (only in the presence of water). Iodine is used as a micro-chemical test for starch and for cellulose. The blue colour which it gives with starch, and the conversion of the faint yellow colour of a cellulose cell-wall stained with iodine into blue when c2 20 PRACTICAL BOTANY. it is treated with sulphuric acid, are characteristic The cellulose reaction is also given with the chloride of zinc mixture. IV. Inorganic salts. Sodium chloride (common salt). This is used both in dilute (10 per cent.), and in saturated solution in water as a solvent for the proteid crystalloids. The 10 per cent, solution is used for producing plasmolysis. Ferrous sulphate. Used in dilute solution in water, to which a drop of nitric acid has been added, as a test for tannin. Potassium bichromate. Used in dilute solution in water as a test for tannin ; used also (in 1 per cent, aqueous solution) for hardening tissues. Potassium chlorate.^Used, together with nitric acid, as a macerating agent. Copper sulphate. Used in very dilute solution in water ; the blue colour of the solution must be only just perceptible. It is used, with potash, as a test for some kinds of sugar, and for proteids. It is used also in the preparation of ammoniacal solution of cupric hydrate, which dissolves pure cellulose. For the preparation of Fehling's fluid the following directions are given in Foster's Practical Physiology : (a). Dissolve 34'65 grm. of pure crystallised cupric sulphate in about 160 c.c. of distilled water. (&). Dissolve also 173 grm. of pure crystallised potassic-sodic tartrate in 600 to 700 grm. of sodic hydrate sp. gr. 1.12. Add (a) to (b) stirring well to cause a thorough mixture, and dilute with distilled water to a litre. Fehling's fluid should be fresh made whenever it is required, since it decomposes on keeping ; it will keep some little time if REAGENTS. 21 kept in a cool place in the dark, and in completely filled, well- closed bottles (Hoppe-Seyler). The solution (6) may be prepared, and kept for adding to (a ) freshly prepared when required. Before using a kept solution to test for sugar, always boil a little of it by itself to see if any reduction will take place. From 1 c.c. of this solution the copper is completely reduced by '005 grm. of grape-sugar. Y. Organic substances. Alcohol. Used as a solvent for various substances, such as fats, oils, resins, colouring-matters, &c., and as a precipitant for various substances. It has a peculiar action upon some proteid crystalloids. Ether. Used as a solvent for wax, fats, resins, &c. Cane sugar. The concentrated aqueous solution is used, together with strong sulphuric acid, as a test A for proteids. dilute (1 per cent.) solution is useful for mounting living cells for observation under the microscope. Alkanet. The alcoholic extract, or better, an alcoholic solution of alkannin, is used as a test for resin and caoutchouc : a fresh solution must be prepared on each occasion. Phloroglucin. Used in alcoholic or aqueous solution as a test for lignin. The section is first treated with hydrochloric acid and then with solution of phloroglucin : the lignified cell-walls assume a bright red colour. If phloroglucin cannot be obtained, it may be replaced by an extract of cherry wood. Shavings of young cherry-branches are extracted with alcohol for twenty-four hours to remove chlorophyll and other substances ; then the alcohol is poured off. The shavings, after being pressed, are extracted for several days with alcohol, 22 PKACTICAL BOTANY. the alcohol extract is poured off and filtered and then evaporated nearly to dryness, until a piece of coarse blotting paper moistened with it and treated with hydrochloric acid turns violet. The extract is then ready for use. It gives a violet colour to lignified cell-walls, as it contains other substances ( especially pyrocatechin) besides phloroglucin. Phenol (carbolic acid). Used, together with hydrochloric acid, as a test for lignin. The best preparation of it is its solution in hydrochloric acid : this is pre- pared by dissolving carbolic acid in warm hydrochloric acid, adding, whilst the mixture is cooling, sufficient hydrochloric acid to dissolve any precipitate that may be formed. Lignified cells, treated with this mixture and exposed to sunlight, assume a bright green colour in consequence of the presence of coniferin. Aniline sulphate and chloride. These salts are also used as tests for lignin in cell-walls ; the chloride is preferable. They may be used in solution either in water or alcohol, but the alcoholic solution gives the best results. The section is first