Digitized by the Internet Archive V in 2007 with funding from ^- ^ IVIicrosoft Corporation http://www.archive.org/details/botanicalmicroteOOzimmrich Botanical Microtechnique A HAND.BO. METHODS FOR THE PREPARATION, ST^^^IfTNG, A^ MICROSCOPICAL INVESTIJuAt'lON y'' OE-VEGETABLE STRUC^T^ES / !;•> BY Dr PRIVAT-DOCENT {fi, T IN t^CyNlVERaSrV AT TUBINGEN \^ E JAi ^/?(9^ r//£ GERMAN BY ELLIS HUMPHREY, S.D. NEW YORK HENRY HOLT AND COMPANY 1893 Copyright, 1893, BT HENRY HOLT & CO. ROBERT DRDMMOND, RLBCTROTTPER AND PRINTER, NEW TORS in PREFACE. The methods brought together in the present volume are, of course, chiefly taken from the Hterature scattered through various original papers and text-books. But the author has always endeavored, so far as possible, to reach an opinion from his own experience concerning the methods described ; and many of the details and modifications of previous methods contained in the book are due to his own investigations. However, the literature used has been as fully quoted as possible at all points, in so far as it seems of present value. Works of merely historical interest are not referred to, since the book is designed only for practical use. If the writer has overlooked many statements of value, it is to be hoped that it will be understood and pardoned by those familiar with the immense extent of botanical litera- ture, especially in recent years. The author will be grateful to any one who will call his attention to such omissions. Regarding the quotation of literature, it may be said that numerals are placed after authors' names in the text, which refer to the literature hst at p. v, the first (Roman) num- ber indicating the work, and the second, the page of the work cited. Where the author was not able to consult the original in the preparation of this book, the abstracts used are referred to. In the arrangement of the organic compounds, Beilstein's 5486:/ IV PREFACE. Handbuch der organischcn Chemie (II. Auflage, Leipzig, 1 886-1 890) has been substantially followed. The illustrations, where the contrary is not expressly stated, are prepared from the author's original drawings. The manuscript was practically completed in July, 1891 ; but I have tried to include the more recent literature, so far as possible, during the printing. TuEBiNGPiN, March, 1892. TRANSLATOR'S NOTE. The need of a good handbook of microscopical methods, as appHed to plants, has for some time been evident. Such as we have had have only partially covered the ground, and are now mostly out of date on account of the rapid advances of the last few years. The appearance of the original edition of this work very satisfactorily met this growing demand so far as concerned students familiar with the German language ; while its evi- dent thoroughness and the familiarity with its subject-mat- ter shown by the author in the selection of the most useful among the innumerable published methods give it especial value for students of less experience. The belief that elementary students should have access, from the first, to the best methods, and the fact that few such English-speaking students read German readily, have led me, with the support of the present pubhshers, and with the cordial consent of the author and publishers of the orig- inal edition, to undertake its translation. In preparing this English edition, I have followed quite closely the original. Certain notes and tables have been added which, it is hoped, will add to its practical usefulness to American and English students ; and certain matters not included by the author, which seem to demand notice, have been discussed in their proper places. V VI TRANSLATOR'S NOTE. I am especially indebted to Dr. Zimmermann for prepar- ing, for this edition, notes on several important results of very recent studies ; and I have added a few annotations of the same sort, thus bringing the present edition as com- pletely as possible up to date. All additions by the trans- lator are enclosed in square brackets. Weymouth Heights, Mass , July, 1893. CONTENTS. PART FIRST. GENERAL METHODS. PAGE 1. The Observation of Living Plants and Tissues. §§ 1-5 i 2. The Investigation of Dried Plants. §§ 6, 7 5 3. Maceration. ^§ 8, 9 6 4. Swelling. § 10 8 5 Clearing. §§11-27 8 A. Chemical Clearing- methods. § 12 9 B. Physical Clearing-methods. §§ 13-27 11 1. The Ordinary Method of Transfer from Water to Canada Balsam. §§ 14-22 12 II. The Transfer from Water to Canada Balsam without Alcohol. §§ 23-25 17 III. The Use of other strongly refractive Mounting Media §§ 26, 27 18 6. Live Staining. § 28 19 7. Fixing and Staining Methods. §§ 29-40 2a A. Fixing. §§ 32-34 21 B. Removal of Fixing Fluids. § 35 22 C. Staining. §§ 36-39 24 D. Fixing and Staining Microscopically Small Objects. § 40 . . 27 8. Microtome Technique. §§ 41-52 29 I. Imbedding in Paraffine. §§ 43-49 31 la. Imbedding in Celloidin. § 4ga 35 II. The Attachment of Sections. §§ 50-52 37 9. Making Permanent Preparations. §§ 53-62 40 PART SECOND. MICROCHEMISTRY. A. Inorganic Compounds. 1. Oxygen. § 63 44 2. Peroxide of Hydrogen. §§ 64-67 45 3. Sulphur. §§ 68-70 47 4. Hydrochloric Acid and its Salts. § 71 48 vii VUl CONTENTS. PACK 5. Sulphuric Acid and its Salts. § 72. 49 6. Nitric Acid and its Salts. §§ 73-76 50 7. Phosphoric Acid and its Salts. § 77 52 8. Silicic Acid and the Silicates. §§ 7S-81 53 9. Potassium, § 82 56 10. Sodium. § 83 56 11. Ammonium. § 84 57 12. Calcium. §§ 85-99 57 a. Calcium Oxalate, gg 86-89 57 b. Calcium Carbonate. §§ 90-92 60 c. Calcium Sulphate. §§ 93, 94 62 d. Calcium Tartrate. § 95 63 e. Calcium Malate. § 95a 64 /. Calcium Phosphate. §§ 96, 97 64, g. Recognition of Calcium in the Ash. §98 66! //. Recognition of Calcium in the Cell-sap. ii 99 66j 13. Magnesium. §§ 100, loi 67] 14. Iron. § 102 6J B. Organic Compounds. I. Fatty Series. 1. Alcohols. § 103 . , Dulcile. § 103 69 2. Acids. §§ 104-106 701 a. Oxalic Acid. § 104 ... 701 b. Tartaric Acid. § 105 70! c. Betuloretic Acid. § 106 .70 3. Fats and Fatty Oils. §§107-112 71 4. Wax. §§ 113-115 74 5. Carbohydrates. §§ 116-125 75 a. Glucose. §§ 1 18-120 77 b. Cane-sugar. § 121 78 c. Inulin. §§ 122, 123 78 d. Glycogen. § 124 80 e. Dextrine. § 125 So •6. Sulphur Compounds. §§ 126, 127 81 a. Garlic-oil. § 126 81 b. Mustard-oils. § 127 81 7. Amido-compounds. §§ 128-130 82 a. Leucin. § 129 82 b. Asparagin. § 130 II. Aromatic Series. I. Phenols. §§ 131-133 84! a. Eugenol. § 131 84I If. Phloroglucin, ^132 84I c. Asaron. § 133 85I CONTENTS. IX PAGE •2. Acids. §§ 134-136 85 a. Tyrosin. §§ 134, I35 85 b. Ellagic Acid. § 136 86 3. Aldehydes. § I37 86 Vanillin. § 137 86 4. Quinones. §§ 138-141 87 a. Juglon. § 139 87 b. Emodin. § 140 87 c. Chrysophanic Acid. § 141 88 5. Hydrocarbons. §§ 142-149 88 a. Ethereal Oils. § 144 89 b. Resins and Terpenes. §§ 145-149 90 6. Glucosides. §§ 150-164 92 a. Coniferin. § 151 ... 92 b. Datiscin. § 152 93 c. Frangulin. §153 93 d. Hesperidin. § 154 93 e. Coffee-tannin. § 155 . 94 /. Potassium Myronate, § 156 95 ^. Phloridzin. § 157 95 h. Ruberythric Acid. § 158 95 i. Rutin. § 159 96 k. Saffron-yellow. § 160 96 /. Salicin. § i6oa 96 m. Saponin. § 161 96 n, Solanin. § 162 97 o. Syringin. § 163 98 /. Glucoside (?) from the Stimulus-conducting Tissue of Mimosa pudica. § 164 98 7. Bitter Principles. §§ 165, 166 99 a. Calycin. § 165 99 b. SperguHn. § 166 99 S. Coloring Matters. ^§ 167-197 . . . . • 100 a. Pigments of the Chromatophores. §§168-179 100 a. Chlorophyll-green. § 169 loi ft. Carotin. §§ 170-172 loi y, Xanthin. §173 103 d. Coloring Matter of Aloe Flowers. § 174 103 6. Coloring Matters of the /"A7;'/«V^ (Phycoerythrin). §175. 103 C. Coloring Matters of the /'/^^^//^/r^^(Phycophcein). §176 . 104 7/. Coloring Matters of the Q/^/;/6'//zjV^^^ (Phycocyanin). §177. 104 0. Coloring Matters of the Z>za^^/«rt!<:^o%, $0%, 70j^, 90j^, and finally in absolute alcohol. The time between the transfers must depend upon the thickness of the tissues. With delicate objects, as, for example, unicellular alga^, intervals of a few minutes each are sufficient. In the case of filamentous algae the transfer can be much iiimplified by binding them together with a thread. 15. Gradual dehydration can be accomplished by a method •devised by J. af Klercker, which consists in allowing abso- lute alcohol to flow slowly into 10^ alcohol through a fine capillary tube. 16. The dehydrating vessel* recommended by Fr. E. Schulze (I) brings about the gradual replacement of water • This may be obtained of Warmbrunn, Quilitz & Co., Berlin, C, Rosen- thalerstr., 40, at the price of Mk. 2.75 (67 cents). GEXERAL METHODS. 1 3 by osmotic action and is especially adapted for small objects. As is shown in the accompanying Fig. 4, in which the bell- shaped cover that closes the vessel is not shown, this consists chiefly of two cyl- inders, broadened at the top and placed one within the other, their lower ends being closed by a membrane which per- mits osmotic exchange between water and alcohol. Schulze recommends for this purpose a thin writing-paper known as '* Postverdruss." " which is glued to the ground lower edge of the cylinder. In the inner cylinder are placed the c- ^ x. ^ •^ ^ Fig. 4.— Dehj-dratine ves- objects to be dehydrated in ver>^ dilute, ^^- After f. e. scTiuize. about 10^, alcohol ; in the outer cylinder is placed a small quantity of stronger, about 50%, alcohol ; and in the vessel containing the cylinders is absolute alcohol, which is kept water-free by a layer of anhydrous copper sulphate on the bottom of the vessel. For complete dehydration a period of twenty-four hours is always sufficient. Further, the rapidity of the osmotic interchange may be largely regu- lated by changes of the differences in level between the different fluids. With less sensitive objects one may find one cylinder sufficient, and then the dehydration can be accom- plished in a few hours. 17. According to the method proposed by Overton (I, 12), dehydration may be conducted by placing the objects first in \o' on the manipulations to be described on the slide rather than on the cover-glass, as Overton recommends^ unless for special reasons culture in the hanging drop is^. necessar>'. In the first place, for fixing the objects in a drop of cul~ -:re fluid an easily removable fixing medium should be used. For this purpose the fumes of iodine are well adapted. They are poured out upon the preparation front a heated test-tube, and are easily driven off again by subse- quent warming (2 to 5 minutes in the paraflfine bath). For the same purpose the fumes of osmic acid may be used. They are applied by holding the slide, with the objects downward, over the mouth of a bottle containing a dilute solution of osmic acid.* * [Where this ifnretskm of the slide cannot be safely risked, the addition to the cnhure Said oC a drop of a i^ solotioa of ocouc acid may save the same ^1 28 BOTANICAL MICROTECHNIQUE. After fixing, the objects are transferred to alcohol. This is accomplished by first adding a drop of 10-20^ alcohol, which causes no collapse, and then placing the preparation in a close chamber saturated with alcohol vapor, to bring about a gradual concentration of the alcohol. For this purpose a flat crystallizing dish may be used, its upper edge being ground so as to be hermetically sealed by a greased glass plate. Its bottom is covered with absolute alcohol and a stand to hold the preparations is placed in it. The latter may be made by simply bending down the ends of some strips of sheet zinc. According to Overton, the cover- glass with the objects is placed, with its wet side upward, on a piece of elder pith, about 3 mm. high and of a diameter less than that of the cover-glass, which, in its turn, rests on an ordinary slide. In this apparatus, which must be pro- tected from sudden changes of temperature and especially from direct insolation, the 20^ alcohol on the cover-glass becomes almost absolute alcohol in a few hours, by diffusion through the air. When this is accomplished, a drop of a dilute solution of celloidin is placed on the cover-glass and evenly spread over its upper surface by tipping it backward and forward. It is of advantage to make the celloidin film as thin as possible, since thicker films both separate more easily and render the subsequent manipulations much more difficult. Therefore pretty thin solutions of celloidin must be used. A suitable one may be prepared by diluting the ordinary ofificinal solution of celloidin with ten times its bulk of a mixture of equal parts of alcohol and ether.* As soon as the celloidin no longer flows evidently, the whole cover-glass (or slide) is placed in 80^ alcohol, wet side up. Here the celloidin film becomes so hard in a few minutes that the objects can be placed in suitable staining fluids without being washed away. A long-continued action of alcohol of more than 90% is to be avoided, as it dissolves * [The thinnest of the celloidin solutions recommended for use in imbed- ding (cf. § 49a) may be diluted with twice its own bulk of the alcohol-ether mixture for this purpose.] GENERAL METHODS. 29 the celloidin film. Therefore Overton dehydrates with 80 to 85 % alcohol and uses for the transfer to Canada balsam, creosote, which will mix with ev^en 70^ alcohol. From the creosote the preparations are either placed directly in Canada balsam or are passed first through xylol. Aniline may be used for the same purpose by passing the objects from 90% alcohol to aniline, then to a mixture of equal parts of aniline and xylol, and then to xylol (cf. § 24). It should be noted also that many stains, as, e.g.. Gentian violet, stain the celloidin film strongly and are, therefore, not to be used with this method. VIII. Microtome Technique. 41. While the microtome has been generally used for years by anatomists and zoologists, it has been used by botanists in a comprehensive way only in recent years. But since, so far as I know, no one who has recently taken the trouble to familiarize himself with the technique of the microtome, has denied the great value of microtome methods, it seems superfluous to discuss here in detail their advantages and disadvantages. I will only remark that the methods described in the fol- lowing sections are not applicable to very hard objects, especially to woods, while they have given me excellent re- sults with all soft structures and also with most leaves and herbaceous stems or roots. How far the methods proposed by Vinassa (I and II) for cutting very hard objects, aided by a firmer microtome, specially constructed for this purpose, are capable of general application, I cannot judge for want of personal experience. At all events it would be desirable, where possible, to so modify Vinassa's methods as to preserve the protoplasmic elements in the preliminary preparation of objects.* * [The Providence microtome, devised and sold by Rev. J. D. King, Cot- tage City, Mass., is especially constructed for cutting hard objects and is said to be well adapted to its purpose, but I am not able to speak from expe- rience of it.] 30 BOTANICAL MICROTECHNIQUE. 42. I refrain from describing here in detail the various microtomes and their manipulation, and merely remark that I have obtained the best results with a relatively small microtome by Schanze, namely, sections of i micron and even of fractions of a micron in thickness. In this instrument, as may be seen in Fig. 14, the move- ment of the knife, as well as the raising of the object, is accomplished by the aid of screws. To obtain very thin Fig. 14.— Microtome by Schanze. sections, one turns the disk which is connected with the object-raising screw by a system of cogs. This shows di- rectly I }x of thickness and permits the estimation of frac- tions of that thickness.* I have also worked for a long time with a more elaborate microtome by Aug. Becker (Gottingen) which was very exactly constructed. [1 have used with great satisfaction for serial sections of objects imbedded in parafifine the Minot microtome. This has the knife fixed, while the object is moved vertically past its edge, being pushed forward by an amount equal to the desired thickness of a section, at each descent, by an auto- matic device. The operation of the instrument consists in ♦ This microtome was constructed from the specifications of Prof . Altmantr and may be had of the mechanician M. Schanze (Leipzig, Brtiderstr. 63.) GENERAL METHODS. 3 1 the rotation of the balance-wheel by hand power or by a water motor, and its work is thus more uniform and more rapid than that of the sledge , microtomes of the Schanze and \ ^^^ ^.- ^ ^ f^mmmm iis^^ ,^, _, . , ., , 1="----- --^-- - ." iPiiiilWiiiiiiiiiiiniiiiiiiiiiniiiiiiiiiiiiniiiiniini Thoma types. It is not suited y"^ . . -y ^^"*— i— ^mjp' for cutting objects in celloi- .„^ ^ ^ o J Fig. 15.— Microtome knife. After ^in.*l Henking. Of the many microtome knives used I have found most suitable that recommended by Henking (I) with a very short €dget (cf. Fig. 15). I must particularly describe the imbedding of objects to be cut and the manipulation of microtome sections, espe- cially their attachment to the slides. But it cannot be my duty to bring together the very numerous methods recom- mended by various authors. It will be better for me to confine myself to the careful description of a few methods whose trustworthiness I have had opportunity to prove. Therefore I will particularly describe, among the various modes of imbedding, only the paraffine method, which is by far the best adapted to vegetable objects. I. Imbedding in Paraffine. 43. For imbedding in paraffine, objects stained in mass or unstained objects may be used. If one is concerned with protoplasmic structures, these must, of course, be carefully fixed and the fixing medium must be washed out before imbedding. The size of the pieces to be imbedded depends naturally upon the nature of the object. In general it is advantageous to use as small pieces as possible, for, on one hand, these are more easily penetrated by the various fluids, and, on the * [This microtome is sold by the Franklin Educational Co., Hamilton PI., Boston, at $60, with knife.] f These are to be obtained of W. Walb (Heidelberg, Hauptstr. 5) under the name of " Henking's microtome knife," at the price of Mk. 4.50 ($i.io) «each. 32 BOTANICAL MICROTECHNIQUE. other hand, it is the easier to obtain very thin sections the smaller the surface to be cut is. 44. It is also in no respect unimportant what sort of paraf- fine is used. I have used in most cases a parafifine recom- mended by Altmann which melts at 58° to 60° C* and is obtained from the drug-store of Franz Wittig (Leipzig). But when the size of the sections is more important than their extreme thinness, as may be the case where one wishes a general view, paraffine to which has been added more or less of the superheated paraffine recommended by Count Spee is more useful. Objects imbedded in this have the advantage of rolling up much less easily during cutting. This superheated paraffine may be prepared by heating ordinary paraffine in an open dish for one to six hours until it has assumed a brownish-yellow color like that of yellow wax, with the evolution of disagreeable white fumes, a slight reduction of its volume, and the elevation of its melting point. Recently such superheated parafifine can be ob- tained directly from Dr. G. Griiblerf and others. 45. In all cases a complete dehydration must precede the transfer to parafifine. This can ordinarily be accompHshed by means of alcohol. Delicate objects are better not trans- ferred directly from water to alcohol, in order to avoid col- lapse ; but one of the methods for dehydration described in §§ 14 to 17 may be used. In general it is sufificient to use between water and alcohol a mixture of equal parts of both fluids, in which the objects are left for an hour or longer. Afterwards they are left in absolute alcohol from six to twenty-four hours according to their size ; or even several days, in some cases. 46. From alcohol the objects are passed to a mixture of * [If paraffine of just this melting point cannot be obtained, it may be readily prepared by mixing two paraffines of respectively higher and lower melting points in proper proportions.] f [Dr. Grtiblcr's stains, mounting media, and other preparations, which are of standard excellence, may now be obtained of Eimer & Amend, Third- Avenue, New York, and of the Franklin Educational Co., Boston.] GENERAL METHODS. 33 three parts by volume of xylol to one part of alcohol, in which they remain twelve to twenty-four hours.* From this they are placed in xylol for twelve to twenty-four hours more. Their complete permeation with xylol may be recog- nized by the preparations becoming transparent. I may here remark that chloroform, oil of turpentine, and • toluol have been used instead of xylol in this transfer from alcohol to paraffine. But I think it doubtful whether these substances offer any advantage ov^er xylol. How- ever, the above-mentioned mixture of alcohol and xylol is decidedly preferable to the clove-oil formerly used between the alcohol and xylol. 47. From xylol the objects are transferred to melted paraffine ; but to prevent the collapse which almost always occurs on a direct transfer from xylol to paraffine, it is better to interpolate a solution of paraffine in xylol. I ordinarily proceed in the following manner with the best results. I place in a so-called bird's trough\ a mixture of xylol and paraffine which is solid, or at least of a thick consistency, at ordinary temperatures ; an exact statement of proportions is not important here. On this cold and soHd paraffine-xylol mixture I place the objects to be cut and pour over them enough pure xylol to cover them. Then I place the dish uncovered on the top of the paraffine oven about to be described, where the mixture melts gradually so that the objects covered with xylol can sink into it. A further con- centration of the xylol-paraffine is brought about by the eva- poration of the xylol. After six to twenty-four hours I place the objects in a porcelain dish filled with melted paraffine, which I place in the paraffine oven, A^ile I allow the dish with the xylol-paraffine mixture to cool for future use in the same way. The objects remain in a dish full of melted paraffine from twelve to twenty-four hours according to their size, but a longer time is seldom necessary. * For all these transfers Sleinach's glass drainers are very useful, f [Any small porcelain dish will serve.] 34 BO TANICA L MICRO TECHNIQ UE. Fig. i6.— Paraffine oven. For ?i. faraffine cn>en I use in Tubingen with the best results tan ordinary double - walled drying- I oven (cf. Fig. i6) with the mantle filled with liquid paraffine, into which ^iH^B projects a Desaga thermostat. This ^^L is so arranged that the temperature '' f^B ^^ ^^^ ^u\d paraffine is about 63° C* f^^ 48. Finally, to enclose the object quite saturated with paraffine in a block of paraffine suitable for cutting, it is convenient to place a drop of glycerine in a watch-glass of about 60 mm. diameter and to rub it over the inner surface of the glass until the glycerine can no longer be seen.f The waich-glass is then somewhat warmed and filled with melted paraffine. When this has cooled to near its melting-point, which may be known by its hardening at the edges when lightly blown upon, the objects to be enclosed are placed in it and so oriented with a heated needle that suitable blocks of paraffine can be cut from the mass when -cool. In order to prevent crystallization in the cooling paraffine •so far as may be, it should be cooled as rapidly as possible. This is best accomplished by placing the watch-glass, as soon as the objects are oriented, upon a large vessel of cold Avater, where it will readily float if carefully placed upon the surface. For the same reason it is desirable to place the objects near the edge of the watch-glass where the par- affine is thinnest. When the paraffine is quite cold it sepa- rates easily from the watch-glass and is then cut into rect- angular blocks two or three centimetres long, each of which ♦ The so-called '• Naples waier-bath " recommended by Paul Mayer (I) is also very convenient. [This bath in various more or less modified forms may be obtained of American dealers and in its best forms is very useful in the work of imbedding and mounting.] f This serves the purpose of aiding the subsequent separation of the paraf- fme from the watch-glass. GENERAL METHODS. 35 lias near one end an object to be cut. The opposite end is to be put into the object-carrier of the microtome. But lirst the end containing the object is to be so trimmed down that, while the object is still wholly enclosed in paraf- iine, the surface to be cut shall be rectangular and as small as possible. In cutting, this rectangle should be so placed that two of its sides are parallel to the edge of the micro- tome knife, which is placed at right angles to the direction of its motion. 