treated with the solu- tion and then with sulphuric or hydrochloric acid re- spectively, or better, the solution may be kept slightly acidulated by one or other ot these acids : the lignified cell- walls assume a bright yellow colour. VI. Mixtures. Schulze's Solution 1 (Chlor. Zinc lod.). Used as a test for cellulose cellulose cell-walls turn blue ; when treated with this mixture ; corky and lignified cell-walls turn yellow, protoplasm brown, and starch blue. 1 Since there has been some uncertainty as to the exact name of the botanist who introduced these reagents, it may be here stated that they were first used by Professor Franz Sclmlze, of Rostock. See Flora, 1850, p. 643. REAGENTS. 23 It is prepared by dissolving zinc in pure hydrochloric acid, and evaporating the solution, on a water bath, in the presence of metallic zinc until it has a syrupy consistence ; it is then saturated with potassium iodide, and then with iodine ; a few grains of iodine should be left in the liquid after it is poured off for use. It may also be prepared by dissolving 25 parts of pure fused zinc chloride and 8 parts of potassium iodide in 8^ parts of water, filtering through asbestos, and saturating with Iodine. On adding Iodine to Schulze's solution till precipitation begins, a fluid is obtained which stains the cell-walls yellow, and the oallus of sieve-plates a deep brown (Russow.) Schulze's Macerating Fluid.1 One gramme of potassium chlorate is dissolved in 50 c.c. of nitric acid ; the tissue is then placed in a small quantity of it, and the whole is boiled for a short time in a test tube the ; liquid is poured off, and the residue is well washed A with water. filter may be used for washing. The cells become isolated in consequence of the solution of the middle lamella. 1 See Note, p. 22. II. THE STRUCTURE AND PROPERTIES OF THE CELL. A. General Structure. CUT longitudinal sections of a parenchymatous tissue, the young shoot of the Elder, for example, mount in water, examine the parenchymatous cells of the pith with a high power ; Note, 1, the Cell-wall, transparent, colourless, and apparently homogeneous ; 2, the Protoplasm, forming a layer (the pri- mordial utricle), closely lining the cell-wall, and connected by bridles with a more centrally placed mass in which 3, the Nucleus, a well-defined, roundish, highly refractive body, is situated ; 4, the Vacuole, filled with colourless fluid, the Cell-sap. Structure of the Protoplasm and Nucleus, Harden a small piece of a young growing shoot or root of Pinus in picric acid or in absolute alcohol ; stain with ammonia-haematoxylin ; mount in dilute glycerine, or stain with Kleinenberg's hsematoxy- lin, and mount in Canada balsam ; examine with a high power : Observe in the protoplasm 1. The Ectoplasm, a hyaline layer, but little stained, next to the cell- wall. STRUCTURE OF THE CELL. 25 2. The Endoplasm, the more internal, deeply stained protoplasm ; note that the staining is confined to fibrillse which form a sort of network in the endoplasra, and to numerous minute particles, the Microsomata. Observe in the. nucleus 1. Stained fibrillae forming apparently a reticulum (ehromatin). 2. The unstained matrix (achromatin) in which the fibrillaj are imbedded. 3. Cell-division. In order to study the process thoroughly the hairs on the stamens A of Tradescantia may be taken. stamen is to be removed from a bud, on a warm day, and is to be placed at once in a drop of 1 per cent, sugar- solution on a cover-slip ; the cover-slip is then A to be placed over a moist chamber as previously described. magnifying power of about 500 diameters is to be used. A terminal cell of one of the hairs, with a large and conspicuous nucleus, is to be observed. It will be seen that the nucleus gradually elongates in the direction of the longer axis of the cell ; it becomes more granular, and the protoplasm of the cell aggre- gates at its poles ; then the nucleus presents a striated appearance, the fibrillse gradually arrange themselves parallel to the longer axis of the nucleus, and approach each other at the poles ; thus a characteristic nuclear spindle is produced ; the fibres are then ruptured in the equatorial plane, and gradually collect at each A pole, so that two new nuclei are found. layer of granular protoplasm (the cell-plate) consisting of microsomata, is now found in the equatorial plane, and it extends on each side until it reaches the wall of the cell ; this layer becomes converted into cellulose, and constitutes the dividing wall between the two cells. Good preparations of nuclei may be obtained by making longi- tudinal sections of growing points (e.g. of the young roots of } and staining with heematoxylin. 4. Structure of Chlorophyll-corpuscles and of Leukop lastids. a. Chlorophyll -corpuscles, or chloroplastids. Mount a thin leaf of a Moss (e.g. Funaria), or the prothallus of a Fern, in water ; note the corpuscles in the cells. 2G PRACTICAL BOTANY. Treat with alcohol ; the green colouring matter (chlorophyll) is gradually dissolved out, and the corpuscle is left colourless. Press out the contents of an internodal cell of Nitella or of Cham on a slide ; put on a cover-slip and examine with a high power. Run in some distilled water : observe that the corpuscles swell up, assuming the form of large hyaline vesicles ; the chlorophyll is con- fined usually to one portion of the vesicle. If chlorophyll-corpuscles, which have been treated with picric acid and decolorised with alcohol, be stained with iodine, Hoffmann's blue, or hsematoxylin, and be examined with a very high power, it will be seen that they have a trabecular structure ; it is from the inter- stices of the trabeculaB that the colouring-matter has been removed. The leaves of Vallisneria afford good material. The same result may be obtained by prolonged treatment with dilute acid (hydrochloric acid mixed with water in the proportion of 1 : 4, is most effectual), or by exposure for one or more hours to steam (Pringsheim). The minute structure of the corpuscles can be very readily made out in cells of the leaves of JEcheveria. If the plants used have been previously exposed to light, it will be observed that the chlorophyll-corpuscles contain granules. If a decolorised corpuscle be treated with iodine, the inclosed granules will turn blue, showing that they are starch -granules (see p. 33). b. Leukoplastids. These are colourless protoplasmic corpuscles of various shape, which are to be found in the cells of those parts of plants which are not exposed to light, and in which starch is deposited. STRUCTURE OF THE CELL. 27 The material must have been previously treated for a short time with picric acid, so as to prevent their swelling up and disappearing when they are mounted in water or in dilute glycerine. The most suitable material is any tissue of which the cells contain but few starch-granules ; the best is the tubers of the orchid Phajus grandifolius, (Bletia Tankervillice). In this starchgrains can be easily seen borne on the leukoplastids. 5. Structure of Thickened Cell-walls and of Starch-grains. a. Cell-walls. Cut a transverse section of an old branch of Clematis Vitalba ; mount in water ; examine with high power. Observe the thick-walled cells of the pith ; the wall appears to consist of a series of concentric layers ; this is described as the stratification of the cell-wall. Strip a piece of the bark from the branch, and remove with a needle some of the fibrous internal layer of the bark ; mount in water, tease out with needles, and examine with a high power. Observe the dark lines running in the wall of the fibre at an acute angle to the longer axis of the fibre note that these lines run in different direc; tions in different layers of the wall of the fibre ; this may be seen by carefully focussing first the surface and then the deeper layers of the wall; these lines are described as constituting the striation of the cell-wall. Observe the canals running transversely across the cell-walls. Some of the cells will present their upper walls (those nearest the observer) : on these pits 28 PRACTICAL BOTANY. will be seen, which are the terminations of canals like those seen in the sections of the longitudinal walls. Pits can be readily seen, without making sections, in the leaves of some species of Trichomanes. Cystoliths may be included here, since they are developed from the cell-wall. Cut a transverse section of a leaf of Ficus elastica : mount in water; examine with a high power. Observe the layer of large clear cells underlying the superficial layer of the epidermis of the upper surface of the leaf : here and there one of these cells is seen to contain a botryoidal body suspended by a stalk from the top of the cell ; this is a Cystolith : it consists of a mass of cellulose developed as an outgrowth from the cell-wall, encrusted with calcium carbonate. Run in a drop of acetic acid : observe that the cystolith becomes gradually transparent, and that an evolution of bubbles of gas is taking place from it. When the calcium carbonate is all dissolved, a mass of cellulose will be seen to remain, presenting both striation (from above downwards) and stratification (parallel with its margin). Apply tests for cellulose (p. 29). 