49. It may be observed here that small blocks of paraffine can be easily attached to rectangular blocks of cork, which may then be fixed in the object-carrier of the microtome. It is only necessary to place a few drops of melted paraffine on one face of the cork and then to quickly put the paraffine block upon it and, by running around its edges a heated metal instrument, to cause it to be completely attached to the block. This proceeding is especially convenient if one wishes to change abruptly the direction of sections, as from transverse to longitudinal. If it is desirable to cut off the paraffine block for this purpose, it may be done with a knife heated over a flame, as in this way the crumbling of the paraffine is prevented. For preserving blocks of paraffine, the boxes used for the so-called Swedish matches are convenient; [or any small pasteboard boxes.] la. Imbedding in Celloidin. [49a. While the above-described imbedding method and medium are unquestionably of the first value in both animal and vegetable histology, the use of celloidin as an imbedding medium has recently become so extended, and the service it renders in many cases where paraffine does not do well, Is so good that some account of the manner of its employ- ment should be given here. It has the advantage that no heat is required in the process of imbedding, and that very large sections may be cut ; and the disadvantage that the knife must be kept wet while cutting, and that the thinnest 5G ^^^FiffAT^TCAL MICROTECHNIQUE. ^^H sections which can be cut from it are relatively thick as compared with those which may be cut from paraffine. Objects to be imbedded in celloidin must first be thor- oughly dehydrated, preferably in Schultze's apparatus (cf. § i6). They are placed in a mixture of equal parts of abso- lute alcohol and sulphuric ether, and then, after a few hours, in a solution of celloidin in the above mixture, which should contain, according to Busse (I), one part by weight of cel- loidin to 15 parts of the solvent. After it is thoroughly penetrated by this solution, which will require from a few hours to a few days, according to its size and nature, the object passes to a stronger solution containing one part of celloidin in 1 1 parts of the solvent ; and finally, after well penetrated by this, to a still stronger one with the propor- tion of one to eight parts. After remaining for a suitable time in the last solution, the object is ready for imbed- ding. For this purpose, a paper strip may be wound tightly about the end of a small block of suitable size and material, preferably of bass-wood or of vulcanized fibre, so as to form the sides of a box whose bottom is the end of the block. This box is now filled with the thick- est solution of celloidin, and in it the object is placed and oriented carefully by means of needles wet with the ether-alcohol mixture. As soon as the solvent has evap- orated sufficiently to form a firm film over the surface of the mass, the whole may be immersed in alcohol, where it becomes quite hard in a few hours. Since very strong alcohol dissolves celloidin, it cannot be used ; and statements vary widely as to the best strength for this purpose. Busse (II) has found, however, that 85^ alcohol gives the best results, both as regards the transparency of the celloidin and the thinness of the sections which may be cut from it. The paper is removed from about the celloidin mass, after it has hardened, leaving it attached to the block. The mass is now trimmed to present a rectangular upper face, and the block clamped upon the microtome so that the object may be cut in the desired plane. To cut successful sections from a celloidin block, it is GENERAL METHODS. 37- necessary to set the knife very slightly oblique, and as nearly as possible parallel, to the direction of its motion, so that the celloidin shall be cut with a long drawing stroke. The knife and the top of the block should also be kept wet,, during cutting, with alcohol of the strength of that in which the block was hardened. With these precautions excellent sections may be obtained. Busse (I) recommends the use of photoxylin instead of celloidin, as it gives a more completely transparent imbed- ding mass. The details of its manipulation are precisely the same as for celloidin. More detailed accounts of the use of celloidin may be found in papers by Eyclesheimer (I) and Koch (I).] 2. The Attachment of Sections. 50. For the purpose of dissolving out the paraffine fron> microtome sections filled with it, these are commonly at- tached to the slide. Although recently a large number of methods for accomplishing this have been proposed, I will restrict myself to describing somewhat in detail four of them, each of which seems to possess certain advantages for some cases. A. ATTACHMENT WITH COLLODION. In the first of these a solution of about 5^ of officinal collodion* is used for attachment. It is conveniently kept in a bottle having a soft brush inserted through its cork. A drop of this solution is first allowed to flow under the sec- tions arranged as desired on a sHde, a piece of filter-paper is then laid upon them, and the sections are pressed down upon the slide with the finger or with a paper-knife or simi- lar instrument. Then the sections are painted over with the collodion solution and it is allowed to dry in the air. When this is done, the slide is warmed over a small flame * [Or a mixture of equal parts of a thin solution of collodion and clove-oil. 1 3« BO TA NICA L MICRO TECHNIQ UE. until the paraffine melts, and then plunged in xylol t^fff solve the paraffine. After this the sections, if from objects stained in mass, can be at once enclosed in xylol-Canada balsam. But if they are to be stained, they must first be brought into water or alcohol according to the nature of the stain to be used. Since the separation of the sections from the slide has often occurred during this transfer, I now perform it by carrying the preparations from xylol successively into a mixture of three parts xylol and one part alcohol, 90^ alcohol and 50^ ^.Icohol, leaving them in each fluid two minutes, or as much longer as is necessary. I use for this purpose vessels with parallel sides, on the bottom of each of which, at one of the short sides, a piece of cork about i cm. high has been fastened. The slides are then so placed in these that one end rests on the piece of cork and the side bearing the sections is turned downward. From 50^ alcohol the preparations can be transferred to water or any suitable staining fluid, without fear of the separation of the sections. At least, I have experienced such a result very rarely even in the use of the most com- plicated staining processes when the above precautions have been observed ; and I have not been troubled by any seri- ous staining of the delicate collodion film by any of the more important methods. [50a. Cclloidin sections, when arranged on the slide, may be attached to it by placing the whole in a close chamber over ether. The ether vapor quickly dissolves the celloidin sufficiently to cause the sections to adhere firmly to the slide on removal from the chamber. Should any difficulty be experienced, the sections may be arranged on a thin collodion or celloidin film on the slide and then treated as above. After they are attached, they may be stained and mounted as described for paraffine sections (§ 50). Objects stained in mass may be imbedded in cellodin as well as in paraffine. For mounting in Canada balsam, celloidin sections may be cleared with a mixture of three parts xylol and one part GENERAL METHODS. 3^ phenol, or with equal parts of phenol and oil of bergamot, or with oil of bergamot alone. The last two are especiall}r recommended. When objects are stained before imbedding-, the whole block may be cleared before cutting.] B. ATTACHMENT WITH AGAR-AGAR. 51. Agar-agar is recommended by Gravis (I) for the attachment of microtome sections. A yL^ aqueous solu- tion of this substance is warmed for some time after the mixture of the ingredients until quite homogeneous, then filtered through a fine cloth or through glass wool, and finally protected from spoiling by the addition of some pieces of camphor. A drop of this solution is placed on the carefully cleaned slide, the sections are laid upon this drop, and the whole is warmed until the paraffine becomes soft without wholly melting. Crumpled sections then spread out completely. After the cooling of the slide the superfluous agar-agar is taken up with filter-paper and the rest is allowed to dr}^ com- pletely. After this the paraffine may be removed by xylols as in the previous method, and the slide may be transferred to alcohol. This method, which I have recently tried many times^ has the advantage that it admits of the use of rolled sec- tions ; and even crumpling due to the imbedding may be wholly or largely overcome. I have seen a troublesome staining of the agar-agar only with haematoxylin. A disadvantage of the method, however, consists in the fact that in pure water the solution of agar-agar, and there- fore the separation of the sections, often occurs. But one can always treat sections attached with agar-agar with solu- tions in strong or 50.^ alcohol, and can usually, with some care, stain them with aqueous solutions. C. COMBINED AGAR-AGAR-COLLODION METHOD. The separation in water of sections attached with agar- agar can be prevented by painting over sections attached by 40 BOTANICAL MICROTECHNIQUE. the method just described, after they have fully dried, with the above-mentioned collodion solution (cf. § $o) and letting them dry in the air. I have used this method often recently, and can recommend it heartily for difficult cases. It unites the advantages of both methods in that it makes possible the recovery of collapsed sections and permits the use of aqueous stains without fear of separation of the sections. D. ATTACHMENT WITH ALBUMEN. 52. According to P. Mayer's (I, II) methods, a solution of albumen is used for attaching sections. This is prepared by mixing 50 cc. of the albumen of hens' eggs with 50 cc. of glycerine and i gram of sodium salicylate, and filtering the mixture after hard shaking. A small drop of this solu- tion, which, according to Vosseler (I, 457), becomes useless in about six months, is placed on a carefully cleaned slide and is rubbed with the finger or a soft cloth until a barely visible film remains upon the slide. The sections are placed upon this and pressed down upon the slide, a dry brush being held between the finger and the sections. If the i5lide is now heated over a small flame until the paraffine melts, the sections become so firmly attached by the coagu- lation of the albumen that the paraffine can be dissolved out with xylol or other solvent without fear of their being washed away. Nor does this occur when they are trans- ferred directly from xylol to alcohol or from alcohol to Avater. Neither have I observed the staining of the albu- men film by any coloring matter ; so that this method may be most conveniently used for most cases. IX. Making Permanent Preparations. 53. One may use very various methods for preserving preparations as long as possible. In nearly all cases prepa- rations enclosed in Canada balsam or some other resin or balsam possess the greatest permanence. But, on account of their high refractive index, which nearly corresponds GEiXERAL METHODS, 4 1 with those of cellulose and of most of the contents of the vegetable cell, these substances are hardly to be used except for stained preparations or such as are intended for observa- tion by polarized light. Besides, the transfer of easily col- lapsible objects to balsam is so complicated that other mounting media are preferable for them. It depends wholly on the nature of the objects to be mounted what mounting medium is best to be used , and it will be neces- sary, in the second and third parts of this book, to re- peatedly indicate what method of preservation is best adapted to the case in hand. Since the methods of mounting in Canada balsam. Dam- mar lac and turpentine have been already described in §§ 14 to 27, only the remaining methods, in which glycer- ine especially plays an important part, need be here brought together. 54. Glycerine. — Pure glycerine in various degrees of dilu- tion, or a mixture of this with an acid, was formerly a much esteemed and almost universally used medium. In its use, however, especial care must be taken that the glycerine used is not diluted by too long exposure to the air, since in that case a gradual drying up of the preparation takes place, if the subsequently applied cement ring (cf. § 62) is not air- tight. Concentrated glycerine often cannot be used, how- ever, on account of its strong clearing and dehydrating power. In such cases a dilute solution of glycerine with a few drops of acetic acid offers great advantages, but must, as already remarked, be very carefully protected against evaporation. (Cf. especially Dippel, II, loio.) 55. Glycerine and Chrome Almn. — P'or preserving prepara- tions of Schizophycece and Floridece in their natural colors, Kirchner uses (I, p. Vll) dilute glycerine, to which is added enough chromium-potassium sulphate (chrome alum) to give the fluid a clear bluish color. 56. Glycerine-gelatine. — This is of late most used and offers undeniable advantages, in most cases, over the fluid glycerine mixtures. It is conveniently prepared from the recipe recommended by Kaiser, as follows : One part by 42 BOTANICAL MICROTECHNIQUE. weight of gelatine is soaked in six parts of water ; seven parts of pure glycerine are then added, and finally a gram of phenol to each lOO grams of the mixture. The whole is then warmed for lo to 15 minutes with constant stirring, until the fluid is quite clear, and is finally filtered through glass wool or filter-paper. This may evidently be best done with the aid of a hot-water filtering apparatus. 57. Less delicate objects, like sections of wood and the like, may be transferred directly from water to glycerine- gelatine ; more delicate preparations should first be brought into glycerine. This may be accomplished, in case of objects which collapse very easily, by placing them in a \o% solution of glycerine, which is then allowed to concentrate gradually by standing in the air. 58. Since the glycerine-gelatine (glycerine jelly) is solid at ordinary temperatures, it must be warmed before use until it becomes fluid ; and for this purpose the parafifine bath may be used (§ 47). Or one may prepare small pieces of the jelly, each of suitable size for one preparation, and melt them upon the slides. Such pieces may be readily prepared by allowing a quantity of the jelly to harden upon a plate in a thin layer, w^hich is then cut into blocks. 59. If annoying air-bubbles occur in the preparation en- closed in glycerine-gelatine, they can be easily removed from objects not too delicate by heating the jelly to boiling. Since preparations in glycerine-gelatine usually shrink pretty strongly when kept for a long time, it is generally advisable to seal them with a cement ring ; but it is best, especially with thick sections, to apply this ring after some time, as otherwise the cover-glass is easily broken by the subsequent concentration of the jelly, and it is easier to remove by warming, any air-bubbles that may appear. For demonstration preparations I apply the cement only after a year. 59a. Chloral-hydrate gelatine is recommended by Geoff roy (I) as a mounting medium. It is prepared by dissolving 3 to 4 grams of good gelatine in 100 ccm. of a 10^ aqueous solution of chloral hydrate, at as low a temperature as pos- GENERAL METHODS. 43 sible. The sections are placed directly in this fluid, and, since a thin layer of gelatine is soon formed at the edge of the cover-glass by the evaporation of water, the preparation may be sealed after a short time with maskenlack or an alcoholic solution of sealing-wax. Many stains, such as those with iodine green or carmine, are well preserved in this medium. 60. Enclosure in Air. — Ash skeletons, crystals easily solu- ble in water, and the like may be often best preserved sim- ply dry. To protect them from dust it is also necessary in such cases to cover the objects with a cover-glass. This may be attached to the slide by wax or paraffine around its edges or even with gummed paper. 61. TJie observation of crystalline precipitates and the like is best conducted in the air by ordinary light ; while in polarized light the interference colors appear most pure on enclosure in a strongly refractive medium like Canada bal- sam. Instructive preparations of both kinds may be pre- pared by placing on the slide a drop of Canada balsam so small that it occupies only a part of the space beneath the cover-glass, leaving a part of the crystals in air. To exclude destructive agencies so far as possible, the edge of the cover-glass may then be surrounded with paraffine or wax. 62. Sealing Media. — Of the numerous sealing media pro- posed by various authors may be mentioned here first the so-called "gold-size," which is well adapted for glycerine and glycerine-gelatine preparations. Since the method of." preparing it is quite elaborate, it is best to obtain it ready- prepared (e.g., from Dr. G. Griibler, Leipzig). For glycerine-gelatine preparations Canada balsam, asphalt varnish, and maskenlack N. Ill are also well adapted. The cover-glass cement containing amber, recommeded by Hey- denreich, affords a very trustworthy medium ; but it should not be colored with eosin, as was the case with a prepara- tion formerly furnished by Dr. G. Griibler, because this gradually goes over into the glycerine-gelatine and ma^^r cause an unpleasant staining of the preparation. IPart Second MICROCHEMISTRY. A. Inorganic Compounds. I. Oxygen, Oa. 63. For the microchemical recognition of oxygen, the method with Bacteria devised by Engelmann (I) may often be used with success. This depends upon the fact that moving Bacteria at once cease their motion if oxygen is withdrawn from them, and immediately resume it on the subsequent renewal of the oxygen supply. Oxygen also affects the direction of motion of Bacteria, since they move toward the fluid which is richest in oxygen. It is easy to satisfy one's self of this by placing a drop of fluid containing moving Bacteria on a slide, and covering it with a large cover-glass. The oxygen of the fluid is soon exhausted and the motion continues only at the edges of the cover, or around included air-bubbles, which are espe- •cially instructive. It may also soon be seen that the Bacteria group themselves in heaps at these places. The sensitiveness of this reaction, which shows very small quantities of oxygen, is naturally dependent in some degree upon the choice of Bacteria. Those which are obtained by letting split peas decay in water are very useful. After a few days innumerable Bacteria appear, which are commonly called Bacterinui tcrvio. It may be added, with reference to the management of the reaction, that it is usually desirable to use large cover- •^lasses, whose edges may be sealed with cacao-butter, wax, 44 MICROCHEMISTRY. 45 or paraffine to prevent the evaporation of the fluid and the access of oxygen. 2. Peroxide of Hydrogen, HjOa. 64. For testing living Spirogyrce for the presence of perox- ide of hydrogen, Bokorny (III) used the two following methods. The first is based on the fact that peroxide of hydrogen in the presence of iron sulphate at once sets iodine free from potassium iodide, so that any starch or starch-paste present is colored blue. He therefore placed Spirogyra cells con- taining starch in a very dilute solution of ferrous sulphate and potassium iodide, and deduced the absence of peroxide of hydrogen from the failure of the starch-grains to become colored blue. This was emphasized by the intense bluing of the starch in threads which had previously been saturated with the peroxide. In the second method, Bokorny acted upon the fact that tannin which gives a blue reaction with ferric salts is at once turned blue by ferrous sulphate in the presence of peroxide of hydrogen, while the blue color otherwise appears only after some time in consequence of the gradual oxida- tion of the ferrous salt in the air. He observed, in agree- ment with the above, that Spirogyra threads containing a tannin that reacts with ferric salts became blue only many hours after being placed in a solution of ferrous sulphate, while the blue color appeared at once in threads saturated with the peroxide. Pfeffer has (IV, 446) questioned the conclusiveness of these experiments and especially doubted whether the dilute reagents used by Bokorny were really taken up by the living cells. But Bokorny has (I and II) recently made observa- tions which show that the ferrous sulphate is really taken up by the living cells, and the conclusiveness of the second reac- tion cannot, therefore, be doubted. 65. Pfeffer (IV) was led by more extended observations to the conclusion that peroxide of hydrogen does not occur A 46 BOTANICAL MICROTECHNIQUE. within the living cell. He showed first that the peroxide may be taken up by living cells without harm and that it often produces in them, even when in very small quantity^ plainly visible reactions, which do not otherwise occur in living cells. Pfeffer used first for these researches plants whose color- less cell-sap is colored by the oxidizing effect of the peroxide, as, e.g., the epidermal cells of the stem and root of seedlings of Vicia Faba or the root-hairs of Trianca bogotensis. In these the peroxide produces a browning of the cell-sap which is usually followed by the separation of red-brown or almost black granular masses, as is shown in Fig. 17. Here is figured a part of an epidermal cell from the stem of Vicia Faba^ which has lain five hours in a solution of peroxide of hydrogen, prepared by mixing ten parts of pure water with one part of a commercial peroxide solution already six months old. 66. Pfeffer also worked with cells which have naturally a colored cell-sap, like the stamen-hairs of Tradescantia virginica. In this case the blue Fig. i7.-Partof ccll-sap is wholly blcachcd by the peroxide or cell *^of ^'t'h^ takeSz-a yellow-brown or vinous-yellow color. \aba, hJe Blcaching by the peroxide taken up may also being placed bc obscrvcd in cclls whosc protoplasm has been hydrogen so- previously colored blue by cyanin. The root- lution. hairs of Trianca bogotensis are well adapted for these experiments. In their protoplasm, when in a very dilute solution of cyanin, prepared by warming that dye with water, various blue differential stains were evident in from three to fifteen minutes, and were destroyed by perox- ide of hydrogen in less than a minute. 67. It may be remarked that Pfeffer worked with solu- tions of from .015^ to i^ of the peroxide. Since the com- mercial peroxide always contains some free hydrochloric acid to increase its keeping quality, it must be neutralized with sodium bicarbonate ; and Pfeffer adds this in slight excess. MICROCHEMISTR V. 47 3. Sulphur, S. 68. The sulphur which occurs in various Bacteria in the form of strongly refractive spheres (cf. Fig. 18, i, a~c) is, according to Cohn (I, 178), insoluble in water and hydro- chloric acid, but soluble in an excess of absolute alcohol, in hot potash, or in sodium sulphite. Nitric acid and potassium chlorate dissolve them at ordinary temperatures, as does car- bon bisulphide ; but the entrance of the latter into the cells of the Bacteria must be aided by previously killing them with sulphuric acid or by drying. According to Wino- gradsky (I, 521), this solubility in carbon bisulphide is not complete, although the insoluble residue in this reagent is always small. According to Biitschli (I, 6), the granules of sulphur are soluble in twenty-four hours in artificial gastric juice, or in a 10^ soda solution. 69. Various observations of Winogradsky (I, 518) ex- plain the accumulation of the sulphur granules. Accord- ing to this author, they are always quite spherical in the living cell and run to- gether on the death of the cells, for example, on heat- very rich hours'" cul- ing to 70° C, into large drops which change into beautiful crystals of sul- phur. This crystallization takes place best when Beggi- atoa threads rich in sulphur are placed for about a min- ute in a concentrated aque- ous solution of picric acid and then washed in a large quantity of water. On such threads beautifully formed sulphur crystals were found after twenty-four hours, partly monoclinic prisms and partly rhom- bic octahedra (cf. Fig. 18, 2). It is therefore to be presumed that these sulphur grains consibt of the modified form of sulphur which is semi-fluid or oil-like at ordinary tempera- FiG. 18.— I. Beggiatoa threads, in sulphur; b, after 24, f, after ture in spring-water; s, granules of sulphur. 2. The same, 24 hours after treatment with picric acid, which largely converts the sul- phur globules into crystals. After Wino- gradsky. 48 BOTANICAL MICROTECHNIQUE, tures. In fact, the precipitate of sulphur which is formed when dilute hydrochloric acid is added to a solution of calcium pentasulphide shows the same relations, on micro- scopic examination, as the sulphur granules of the Beggia-- toas. It is possible, according to Winogradsky, that these,, as well as the granules of the precipitated sulphur, gradually- pass over into the solid condition, and that, especially in slowly growing threads, all the stages from the fluid to the almost solid condition occur. 70. It should be observed here that Jonsson (I) has seen in a mycelium of Penicillium, growing on dilute sulphuric acid, strongly refractive bodies which correspond in many of their reactions with the sulphur granules of the Beggia- toaSy and consist, according to Jonsson, of a mixture of suU phur and an oil-like substance. 4. Hydrochloric Acid, HCl, and its Salts. 71. For the recognition of hydrochloric acid Schimper (II, 212) found the two following methods especially useful. I. The addition of silver nitrate causes the formation of amorphous silver chloride, but this may be obtained in crys- talline form by dissolving the precipitate arising from the addition of silver nitrate in as little ammonia as possible and allowing the fluid to evaporate. Regular crystals of silver chloride are thus formed, consisting chiefly of hexahedra,^ octahedra, and rhombic dodecahedra, as well as combina- tions of these (cf. Fig. 19). These crystals gradually become violet-col- ored in the light ; but in the presence of reducing plant-juices they often be- come very rapidly colored. Formed silver chloride may also be recognized Fig. i9.-crystais of silver bv its ready solubility in potassium chloride. After Haushofer. ^ '' ' * cyanide, in sodium hyposulphite, and in a concentrated solution of mercuric nitrate. It is also somewhat soluble in concentrated solutions of the alkaline metals and in concentrated hydrochloric acid ; and, accord- oo2 MICROCHEMISTRY. 49 ing to Borodin's method,* silver chloride may be tested with a concentrated solution of silver chloride in concentrated hydrochloric acid or salt solution (sodium chloride), II. Thalliiun sulphate causes at once, or at least on evap- oration, the formation of regular octahedra or variously shaped skeletons of thallium chloride, which may be tested, according to Borodin's method, with a concentrated solu- tion of thallium chloride. 5. Sulphuric Acid, H2SO4, and its Salts. . 72* For the recognition of sulphuric acid we still lack a completely trustworthy method. The following methods have been used by Schimper (II, 219) : 1. Barhun chloride always causes a precipitate of barium sulphate, but this is rarely crystalline and its positive deter- mination is therefore rarely possible. 2. Strontium nitrate causes the formation of small, thick crystals of a mostly roundish-rhombic form, though some- times sharp and with straight outlines, which are insoluble in water. 3. Potassium sulphate often crystallizes out of a solution of ash in water in the form of hexagonal plates, which fall into colorless granules on the addition of barium chloride. * According to Borodin's method (II, 805) a given precipitate soluble in water is tested with a completely saturated solution of the substance that is suspected in it. If the suspicion is correct, the precipitate will not be dis- solved, while any other substance, unless some reaction occurs, will be solu- ble. If, for instance, we have to do with a mixture of asparagin and saltpeter (potassium nitrate) the asparagin crystals will, of course, be insoluble in a con- centrated solution of asparagin, but the saltpeter crystals will be dissolved. On the subsequent addition of water, asparagin crystals will be dissolved also. So, as in. the above-mentioned case, silver chloride will be insoluble in a con- centrated solution of silver chloride in strong hydrochloric acid (or NaCl), while it must dissolve on the addition of more acid (or NaCl solution). In case of substances not too easily soluble, this method renders good service ir» microchemistry ; but great care must be taken in each case that the solution employed is really completely saturated, and that it does not become capable, through changes of temperature, of dissolving more of the substance concerned. -50 no TA NICA L MICRO TECHXIQ UE v>r into heaps of red granules on the addition of phitinum chloride. 4. Sodium and potassium sulphates may often be recog- nized in the living tissues by means of nickel sulphate. With this they form well crystallized double salts of the composition NiSO, + NajSO, + 6H,0 (or the correspond- ing K salt) ; these occur mostly in the form of the mono- clinic prism combined with the basal plane, but are pretty easily soluble in water. 6. Nitric Acid, HNOa. Nitrous Acid, HNO,, and their Salts. 73. Diphenylaminc was first recommended by Molisch (I) for the recognition of the nitrates, and he used for fresh rsections a solution of from jL^ to -^^ of a gram of it in 10 ccm. of pure concentrated sulphuric acid, or for dried sec- tions a concentrated solution of it in concentrated sulphuric acid. In the presence of nitrates there occurs immediately after the addition of this reagent a deep-blue coloring which, after a time, disappears or passes into brownish yellow. This reaction occurs in the same way in the presence of nitrites, and it can therefore be used for the recognition of nitrates only when the absence of nitrous salts is proved. But in fact all investigations on the subject heretofore have led to the conclusion that nitrous salts do not occur Avithin the living plant ; and therefore this objection to the applicability of diphenylaminc as a reagent for nitrates falls, so far as the microchemical study of the plant is concerned. It should be remarked that other compounds than nitrates and nitrites give the same reaction, as, for example, man- cjanese peroxide, potassium chromate and chlorate, hydro- <^^en peroxide, ferric oxide and its salts (cf. Frank I and II, and Kreusler I). But these substances appear to be as rare in the plant as nitrites ; at all events, plants freed from nitrates never give a blue color with diphenylamine, accord- ing to the confirmatory researches of Frank and Schim- perfll, 217). MICROCHEMISTRY. 5*1 It is more important to note that the reaction may entirely fail, even with large quantities of nitrates, in pres- ence of various substances, as, for example, lignified cell- membranes (cf. Schimper II, 217). It follows, therefore, that the absence of nitrates can never be deduced from a negative result of this test. 74. Brucin gives a bright red or reddish-yellow color with nitrates and nitrites, but this gradually disappears. Molisch (VI, 152) uses for microchemical purposes a solution con- taining .2 gram of brucin in 10 ccm. of concentrated sul- phuric acid, but remarks that this reaction is inferior in clearness to the diphenylamine-reaction. 75. According to Arnaud and Fade (I), the alkaloid cinchonamin (CigH^^N^O), obtained from the bark of Remijia purdieana, may be used for the microchemical recognition of nitrates. Its nitrate is almost absolutely insoluble in acidified v/ater and forms beautiful, readily recognizable crystals whose form is, unfortunately, not described by these authors. They immerse fresh sections of the parts to be tested directly in a .4^ solution of the chloride of cin- chonamin which is slightly acidified with hydrochloric acid. The crystals of nitrate of cinchonamin will then be formed within the cells containing nitrates. 76. Potassiitin nitrate (KNO3) ^nay also be recognized by covering the sections with a cover-glass, adding alcohol, and then allowing them to dry. The saltpeter then usually crystallizes, chiefly in the form of rhombic plates (cf. Fig. 27, § 1 30), which stand out sharply, especially in polarized light. Asparagin also forms similar crystals, but these may be easily distinguished from saltpeter crystals by measuring their angles (cf. § 130). Besides, the latter are, of course, easily soluble in a concentrated aqueous solution of aspar- agin, and are not destroyed by heating. They can also be readily tested with a solution of diphenylamine. Borodin's method (cf. § 71, note) is inapplicable, on the other hand, on account of the ready solubility of potassium nitrate. 52 BOTANICAL MICROTECHNIQUE. 7. Phosphoric Acid, H3PO4 , and its Salts. 77. The following reactions are adapted for the micro- chemical recognition of phosphoric acid : 1. Nitric acid and amtnonitim molybdate. This reagent, first introduced into microchemistry by Hansen (I, 96), causes the formation of regular crystals which represent chiefly a combination of the octahedron and the cube, and are colored an intense yellow. There is commonly no danger of confusing these with the isomorphic compounds of arsenic acid, so far as the study of vegetable objects is concerned. It is convenient to use as the reagent a solution which contains 12 ccm. of officinal nitric acid of specific gravity 1. 1 8, to one gram of ammonium molybdate. In the pres- ence of small quantities of acid the precipitate is formed only after slight warming (to 40°-50° C), and then oftea only after some time. The sections to be tested are best burned before the addition of the reagent, since otherwise the reaction may be hindered by the presence of certain organic substances, as, for example, potassium tartrate. Besides, the phosphoric acids combined with the nuclein or otherwise organically united, as, for instance, the phosphoric acids contained in the globoids, are not directly shown by this reagent, but only in the ash (cf. Schimper II, 215). This reagent may be applied directly to the ash prepared by heating upon the cover-glass. Thus is obtained at once with the ashes of sections of not too young stems of Stapclia picta a strong reaction, which occurs only after some hours in sections prepared from alcoholic material which contain spha^rites of calcium phosphate (cf. ^ 96). 2. The addition of magnesium sulphate and ammonium chloride produces with salts of phosphoric acid a crystalline precipitate of ammonio-magnesium phosphate, w^hich is practically insoluble in ammonia and ammonium chloride solutions. These crystals, some of the most characteristic MICROCHEMIS TRY. 55 of which are illustrated after Haushofer (I, 92) in Fig. 20^ belong to the rhombic system. A similar salt is also formed by arsenic acid. A suitable reagent may be obtained by mixing 25 vol- umes of a concentrated aqueous solution of magnesium sulphate, 2 volumes of concentrated aqueous solution of ammonium chloride, and 15 volumes of water. If there be placed in this solution sections from alcoholic material of the stem of Stapelia picta, which have previously F'g. 20. — crystals of ammonio- J: ^ ' A • magnesium phosphate. After been soaked for a time in water to Haushofer. prevent the formation of a precipitate by the alcohol, there appear after a time in the immediate vicinity of the sphae- rites of calcium phosphate, in consequence of their gradual solution, well formed crystals of ammonio-magnesium phos- phate, among which the X-shaped skeleton-crystals appear to be especially characteristic. This reaction may be hastened by warming, but the crystals are then less regu- larly formed. For the recognition of phosphoric acid within the tissues^ this reaction is, according to Schimper (II, 216), preferable to the previously described one, since it is not interfered with by the presence of organic compounds and is very delicate. 8. Silicic Acid, SiO^, and the Silicates. 78. Silicic acid occurs in the vegetable kingdom partly in incrustations of cell-membranes and partly in the form of variously-shaped silica masses in the interiors of cells (cf. Kohl's compilation, II, 197). For the microchemical recognition of silicic acid, one may utilize its peculiarity of not being changed by heating. Its insolubility in all acids except hydrofluoric acid serves to distinguish it from other inorganic substances. In case of some strongly silicified organs it is possible by the combined 54 BOTANICAL MICROTECHNIQUE. action of acids and heat to obtain completely coherent siliceous membranes, the so-called silica skeletons. From the membranes of the diatoms, which are peculiarly rich in silicic acid, or from the epidermis of the Graminece or Eqtiu jetacece*j beautiful siliceous skeletons maybe obtained by treating them as proposed by Sachs. This method consists in heating the organ or organism on a cover-glass, or on a bit of mica to prevent the residue from adhering, with a drop of concentrated sulphuric acid until the ash remaining after the evaporation of the acid has become quite white. In case of objects poorer in silicic acid, satisfactory siliceous skeletons cannot usually be obtained by this simple method. It is then commonly better to remove the soluble inorganic substances from the pieces before burning by treatment with hydrochloric or nitric acid. In this way pure white skeletons may be much more easily obtained and may be freed from foreign admixtures by renewed treatment with hydrochloric acid. 79. Besides, siliceous skeletons may be very well prepared wholly in the wet way by the method proposed by Mil- iarakis (I). The object is first treated in a beaker with con- centrated sulphuric acid until it is quite black and then a 20% aqueous solution of chromic acid is added. In this mixture suberized membranes are also wholly destroyed, and only the siliceous skeletons remain behind. They may then be easily isolated, after the addition of water, by de- canting, and may be completely cleaned by repeated wash- ing with water and alcohol. The siliceous skeletons of perhaps of silicium with cellulose, is not yet certain. 81. Besides its solubility in hydrofluoric acid, one may use for the recognition of silicic acid the formation of crys- tals of sodium silico-fluoride, which are with great difficulty soluble in water. To obtain these crystals hydrofluoric acid and some sodium chloride are added to the ash and allowed to slowly evaporate. The crystals of sodium silico-fluoride which then form if siHcium is present belong to the hex- agonal system and represent chiefly combinations of prisms and pyramids, or of these with six-sided plates also. In stronger solutions six-rayed stars and rosettes are also ob- served as skeleton forms (Haushofer, I, 98). 56 BOTANICAL MICROTECHNIQUE. 9. Potassium, K. 82. Since ammonium cannot occur in the ^s\\, platinum chloride may well serve for the recognition of potassium. The potassium-platinum chloride thus formed crystallizes in regular octahedra and cubes. According to Schimper (II, 213) the ash is dissolved in a drop of acidified water, is warmed until dry, and the reagent is added before or after cooling. But the reagent used must first be tested with much care to show that it is really free from potassium. This may be done by letting a drop of the reagent slowly evaporate on the slide. 10. Sodium, Na. 83. The nranyl-magnesiiim acetate recommended by Streng (I) serves excellently for the recognition of very small quantities of sodium. It forms with sodium a double salt of the composition CH,CO,Na + (CH,CO,XUO,+ {CH,COO),Mg + (CH,COO),UO + 9H,0. This compound, very poor in sodium and therefore formed in the presence of very small quantities of sodium, forms small colorless or very pale yellowish rhombohedral crystals, which are little soluble in water and almost insoluble in alcohol. Since the solution of the uranium salt extracts sodium from glass vessels on long standing, Streng (III) recom- mends the direct addition of the solid magnesium-uranyl salt. . Schimper (II, 215) used tiranyl acetate for the recogni- tion of sodium, as it causes the formation, on evaporating, of sharply developed tetrahedra of sodium-uranyl acetate (CH,COONa+(CH3COOXUO), of which the larger ones appear faintly yellowish. In the presence of very small quantities of sodium simultaneously with magnesium there is formed, of course, the above mentioned uranyl-magnesium- sodium acetate. MICROCHEMIS TR V. $7 II. Ammonium, NH*. 84. The so-called Nesslers reagent may be used for the recognition of ammonium, according to Strasburger (I, 74). It is prepared in the following manner : 2 grams of potas- sium iodide are dissolved in 5 ccm. of water, and then mercuric iodide is added to the solution while warm, until a part remains undissolved. After the fluid is cooled it is diluted with 20 ccm. of water, allowed to stand for a time, filtered, and 20 ccm. of the filtrate are diluted with 30 ccm. of a concentrated caustic potash solution. If the fluid then becomes turbid, it must be filtered again (Nickel I, 94). In the presence of ammonium this solution takes a yel- low color, and with more ammonia a brown precipitate is formed. But various organic compounds give the same reaction (Nickel I, 94). 12. Calcium, Ca. 85. Calcium occurs very often within the living plant in crystalline form, and these crystals, which are met with sometimes in the cell-sap, sometimes within the membrane, consist most commonly of calcium oxalate ; crystals of calcium carbonate, gypsum, and calcium tartrate are less often observed. Besides, calcium carbonate often incrusts cell-membranes in greater quantities ; and finally, calcium phosphate has been recognized in the vegetable organism. We will describe first the methods for recognizing the various calcium salts, and then the methods of recognizing the presence of calcium in the ash and in the cell-sap. a. Calcium Oxalate, Ca(COO)a. 86. Nearly all crystals which occur within the plant-cell consist of calcium oxalate. They are found partly in the cell contents, and are partly within or upon the wall. They belong partly to the tetragonal, partly to the monoclinic crystal system. Their most important forms are illustrated 5« BOTANICAL MICROTECHXIQUE. in Kig. 21. Here Fig. I shows a tetragonal pyraniid, Figs. II and III combinations of pyramid and prism, Fig. IV a monosymmetric rhombohedron, Fig. V a rhombic plate. Fig VI probably a combination of positive and negative hemipyramidswith the basal plane, Fig. VII a combination of the rhombic plate (Fig. V) with the clinopinacoid, Fig. H ^1 ^ Fig. 21.— Crystals of calcium oxalate. I-III. from the spongy parenchyma of Tra^ dttcantia discolor ; IV, from Cycas circinalis ; V. Alusa paradisiaca : VI, Citrus vul- garis: VII and IX, Cuaiacum ojfficinale : VIII, Citrus medica. IV, V, VII, IX after Holzoer ; VI after Plitzner. VIII a combination of the rhombohedron (Fig. IV) with a hemipyramid, Fig. IX a twin crystal whose angle xyz measures 141° 3', according to Holzner (I, 34). Calcium oxalate is also especially common in the form of fine needles (" raphides ") or tiny slivers (" crystal sand ") on which no crystallographically determinable faces or angles can be recognized ; and spha^rocrystals have been seen (cf. Kohl's compilation, II, 15). Calcium oxalate is insoluble in water and acetic acid ; but in hydrochloric acid it is soluble, though the solution of the larger crystals, especially if they are imbedded in mucilage, does not occur at once. It is best to place the preparations MICROCHEMISTRY. 59 in concentrated hydrochloric acid and to follow the solution with a polarizing microscope. The action of the acid can be very much hastened by warming. With nitric acid calcium oxalate behaves essentially as with hydrochloric acid. It is readily soluble in the former, especially on warming. 87. By sulphuric acid calcium oxalate is changed into calcium sulphate (gypsum), which is little soluble in water or sulphuric acid, and separates chiefly in the form of needles. An immediate transformation of the calcium oxalate into gypsum occurs if the sections containing it are placed directly in concentrated sulphuric acid or in a mix- ture of equal parts of water and concentrated sulphuric acid, and heated nearly or quite to boiling. The gypsum is then formed within the same cells which formerly con- tained the calcium oxalate crystals ; and each more or less, opaque mass of sometimes plainly needle-shaped, sometimes more granular, particles of gypsum usually possesses exactly the same form as the original crystal. These crystalline conglomerates glisten brightly under the polarizing micro- scope. For distinguishing calcium oxalate from calcium sulphate,. Kohl has recently (II, 194) proposed a solution of barium chloride, which leaves the oxalate unchanged, while gypsunV crystals become covered by a finely granular layer of barium sulphate. In a mixture of barium chloride and hydrochloric acid, gypsum is rapidly converted into barium sulphate,, while calcium oxalate crystals disappear in the same mix. ture without forming any precipitate. On treatment with caustic potash solution, calcium oxalate at first remains unchanged; but, as Sanio (I, 254) first observed, its crystals are suddenly dissolved after some time, usually after several hours, and new crystals are formed in the fluid, which have the form of six-sided plates whose chemical composition is not yet determined. 88. On burning calcium oxalate crystals, which can oest be done on a cover-glass laid on platinum foil, the oxalate is changed first into calcium carbonate and then into caU 6o BOTANICAL MICROTECHNIQUE. cium oxide. The crystals preserve their original form, but become opaque and therefore appear black by transmitted light, but pure white by reflected light (or dark-field illumi- nation). If the crystals dissolve, after the burning, in dilute acetic acid or concentrated hydrochloric acid, without the formation of gas-bubbles, this shows that an oxalate has been changed to the oxide ; while the carbonate dissolves in hydrochloric acid with the liberation of carbonic acid. 89. The finding of calcium oxalate crystals can be made much easier by examination by polarized light. They are distinguished in general by their strong double refraction, which is, however, much greater in those of the monoclinic system than in those of the tetragonal system. The latter, naturally, cannot glisten in the polarizing microscope with crossed nicols, when their optical axes stand vertical. To make visible the crystals of calcium oxalate within large organs, for example whole leaves, without cutting them into sections, these may be made quite transparent. For this purpose chloral hydrate, which does not attack calcium oxalate, has been used ; and phenol can also be em- ployed. If the pieces are heated to boiling in one of these fluids, they usually become wholly cleared in a short time. The alcoholic solution of sulphurous acid used by Wehmer (I, 218) for decolorizing leaves will certainly be of much service in many cases. For \\i^ preservation of such preparations Canada balsam is best adapted. They may be transferred directly from phenol to xylol and xylol-Canada balsam. The study of these cleared preparations is best conducted by polarized light. b. Calcium Carbonate, CaCO,. 90. Calcium carbonate rarely occurs in the interior of cells, but is usually deposited in or upon the cell-wall (cf. Zimmermann I, 104). For the recognition of the carbonic acid in calcium car- bonate, acetic or hydrochloric acid may be used. After the addition of one of these, the carbonic acid is set free in MICROCHEMIS TRY. 6 1 l>ubbles, as can be directly observed under the microscope. It has been pointed out by Melnikoff (I, 30) that a concen- trated acid* should be used for the recognition of small quantities of carbonic acid, and that care should be taken that it reaches the bodies to be tested as quickly as possible. Evidently, the more slowly the evolution of carbonic acid occurs, the more readily will it be absorbed by the surround- ing water and carried away by diffusion without being given off in bubbles. 91. For the recognition of calcium a solution of animo- niiim oxalate acidified with acetic acid may be used. The manner in which this solution reacts with calcium salts is largely dependent upon its strength. For example, I obtained, in sections of the leaf of Fiats elastica, abundant masses of crystals grown together in gland-like masses within and near the cystolith cells, by placing them in a solution containing .5^ of ammonium oxalate and i^fc of acetic acid. This reaction took place at once on placing the sections in the solution, which had previously been heated to boiling. The crystals thus formed are strongly doubly refractive. But when a solution containing 10^ of ammonium oxalate and \io of acetic acid was used, the oxalate was precipitated very easily soluble in caustic potash solu- I i c^ SX^A) tion, almost instantly so in a 10^ solution, |a "^ *^ which does not attack calcium oxalate, p^^, 23._caicium tar- Their behavior with acetic acid is also ISl^SfXy^^S'l characteristic. Calcium tartrate crystals ?J«?J^, glth^/ed Oc" are easily soluble in dilute solutions con- ''^' taining about 2^ of glacial acetic acid, while in the pure glacial acid, or even in a 50^ solution of it, they are insoluble. In consequence of this, it may be observed, in sections to which concentrated acetic acid has been very gradually applied, that a recrystallization of previously dissolved crys- tals occurs. Calcium tartrate crystals are doubly refractive, but this power seems to me much less than that of the monoclinic crystals of calcium oxalate. On burning they are converted 64 BOTANICAL MICROTECHNIQUE, into globular masses which dissolve in lo^ acetic acid with the formation of bubbles. e. Calcium Malate, Ca(COO),.CH,.CHOH. 95a. Calcium malate is thrown down in large quantity hy alcohol in the stipes of the fronds of Angiopteris evectUy according to Belzung and Poirault (I). It often forms prisms of considerable size, which belong to the rhombic system, and are with difficulty soluble in water, but readily so in acids. With sulphuric acid they form needles of gypsum. On heating on platinum foil they are first black- ened, then show a striking increase in volume, and are finally converted into pure white lime. On being heated in the reducing flame they give off the characteristic odor of succinic acid. By the aid of Borodin's method it may be shown that they are completely insoluble in a saturated solu- tion of neutral calcium malate. f. Calcium Phosphate, (CaOjijiPO), ? 