1. Starch-grains. Scrape lightly with the blade of a knife the freshly cut surface of a piece of a potato ; mount the scrapings in a drop of water ; examine with a high power. A number of somewhat ovoid bodies of various sizes will be seen ; these are Starch-grains. Near the pointed end of a well-developed grain will be seen a small, round, clear spot, the hilum. MICRO-CHEMISTRY OF THE CELL. 29 On each side of the hilum a number of layers will be seen, constituting the stratification of the grain. The layers near to the hilum are concentric with it, and are complete ; the more external layers are excentric, and many of those between the hilum and the broad end of the grain will be seen to be incomplete ; hence the layers are more numerous between the hilum and the broad end than between the hilum and the pointed end. Here and there will be seen a compound grain, consisting of two small grains in contact b}r their broad ends, and invested by several layers common to both. !>. The Micro-Chemistry of the Cell. I. The CELL-WALL. a. Cellulose cell-walls, i. Coloured faintly yellow by iodine. ii. Coloured violet on treatment with Schulze's solu- tion (p. 22). iii. Coloured blue on treatment with iodine and sulphuric acid. In some cases the cell-wall turns blue when it is treated with iodine alone, a substance allied to starch being probably present (amyloid) ; instances of this are, the asci of Lichens, the bast in the stem of Lycopodium and in the root of Ruscus, the endosperm-cells of Pceonia, and the cells of the cotyledons of various Leguminous seeds. In other cases the characteristic reactions are not given on treatment with Schulze's solution, or with iodine and sulphuric acid ; instances of this occur in the tissues of young seedlings, of growing-points, of the cambium, and of Fungi. In the case of young tissues it suffices to treat them previously with hydrochloric acid or with solution of potash for a short time ; they then give 30 PRACTICAL BOTANY. the reactions mentioned above ; the tissues of Fungi require a long treatment (three or four weeks) with potash. It appears that in these cases other substances are present which must be extracted from the cell-walls before the characteristic cellulose-reaction can be obtained. iv. Dissolved by ammoniacal solution of cupric hydrate and by strong sulphuric acid. v. Stained by solutions of carmine and of hsematoxylin which contain a mordant, by methylene blue, and in various degrees by other aniline colours. 1. Lignified cell-walls i. Coloured yellow by iodine and Schulze's solution, ii. Coloured deep brown by iodine and sulphuric acid. iii. Coloured bright yellow when treated with solution of aniline chloride or sulphate, the colour being intensi- fied by subsequent treatment with hydrochloric or sulphuric acid. iv. Coloured red when treated with solution of phloroglucin (p. 21), and with strong hydrochloric acid. v. Coloured green when exposed to light (\ 1 min.), after treatment with carbolic and hydrochloric acids (p. 22). vi. Swollen and slowly dissolved in strong sulphuric acid ; dissolved slowly in concentrated chromic acid ; soluble in Schulze's macerating fluid (p. 23). When the lignification is not complete the cell-wall becomes disorganised and dissolves partially in strong sulphuric acid ; this is due to the presence of a considerable proportion of cellulose. Lignified cell-walls give the characteristic cellulose-reactions after maceration in Schulze's fluid. The solubility of lignin in this fluid affords a means of isolating the cells of a woody tissue. vii. Stained slightly or not at all by solutions of carmine and hsematoxylin, but readily by aniline colours MICRO-CHEMISTRY OF THE CELL. 31 c. Cuticularised cell-walls (including cork) i. Coloured yellow by iodine, by Schulze's solution, and by iodine and sulphuric acid. ii. Coloured yellowish by concentrated solution of potash ; on gradually warming (without boiling), it becomes bright yellow ; on boiling, yellow drops of suberin escape. iiii. On treatment with Schulze's macerating fluid, the cuticularised cell-walls become conspicuous ; on boiling, viscous drops (impure suberic acid) escape, which are soluble in hot alcohol, ether, benzol, chloroform, and dilute potash solution. Traces of cuticularisation may be detected by treating the tissue for a short time with Schulze's fluid without heating, and then with potash ; the cuticularised cell-walls become conspicuous and turn yellow ; the colour may be intensified by gently warm- ing in