96. Calcium phosphate has been observed only in solu- tion in the cell, except in case of globoids (cf. § 388) and of a single instance which requires confirmation (cf. Nobbe Hanlein and Councler I), It separates in the form of beau- tifully formed sphaerocrystals in the interior of many parts of plants, after they have been placed in absolute alcohol ; for instance, in the stems of Euphorbia caput-meduscB and Stapelia picta, as well as in the stalk of the frOnd of Angi- opteris cvccta. These sphaerites are usually formed only after a considerable time (weeks or months).^ They have usually a yellowish or brownish color and are very slowly soluble in cold water. In hot water, too, they are only dissolved after a long time ; at least, the solution of large sphaerites was not complete after several minutes, when they had been heated to boiling in water on the slide. With ammonia they behave as with water ; they are only • I have lately found globular or clustered bodies consisting at least chiefly of calcium phosphate in the living epidermal cells of a species of CyperusM. Zimmermann VI, 311). MICROCHEMIS TRY. 65 slowly soluble in acetic acid, but readily so in nitric and hydrochloric acids, of course without any evolution of gas. In sulphuric acid they are quickly dissolved with the for- mation of gypsum needles. If sections are quickly heated on the slide in a mixture of two parts concentrated sul- phuric acid to one part water, the masses of gypsum needles then formed show the same outline as the sphaerocrystals previously present. They may be distinguished from the latter by being wholly opaque and therefore black by trans- mitted light, and white by reflected light. But if the sec- tions are placed in dilute, e.g., i^, sulphuric acid, the gyp- sum needles are gradually formed in the vicinity of the sphaerites. On burning, the calcium phosphate sphaerites at first become black in consequence of the organic admixture to be mentioned in the next section, but on further heating they yield a pure white ash. With nitric acid and ammonium molybdate, as well as with magnesium sulphate and ammonium chloride, they give the reactions for phosphoric acid (cf. § 77). When examined with a polarizing microscope these sphaerocrystals show the well-known dark cross, with crossed nicols. By the interposition of a gypsum plate it can be determined that the orientation of the optical axis is the same in them as in starch-grains and in the sphaerocrystals of inulin. In Canada balsam these sphaerites may be preserved for as long as one wishes, and in glycerine gelatine at least for a considerable time. 97. The various sphaerocrystals do not represent an even approximately chemically pure compound, but always con- tain a considerable quantity of organic substance, which often forms an amorphous nucleus at the centre of each, but is also often contained in the separate layers. It is to be ascribed to this circumstance that calcium phosphate sphaerites take up pretty freely various coloring matters like methylene blue and borax-carmine (cf. Leitgeb III). The chemical composition of these organic substances is 66 BO TA XICA L MICRO TECIJNIQ UE. fstill as uncertain as the molecular formula of the calcium phosphate contained in the sphaerocrystals. g. Recognition of Calcium in the Ash. 98. For this purpose Schimper (II, 211) recommends especially the sulphuric acid reaction, which may be con- ducted by dissolving the ash directly in about 2^ sulphuric acid and then letting it dry slowly. There are thus pro- duced, especially at the edge of the drop, crystals of gyp- sum which belong to the mono- clinic system. Among these, plate-like crystals, whose obtuse angle (^, Fig. 24) measures 127° 31', according to Haushofer(I, 33), are especially characteristic. Be- sides, twin crystals are very nu- merous, whose edges form an angle of 104° or of 130° with each other (cf. Fig. 24). But the most various fusions are also found, on whose projecting ends pretty accurate determinations of the angles may be made. The crystals of gypsum are also distinguished by the fact that they are transformed instantly into small needles on lieating in concentrated sulphuric acid. The masses of needles preserve the form of the original crystal, but appear quite opaque if of much thickness. These needles may probably represent the anhydrite of gypsum. If calcium is present in the ash as calcium sulphate, it will, of course, form its characteristic crystals if the aqueous solution of the ash is allowed to slowly evaporate. Fig. 24.— Crystals of calcium sul phate. After Haushofer. h. Recognition of Calcium in the Cell-sap. 99. For the recognition of calcium in the cell-sap, Schim- per employed chiefly the two following reactions: I. On the addition of ammoniitin oxalate, calcium oxalate U formed in the cells containing calcium, in the form of MICROCHEMISTRY. 6/ tetragonal pyramids at ordinary temperatures, but in the monoclinic form in a boiling solution. 2. Fresh sections are placed directly in a solution of a^n- rnoniiim carbonate ; if calcium is present, small, strongly doubly refractive rhombohedra of calcium carbonate are formed within the cells. If the cell-sap is strongly acid, it should first be neutralized with ammonia. 13. Mag^nesium, Mg. 100. Schimper recommends (II, 214) the addition to the sections or to the ash, for the recognition of magnesium, of a solution of sodium phosphate or of microcosinic salt {NaNH^HPO,) reduced with a little ammonium chloride. There are then formed rhombic crystals of ammonio-mag- nesium phosphate (MgNH^PO^) which have in sections the form of coffin-lids, but in the ash are chiefly the X-shaped skeletons (cf. Fig. 20, § 77). Uranyl acetate causes, if sodium is also present, the forma- tion of the crystals of magnesium-sodium-uranyl acetate, already referred to (cf. § 83). 101. Magnesmin oxalate, Mg(COOX. Monteverde (I) found, in the epidermis of fresh leaves of Setaria viridis and in dried leaves of numerous PanicecBy radially striped sphae- rocrystals or irregular aggregates, which probably consist of magnesium oxalate. These were, according to his state- ments, with difficulty soluble in water, insoluble in acetic acid, and soluble in hydrochloric, nitric, and sulphnric acids, in the latter without formation of gypsum needles. After the addition of an ammoniacal solution of sodium phosphate and ammonium chloride, crystals of ammonio-magnesium phosphate were formed ; after heating, these dissolved with- out evolution of gas ; gypsum-water caused the formation of calcium oxalate crystals ; and after treatment with caustic potash solution the sphaerocrystals lost their striping and double refraction and became soluble in acetic acid. Magnesium phosphate (MgO,)3(PO)3? According to Han- sen (I, 115), crystals of magnesium phosphate are precipe itated in the stem of the sugar-cane by alcohol. These 68 BOTANICAL MICROTECHNIQUE. have partly the glandular form and are partly more or less regularly formed sphaerocrystals. They are soluble in cold water with difficulty, but more easily so in hot water. They are also hardly soluble in acetic acid, but readily so in mineral acids, in sulphuric acid without the formation of gypsum needles. Ammonium carbonate gave no precipitate with them, but the ammoniacal solution of ammonium chloride and sodium phosphate produced a crystalline pre- cipitate. The phosphoric acid was recognized with ammo- nium molybdate (cf. § 77, i). 14. Iron, Fc. 102. Weiss and Wiesner (I) have shown microchemically that iron incrusts especially the thicker cell-membranes of the higher plants in the form of insoluble ferric and ferrous compounds, and that it also occurs in the contents of the cells. The authors mentioned used as a reagent an alco- holic solution of potassitim sulphocyanide added directly to sections cut with a silver or platinum knife. If a red color appears at once, it shows the presence of a soluble ferric compound ; but if it appears only after the addition of hy- drochloric acid, the presence of a ferric compound insoluble in water is shown. In the same way sections were treated with potassium sulphocyanide and chlorine-water or nitric acid to demonstrate soluble or insoluble ferrous compounds. Large quantities of iron compounds also occur as incrus- tations of the membrane in various Schizomycetes {Clado- thrixy Crcnothrix, etc.) and in Closteritim. It also forms thick crusts, in the form of ferric hydroxide, on the mem- branes of many ConfervacecB (cf. Hanstein I). For the microchemical recognition of iron, a 10^ solution of potas- sium ferrocyanide, to which a little hydrochloric acid is added, may be used in these cases. The reagent causes the immediate formation of Berlin blue in the presence of ferric oxide. The presence of ferrous oxide may be recog- nized in the same way by the use of potassium ferricyanide.* * A method for the recognition of organically combined iron, the so- called " masked iron," has been given by Molisch (VII). But the same MICROCHEMISTKY. 6^ For Leptothrix ochracea Winogradsky (II, 268) has shown by recent investigations that the iron is first deposited in soluble form in the gelatinous envelopes, most probably as. a neutral ferric salt of an organic acid. This then gradually passes over into a basic salt insoluble in water, and finally into almost pure ferric hydroxide, which is transformed by long submergence in water into a modification somewhat- less soluble in hydrochloric acid. B. Organic Compounds. I. FATTY SERIES I. Alcohols. Dulcite (Melampyrite) {C^lOYi\. 103. Dulcite has been recognized by Borodin (I) by^ adding one or a few drops of alcoJiol to sections of the plant- under investigation, covering with a cover-glass, and allow- ing them to dry slowly. Dulcite then crystallizes in the form of large prismatic or irregular flattened crystals which may be distinguished from saltpeter and asparagin crystals by being insoluble in a concentrated solution of dulcite and by being transformed on heating to 190° C. into frothy dark brown masses, with complete decomposition. Dulcite crystals also differ from the very similar saltpeter crystals in dissolving without color in diphenylamine-sulphuric acid (cf. § 73). Suitable objects for study are furnished by one-year-old stems of Evonymus japonicus, author has recently shown (VIII) that the iron observed by him came frontv the caustic potash used for the reaction, and that therefore the results ob- tained by his method are untrustworthy. [Carl Miiller (I) has still more recently concluded, not only that Molisch's proposed method is untrust- worthy, but that his explanation of the source of the iron he found is-- equally so. Miiller finds that the commercial hydroxide in stick form con- tains no iron, and that the iron found in solutions of caustic potash comes- from the glass of the vessels in which they are contained. He believes also- that the "masked" iron of Molisch is accumulated by plant specimens fromi the glass vessels in which they are kept, and rejects Molisch's view that most of the iron in the plant is organically combined.] yo BOTANICAL MICROTECHNIQUE. 2. Acids. a. Oxalic Acid {COOH\ 104. For the recognition of oxalic acid and its soluble salts Schimper recommends (II, 215) : 1. The addition of a solution of calcium nitrate, when <:rystals of calcium oxalate are formed (cf. § 99, i). 2. The addition of tiranyl acetate causes the formation of rhombic crystals of mostly rectangular form, which, when large, are plainly yellow and strongly doubly refractive, but whose composition is still unknown. 3. Acid potassiwn oxalate, when present in considerable quantity, is often directly recognizable in dried preparations by its crystalline form and strong double refraction on com- parison with a dried solution of the same salt, as well as by the aid of Borodin's method. b. Tartaric Acid, C,H,(OH),(COOHX. 105. Streng (III) has recommended for the recognition of tartaric acid the addition of a solution of barium chloride and anti7no7iic oxide in hydrochloric acid. This causes the formation of rhombic plates of antimonyl-barium tartrate whose obtuse angles measure 128°. Schimper (II, 220) recommends the use of the two follow- ing reactions : 1. The addition of potassium acetate produces rhombic- hemihedric crystals of the hardly soluble acid potassium tartrate. 2. Neutral solutions are treated with calciinn chloride. There are then formed rhombic crystals of calcium tartrate which represent chiefly a combination of an elongated prism with the dome. Concerning their reactions see § 95. c. Betuloretic Acid, C„H„0,. 106. This acid is secreted by the trichome-glands on the leaves of Betula alba. It is insoluble in water, but soluble in alcohol, ether, alkalies, alkaline carbonates, and concen- trated sulphuric acid, in the latter with a red coloration (cf. Behrens III, 379). ICROCHEMISTRY. 'JX 3. Fats and Fatty Oils. 107. Under the names fats and fatty oils are included, ac- cording to their consistencies, the glycerine ethers of various organic acids of high molecular weights, especially those of palmitic acid, C.sHgjCOOH, stearic acid, C^Hj.COOH, and oleic acid, C^HgaCOOH. But beside these, a whole series of acids still partly but little studied have been isolated from the various oils of vegetable origin (cf. Beilstein I, 427). An exact microchem- ical separation of these compounds is not yet possible. Even those reactions which should show whether doubtful substances belong to the group of fats still leave much to be desired in the matter of exactness, since they nearly all occur in the presence of other substances. 108. In general, however, the fatty oils show the following reactions : They are insoluble in cold and hot water and slightly soluble in alcohol ; but castor-oil forms an exception in being pretty readily soluble in alcohol. They are easily soluble in carbon bisulphide, ether, chloro- form, petroleum ether, '^ phenol, ethereal oils (as, e.g., clove- oil), acetone, and wood-siprit (methyl alcohol). According to A. Meyer (II), most fatty oils are insoluble m glacial acetic acid, if the quantity of acid is not too great, as, for instance, when the reaction is conducted under a cover-glass. An aqueous solution of chloral hydrate acts in the same way as glacial acetic acid, according to A. Meyer (II). 109. Alcannin, the coloring matter contained in the roots of Alcanna tinctoria, colors the fats deep red. The solution used for this reaction may be prepared by dissolving the commercial alcannin in absolute alcohol, adding the same volume of water, and filtering. In this solution the sections to be tested are left for one or two, or better, six to twenty- four, hours. All oil-drops then appear deeply colored ; but, on the other hand, ethereal oils and resins show the *[This is the benzinuin of the U. S. Pharmacopoeia.] 72 BOTANICAL MICROTECHNIQUE. same reaction. The staining with alcannin may be much hastened by warming. This is especially to be recommended when one has to deal with fats which are solid at ordinary temperatures, as in the cocoa-bean. If cross-sections of this seed are heated to the boiling point in a considerable quantity of the above solution, the crystals of cocoa-butter melt and fuse into drops, which become colored deep red at once. 110. Ranvier has used (I, 97) cyanin (identical with Since wax is not wetted by water, the study of the various rods, granules, etc., is better conducted in cold alcohol which wets the wax without dissolving it at once, to say the least. 115. The waxy incrustations of suberized membranes, observed especially on various epidermal cells, as, for ex- ample, those of Aloe verrucosa, become at once visible, according to DeBary (I), if the sections are warmed under a cover-glass to near the boiling point of water. They then separate from the membrane in the form of drops. These drops are soluble in boiling alcohol and behave chemically like the wax coverings described. Wax may also be ex- tracted from the incrusted membranes by boiling alcohoL The membranes always suffer in consequence a correspond- ing reduction of volume, which cannot be made good by- subsequent immersion in water. 5. Carbohydrates. 116. The carbohydrates are characterized, as is well known, by the fact that they contain, besides carbon, hy- drogen and oxygen in the same proportion as in water, so- that their general formula may be written CxH2yOy. Of course not all organic compounds which show this- empirical formula are included in the carbohydrates,, and already various substances which were formerly included here have been transferred to other places in the natural system of organic compounds, after their constitution has; become more exactly known. And the recent investigar- tions of Emil Fischer (I) have introduced a more rational classification of the carbohydrates. But these investigations have at present no significance for microchemical methods, since the certain microscopical separation of the compounds of this group is as yet possible only in very rare cases. I will therefore restrict myself here to the description of microchemical methods for recog- nizing some soluble carbohydrates. The solid carbohy- drates, cellulose and its derivatives, as well as starch and 76 BOTANICAL MICROTECHNIQUE. the related compounds, will be discussed in Part III of this book (cf. §§ 242-297 and §§ 400-415). 117. But before we enter upon the special reactions of the soluble carbohydrates, two reactions common to many car- bohydrates may be described. These were introduced into microchemistry by Molisch (V), and at first especially for the recognition of species of sugar. The reagents used are rt.naphtol and thymol. Molisch uses a-naphtol by treating sections not too thin, on the slide, with a drop of a 15-20^ alcoholic solution of the compound and then adding two or three drops of con- centrated sulphuric acid, so that the sections are wholly covered. In the presence of cane-sugar, milk-sugar, glucose, Ixvulose, maltose, or inulin, the section becomes colored a beautiful violet in a short time (about two minutes), while this reaction does not occur with inosite, mannite, melam- pyrite, and quercite. If thymol be used in the same way intead of o'-naphtol, a carmine-red color is produced. Concerning this reaction it should be said that the con- centrated sulphuric acid contained in the reagent may split off sugars from glucosides, starch, cellulose, and various other substances, and these may then give the reaction in- ^' 1 s-s 1 1 ^ £ii : s . g^^ r '^ -1 1 s'p It € "1 s 1 c 30-0 .^ •= ^« -So o oa B rt w E E o liiilf' iii.h ii ? « ^ "o *J u iMMt«be.2 -^ u|3 SS-5 oc ^-£3 1 " ^ - ^ 1 1 1. 1 1 1 s si 1 ,i ^ ' s mil E i i it|!ii-i|i 1 i •Mill 1 1 ||.-liiir|i| i 112 BOTANICAL MICKOTECHNIQUE. g. Coloring Matters which are deposited upon the Ccll-ivall. 192. In many lichens colored compounds occur which are attached externally to the membranes. They are mostly crystalline, more rarely amorphous. Amorphous excretions of pigment were found by Bachmann (IV, 27) only in two lichens, and he has named them, from the lichens in which they occur, Arthonia-violet and Urceo- laria-red. Arthonia-violet occurs in all parts of Arthonia gregaria and is especially distinguished by being somewhat soluble in cold, but readily so in hot, water. It is also solu- ble in alcohol with a wine-red color. It is dissolved by a solution of caustic potash with a violet color, but is insoluble in lime and baryta waters. It dissolves in sulphuric acid with an indigo-blue color, passing later into mallow-red ; and in nitric acid with a red color. Urceolaria-red occurs in the thallus of Urceolaria ocellata. It is characterized microchemically by not being changed by alcohol, lime-water, or ammonium carbonate. It is dissolved with a greenish-brown color by caustic potash solution and baryta-water, as well as by concentrated nitric and sulphuric acids. A solution of calcium chloride decolorizes it. 193. The substances already described elsewhere, emodin,. chrysophanic acid, and calycin (cf. §§ 140, 141, and 165),. belong to the crystalline excretions of the lichen-fungi, as- also a series of other so-called lichen acids, which have here- tofore been studied almost exclusively macroscopically (cf. Schwarz II and Zopf II, 401). But I will discuss somewhat more in detail certain recently described fungus-pigments. ix. Thelephoric Acid. 194. Zopf (V, 81) designates as thelephoric acid a pigment extracted from various species of Thelephora, whose solu- tions are of a beautiful red color, while the solid crystalline pigment has a violet-blue or indigo-blue color. This partly forms an incrustation of the membrane, partly a crystalline deposit upon it. MICR O CHEMIS TK Y. I I 3 Its behavior with aniniojiia, which gives it a splendid blue color, is especially characteristic of thelephoric acid ; while caustic potash and soda produce a more bluish-green color. Thelephoric acid is also, according to Zopf, insoluble in water, ether, chloroform, petroleum-ether, carbon bisul- phide, and benzol, but is pretty readily dissolved by warn-^ alcoJiol. Concentrated sulphuric or hydrochloric acid neither changes the color nor dissolves the solid pigment ; but con- centrated acetic <^^/^ dissolves it with a rose-red or wine-red color, nitric acid with a yellow color, and dilute chromic acid with a dark chrome-yellow color. ft. Xanthotrametin. 195. Zopf calls by the name xanthotrametin a red pig- ment which is deposited on the membranes of Tranietes cinnabarina in the form of granular brick-red incrustations. This dissolves in concentrated nitric acid with a deep orange- red color, in JiydrocJdoric acid with an orange-yellow, and then more reddish, shade, in srdpJmric acid with a rose-red color, and in glacial acetic acid with a yellow color. Dilute sulphuric acid dissolves it with an at first orange-yellow color which then becomes redjder. Xanthotrametin is dissolved by ammonia and sodium car- bonate with a yellow color, by dilute caustic soda and lime- water with a yellow color, becoming paler, and by dilute caustic potash with a yellow color which quickly passes into reddish. In ferric chloride it is insoluble. y . Pigment of A g ar i c u s a r m i 1 1 a t u s . 196. This forms, according to Bachmann (II, 7), crystal- line cinnabar-red slivers or lamellae which are insoluble in alcohol and ether, but dissolve in an aqueous or alcoholic solution of caustic potash with a red-violet color which soon goes over into dark yellow. 8. Pigment of Paxillus atr ot oniento sus . 197. Thorner (I) has extracted a pigment from the above- named fungus which is deposited in crystalline form upon 114 BOTANICAL MICROTECHNIQUE. the hyphc-K. These crystals are colored brown only at the surface of the fungus, those in its interior being at most pale gray or yellowish. In the air the colorless crystals gradually assume a brown color. According to Thorner, they represent a hydroquinone-like body which gradually passes over into the corresponding quinone. For the recog- nition of the quinone, Bachmann (II, 7) recommends strongly dilute caustic potash or soda^ which instantly dissolves it with a greenish-brown color. 9. Tannins. 198. All those substances which give a blue-black or green color with iron salts are commonly designated as tannic acids or tannins. There belong here, of the better known compounds, especially : Pyrocatechin, C.HXOH),; Pyrogallic acid, C.H3(OH)3 ; Protocatechuic acid, C,H3.(OH),.COOH ; Gallic acid, C,H,.(0H)3.C00H ; Gallo-tannic acid, C,,H,„0, (= digallic acid ?) ; and besides these there are many other compounds whose constitution is not yet certainly determined (cf. Beilstein, III, 431 ff.). We have no trustworthy methods for the certain micro- chemical distinction of these substances, although this is the more to be desired since we have certainly to do with very different .substances and, as Reinitzer (I) has lately shown, it is very hazardous to assume a common physio- logical function for this whole group of compounds. But a detailed account of the methods used for the recog- nition of the whole group of tannins seems to be demanded by their wide distribution in plants. The following reac- tions have been made use of in their study : a. Iron Salts. 199. Of the iron salts, an aqueous solution of ferric chlo- ride was formerly chiefly used ; but it has the disadvantage that it has nearly always an acid reaction, and when used in MICROCHEMIS TR V. 1 1 5 excess with the tannins which give a green color with iron, it very quickly stops the reaction. H. Moeller (I, LXIX) used, however, a solution of anhydrous ferric cJilo^ide in water-free ether as a reagent for tannin. This is especially adapted to the study of large parts of plants, such as whole leaves or pieces of them. Loew and Bokorny (I, 370, note) have recently used for the recognition of tannin in algae a concentrated aqueous solution of ferrous sulphate^ in which the algae were allowed to be exposed to the air for from twelve to twenty-four hours. I have obtained in this way a very intense reaction in Spirogyra and Zygnema. This may be hastened by warm- ing to 60° C. A very rapid reaction is produced hy ferric acetate, accord- ing to Moeller (I, LXIX), in the form of the concentration of the ofificinai tincttira ferri acetici, which contains about ^% of iron or about 20^ of Fe3(C2H30aX. (^. Cuprlc Acetate. 200. Cupric acetate, which was introduced into micro, chemistry by Moll (I), has the advantage that it forms an insoluble precipitate with tannins. This is brownish in color, but takes, on subsequent treatment with iron salts, a blue or a green color according to the kind of tannin con- cerned. Moll places the tissues to be studied in a concen- trated aqueous solution of cupric acetate and leaves them in it from eight to ten days, or as much longer as may be de- sired. Sections prepared from this material are treated for a few minutes with a 5^ solution of ferric acetate, and, after it is washed out, may be preserved in glycerine or glycerine- gelatine. If it is desired at the same time to fix the cell-contents, one may use, according to Klercker (I, 8), a concentrated alcoholic solution of cupric acetate* instead of the aqueous solution. The pieces of tissue should be left several days, at least, in it. * This solution must be kept in the dark. 1,6 BOTANICAL MICROTECHNIQUE. y. Potassium Bichromate and Chromic Acid. 201. Most tannins form with potassium bichromate a volu- minous precipitate which is bright brown or blackish brown according to the quantity of tannin present, and which is insoluble in water or in an excess of the salt, and, according to Moeller (I, LXIX), probably consists of purpurogallin. Potassium bichromate is most conveniently used by placing large pieces of the tissue to be examined for one or several days in a concentrated aqueous solution of this salt, and then preparing sections after washing out the bichromate. These sections generally show the precipitate in the places where the tannin was formerly present. They may be pre- served unchanged in glycerine or glycerine-gelatine. Thicker sections or larger fragments may also be transferred to Can- ada balsam. But of course they must first be dehydrated with alcohol and cleared with clove-oil or the like (cf. §§ 14-22). The precipitate produced by potassium bichromate does not change its color with iron salts, or, as Overton (II, 5) has shown, with sulphurous acid. Even hydrogen peroxide does not attack it. To obtain a rapid reaction J. af Klercker (I, 8) recom- mends for many cases that the objects be plunged in a boil- ing solution of potassium bichromate. In fact an immediate reaction may thus be obtained with algae and sections of higher plants. According to Moeller (I, LXX), the diffusion of the bichro- mate is much hastened by the addition of a few drops of acetic acid. 202. Dilute chromic acid of about i^ appears to give a reaction similar to that of potassium bicarbonate, and may be used, as well as mixtures of chromic and osmic acids, for the recognition of tannins. These have the advantage of fixing the cell-contents well at the same time. But it should be noted that, according to Nickel (I, 74),. various compounds not related to the tannins give similar precipitates with potassium bichromate. MICR O CHE Alls TRY, 11/ 6. Osmic Acid. 203. Osmic acid is rapidly reduced by tannic acids and very soon forms with them a solution sometimes bluish and sometimes brownish, and finally a black precipitate. For the reaction a i^ solution should be used. The osmic acid is regenerated and the preparation wholly decolorized by peroxide of hydrogen. If hydrochloric acid be first added to the preparation and then \io osmic acid, there appears, according to Dufour (I, 3 of separata), in a few minutes a blue color, and soon, if much tannic acid be present, a blue precipitate. As Overton (II, 5) has shown, albuminoids saturated with tannins are browned ; thus he obtained a beautiful brown coloring of protein crystalloids on leaving sections from the endosperm of Ricinus, from which the fat had been removed,, for about ten minutes in a dilute solution of tannin, and transferring them, after careful washing, to 2 solution of ferrocyanide containing 5^ of acetic acid, then washed in cold water, and finally let them remain for 12 hours in a not too dilute solution of ferric chloride. Certain differentiations of the cytoplasm then showed an evident blue color. MICROCHEMIS TR V. 13$ I. Pepsin and Pancreatin. 232. Recently, the ferments secreted by the stomach and pancreas, which have the power of converting proteids into soluble compounds (peptones), have been used for their mi- • crochemical recognition. Both ferments can now be obtained in very stable form, as pepsin-glycerine and pancreatin-gly- cerine, of Dr. G. Griibler, Leipzig. 233. Digestion with pepsin maybe accomplished bykeep^ ing the objects in a mixture of one part pepsin-glycerine and three parts water, acidified with .2^ of its weight of chemically pure hydrochloric acid, for an hour, at a temper- ature of 40° C. The effect of hydrochloric acid may be ob- served in control-experiments containing the acid alone. 234. Pancreatin-glycerine maybe diluted with three times- its volume of water and then used in the same way. The previous treatment of the objects has an important influence upon the reaction. The solution of the proteids takes place most easily in sections taken directly from the living plant. But, in general, alcoholic material is to be preferred, since the digestibility of the substances soluble in water may thus be determined, and clearer images are usually obtained.. But the alcohol should act for as short a time as possible (24. hours), since it may influence the digestibility by its longer action. d, Nucleins. 235. Nucleins have been prepared especially from yeast,, from the thymus gland of the calf, from the yolk of eggs, and from salmon-sperm. These are distinguished from pro- teids especially by the fact that they contain phosphorus.- In other respects the analyses of nucleins from differ- ent sources show considerable differences. Altmann (II) has lately isolated from these nucleins bodies of uniform compo- sition which he calls nucleic acids. These contain about 9^ of phosphorus and are quite free from sulphur when pure. They are precipitated by albumen, and Altmann regards the 1 34 BO TA XICA L MICRO TECHNIQ UE. iiuclein preparations of various authors as compounds of nucleic acids with various amounts of albumen. [Malfatti (II) has prepared an artificial nuclein from syn- tonin and metaphosphoric acid, which yields nucleic acids when treated by Altmann's method.] 236. Zacharias has recently (I and II) attempted to recog- nize microchemically the general distribution of nucleins, especially in cell-nuclei. He gives as a characteristic reac- tion for them, their, insolubility in pepsin and hydrochloric acid (cf. § 233), in which, as well as in .2-.3;^ h}'drochloric acid, they take a sharply defined and peculiarly glistening appearance. But the nucleins are, according to Zacharias, soluble in a \oi solution of common salt, in a concentrated solution of sodium carbonate, in dilute caiistic potasJi'^oXyiXAon, in concentrated hydrochloric acid, and in a mixture of four parts concentrated hydrochloric acid with three parts water. Further investigations must show how far the macrochemi- cally prepared nucleins and those recognized by the above reactions correspond with each other. [According to Malfatti (I) and Zacharias (V), the nucleins seem to constitute the so-called chromatin-bodies of the nucleus (cf. § 239).] c. Plasiin 237. Reinke (I) prepared a nitrogenous compound from the Plasmodia of ^thalinm septicum^ which he calls plas- tin. According to Zacharias (I and II), this compound rep- resents the fundamental substance of the cytoplasm and agrees in its microchemical behavior with nuclein, in not being attacked hy pepsin with hydrochloric acid and in being soluble in concentrated hydrochloric acid. But plastin differs from nuclein in not swelling in a 10^ solution of salt after treatment with pepsin, and in being less readily soluble in alkalies and insoluble in a mixture of four parts of pure con- centrated hydrochloric acid and three parts water. It remains to be learned whether plastin is really a single compound or includes a group of related compounds. Ac- MICR CHEMIS TR V. 135 cording to Loevv (I), the plastin prepared macrochemically byReinke is to be regarded simply as an impure albuminoid preparation. This author also showed that the cytoplasm regarded by other authors as free from albumen gives the protein-reaction after being first treated for a time with caustic potash (cf. § 228 and Loew, II). i/. Cytoplasthi, Chloroplastin, Metaxin, Pyrenin, Amphi- pyrenin, Chromatin^ Linin, and Paralinin. 238. According to the investigations of Schwarz (I), the protoplasmic body is made up of eight different proteids which are limited in their distribution to very special differ- entiations of the protoplasm, and which should be compara- tively easy to distinguish microchemically according to the tables of their reactions prepared by this author. But if one examines the separate results of his observations, as described in detail, it is found that the most of the reagents used have given very different results even with the few objects ex- amined, and that the author's own observations do not at all always correspond with the special statements of his tables^ There can no longer be any doubt that the substances distin- guished by Fr. Schwarz do not represent uniform, chemically definable substances. Further studies must show whether the reagents used by him are capable of rendering good service in the study of the morphological elements of the protoplasm. This seems most probable in case of the " pretty concentrated " solution of copper sulphate used by Schwarz (I, 116), which dissolves only the chromatin* in the nucleus and fixes all its other constituents well. A mixture of one volume of a 10^ aqueous solution oi pot as shun ferro- cyanide, two volumes of water, and half a volume of glacial acetic acid also dissolves only chromatin ; but this reagent is not adapted to fixing, since it causes swelling. 239. But, since the nomenclature introduced by Fr. * [Malfatti (I) states that copper sulphate does not dissolve chromatin, but lorms an unstainable precipitate with it.] 136 BOTANICAL MICROTECHNIQUE. Scliwarz has been used elsewhere in the literature, at least the names of his eight compounds may be given here. Two of them occur in the chlorophyll granules, chloro- plastin and metaxin. The first of these represents the green iibrillse within the chloroplasts, and between them is the water-soluble metaxin. In the nucleus Schwarz distinguished five substances, amphipyrenin, which forms the nuclear membrane; pyrenin, the substance of the nucleoli ; chromatin, the strongly stain- ing material of the nuclear framework ; and linin and para- ]inin, the former of which forms a fibrillar network in the nucleus, while the latter fills the meshes of this net. In the cytoplasm Schwarz finds only one proteid, which iie calls cytoplastin. 13. Ferments. 240. It was stated by Wiesner (IV) that pepsin, diastase, and the gum-ferment described by him give characteristic color-reactions with orciyi and hydrochloric acid, which are jnicrochemically applicable. But Reinitzer (II) showed that these reactions occur with various carbohydrates, and most probably depend upon the fact that the reagent splits off from them furfurol or related compounds. But Guignard has recently tried to determine the location of emulsin and myrosin, partly by the use of orcin. a. Emulshi. 240a. Emulsin splits the glucoside amygdalifi, contained in bitter almonds, into prussic acid, oil of bitter almonds, and sugar. It occurs, according to Guignard (III), in the leaves of Primus Lauro-cerasus, exclusively in a parenchyma- tous sheath surrounding the vascular bundles. This author Teaches this conclusion from the fact that only these cells form prussic acid with a solution of amygdalin ; while the spongy and palisade parenchyma, which, on the other hand, forms prussic acid with a solution of emulsin, is plainly to be regarded as the seat of amygdalin. MICROCHEMISTRY. 137 The contents of the cells containing emulsin become red with Millons reagent, and violet with copper sulphate and caustic potash. It seems probable that these reactions are due to the emulsin, since the corresponding cells of the emulsin-free leaves of Cerasus liisitanica do not give them. b. Myrosin, 241. For the microchemical recognition of myrosin, which is contained in many Crticiferce, Guignard recommends (II) concentrated hydrochloric rt:«<^ which contains a drop of a loio aqueous solution of orci7i in each ccm. If the sections are heated in this solution to near 100° C, a violet color appears in the cells containing myrosin. In the seeds of black mustard and in other parts of various CrucifercB, this reaction occurs in specialized cells rich in proteids, which alone, as Guignard has experimentally shown, are able to decompose potassium myronate (cf. § 156). [Spatzier (I) finds myrosin also in the Resedacece and in the seeds of the Violacece and Tropceolacece. Where it occurs in vegetative organs he finds it in a dissolved condition ; but in dry seeds it is in the form of solid homogeneous granules of about the size of aleurone grains.] part Z\)ivi>. METHODS FOR THE INVESTIGATION OF THE CELL-WALL AND OF THE VARIOUS CELL- CONTENTS. A. The Cell-wall. 242. Since vegetable cell-membranes belong to the class of absorbent bodies and, as their osmotic relations show, can readily give passage to the most various substances which are soluble in water, it may be assumed that they never consist simply of cellulose and water, in the living plant. Rather, they are always incrusted, within the plants with a greater or less amount of foreign substances, accord- ing to their age and position. How far the varying chemi- cal and physical relations of vegetable cell-membranes are to be attributed to such incrustations of organic or inorganic nature cannot at present be certainly determined. But it is well established by modern researches that pure cellulose is much less than the other constituents in many membranes or parts of membranes. Indeed it is very probable that cellulose is entirely wanting in many parts of membranes. Positive conclusions concerning these questions can only be reached when we have obtained, by macroscopic re- searches, a sure means of distinguishing the different con- stituents of the cell-wall. But our knowledge in this re- spect is still too fragmentary to make possible any uniform U3 SPECIAL METHODS. I39 system of the constituents of the cell-wall, although the macrochemical study of the cell-wall has been taken up in various aspects in recent years. 243. I prefer, then, to discuss first in the following pages the kinds of membranes which may primarily be distin- guished by their microchemical relations. For some time there have been pretty generally distinguished the pure cellulose wall, the lignified wall, the cuticularized or su- berized wall, the gelatinized wall, and fungus-cellulose. In connection with the gelatinized wall may be discussed the remaining plant mucilages and gums and the jelly-formation of the Conjugates. As related to these various modifications of the membrane may be mentioned the paragalactan-like substances which serve as reserve-materials, callose, and the pectins. Finally, this chapter may describe the preparation of ash and siliceous skeletons, and some methods which have been used in the investigation of the development and finer structure of the cell-walls. I. The Cellulose Wall. 244. Cellulose is a carbohydrate whose empirical compo- sition corresponds to the formula CeHjoOj. It is especially characterized by its solubility in ciiprammonia and in con- centrated sulphuric acid, by its blue or violet color with iodine and sulphuric acid ox with chloroiodide of zinc, and by the fact that from its hydrolytic splitting with sulphuric acid there finally results a fermentable sugar (glucose). 245. But it should be remarked that there are very prob- ably different substances which give the same reactions, and which perhaps represent nearly related isomeric compounds. Thus, according to W. HofTmeister CI and II), cellulose shows very varying relations, especially toward a 1-5^ caustic soda solution, in which it is partly soluble, partly insoluble. But an exact microchemical distinction of the kinds of cellulose has not yet been possible, and it is there- fore best for the present to call all membranes or parts of membranes which show the above reactions pure cellulose 140 BOTANICAL MICROTECHNIQUE. membranes, any ash constituents being, of course, quite disregarded. 246. For the microchemical recognition of the cellulose membranes the following reactions are used : 1. Solubility in concentrated sulpJiuric acid. This begins with a strong swelling, which finally passes into complete solution. 2. Solubility in cupr ammonia. This reagent, which is also known as Schweizers reagent, may be prepared by precipi- tating cupric oxyhydrate from a solution of cupric sulphate with a dilute solution of caustic soda, then washing the pre- cipitate with water by repeated decantation and filtering, and finally dissolving it in the most concentrated ammonia- water. A very good reagent may be more simply prepared by pouring 13-16^ ammonia-water over copper turnings and letting the whole stand in an open bottle (cf. Behrens II, 55). Cuprammonia can be preserved for only a limited time. To test its fitness for use, one may use cotton, which it should completely dissolve at once. 3. The blue color with iodine and sulphuric acid. Ac- cording to Russow, this reaction may be best conducted by treating the sections first with an aqueous solution of \i> iodine and \\^ potassium iodide, and then adding a mixt- ure of two parts concentrated sulphuric acid and one part water. 4. The violet color with chloroiodide of zinc. This re- agent is usually prepared by dissolving an excess of zinc in pure hydrochloric acid and then evaporating the solution to the density of sulphuric acid in the presence of an excess of metallic zinc ; the solution is then saturated with potassium iodide and finally with iodine. The chloroiodide may be more simply prepared by dissolving 25 parts of zinc chloride and 8 parts .of potassium iodide in 8.5 parts of water, and then adding as much iodine as will dissolve (Behrens II, 54). [I have used with excellent results a preparation obtained by dissolving solid commercial chloroiodide of zinc, a moist SPECIAL METHODS. I4I Avhite salt, in somewhat less than its own weight of water, and then adding sufficient metallic iodine to give the solu- tion a deep sherry-brown color. I prefer Griibler's prepara- tion of the chloroiodide.] These solutions remain for a long time unchanged, especially when kept in the dark. The reaction succeeds best when the sections are placed directly in the concentrated reagent. 5. Recently a number of reagents containing iodine have been recommended by Mangin (VII), which act in the same manner as chloroiodide of zinc and seem to be, in part, more delicate than it. Of these reagents I have used with good results a calciiim- cJdoride-iodine solution, and, instead of following Mangin's somewhat more elaborate method, have prepared it by adding about .5 gram of potassium iodide and .1 gram of iodine to lo ccm. of a concentrated solution of calcium chloride and then, after gentle warniing, separating the solution from the excess of iodine by filtering through glass-wool. This solution, in which the sections should be placed directly, colors lignified membranes yellow to yellow-brown, but pure cellulose walls become first rose-red and, after a time, violet. According to Mangin, it should be kept in the dark. By the aid of iodine-phosphoric acid recommended by Mangin, one obtains a very deep violet coloring of cellulose walls, while lignified and suberized walls are colored yellow or brown. This reagent is prepared by adding a small quantity of potassium iodide (about .5 gram to 25 ccm.) and a few crystals of iodine to a concentrated aqueous solution of phosphoric acid, and gently warming the whole. The sections should be freed of all water adhering to their sur- faces, by means of filter-paper, before being placed in this solution. Mangin also recommends mixtures of aluminium chloride or stannic chloride with iodine and potassium iodide. For the manner of preparing and using these solutions, Mangin's work may be consulted. 142 BOTANICAL MICROTECHNIQUE. [Mangin states (VIII) that the most important and most characteristic reaction of cellulose is its conversion into hydrocellulose or amyloid. This conversion is not certainly accomplished by acids, but the best results are obtained by treating the cellulose with a saturated alcoholic solution of sodium or potassium hydroxide and then transferring it to absolute alcohol. Cuprammonia also produces the same result. The above-described reagents for cellulose act promptly with hydrocellulose.] 247. Their behavior with staining media can also be used for the recognition of pure cellulose membranes. These also serve in delicate sections, microtome sections or the like, to bring out better the network of cell-walls. HcBmatoxylin is especially adapted to this purpose, Giltay (I) having first observed the fact that it stains deeply only the unlignified and unsuberized membranes. It may be used in very various solutions (e.g. in the so-called Bohmer's (cf. § 315) or in Delafield's (cf. § 314) solution). These stain pure cellulose walls deep violet, while lignified and suberized membranes remain at first uncolored, or are stained yellow or brown. In most sections an exposure of a few minutes is sufficient for a deep staining of the membranes. 248. The writer has used haematoxylin with the best results for the recognition of the closing membrane of bor- dered pits (Zimmermann IV). With the wood of Coniferce it is sufficient to leave the sections for fifteen minutes in Bohmer's haematoxylin solution (cf. § 315) to obtain a deep staining of the " tori " of the bordered pits, which, naturally, come out most sharply after clearing in Canada balsam (cf. §§ 14-22). 249. Aniline blue and methyl blue may also be used for staining cellulose walls. These produce an intense stain in an hour with microtome sections, which is not affected by alcohol, clove-oil, or xylol, so that the preparations may be mounted in Canada balsam. A solution of Berlin blue acts in the same way. This is prepared by allowing i gram of soluble Berlin blue and .25 gram of oxalic acid to stand sev- eral hours with a little distilled water, then adding 100 ccm. SPECIAL METHODS, 1 43 of water and filtering (cf. Strasburger I, 622). To obtain a sufficiently deep stain, the solution must usually be allowed to act for several hours. It is not washed out by alcohol. [According to Mangin (VIII), pure cellulose is readily stained by many of the azo-colors, as by orseillin BB, crocein and naphtol black in an acid solution, or by Congo- red and benzo-purpurin in an alkaline solution. Several of the dyes recommended heretofore for cellulose walls really stain only the pectic constituents of cell-membranes (cf. § 292). Such are methylene blue, aniline brown, and chino- lin blue.] 250. A very deep and permanent staining of the wall is obtained, according to Van Tieghem and Douliot (I), by placing sections, after all cell-contents have been removed by eau de Javelle and caustic potash, and after thorough washing, first in a dilute solution of tannin for one to two minutes and then, as quickly as possible, in a very dilute solution oi ferric chloride. They are -at once removed from the latter solution and enclosed in glycerine or Canada bal- sam. All the membranes are then stained a deep black. For staining the younger membranes of microtome sec- tions, I have lately found Congo-red well adapted. I allow it to act in concentrated aqueous solution, for 24 hours, upon the sections, and then wash them in alcohol and mount in Canada balsam. 2. Lignified Membranes. 251. Lignified membranes are distinguished from those of pure cellulose by being insoluble in cuprammonia and by being colored yellow or brown by iodine and sulphuric acid or chloroiodide of zinc. It was formerly generally believed that this difference in chemical relations of lignified walls is due to the incrustation of the cellulose with a substance richer in carbon, lignin. And in fact lignified membranes give the reactions of pure cellulose after treatment with Schulze's macerating mixture (cf. § 9). According to Man- gin (VII), the same thing occurs after treatment with eau de Javelle. 144 BOTANICAL MICROTECHNIQUE. 252. Recently the attempt has been made in several quarters to reach more accurate conclusions concerning the chemical composition of lignified membranes, and especially as to the constitution of lignin. Wholly trustworthy results can, of course, only be reached by macrochemical investigations with exact quantitative an- alysis. In this connection should be mentioned the recent researches of Lange (I and II), who has isolated from the woods of the beech, oak, and fir, two compounds of an acid character, '^ lignic acids,'' which may, however, possibly come from a single substance. Lange also obtained various by-products concerning whose significance nothing is yet known. 253. There is also widely distributed in lignified mem- branes a gum-like substance which Thomsen has called wood- gum. It may be extracted with a 5^ solution of caustic soda and then precipitated from this solution with 90^ alcohoL On hydrolysis wood-gum yields either arabinose, CgH^O^ ,. or xylose, C^H.