potash. iv. Dissolved very slowly in concentrated chromic acid ; hence on treatment of a section with this reagent the cuticularised cells are the last to disappear. v. Not stained by solutions of carmine or hsematoxy- lin ; stained by aniline solutions. The cuticle may be isolated, from the surface of a leaf for instance, by boiling for a few minutes in hydrochloric acid, and then washing with water. d. Callus. To be found on the plates of the sieve- tubes. i. Soluble in sulphuric acid. ii. Stained by Hoffmann's blue, and by hsematoxylin. The most delicate reagent for callus is the following : to a quantity of chlor. zinc, iod., add an equal volume of the ordinary solution of iodine in potassium iodide ; to 32 PRACTICAL BOTANY. the mixture add a saturated solution of iodine potassium iodide drop by drop, until precipitation begins, This mixture stains the callus a deep brown. e. Mucilaginous cell-walls. Resemble cellulose cell-walls in their reactions. On treatment with iodine and sulphuric acid they sometimes assume a brownish colour in addition to the blue. Cell-walls which have become converted into gum do not turn blue on treatment with iodine and sulphuric acid : Hanstein's aniline-violet colours them red. Both mucilaginous and gummy cell-walls are stained by inethylene blue. Mucilages stain pink with corallin solution ; certain kinds stain with Hoffmann's blue. Gums stain with neither of these reagents. f. Mineral deposits in cell-walls. i. Silica. On heating a section of tissue containing silica on platinum foil with nitric acid, a complete skeleton of the silicified cell-walls remains. (ii.) Calcium oxalate. Occurs in the form of crystals: insoluble in acetic acid ; soluble, without evolution of gas, in nitric acid. (iii.) Calcium carbonate. Occurs either in distinct crystals, or, apparently, as granules : soluble in acetic acid with evolution of bubbles of gas The most characteristic form in which it appears is in special outgrowths of the cell- wall which are incrusted with it ; these are termed cystoliths (see p. 28). MICRO-CHEMISTRY OF THE CELL. 33 II. The CELL CONTENTS. a. The Protoplasm. i. Coloured yellow by iodine, and by Schulze's solution. ii. Coloured yellow by nitric acid, the colour becoming more intense on warming ; on the addition of potash or ammonia a bright yellow colour is produced (xantho- proteic reaction). iii. Coloured violet after treatment with dilute solu- tion of copper sulphate on the addition of potash. Fehling's solution may be used. See page 20. iv. Coloured pink after treatment with syrup on the addition of dilute sulphuric acid. v. Stains readily with solutions of carmine, hgema- toxylin, and Hoffmann's blue ; bright red with Hanstein's aniline violet. These reactions are given by all bodies consisting of proteids. I. The Chlorophyll-corpuscles. On treatment with alcohol the green colouring- matter (chlorophyll) is dissolved, and the substance of the corpuscle is left: this gives the reactions enumerated above as being characteristic of proteids. The orange colour of many fruits and flowers is due to the presence of coloured granules which appear to be modified chlorophyll-corpuscles (chromoplastids). These may be well observed in the petals of Tropceolum. c. The Starch-grains. Coloured blue on treatment with iodine. Coloured pink with corallin solution (p. 12). In order to detect the presence of minute starch-grains in chlorophyll-corpuscles, the tissue must be kept in alcohol exposed D 34 PRACTICAL BOTANY. to light until the whole of the chlorophyll is dissolved out ; it must then be treated for several hours in strong solution of potash ; after neutralisation with acetic acid the tissue may be treated with iodine. d. Oil-drops. i. Coloured black on treatment with osmic acid, ii. Soluble in alcohol, in ether, and in potash (sapo- nified). e. Mineral substances. i. Calcium oxalate : occurs with two molecules of water of crystallisation in crystals belonging to the clinorhombic system (including raphides), or with six molecules in crystals belonging to the quadratic system. Clusters of crystals and sphsero-crystals may consist of crystals belonging to either system. Insoluble in acetic acid ; soluble in nitric acid, without evolution of gas. ii. Calcium carbonate : occurs usually in small crystals, the crystalline nature of which can only be ascertained by means of the polariscope. Soluble in acetic acid, with evolution of bubbles of gas (CO 2). iii. Calcium phosphate (also magnesium phosphate) : occurs in the form of granules (e.g. the globoids). Soluble in acetic acid without