^O,. Wood-gum and both of its derivatives above mentioned take a cherry-red color on warming with phloroglucin and hydrochloric acid. But Allen (I, 39) has shown that the phloroglucin reaction about to be described is not to be referred to the wood-gum ; for, on one hand, the reaction takes place with lignified membranes in the cold, and, on the other, the colors which appear in the different reactions show very different spectroscopic relations. 254. Attempts have also been made to determine the chemical constitution of lignified membranes bymicrochem- ical studies. Especially Singer (I) and, more recently, Heg- ler (I) have tried to prove that coniferin and vanillin always occur in lignified walls. This view is based chiefly on a series of color-reactions which lignified walls give with vari- ous aromatic compounds. I give a compilation of the chief of these reactions with remarks on their application, which should receive notice here because the reactions may be used with good results for the microchemical recognition of lig- nification. SPECIAL METHODS. 145 ^ ^ ^^ 1 V ^ - &0O , H 1 C ro In ^ oo>-r 4> .,1^ o" >n -3 N "^ 3 ro vO m ns =i «' ;f S 2' ;r; ^^ ro HH (^ <^ U p ►-1 '-' •"^ «-• •"*__ "u ^ d i-T 6 J c „ c J3 c G :0 _U "^ 1 .5 E^:^' % ^ S 5 s ^ > I! ii S E S "c . ^ . & ^ 0) ■£^ _b£^ a u 3 5 •a 4> ^ _o bo c 1 rt 5 1-T3 u (U bfi 3 2 2 > _3 •6 bi) i U 3 bo bo >>o x;-o ^ c C A >, •0 « G M '? >> en 3 S u G E 1° u bo c "cfi u c •a c G ill 1 Hi ■2 --23 •0 u " a 1 1 a in •a c .0 _>, 3 lU G a ■35 •a 3 8 1 3 a W C .0 3 1 tfi 3 u 3 0* 1 !l C a G 3 1 3 G X + 3 G W c 3 > 3 a •a u 3 ■'3 G 3 x: . n li at^Ji p So §0 "G It! w c '^ tij 2 03 > s cA 8 5o-5 ^ua II.SH G + 3 1 "5 G + c 3 1 1 8 3 1 Si •a ■% c .0 3 yO •§^ 3 § Ig -o-S ^•5 '0 in v> 3 0) 3 CT' rt -s i c c ^ as SO 3 J3 3 c c 4;^ O" ^ T5 CT' H < < < < U < U < U u £ a I q q u E U g q 6 u a: ^ E Z E IS CI E E E u n en E -^ E E 3 a J u cJ •a u X <0 X u X^ E 8 E E U G u u CJ » 0» U dJ u « c a» rt •0 a rt a J3 a ^ .2 •a be 3 "5 x: c 3 s ■3 a c '0 c t r. c 1 •a '5 3 c f c cQ 3 X! 4J J2 x: u 2 z c c x: •a c c« CU H Oi 0, ti smic acid 2iV\A cyanin maybe also used for the recognition of suberized membranes. For this purpose, I dissolve cya- nin in 50^ alcohol and add an equal volume of glycerine. Preliminary treatment of sections with eau de Javelle is gen- erally to be recommended, as it destroys the tannins which hinder the staining. It also causes the lignified walls to lose their power of staining, while suberized ones are as deeply stained as in the fresh condition, even after being exposed for a day to its action (cf. Zimmermann VII). 268. In their behavior with staining media, the cuticular- ized and suberized membranes show in many respects an agreement with lignified ones. This is especially true of the •so-called suberin lamella of cork-cells; but the true cuticle is often less easily stainable. But it is usually easy to stain the cuticular layers differentially, especially in thick-walled epidermal cells ; and double stainings, in which these layers are differently colored from the cellulose layers lying be- neath, may be obtained. But it must be remarked that these stains do not always act with the same precision, in all cases, as with lignified walls ; and different plants do not appear to behave in the same way in this respect. I recommend as suitable objects for study, the leaves of Clivia nobilis or Agave americana. On these the following stainings and double stainings may be readily carried out. The statements as to time refer to microtome sections. a. Safranin. 269. The best staining medium for suberized walls is ani- line-water-safranin, prepared by mixing equal volumes of ani- line-water and a concentrated alcoholic solution of safranin. I allow this to act for half an hour or longer on the sections, then cover them with acid alcohol,* which is quickly replaced * Thai Is, alcohol to which is added about .55^ of the ordinary chemically pure HCI. SPECIAL METHODS. 153 by alcohol. They are washed with the latter until no more color is given off, and then transferred to Canada balsam in the usual way. Especially if the washing with acid alcohol is just right, only the lignified and suberized cell-walls are stained in these preparations, in which the former show a bluish, the latter a rather yellowish, color. If it is desired to stain the cellulose walls also, this maybe done by one of the following methods : a. Methyl Blue. The sections, stained with safranin and washed with alco- hol, are placed in a concentrated aqueous solution of methyl blue, in which they remain a quarter of an hour or longer. They are then washed in alcohol and mounted in Canada balsam. The cellulose walls are then stained blue, the su- berized and lignified ones, red. /?. Aniline Blue This must be used in aqueous solution which must be first washed off with water after the staining, since turbidity readily results from the direct addition of alcohol. Other- wise it is used like methyl blue. y. Haematoxylin. , Bohmer's haematoxylin (§ 315) may well be used for double staining with safranin. This is allowed to act for a few min- utes on sections stained with safranin and washed, is then washed off with water, and the sections are mounted in Canada balsam. Sections thus treated show the lignified and suberized walls red, and the cellulose walls violet. b. Gentian Violet and Eosin. 270. In the so-called Gram's staining process (§ 321) with gentian violet, only the lignified and suberized walls remain stained after thorough washing with clove-oil ; but a fine double staining may be obtained by proceeding according to Gram's method and adding to the clove-oil used in washing 154 BOTANICAL MICROTECHNIQUE. a little eosin, which dissolves readily in it. The eosin at once stains the cellulose walls a beautiful red, while not changing the staining of the other walls. c. Ammonia-fuchsin. 271. As was first recognized by Van Tieghem, ammonia- fuchsin is well adapted to staining suberized and lignified membranes. It is prepared by adding ammonia to a not too concentrated alcoholic solution of fuchsin, until the solu- tion becomes straw-yellow after a little shaking. The solu- tion should be filtered after a few days, but can be kept only a few weeks, even in well closed bottles. A double staining may be had by placing the sections first in the above described ammonia-fuchsin solution for a few minutes, and then passing them directly to an aqueous solution of methyl blue, in which they are left a quarter of an hour or longer, then washing with alcohol and mounting in Canada balsam. d. Cyanin and Eosin. 272. If sections are placed for several hours in a freshly prepared, very dilute aqueous solution of cyanin, which may be prepared by adding 20 drops of a concentrated alcoholic solution to 100 ccm. of water, the lignified and suberized membranes appear beautifully blue after washing in alcohol. If clove-oil containing eosin be used in transferring to Can- ada balsam, a fine double staining is obtained. The modified walls are blue, the cellulose walls red. I obtained also a deep staining of the cuticle by leaving sections for a considerable time in a solution of cyanin in 50j^ alcohol and then washing out the .stain with glycerine. 4. Gelatinized Cell-walls, Plant-mucilages, and Gums. 273. The so-called gelatinized membranes are distin- guished from cellulose walls chiefly by their different physi- cal character, their strong power of swelling; and indeed there occur all stages between pure cellulose, which takes up little water, and the gums which are wholly soluble in water, SPECIAL METHODS. I 55 like gum arable. Part of these substances are formed from cellulose, but most of them are formed by the plant directly as mucilages. It should be observed that the occurrence of vegetable mucilages and gums within the plant is not at all restricted to the cell-wall, but they may also be formed within the protoplasm. But it has seemed to me best to discuss all these bodies together here, in view of their un- doubted relationships, which may lead one, with Beilstein (I, 877), to group them under the designation^?/;;/^. 274. So far as the chemical relations of the gums are con- cerned, it should first be observed that most of them, so far as they have been analyzed, agree in their percentage com- position with cellulose and thus correspond to the formula CgHjoO^. But, on the other hand, they differ considerably from cellulose in their chemical relations, and also show^ great differences among themselves. Thus some of them are colored blue by iodine alone,, others only by iodine and sulphuric acid or chloro'iodide of zinc, and still others are colored only yellow or not at all by iodine preparations. A part of the gums are soluble, a part quite insoluble, in. c'lipranunonia. On oxidation with nitric acid, a part of them give oxalic acid, (COOH),, a part, mucic acid, (CHOHX.(COOH}^, a. part, both acids. Unfortunately the chemical characters of the various gums are not determined with sufficient exactness to make possible a strictly scientific grouping of them. But in the following account some remarks on the general methods o£' recognizing the gums may be in place, and then the chief chemical characters, and especially the microchemically applicable reactions, of the gums which have been studied in detail may be brought together. 275. For the microchemical recognition of the gums their strong power of szvclling in water may first be used. To follow the process of swelling exactly with the microscope, one may first place the objects in absolute alcohol, in which all the gums are insoluble and do not swell, and then 156 BOTANICAL MICROTECHNIQUE. gradually allow water to enter from the edge of the cover- glass. The dissimilar behavior of the gums with iodine solutions and with cuprammonia has already been mentioned. Be- sides these, corallin may be used in many cases in the study of gelatinized cell-walls and gums, since many of them are deeply stained by it. Since it is practically insoluble in water, it may be dissolved in a concentrated solution of soda. This solution gradually decomposes, but preserves its staining power for a long time (cf. also § 289). Characteristic stainings of plant-mucilages are often ob- tained with Hanstein's aniline mixture.* a. Amyloid. 276. The substance known by the name amyloid occurs in the seeds of various plants {TropcBoltnn majus, Impatiens Balsavtina, PcBonia officinalis, many Prirnulacece, and others) and constitutes a reserve material which goes into solution on the germination of the seed. Amyloid is characterized by being colored blue by iodine solutions, the best adapted for this reaction being, according to Nadelmann (I, 616), a dilute solution of iodine and potassium iodide, since a concentrated solution of the same substances colors it brownish orange, and fresh tincture of iodine does not generally color it at all at first. In cuprammonia amyloid is insoluble. Its behavior with nitric acid is also characteristic. In an acid which contains 30^ of HNO3 (spec, gravity 1.285), amyloid at once swells strongly, and after a time becomes entirely dissolved (cf. Reiss I, 735, 737, 739). The amyloid contained in the seeds named is not identical with the compound prepared from cellulose by treatment with acids (§ 246), which has often been termed amyloid (cf. Beilstein I, 863, 882). Amyloid is distinguished from reserve-cellulose (cf. § 286) by the reactions already de- * [This consists of an alcoholic solution of equal parts of fuchsin and methyl violet.,] SPECIAL METHODS. 15/ scribed and by the fact that its hydrolytic spHtting with sulphuric acid yields no seminose, but most probably glu- cose (cf. Reiss I, 761). [Winterstein's (I) recent researches give results which differ in several respects from those of Reiss. He finds amyloid soluble in cuprainmonia after a day. From this solution it is not precipitated by acids, but is thrown down by alcohol. Its composition seems to correspond to the formula C^HgoOj^, and it appears to belong to Tollens' group of Saccharo-colloids, though it is not certain that it is a single compound. In spite of its bluing with iodine, it cannot be regarded as very nearly related to starch.] b. Wound-gum. 277. The name wound-gum is commonly given to a substance which, according to Temme's researches (I), is very abundantly secreted in the vessels by the surround- ing starch-cells, in natural and artificial wounds, and, like tyloses, closes their cavities. This wound-gum agrees, ac- cording to Temme, with many sorts of gums in that it yields oxalic and mucic acid on oxidation with nitric acid. But it differs essentially from all gums in not swelling in water and in being insoluble even in caustic potash and sul- phuric acid. As has been recognized by Temme, wound- gum is stained deep red by pJiloroglucin and hydrochloric acid. Molisch showed later (IV, 290) that it behaves just like lignified membranes with aniline sulphate, metadiainido- henzol, orcin, and thymol ; and he believes that wound-gum contains vanillin in solution (cf. § 254). c. The Gelatinous Sheaths of the Conjugatae, 278. In many Zygnemacece the whole surface of the cell- filaments is surrounded by a colorless covering, a ''gelatin- ous sheath," while in the DesmidiacecB the excretion of jelly is often limited to distinct regions on the membrane (cf. Klebs II, and Hauptfleisch I). Since the refractive index of these jelly-sheaths differs but little from that of water, they can be well recognized. 158 BOTANICAL MICROTECHNIQUE. when unstained, only by the aid of strong objectives. But with lower powers they stand out clearly when the algae are placed in very finely rubbed India ink, according to the method proposed by Errera (III). For this purpose, enough of the genuine Chinese '* India ink" may be rubbed up directly on the slide to give the drop a dark-gray appear- ance, and then the alga to be studied is placed in it. No trustworthy statements can yet be made as to the chemical composition of these jelly-masses ; and it need only be said that they give no cellulose reactions either with iodine and sulphuric acid or with chloroiodide of zinc, and that they are always sharply defined against the cellu- lose wall and are not in genetic connection with it. A number of observations, made especially by Klebs (II) on the gelatinous sheaths of the Zygnernacece, deserve more detailed notice, as they show that these must possess a very complicated organization. 279. Klebs first established the fact that the gelatinous sheaths always consist of two different substances, one of which can be extracted with hot water and is pretty deeply stained by certain dyes, like methylene blue, methyl violet, and vesuvin ; while the substance which is insoluble in hot water remains quite colorless with these stains. After staining with one of the colors above named, delicate rods are seen in the sheaths, which often appear united into a fine network at the ends which are directed toward the cell-lumen (cf. Fig. 33, /). The same structure can also be made visible by other means, especially by alcohol. It is evidently due to the fact that the different substances are unequally distributed in the jelly-sheath. 280. A further remarkable character of the jelly-sheaths consists in the fact that, after the deposition in them of certain precipitates, for instance, of Berlin blue, these are thrown out, together with a greater or less part of the water-soluble substances of the sheath, with swelling of the latter (cf. Fig. 33, // and ///). This " throwing off of the gelatinous sheath" begins with an accumulation of the pre- viously evenly scattered particles into evident granules (cf. SPECIAL METHODS. 159 Fig. 33, ///), which are held together by the mucilage sepa- rated with them and finally thrown off with them. This expulsion may be caused by various precipitates. A very suitable one is chrome yellow (PbCrOJ, which is precipitated in the membranes by placing the algae, held together by a thread, in a .25^ solution of potassium chro- mate (K^CrOJ, then rinsing quickly in water, and finally transferring them to a .25^ solution of lead acetate. To obtain a heavy precipitate, this proceeding may be several Fig. ^3. — /, membrane and gelatinous sheath of Zygnenia sp. (X 580). //, two Zygnema- cells after deposit of chrome yellow (X 245). ///, membrane and gelatinous sheath of Zygnetna after deposit of chrome yellow (X 245). IV, the same of Pieurot^nium Tra- becular after staining with fuchsin (X 950). F, the same of Staurastrum bicorne, after staining with gentian violet (X 950). z, cell-membrane; ^, gelatinous sheath. / to /// after Klebs; /Fand F after Hauptfieisch. times repeated. But the expulsion takes place the more rapidly the less chrome yellow is deposited, and may not be completed for several days if the deposit be large. Finally, it may be remarked that this expulsion is not directly dependent on the life of the protoplasm, and may occur in dead individuals, under some circumstances. 281. Klebs has also established the remarkable fact that the gelatinous sheaths increase markedly in density in a [6o ^^^^ANICAL MICROTECHNIQUE. solution of glucose and peptone by the deposit of a sui stance whose composition is not yet known. This " thickening " of the gelatinous sheaths occurs, how- ever, only when soluble albuminoids and a sugar are simul- taneously present in the surrounding fluid, and, like the expulsion described, is independent of the life of the proto- plasm. 282. According to the investigations of Hauptfleisch (I), the gelatinous formations of the Desmidiacece consist, on the other hand, of single prisms or caps, each of which covers a pore in the cell-wall. These pores are occupied by threads of protoplasm which commonly terminate externally in globular swellings which penetrate to a greater or less dis- tance into the gelatinous covering, in different species\(cf. Fig. 33, /Fand V), For the observation of these structural relations, this author recommends that at first dilute, and then gradually more concentrated solutions of safranin, fuchsin, gentian violet, methylene blue, or methyl violet, be allowed to run from the edge to the living algai under a cover-glass, and that the changes in the jelly during the action of the stain be followed. Then the changes may be followed backward by careful washing of the specimens. The presence of two different substances in the gelat- inous covering has been disputed by Hauptfleisch for tl.e Desmids. 5. Fungus-cellulose. 283. The membranes of the fungi show very varying relations. In a number of species they give the normal cellulose reactions, and this is especially the case in young- stages (cf. de Bary II, 9). But in most fungi they differ from pure cellulose membranes in being insoluble in cupram- monia and in being colored only yellow or brown by iodine and sulphuric acid or by chloroiodide of zinc. They also show great powers of resistance to alkalies and acids in general. But since, on the other hand, they do not show the reactions for lignification or suberization, we are compelled SPECIAL METHODS. l6l at present to regard them as a special modification of cellu- lose, which is commonly termed fungus-cellulose. It should be remarked that, according to the researches of K. Richter (I), the membranes of a large number of fungi giv^e the reactions for pure cellulose after being first treated for a long time with caustic potash. But in many cases the caustic potash must act for a week. On the other hand, W. Hoffmeister (I, 254) has lately ob- tained from the fructification of Boletus edidis, by the use of methods always successful with the higher plants, no com- pounds giving the reactions of cellulose. The membranes of this fungus are, according to his researches, characterized by being completely soluble in concentrated hydrochloric acid and caustic potash. 284. The Membranes of the Bacteria. — There can now be no doubt that the Bacteria possess a solid membrane. In most cases its presence may be readily demonstrated by plasmolyzing the organisms (cf. §§ 431 and 463). No reliable statements can be made at present as to the chemical constitution of these membranes. They seem, moreover, to consist in part of cellulose; at least, W. Hoff- meister (I, 253) has isolated a substance reacting like cellu- lose from a species of Bacillus not exactly determined. 6. Paragalactan-like Substances (Hemicelluloses). 285. Reiss and E. Schulze have shown that, especially in the cell-walls of seeds with considerable thickenings of the walls, carbohydrates occur which differ essentially from cellulose and are dissolved at germination, like the other reserve materials of the seed. One of these substances is called by Reiss reserve-cellulose, another, by Schulze, para- galactan. But it is probable that various related com- pounds exist. All these substances can at present best be grouped under the name proposed by E. Schulze, '* Paraga- lactan-like compounds." [Schulze's later studies (II) afford ground for distinguish- ing this group of substances from cellulose as hemicelluloses. He finds that they become soluble, with the formation of j62 BOTAMCAI. MJCA'OT/'.CHXIQUE. 'glucose, through the action of hot dilute mineral acids, by which true celluloses are not affected. They are dissolved by dilute alkalies and by cuprammonia after brief treatment with hot dilute hydrochloric acid, too short to cause their solution.] a. Reiss' Reserve-cellulose. 286. The so-called reserve-cellulose has been prepared by Reiss (I) from the endosperm of Phoenix daciylifera, Phyt- i'lephas, and various other seeds with strongly thickened cell-walls. It differs from the ordinary cellulose especially in the products resulting from hydrolysis with sulphuric acid. There is first formed a compound corresponding to ■dextrine, but Isevo-rotary {seminin), and then a dextro-rotary sugar which reduces Fehling's solution and is fermentable {seininose) and especially characterized by the fact that It forms with phenylhydrazin acetate (C6Hj,NH.NH2) an hydrazon, which may be obtained in crystalline form, of the composition C^Hj^N^Oj, probably according to the reaction: C.H.,0, + QH,N, = C„H„0,N, + H,0. Reserve-cellulose cannot be distinguished microchemically from ordinary cel- lulose and behaves quite like pure cellulose with iodine solu- tions and cuprammonia. An exception is shown only by the cell-walls of the endo- sperm of Paris qiiadrifolia and Fceniculum officinale, which are insoluble ifi cuprammonia, although they give seminose on hydrolysis and must therefore be regarded as reserve cellulose. [Schulze finds (II) that this substance shows the characters of other hemicelluloses (cf. § 285) and should be placed .among them, with the name mannose.'] b. Paragalactan. 287. The name paragalactan has been given by E. Schulze (cf. Schulze I, and Schulze, Steiger, and Maxwell. I) to a compound recognized in the thickenings of the walls of the cells of the cotyledons of Liipimis luteus, with true cellulose, and which very probably occurs in other Legumitiosa:. It SPECIAL METHODS. J63 yields, on oxidation with nitric acid, mucic acid ; on heating with dilute sulphuric acid, galactose (CeHj^Og) and a penta- glucose. It is also characterized by giving a cherry-red fluid on heating with phloroglucin and hydrochloric acid, while no color is produced in the cold. On heating, para- galactan is transformed by i^ hydrochloric acid or i^ sul- phuric acid into sugar, while cellulose is attacked only by pretty concentrated solutions. It is an important fact for the microchemical recognition of paragalactan that it is insoluble in cuprammonia and prevents the solution of the cellulose which occurs in the same membranes, while the latter is readily dissolved by 'Cuprammonia after the removal of the paragalactan by boiling 2.5^ hydrochloric acid. Paragalactan does not seem to be colored by chloroiodide of zinc ; at least, membranes treated with this reagent showed only a slight bluing, while the remains of the membrane are deeply colored after the solution of the paragalactan. [This substance also shows the characteristics of the hemicelluloses (cf. § 285). The pentaglucose which it yields besides galactose is probably arabinose, and it may there- fore be called paragalacto-araban. It is very possible that it is a mixture of two substances, galactan and araban.] c. Arabanoxylan. [287a. Schulze finds (II) a hemicellulose in wheat and rye bran which yields, on hydrolysis, an arabinose and a xylose, and may therefore receive the above name.] 7. Callose, the Callus of the Sieve-tubes. 288. Until recently the name callus was generally given to a pretty strongly refractive substance which causes a more or less complete closing of the sieve-poles in old sieve- tubes, and finally covers the whole sieve-plate with a thick mass. Mangin has lately recognized (I-III) the more gen- eral distribution of this substance, especially in the mem- branes of various pollen-grains and pollen-tubes and in many fungi. It is, for instance, widely distributed in the l64 BOTANICAL MICROTECHNIQUE. mycelium of the Pcronosporacece, where it partly incrusts the cellulose wails and partly occurs in more or less pure condi- tion in the interiors of the hypha^ and of the haustoria (cf. Mangin III). [The same author has also shown (IX) the presence of this substance in various cell-walls of Phanero- gams which are incrusted with carbonate of lime, especially those of the cystoliths of the Urticales and of the calca- reous hairs and pericarps of several Borraginacece. In the achenes of Lithospenmim, Cynoglossurn, etc., where it occurs without a deposit of lime, its occurrence seems to be related to the disappearance of the cell-contents and the gradual destruction of the parenchyma. He has also observed it in the walls of cells bordering tissues which have become suberized in consequence of injuries.] This author calls this substance callose, a term w^hich deserves preference, since the word "callus" is used, as is- well known, in quite another sense. 