evolution of gas. iv. Calcium sulphate : occurs in the crystalline form. Soluble with difficulty in acetic or nitric acid. /. Crystalloids : may be seen in the more external cells of potato-tubers, in the form of cubes. i. They give the reactions characteristic of proteids. ii. Soluble in potash. iii. Soluble in saturated solution of common salt. g. Aleurone-grains : occur most prominently in oily seeds. MICRO-CHEMISTRY OF THE CELL. 35 i. Give the reactions characteristic of proteids. ii. Soluble, usually, in potash. The reactions of these bodies are very different in different seeds ; the following will serve as types : 1. Grains without crystalloids. (a). Soluble in water : peony, almond, cherry, apple. (6). Partially soluble in water ; more or less readily soluble in 10 per cent, solution of common salt. a. Soluble in saturated solution of common salt : lupine, pea, bean, scarlet runner. j8. Soluble in saturated solution of common salt only after treatment with alcohol : sunflower, turnip, cress, 2. Grains containing crystalloids. (a). Partially soluble in water ; more or less readily soluble in 10 per cent, solution of common salt. a. Soluble in saturated solution of common salt : Brazil nut, pumpkin. . Soluble in saturated solution of common salt only after treatment with alcohol : castor-oil plant, walnut. In all cases a mass (globoid) of mineral matter remains behind after the solution of the grain ; this is soluble in acetic acid. The sections should be examined in alcohol. h. Tannin : gives the cells in which it is present a brownish colour. i. Coloured deep brown by potassium bichromate, or chromic acid. ii. Coloured greenish-blue by dilute solution of iron sulphate. iii. On treatment with a solution of ammonium molybdate in a strong solution of ammonium chloride, either a voluminous yellow precipitate is formed (showing presence of tannin), or a red colour is produced (showing presence of tannic, i.e., digallic acid). i. Resin : occurs in drops in the cells bounding resin-passages as well as in the passages themselves. D2 36 PRACTICAL BOTANY. i. Coloured red by tincture of alkanet. ii. Coloured blue by Hanstein's aniline violet iii. Decomposed by potash. iv. Soluble in alcohol and ether. 7c. Caoutchouc : occurs in the laticiferous vessels in the form of granules of different size in different plants : stains red with alkannin solution. By means of this reaction good preparations of laticiferous vessels can be made. I. The Cell-sap may contain in solution : 1. Colouring matters. H O 2. Cane-sugar (as in the Beet-root) (C12 22 n), which does not give a reaction with Fehling's solution. See p. 20. If much is present it may be made to crystallise out by treatment with absolute alcohol. H O 3. Grape-sugar (Glucose) (C6 12 6). If a section be boiled in dilute Fehling's solution, it will, if the cells contain glucose, turn yellow, owing to the reduction of the copper. See p. 20. The precipitate (cuprous oxide) appears in the cells under the microscope as small black granules. H O 4. Inulin (C6 10 5). When the material or the section has been treated with alcohol, the inulin is precipitated in the form of sphsero-crystals, which may be readily observed. These crystals are insoluble in cold, but readily soluble in warm water, and in dilute acids and alkalies. Coloured slightly brown by iodine. H N O 5. Asparagin (C4 8 2 3). When a section of a tissue containing asparagin is treated with absolute alcohol for some time, the asparagin is precipitated in the form of prismatic crystals, MICRO-PHYSICS OF THE CELL. 37 either in the cells or at their edge, which are readily soluble in water. The best method is to maintain a stream of alcohol under the cover-slip by means of blotting-paper. A saturated solution of asparagin may be used as a further test ; the precipitated crystals will not dissolve in it, but will be dissolved on the addition of water. In performing these tests it is better to use longitudinal than transverse sections. C. The Micro-physics of the Cell. I. Imbibition. This term is used to express the fact that the cell-wall and certain of the cell-contents (proto- plasm, starch-grains, aleurone-grains, crystalloids) usually contain a certain amount of water, termed the water of imbibition. The amount of water of imbi- bition may be made to vary by appropriate re-agents, and this involves variation in size of the body observed. These phenomena are best seen in cell-walls and in starch-grains ; the cell-walls should be such as are thickened, and consist of cellulose; those which are chemically altered (either cuticularised or lignified) cannot be made to vary to any considerable extent. Cut a transverse section of the petiole of the Sun- flower (Elder or Mallow will do as well) ; mount in water ; examine with high power. Observe just beneath the epidermis, several layers of cortical cells, the walls of which are thickened at their point of junction (collenchyma). Bun in some potash solution, or some moderately 38 PRACTICAL BOTANY. strong sulphuric acid ; notice the swelling-up of the thickened cell-walls. The swelling-up of starch-grains may be observed in the same way. The amount of the swelling-up may be estimated by using a micrometer-eye-piece. The thickened cell-walls of pith-cells of Vitalba, or those of seeds (Lupine, Date) Clematis are also suitable material for this purpose. II. Osmotic Properties. These can be most easily studied in cells which have coloured cell-sap. Cut a rather thick section of a piece of fresh beetroot, and mount in water ; Observe, the thin cell-wall ; the layer of protoplasm (primordial utricle) which lines the cell-wall ; the red cell-sap filling the cavity of the cell (vacuole). Note that the red sap does not escape from unin- jured cells. Examine a similar section which has been dipped for a moment into alcohol ; the red sap diffuses out of the cells. Hence it is evident that the colouring-matter cannot diffuse out of a living cell, but diffuses readily out of a dead cell. Mount another section in water, and run some 10 per Na cent. Cl solution under the cover-slip ; it will be seen that the red sap collects as rounded deeply- coloured bodies in the centre of the cells. This is due to the contraction of the primordial utricle. MICRO-PHYSICS OF THE CELL. 39 A cell in this state is said to be plasmolytic. The contraction is due to the withdrawal of water from the cell-sap by the strong salt solution, this withdrawal not being compensated for by the entrance of salt solution into the vacuole. The salt solution diffuses through the cell-wall, and occupies the space between the cell- wall and the contracted primordial utricle, but it cannot pass through the primordial utricle to any considerable extent. On washing the section with water, the plasmolytic cells gradually reassume their normal appearance. From these observations it is evident that the passage of substances in solution into or out of the vacuole is controlled by the primordial utricle so long as the cell is living. Plasmolysis can also be well demonstrated on a Fern- prothallus by treating it as above with salt solution ; it will be seen that the contracted primordial utricle is connected to the cell-wall by a great number of delicate protoplasmic filaments. III. Optical Properties. 1. Double Refraction. In order to study this subject, apparatus for polarising light must be adapted to the microscope. This consists of two Nicol's prisms, one of which is fitted into an eye-piece, the other being fixed below the stage of the microscope, so that the light which is reflected from the mirror must pass through it : the former prism is termed the analyser, the latter the polariser. The sections to be examined may be mounted in water or in glycerine, but the best results are obtained A with sections mounted in Canada Balsam. twig of a A tree affords good material for observation. thin, nearly median, longitudinal section is to be made and mounted : a high power must be used. 40 .., PRACTICAL BOTANY. The examination is to be commenced by rotating the analyser, so that the field of the microscope is bright : the section will then appear much as it does when examined with an ordinary microscope. The analyser is now to be rotated until the field is quite dark : it is then seen that the outlines of the cells appear bright, the thick, dense cell-walls (those of the fibres and vessels, for instance), being brighter than the thin cell-walls (those of parenchymatous cells). This observation teaches that the cell-walls, but not the protoplasmic cell-contents or the cell-sap, are doubly refractive, and that the denser the cell-wall the more highly refractive it is. A thin transverse section examined in the same way is seen to present similar appearances. It will be observed, in addition, that the transverse sec- tion of a much thickened cell-wall (that of a bast-fibre, for instance), presents, when the field is dark, a dark cross : when the analyser is rotated through an angle of 90, the dark cross is replaced by a bright one the field being also bright. For the explanation of this phenomenon reference should be made to textbooks of Physics. Mount some