288a. Callosc gives the following reactions, according to Mangin (II) : It is insoluble in water, alcohol, and cupram- monia, in the latter even after previous treatment with acids. But it is readily soluble in a cold i^ solution of caustic soda or potash, and is also soluble in the cold in concentrated sulphuric acid, as well as in concentrated solu- tions of calcium chloride and stannic chloride. Cold solu- tions of alkaline carbonates and of ammonia make it swell and give it a gelatinous consistency, but without dissolv- ing it. Callose also differs from cellulose in its behavior with various coloring matters. Mangin gives (V) a number of azo-colors which deeply stain cellulose in a neutral or feebly acid solution, but leave callose uncolored ; they are espe- cially orsciUin BB, azorubin, ftaphiol black, and the crocci?ts. On the other hand, callose is distinguished by its strong; staining capacity with coralliii and aniline blicc and certain dyes belonging to the benzidines and tolidines. 289. Corallin or rosolic acid is best dissolved in a 4^ or concentrated aqueous solution of soda (Na,CO,). The sec- tions are placed for a short time in this solution and then SPECIAL METHODS. IO5 examined in glycerine, when, if the staining has taken place properly, the deep-red pads of callose stand out sharply in the sieve-tubes. I have found it very useful to first over- stain the sections with corallin solution and then to wash them out with 4^ soda solution, which quickly decolorizes- all parts except the callose. This method has given es- pecially good results with the fungi. Preparations stained with corallin cannot be long preserved. 290. Aniline blue has been recommended by Russow (I)> for staining sieve-tube callose. It may be used in a dilute aqueous solution, which is allowed to act half an hour or longer on the sections. Overstained sections may be washed out with glycerine. They are properly stained when only the callose masses appear deeply colored. The •' Schlauch- kopfe" of young sieve-tubes (§45S) ^1*^ also pretty deeply^ colored by aniline blue. To distinguish these from the callose, the preparations may be subsequently stained with an aqueous solution of eosin, in which they are left for a few minutes. After a brief washing in glycerine the entire contents of the sieve-cells, including the protoplasmic threads which penetrate the sieve-plates, are- colored violet or red^ while the callose pads remain deep blue. These prepara- tions, as well as those with aniline blue ^lone, can be well preserved in glycerine-gelatine ; or they may be transferred to Canada balsam in the usual way. Since it often stains the protoplasm pretty deeply, aniline blue has usually given me much less instructive preparations than rosolic acid with fungi. [290a. Mangin recommends (IX), for staining the callose of calcified membranes, a mixture of soluble blue extra 6B- and vesuvin, or of the same blue and orseillin BB. These mixtures stain callose blue in a short time, the protoplasm and lignified elements being brown or violet, according to the mixture used. Where incrustations are not numerous^ as on many leaves, large pieces of tissue may be freed from air by boiling alcohol, then placed in cold nitric acid until frothing ceases, then in cold water, in boiling alcohol, and finally in cold ammonia, to remove xanthoprotein and its. l66 BOTANICAL MICROTECHiYIQUE. derivatives. When the tissue is transparent enough, the ammonia may be neutraHzed with acetic acid, and the tissue placed in the staining fluid.] 291. The behavior of callose with iodine reagents, which has been exactly determined only for the callose pads of sieve-tubes, is also characteristic, and, according to Lecomte (I, 268), best brings out their intimate structure. Chloro- iodide of zinc stains callose brick-red or red-brown according to the proportion of iodine it contains, calcium chloride and iodine solution (cf. § 246, 5) stains it rose-red, or wine-red after previous staining with aniline blue, while the sieve- plates are colored violet. 8. Pectic Substances. 292. Mangin (IV-VI) has lately shown microchemically that pectic substances (pectin, pectose, pectic acids) are very widely distributed in the cell-walls of the most different plants, and that they form especially the so-called intercel- lular substance of unlignified and unsuberized membranes. 293. For the microchemical recognition of pectic sub- stances Mangin uses (IV and VI) chiefly various coloring matters, phetiosafrafiin, methylene blue, Bismarck brown, fnc/isin, Victoria blue, violet de Paris (= methyl violet B), and rosolan (= mauvcin), and others. These do not color pure cellulose, but do stain pectic substances, as well in neutral solution as after slight acidification with acetic acid. But lignified and suberized membranes are also stained by these dyes. However, there remains a distinction between them and pectic substances in that the latter are quickly decolorized by alcohol, glycerine, and acids, while the for- mer retain their color in these fluids. Mangin also gives a number of dyes which leave pectic substances uncolored in a neutral solution, while they deeply color lignified and suberized walls. Such colors are : acid green, acid brown, nigrosin, indulin, the croceins, and the ponceaux. Mangin obtained instructive double stainings by mixing one of these dyes with one of the previous group. On the other hand, Mangin (V) has lately named a num- SPECIAL METHODS. 16/ ber of dyes which leave pectic substances uncolored, but stain cellulose or both cellulose and callose. To the former belong orseille red A, naphtol black, and the croceins ; while Congo-red, azo-blue, and benzopurpurin stain cellulose and callose. 294. In order to show that the first described stainings really depend upon the presence of pectic substances, Man- gin treated thin sections for 24 hours with cuprammonia and then washed them in water and in 2^0 acetic acid. On this treatment the cellulose is removed from the membranes and fills the intercellular spaces and the cell-cavities as a gelatinous mass. In consequence of it the membranes are colored not at all or but slightly yellow on the addition of chloroiodide of zinc, while a deep blue color appears in the interiors of the cells. The membranes, which now consist of pure pectic acid, are, however, deeply stained by safranin or methylene blue. It is sufficient to add a few drops of a solution of ammonium oxalate to cause the solution of the pectic acid membranes. 295. In order to show that the middle lamella of the so-called cellulose membranes consists of pectic acid or an insoluble salt of it, Mangin (VI) lets a mixture of one part hydrochloric acid and 4 to 5 parts alcohol act for 24 hours on thin sections, then washes them with water, and treats them with a weak (about 10^) solution of ammonia. After this has acted a short time, the sections may be separated into their constituent cells by gentle pressure. Mangin explains this by the supposition that the pectic acid is set free from its originally insoluble compounds by the action of the acid-alcohol, and is then dissolved by the ammonia solution. In fact, a gelatinous mass is precipitated from the ammoniacal solution on the addition of acid, which has all the characters of pectic acid. On the other hand, sections which were placed in lime- or baryta-water after the action of the acid-alcohol, showed no separation into their cells on subsequent treatment with ammonia, because the pectic acid had recombined into an insoluble salt with the alkaline earth. 1 68 BOTANICAL MICROTECHXIQUE. 296. Mangin (VI) obtained a deep staining of the middle lamella on placing thin sections of adult plant-organs in i)henosafranin or methylene blue after treatment with the above mentioned acid-alcohol mixture. The middle lamella of pectic acid stains much more deeply than the pectic com- pounds mixed with cellulose of the thickenings of the cell- wall. 9. Ash- and Silica-skeletons of the Cell-wall. 297. The inorganic salts which incrust all vegetable cell- Avalls are in many cases present in such quantity that, after the destruction of all organic substances by burning, they still preserve the form of the original membranes. Such ash-skeletons may easily be obtained by burning •cross-sections of the stem of Citcurbita Pepo on the cover- ^lass. But they must be examined in the air, as they are at least partly soluble in water. These ash-skeletons consist chiefly of potassium and calcium salts. In other cases silicic acid also occurs deposited in great quantity in the mem- branes. For methods of recognizing this, see §§ 78-81. 10. On the Developmental History of the Cell-wall. 297a. In the study of the growth of cell-walls it is often important to stain the membranes without affecting the vitality of the cells. If the objects thus treated are then allowed to develop further in pure water, it would seem pos- sible to distinguish the newly formed membranes or parts of membranes from those previously formed, with certainty. Noll (I) proceeded, with this object, with Caiilerpa and some other marine algae by producing a precipitate of Ber- lin blue or TurnbuU's blue in the membranes of the plants under investigation without injuring their vitality, and then allowing them to grow more, under favorable conditions. The newly-formed membranes must then, plainly, be color- less \ and those which have grown, perhaps by intussuscep- tion, must show a lighter color. 297b. To produce a precipitate of Berlin bltie in the membranes, Noll (I, iii) placed the algae first, for one or a SPECIAL METHODS. 1 69 few seconds, in a mixture of one part sea-water and two parts fresh water to which was added enough potassium ferrocyanide to give the solution the specific gravity of sea- water. Then the algae were rapidly passed through a vessel of pure sea-water and placed for one half to two seconds in a mixture of two parts sea-water, one part fresh water, and a few drops of ferric chloride.'^ The depth of the coloring is increased markedly by repeating the proceeding several times. For producing Turnbiiirs blue, which Noll thinks less suitable, he used the corresponding solutions of potassium ferricyanide and ferrous lactate. 297c. But it should be remarked concerning these stain- ings that they are gradually destroyed, probably by the excretion of alkali. But they can be renewed at any time by placing the algae in a solution of potassium ferro- (or ferri-) cyanide acidified with pure hydrochloric acid. Finally it may be observed that, according to Noll's researches, the vitality of the algae is not destroyed by these manipulations and the precipitate is in this way very uniformly deposited in the 'membranes, provided they pre- sent no chemical differences, so that they show the same depth of color in all the layers. 297d. Zacharias (IV, 488) has lately used Congo red in the same manner as Berlin blue. He worked with root-hairs of Lepidiiim, which he placed for 15-30 minutes in a solution of Congo red in water from the public supply and then allowed to grow further in moist air. But, since a decom- position of Congo red takes place in light, the seedlings must be cultivated in the dark. 297e. Congo red was earlier used by Klebs (III, 502) in the investigation of the growth of the membranes of various algae. This author found that Congo red has the remark- able property of leaving membranes already formed color- less 01 almost so, while it gives a red color to forming * This solution must be freshly prepared each time it is used, as it de- composes in a short time. I/O BO TA NIC A L MICRO TECIINIQ UE. membranes. Klebs used in these studies a .01^ solution of Congo red or a suitable culture fluid to which the same pro- portion of the dye (i : 10,000) was added. But it should be observed that the Congo red deposited in the membranes strongly hindered their superficial growth in Klebs' experi- ments, while their growth in thickness was so much the more increased, and the vitality of the cells was in no wise destroyed. II. The Finer Structure of Cell-walls. 297f. Many cell-membranes, especially those of consider- able thickness, are well known to be made up of various lamellae or layers parallel to their surfaces {stratification). In many there occur band-like differentiations within the same layer, which, according to Correns (III, 324), always have a spiral course {striation). Finally, one finds not uncommonly radially arranged lamellas of varying optical properties {transverse iaineilation). The observation of these differentiations may in many cases be conducted on the unchanged membranes. But in general they come out much more plainly if the membranes are treated with swelling media ; and, besides those men- tioned in § 10, chloroiodide of zinc is in many cases very useful. 297g. Three factors may enter into the problem of the cause of the optical appearances described, which have been thoroughly discussed by Correns (III): I. Sculpturing of the wall ; II. Differentiation of the wall into strips or layers of unequal water-content with similar chemical constitution ; and III. Differentiations of the wall which possess, with similar water-content, unequal refractive power, and there- fore depend upon material differences. Besides these, only combinations of these three factors are possible. 297h. Sculpturing of the wall may produce especially stri- ation. This then falls into the category of partial thicken- ings of the membrane, and deserves to be considered here only because, when very delicate, it cannot be distinguished SPECIAL METHODS. l/I from true differentiations of the membrane without much difficulty, and often accompanies these. Striation due to sculpturing of the membrane is, plainly, only visible when the membrane and the mounting fluid have different refractive indices. And it becomes the plainer as this difference is the greater, disappearing en- tirely as the refractive indices become equal. For example,, Canada balsam has almost the same optical density as cell- walls ; on the other hand, Correns used methyl-alcohol with good results, on account of its low refractive index. This is only 1. 321 and therefore less than that of water (1.336). 2971. Further, it is evident that it is unimportant in case of differentiations depending wholly on sculpturing of the wall, in opposition to those which are to be referred exclu- sively to unequal water-content, whether the membranes, are placed in a mounting fluid of similar refractive index in a dry or swollen state. But the use of this criterion en- counters difficulties, as Correns has shown (III, 260), if rifts or canals in the interior of the membrane are involved, as in the bast-cells of Nerhun, where the different layers have different systems of striation. In this case it does not seen> practicable to fill these capillary spaces with ethereal oils or- with balsam without the removal of imbibed water.. But even in this case, the behavior of the dried membranes on being imbedded in Canada balsam or the like may permit positive conclusions as to the nature of the differentiations; in question, since only a slow expulsion of the enclosed air from capillary spaces in the interiors of membranes can take- place. For distinguishing water-bearing clefts from substances rich in water, chloroiodide of zinc and various dyes may be used. Clearly, the capillary spaces must always stand out as colorless streaks on suitable sections, while the parts richer in water may show a more or less deep stain. 297k. Differentiations due to unequal zvater-content must, plainly, disappear on drying, as a rule. The presence of such differentiations may therefore be recognized by exam- ining the objects in the same anhydrous mounting fluid (sucK 172 BOTANICAL MICROTECHNIQUE. as Canada balsam), a part dry and a part moist. But it should be observed that the complete removal of water can only be accomplished by drying at a temperature of 50° to lOO** C, according to the nature of the object. The deh)-- drating media used by various authors for the same purpose, especially absolute alcohol, do not give demonstrative results (cf. Zimmermann I, 87). 297I. It is also to be noticed that, where the water-content is unequal, changes in form must occcur on drying, and therefore, as Correns (III, 262) has specially observed, a cer- tain distinction between differentiations due to sculpturing of the wall and those due to unequal water-content cannot always be drawn from the comparison of dried and moist membranes. Concerning the possibilities in this respect, Correns' work (III) may be consulted. 297m. The presence of differences in water-content was demonstrated by Correns (III, 294) by impregnating the membranes with a salt-solution (NaCl), which is not recog- nizably accumulated. The conclusion is then justified that where the salt occurs in greater quantity this is in conse- obtain it ready prepared from a chemist (e.g., from Dr. G. Griibler, Leipzig). 315. The so-called BoJuners hcematoxylin is also very useful. It is prepared from a concentrated alcoholic solution of haematoxylin which contains .35 gram of haematoxylin to lo- grams of alcohol and will keep indefinitely. A few drops of this are added to a solution of .10 gram of alum in 30 ccm. of water. This mixture is allowed to stand for a few days» and is filtered befo;-e use. P. Mayer (HI) obtained an haematoxylin solution that may^ be used at once by dissolving i gram of hcematein or JicEinatein^ ammonia in 50 ccm. of 90^ alcohol by warming, and then adding the whole to a solution of 50 grams of alum in a litre of water. This solution may be diluted with distilled water for staining, as desired. Concerning the other solutions of haematoxylin which ma)^ be valuable in special cases, and may in part be obtained ready for use from various chemists, reference may be had to the compilation of Gierke (I, 32-35). 316. If it is desired to stain sections with haematoxylin,. they are best placed in a very dilute solution and left in it for a considerable time (i to 24 hours). With alcoholic material it is advisable to place it in water for a short time- before staining it, as otherwise precipitates are readily formed. Beautifully differentiated stainings- may usually be ob- tained by staining sections too deeply (*' overstaining") and then washing them out with a suitable fluid. With an haematoxylin stain, a solution of alum (about 2^) is com- monly best ; but it must be thoroughly washed out before the transfer to alcohol or to Canada balsam, as otherwise alum crystals will be formed in the preparation. Acid alcoJiol has also been recommended for washing out haematoxylin; but the acid must be completely removed with pure alcohol before the final mounting. Very good nuclear staining may often be obtained by ]82 BOTANICAL MICROTECHNIQUE. placing objects stained with ha^matoxylin for a short time in a i^ sohition of potassiiivi bichromate or a concentrated aqueous solution oi picric acid. Both fluids must, of course, be carefully washed out before the transfer to Canada bal- sam or glycerine-gelatine. 317. In dealing with objects to be sectioned with the microtome, very pure nuclear stains may usually be obtained by staining the objects in toto (** staining in mass") before imbedding in paraffine. Since haematoxylin cannot pene- trate the cuticle and therefore only penetrates from cut surfaces, very different depths of staining are obtained, and, at some distance from the original surface, only a nuclear stain. Large objects must sometimes be left in the staining fluid, which in this case should be used pretty dilute, for some time (often several days), in order to be sufficiently stained. Very good mass-staining may be obtained by first staining large pieces of tissue deeply with haematoxylin and then placing them for a considerable time in a i^ solution of potassium bichromate. (i. Carmine. 318. Only a few of the numberless different carmine solutions can be described in detail here. These, as well as various other staining media containing carmine, can be obtained ready for use from chemists. 1. Grenadier s horax-carmine can be prepared by dissolv- ing 4 grams of borax and 2 to 3 grams of carmine in 93 ccm. of water, then adding 100 ccm. of 70^ alcohol, shaking and filtering. This solution is used for staining in mass as well -iis for sections. For washing, acid alcohol and a solution of borax or oxalic acid in spirit are recommended. 2. Beales Carmine. — .6 gram of carmine is shaken up with 3.75 grams of liquor ammonii canst, [aqua ammoniae, U. S. P.], then boiled for a few minutes ; after an hour, 60 grams of glycerine, 60 grams of water, and 15 grams of alcohol are added, and the whole is finally filtered. 3. Ammonitim carminate is best prepared by dissolving in ^vater to which a little (about .5^) ammonium carbonate has SPECIAL METHODS. 1 85 been added, the commercial dry ammonium carminate (the so-called Hoyer's ammonium carminate). Alcohol or acid alcohol is best used for washing. 4. Sodium carminate can be obtained in solid form, and may be dissolved in an aqueous .5^ solution of ammonium carbonate. 5. P. Mayer s carmine solution is prepared by rubbing up 4 grams of carmine in 15 ccm. of water, then adding 30 drops of hydrochloric acid while warming, and finally adding 95 ccm. of 85,^ alcohol, boiling, neutralizing with ammonia, and filtering when cold. Is used both for staining sections and for staining in mass. 6. Carminic Acetate. — Ammonium carminate is decom- posed with acetic acid added drop by drop in the least possible excess until the cherry-red fluid has become brick- red, when it is filtered. For washing, a mixture of one part hydrochloric acid in 200 parts glycerine or one of one part formic acid in lOO parts glycerine is recommended. 7. Picrocannine is the term applied to variously prepared mixtures of picric acid and carmine. Only the simplest recipe (Hoyer's) need be given here. According to this,, pulverized carmine is dissolved in a concentrated solution of neutral ammonium picrate. P. Mayer (HI) now uses pure carminic acid for the prep- aration of carmine solutions, of which he especially recom- mends the following: 8. Carmalnni is prepared by dissolving i gram of carminic acid and 10 grams of alum in 200 ccm. of distilled water, with heat. The solution may be decanted off or filtered. To protect it against decomposition, this author finally adds a few crystals of thymol, or .1^ of salicylic acid or .5^^ of sodium salicylate. On washing with water the protoplasm remains somewhat colored. To obtain a purely nuclear stain, the washing must be carefully done with a solution of alum or a weak acid. 9. Paracarmine is prepared according to the following recipe: i gram of carminic acid, \ gram of aluminium chlo- ride, and 4 grams of calcium chloride are dissolved in 100 BOTANICAL MIL •ccm. of 70^ alcohol with or without heat, the whole -allowed to stand and then filtered. Washing with aci alcohol is usually unnecessary, but a weak solution of alu- minium chloride in alcohol, or alcohol containing 2^^ of dacial acetic acid, is sufficient for all cases. According to P. Mayer's statements, only Grenacher's borax-carmine, of the numberless solutions heretofore rec- ommended, presents any advantages over carmalum and paracarmine. A. Meyer (V) recommends especially for staining the jiuclei of pollen-grains: 10. Chloral Carmine. — This is prepared by heating for 30 minutes on the water-bath .5 gram of carmine, 20 ccm. of -alcohol, and 30 drops of officinal hydrochloric acid,^ and then adding 25 grams of chloral hydrate. After cooling, the solution is filtered. It stains the nuclei of pollen-grains ,().— Lilium Marta^on. I, tip of the embryo-sa*; II, the same, later stage; III and IV, older karyokmeiic figures from the same source; a, centrospheres. After Guignard. also used the vapor of osmic acid, but allows it to act only a short time, in order not to lessen the staining capacity of the objects, and then places them in Flemming's solutioa (§ 309) for half an hour to an hour, and then in alcohol. For staining the attractive spheres Guignard uses especially hsematoxylin ; but he first treats the sections hardened with alcohol with a lO'fc solution of zinc sulphate or ammonia alum. He has yig. also treated the preparations success- -Nuclei from the fer- tilized egg-cell of Liliuvt Martagon during their fu- 1 •.■! jM ^ 1 4.: ^C sion. w, male, ^, female IVely With a dilute aqueous solution 01 nucleus; «, centrospheres. orseillin and eosin-haematoxylin.* '^^^ '^"^ uignar . The * This probably means the eosin-haematoxylin mixture recommended by Renault. It is prepared, according to Gierke (I, 86), by mixing equal parts of glycerine, containing common salt and saturated with eosin, and a saturated solution of potash alum in glycerine. This mixture is filtered and then an alcoholic solution of haematoxylin or Delafield's haematoxylin (§ 314) is added. :20O BOTANICAL MICROTECHNIQUE. interior of the centrosphere is stained deep red by tliis treatment. For moiniting such preparations, Guignard recommends especially glycerine-gelatine and a lO^ solution of chloral hydrate thickened with gelatine. The latter has the advan- tage of clearing the preparations, but gradually destroys most dyes. 348c. Guignard has succeeded in bringing out the attrac- tive spheres especially in various sexual cells. In the grow- ing stamen-hairs of Tradescantia he also succeeded by treating them successively with osmic acid vapor, Flem- ming's chrom-osmic-acetic acid mixture, and alcohol, and then staining with a mixture of fuchsin and methyl green. If this mixture is rightly prepared, the centrospheres are -colored bright red in the pale red protoplasm. 348d. Hermann (II, 583) has recently used the following method for making visible the centrospheres and the radi- ating structures around them, in animal cells. The ob- jects, fixed with platinum-chloride-osmic-acetic acid and then reduced with wood-spirit in the manner described in § 313, are placed whole in the dark in a haematoxylin solution con- taining one part haematoxylin, 70 parts alcohol, and 30 parts water. They remain in this solution 12 to 18 hours, are then treated for the same time, also in the dark, with 70^ alcohol, and are then imbedded and sectioned with the microtome. The sections are then extracted with a solu- tion o{ potassium permanganate so dilute that it has a bright rose color, until they have an ochre-colored appearance. After rapid rinsing in water, the manganese peroxide is dis- solved out with a solution of one part oxalic acid and one part potassium sulphate to 1000 to 2000 parts of water, and the sections are then stained for three to five minutes with safranin. The attractive spheres and the structures sur- rounding them appear deeply blackened, while the nuclear elements have a bright red color. How far this method can be used with success for plant- cells remains to be shown. But I will remark that the methods used by Flemming on animal cells with the best SPECIAL METHODS. 20I results (cf. § 323) are poorly suited to plant-cells, according to Guignard (IV, 167). I have obtained, also, in some not very extended experiments with Hermann's methods, no staining of the centrospheres, while the spindle-threads of such preparations stood out very sharply, especially after staining with gentian violet. 3. The Chromatophores and their Inclusions. 349. Under the name chromatophores are commonly in- cluded at present three different kinds of bodies ; the green chlorophyll-bodies, chloroplasts, and the corresponding bod- ies in the algae which are not green, the mostly yellow or red bodies which carry coloring matters, chromoplasts, oc- curring especially in the bright-colored parts of flowers and fruits, and the colorless leucoplasts^ which are found chiefly in subterranean and young parts of plants. The grouping of these different bodies together is justified, aside from their chemical similarity, by the fact, recognized especially by Schimper, that they stand in genetic relations with each other, and may pass over into each other in the most vari- ous ways I. Methods of Investigation. 350. The study of chromatophores has been conducted chiefly in the living cell. Of course this is only possible in sections which are at least several cell-layers in thickness; and the most rapid preparation possible is necessary, since chromatophores are very sensitive to the most varied harm- ful influences. Since most cells also die very quickly in pure water, and the chromatophores especially suffer pro- found structural changes in this medium, it is advantageous to use a dilute solution of salt or sugar as a medium for their study. I have used with good results a 5^ solution of sugar, with which I injected the tissues to remove the air from the intercellular spaces, which may usually be easily done by means of a filter pump (cf. also § 5). 351. In difficult cases one must have recourse to staining methods. I have found a concentrated alcoholic solution of 202 BO TANICA L MICRO TECHNIQ UE. corrosive sublimate well adapted for fixing (cf. § 310); and a concentrated alcoholic picric acid solution often does, well. According to ray own most recent experiments, a saturated solution of picric acid and corrosive sublimate in absolute alcohol seems to be best for fixing chromatophores. I allow it to act about 24 hours on the objects to be fixed and wash it out with running water. The use of an iodine solution for the removal of the sublimate seems unnecessary here,, as I have seen none of the well-known sublimate needles in my preparations. Krasser (II, 4) recommends the use of a \ic alcoholic so- lution of salicylic aldehyde for fixing chromatophores. He lets it act for 24 to 48 hours on small pieces of tissue. After hardening in alcohol, the sections may be mounted in gly- cerine, glycerine-gelatine, or balsam. If in the latter, the clearing in clove-oil must be made as brief as possible. 352. Schimper used haematoxylin and gentian violet for staining chromatophores; but I have found iodine green» fuchsin, and acid fuchsin better (cf. Zimmermann V, 6). Staining with acid ftichsin is best accomplished by one of the three methods described in §§ 345 to 347. It is easy to make clearly visible the relatively small leucoplasts on each starch-grain in the outer layers of a ripe potato, by the aid of method B (cf. Fig. 38, /). 353- Iodine green is used in concentrated aqueous solution and is either allowed to act for only a short time {^ to a few minutes) on microtome sections, which are then washed with water and exam- ined in glycerine ; or it is al- lowed to act longer and the a few layers removed from the cork. After sectionS are thcn placed in a Iixinjjr with sublimate-alcohol and staining ^ •euco- solution prepared by mixing two parts of common ammo- In this the sections were left Fig. 38. — Cell-contents from a parenchyma celf of a tuber of Solanum tuberosum, but with acid fuchsin (Method B). /, plasts ; J, starch-grains ; z, nucleus crystalloid. nia with 98 parts of water. SPECIAL METHODS. 203 from a few minutes to several hours, according to the depth of the staining and the character of the preparation. The examination may be made in glycerine. Such preparations keep only a very short time, while very permanent prepara- tions may be made by transferring to Canada balsam even sections stained with iodine green. In this transfer alcohol must be wholly avoided, since it decolorizes the chromato- phores. Phenol and aniline also are not suited for this use. Therefore I simply allowed the sections to dry, after wash- ing them with water, and then treated them with xylol, and finally added xylol-balsam. 354. For staining with /7/<;//5/;/, I have used the ammonia fuchsin mentioned in § 271. I let it act only a short time on the sections, until they begin to become red. Then I wash it out with water, and examine the sections in glycer- ine or transfer them in the above described manner, by dry- ing, to balsam. Alcohol decolorizes the chromatophores in this case, also. II. The Finer Structure of the Chromatophores. 355. Opinions are at present divided as to the intimate structure of the chromatophores (cf. Zimmermann I, 56 and Bredow I, 380, for the early literature). It is only as to the. P I jr M pi A @ Fig. 39. — I, chromoplasts from the flower of Neottia nidus-avis \ /, protein crystalloids ; fy pigment-crystals. II, the same, from the root of Daucus Carota. Ill, the same, from the fruit of Sorbus aucuparia. s, starch-grains.— After Schimper. chromoplasts that it may be regarded as settled that the pigment occurs partly in crystalline, partly in amorphous form. 356. In the former case, it forms more or less regular 204 BOTANICAL MICROTECHNIQUE. rhombic plates or peculiar cylindrically curved bodies, as, for example, in the parenchyma of the carrot (cf. Fig. 39, II); or it occurs in the form of delicate needles which are imbedded in the colorless stroma in small numbers, as in Neottia nidus-avis (Fig. 39, I), or in large quantity, as in the pericarp of Sorhus auaiparia (Fig. 39, III). All these pig- ment-crystals, which consist of carotin, according to the prevailing nomenclature, are characterized by strong pleo- chroism *), and this peculiarity has been used by Schimper J in surface view; S, in profile view. /, iridescent plate ; c, chromato- phores (X 250). After Berthold. 372. ¥ or fixiftg the protoplasmic plates Berthold recommends chiefly a concentrated solution of iodine in sea-water (cf. § 301) or osmic acid (§ 308). Neither of these, however, completely preserves the structure. Whether these bodies are to be included with elaioplasts,. as has lately been held to be probable by Wakker (I, 488), must be determined by further researches. 7. Microsomes and Granula. 373- Under the term microsomes are usually included all those small and mostly globular bodies which are distin- guishable by their different refractive power from the main mass of the cytoplasm. But it can no longer be doubted that these include bodies of very different composition, and it is not possible to speak of special reactions of the micro- somes. SPECIAL METHODS. 2 1 5, On the other hand, it has been shown by Altmann (I) that bodies of definite reactions may be quite generally recog- nized in the cytoplasm of animal cells, which this author terms granula and regards as the elementary organisms of the cell. Altmann used, for the demonstration of these granula, chiefly a fixing mixture of osmic acid and potassiunrt bichromate and the acid fuchsin staining method A, de^ scribed in § 345. 374. How far the cytoplasm of plant-cells possesses a sim- ilar granula-structure cannot at present be said. The writ- er's investigations on this point have not yet reached any^ conclusive results. But, by the aid of Altmann's methods,, it can be shown that certain bodies are widely distributed in the cytoplasm of the cells of the assimilating tissue, which correspond in many respects with Altmann's granula and have been termed at first simply granula (cf. Zimmermann,. n, 38)- These are always colorless and are mostly little spheres^ which have, at most, about the size of the nucleoli, in adult cells (cf. Fig. 48, g). Their chemical relations indicate that they consist of protein-like substances. 375. Y ox fixing the granula a concentrated alcoholic solu-^ tion of corrosive sublimate or of picric acid may be used (cf. §§ 310 and 303). Very good results have been obtained also with dilute nitric acid, and I used in this case a solution COntainine:, in O? volumes Fig. 48— a cell from the lowest mesophyll layer ^ ■^' of Trade sca.ntia- albijlora, from a prepara- of water, three volumes of ^^'on ^^^^ wUh alcohoUc picric acid and stained by Altmann's acid fuchsin method, c. chemically pure nitric acid chloroplasts; k, nucleus; g, granula. of specific gravity 1.3, which therefore contained about 1.5^ of HNO5. I allowed this solution to act for 24 hours, and then washed the objects in running water for 24 hours. ¥ or s taming the granula I formerly used almost exclusively Altmann's acid fuchsin method (§ 345), but have lately con^ vinced myself that the other acid fuchsin methods (§§ 346 :2I4 BOTANICAL MICROTECHNIQUE. incr ■ ^nd 347) give very good results. Especially after fixing -with nitric acid, preparations may be pretty easily obtained in which the granula are still deeply colored, while the much 4arger chromatophores are wholly decolorized. I can recom- jnend the leaves of Tradescantia albiflora as suitable objects for study, as they contain comparatively large granula, especially in the spongy parenchyma (cf. Fig. 48, g), [Crato (I) has lately observed, in Chcetopteris and other plants, certain structures, hitherto included under the gen- -eral term microsomes, which he regards as special organs of the cell and calls physodes. For further details, his -account of them may be consulted.] 8. The Cilia. 376. The cilia, which occur on most of the freely motile lower organisms and are always directly connected with the protoplasm, are often so fine that during their active motion they can be recognized with difficulty or not at all, even with the best objectives. 377. In many cases the cilia may be made better visible by bringing the organisms to rest by quickly killing them. For this purpose, the vapor of osniic acid or i^ osmic acid, \<^ chromic acid, or the solution of iodine and potassium iodide may be used. The cilia often appear sharpl}^ also, if a drop containing the organisms be allowed to dry upon the slide. 378. If the position of the cilia is to be determined while jn motion, fine granules of carmine, or the like, may be -added to the fluid containing the organisms, according to the method proposed by Butschli (I, 7). The movements of these granules will show the cilia-bearing end. 379. Recently staining methods have also been used for the recognition of cilia. Migula (I, 76) obtained a fine staining of the cilia of 4^onium pectorale by using the following method : A very rsmall drop of a concentrated alcoholic solution of cyanin was added to the living specimens, and, after a time, enough SPECIAL METHODS. 2\% water was added to precipitate the cyanin not taken up by the organisms, in granular form. The cilia, as well as the rest of the protoplasm, are colored at first pale blue, but after the addition of water, deep violet. These methods have given me no favorable results, when used on various algae. 380. But I have obtained a very deep staining of the cilia in ChlamydomonaSy Pandorina^ and Chromophytum by the following method, which is essentially similar to methods, used for staining the cilia of Bacteria (cf. § 476). The objects were first fixed in the hanging drop on the slide by the fumes of osmic acid (cf. § 308), and then allowed to- dry ; then a drop of a 20^ aqueous tannin solution is added, and washed off with water in five minutes or later. The slide is then plunged in a concentrated aqueous solution of fuchsin,* in which it remains a quarter of an hour or longer. The fuchsin solution is now washed off with water, the: preparation is again allowed to dry, and finally a drop of balsam and a cover-glass are placed upon it. I have ob- tained in this way very beautiful permanent preparations \\x which the cilia were stained bright red. [I have obtained very satisfactory stainings of the cilia of the zoospores of various algae and fungi by adding to the water containing them a drop or two of a i^ solution of osmic acid, and then the same amount of a strong solution in alcohol of equal parts fuchsin and methyl violet. This stains the cilia deep red almost at once ; and the osmic acid- need not be removed before adding the stain.] 9. Protein Grains. 381. The investigation of the aleurone or protein grains which occur in the seeds of all the higher plants is best con- ducted, in oily seeds, after the removal of the oil, which is often a great hindrance to their study. This may be ac- * Carbol-fuchsin (§ 468) is especially useful, as it stains deeply in a few minutes. 2l6 BOTANICAL MICROTECHNIQUE. complished in many cases, as in Ricifius, by placing the ■sections to be studied, for five minutes or longer, in absolute alcohol. Less soluble fats may be extracted with ether or with a mixture of equal parts ether and alcohol. Further methods of study will depend upon the constituent of the protein grain which it is chiefly desired to make visible ; for there may be distinguished in them a proteid funda- mental mass and various inclusions, protein crystalloids, globoids, and crystals of calcium oxalate. These separate constituents, which are not found simultaneously in all protein grains, but yet are widely distributed, will be dis- cussed in order. [But it may be well to give first two general methods recommended by Krasser (III) for making permanent prep- -arations to show the grains and their inclusions in general. 1. The sections are fixed with an alcoholic solution of picric acid, rinsed with alcohol, stained in an alcoholic €Osin solution, ** toned" with alcohol, cleared with clove-oil, and mounted in balsam. The fundamental mass is stained •dark red, the crystalloid is yellow, and the globoid is colorless •or reddish. 2. The sections are simultaneously fixed and stained in a •concentrated solution of nigrosin in an alcoholic picric-acid solution. When the fundamental substance appears blue, which may be determined by examining a section in abso- lute alcohol with the microscope, the sections are washed in alcohol, cleared quickly in clove-oil, and mounted in balsam. This treatment stains the fundamental mass blue, the crystalloid yellowish green, and leaves the globoid color- less.] a. The Fundamental Mass. 382. The fundamental mass of the protein grains, which sometimes forms the bulk of the whole grain and some- times only a thin envelope about the various inclusions, consists of proteid substances and is well suited for the study of the reactions of protein, detailed in §§ 224 to 234. SPECIAL METHODS. 21/ It is also always readily soluble in dilute caustic potash or ammonia solution and in sodium phosphate. The last- named reagent in concentrated aqueous solution is especially recommended by Liidtke (I, 73). The fundamental mass of the protein granules behaves very differently in different plants. In many, as m Pceonia, it is soluble in water ; in others it is insoluble in it. It shows similar relations with a lofc solution of common salt and a i^ sodium carbonate solution, differing with the species of plant. The protein grains which are soluble in water are best examined in alcohol or glycerine ; and, by the gradual addition of water, their solution may be observed under the microscope. 383. But the protein grains may be made insoluble by fixing media, for instance by an alcoholic solution of corro- sive sublimate or of picric acid. Objects fixed in the latter fluid may be directly preserved in balsam. But it is also ■easy to stain the protein grains after washing out the fixing fluid, and for this purpose an aqueous solution of eosin is very useful. 384. The fundamental mass of the protein grains is bounded externally, as well as against the inclusions, by a delicate pellicle which is distinguished from the remaining substance of the protein grain by its insolubility in dilute alkalies and acids, but, as has been shown by Pfeffer (I, 449), also consists of albuminoid materials. According to Pfeffer, it may be well observed by gradually dissolving the funda- mental mass or the inclusions by the addition of very dilute caustic potash, acetic acid, or hydrochloric acid. Ludtke has lately recommended lime-water for the same purpose, as it first dissolves the fundamental mass of the grain, while the membrane becomes sharply visible and then dissolves after a preliminary swelling. b. The Protein Crystalloids. 385. The crystalloids observed in the protein grains of many seeds consist always, like the fundamental mass, of 2l8 BOTANICAL MICROTECHNIQUE. proteids, as may easily be shown by the aid of the reactions described in §§ 224 to 234. When examined in alcohol or glycerine, they are usually hardly or not at all distinguish- able from the fundamental mass, since they have about the same refractive index as it (Fig. 49, II, a). But after beings placed in water, in which the crystalloids are always insoluble, they show clearly in consequence of their greater density (Fig. 49,. II, b). To distinguish them from the globoids and calcium oxalate crystals, one may make use of their ready solubility in very dilute caus- tic potash and their power of be- coming yellow or brown in a solu- tion of iodine and potassium iodide,, according to its strength. ^'onS'^^WoTt^J^^^^ Ludtke (I, J'j) has lately recom- layerr^^Dl^awn froS picric add mended a Concentrated aqueous I I^Proteln grains from the endo- Solution of Soduini pJlOSpkate for sperm of Ricinus communis., . . . /- ^ 11 • i «rin alcohol, b, on the addition the rCCOgUltlOn Of Crystalloids, SlUCC of iodine-potassium-iodide solu- ^. . 1 1 1 • -^ 1 -i n tion after treatment with aico- they are insoluble m it, while all hoi. A, crystalloid; ^, globoid. . ^-.^ ^ e ^\ . - other constituents of the protein grain are dissolved by it, though sometimes only after several hours. The crystalloids with distinct faces belong, according to Schimper (I), partly to the isometric and partly to the hex- agonal crystal-system (cf. Figs. 49, II, <^ and 50, II and III) ; and those of the latter system have a feeble doubly refractive power. 386. Eosin is very well adapted for staining the fixed crystalloids. Acid fuchsin may also be used for the same purpose according to one of the methods given in §§ 345- 347. These dyes give a very pure and deep staining of the crystalloids, especially after fixing with corrosive sublimate. 387. Recently Overton (II, 5) and Poulsen (II) have given methods for staining crystalloids. Overton places sections of the endosperm of Ricinus, hardened with alcohol, first ia SPECIAL METHODS, 219 a dilute aqueous solution of tannin for ten minutes, and then, after careful washing, in 2^ osmic acid. The crystal- loids are stained a beautiful brown by these reagents. After washing out the osmic acid, the preparations may be pre- served in glycerine. Poulsen (II, 548) places the sections first In alcohol for 24 hours, then for an hour in a 25^ aqueous solution of tannin, and finally, after washing this out with water, in an aqueous solution of potassium bichromate, in which he leaves them until they are brown or yellowish. For the preservation of these preparations, in which the aleurone grains should be quite transparent, Poulsen recommends glycerine. According to another method also recommended by Poulsen, the sections, treated in the same way with alcohol and tannin, are placed, after washing, for an hour in a lO- 2oio aqueous solution of ferrous sulphate. The preparations are then washed and transferred to balsam in the usual way. The crystalloids then appear deep blue, almost black. c. The Globoids. 388. The globoids consist, according to Pfeffer's researches (I, 472), of the calcium and magnesium salt of an organically combined phosphoric acid. They do not occur in all protein grains but, according to Pfeffer's investigations, are not wholly absent from any seed. They are sometimes more or less precisely globular in form, as in Pceonia and Ricinus (cf. Fig. 49, I and 11,^), sometimes irregular, biscuit-shaped, or clustered, as in Bertholletia excelsa (Fig. 50, I). The rela- tive and absolute size may vary very greatly in the same seed. For example, the protein grains in the innermost layers of the endosperm of Pmonia are quite free of glo- boids, while their size increases regularly toward the outside (cf. Fig. 49, I, a-c). 389. In oil or Canada balsam the globoids have the 220 BOTANICAL MICROTECHNIQUE. \3 appearance of vacuoles (Fig. 49, II, a\ because they have a lower refractive index than these / "K media. They may be best ob- ^ d dT^^ served by dissolving the funda- C^ © cjr W J mental mass of the protein-grain ^^j^ and the crystalloids contained in it Jf jfT with dilute, about i^, caustic potash ^^ /^ solution, from sections previously r^ ^^ ^^ deprived of their fat by alcohol or •^ ether-alcohol. There remain then """oidl?-:^?' §lwS^/^^^ in the space formerly occupied by III, proteln-Krain of Eleeis gut- . ... . 1.1 1 1 • 1 neensis. ivf protein grains of the protcm-gram Only the globoids Vitis vini/era : g, gloBoid with , , . , , ^ , calcium oxalate crystal in the and any calcmm oxalatc crystals that may be present. To distin- guish between these two constituents, polarized light may be used. The globoids are amorphous and therefore iso- tropic, while the oxalate crystals (cf. § 392) are strongly ■doubly refractive. For the same purpose, a dilute, about i^, acetic acid may be used, in which the crystals are insoluble, while the globoids are quickly dissolved by it. In concentrated acetic acid the globoids are soluble with much greater difficulty. In a concentrated aqueous solution of sodium pJiosphaie the globoids are completely soluble, according to Liidtke (I, 79), even after treatment with corrosive sublimate. But this solution requires several hours, and the larger globoids, like those from the seed of Vitis vinifera, show during solu- tion an evident stratification which gradually penetrates from without inwards. Ludtke also observed similar stratifica- tions when he allowed dilute caustic potash or lime-water to act for a long time on the globoids. It may be remarked here that the globoids are also dis- solved by picric acid. The protein-grains, however, preserve their original form completely in this fluid, and cavities may be seen in them which have exactly the forms of the dis- solved globoids. 390. Pfeffer (I, 472) used the following reactions for the recognition of the chemical composition of the globoids. SPECIAL METHODS. 221 The presence of organic substance in them is shown by the fact that isolated globoids blacken strongly on heating. They may be easily obtained by moving about on the cover- glass sections which have been freed from fats and proteids by successive treatment with ether-alcohol, i^ caustic pot- ash, and water. To obtain a pure white ash from the globoids, very strong heating is necessary. If the residue left after strong heating be treated with an ammoniacal solution of ammonium chloride, the character- istic crystals of ammonio-magnesium phosphate are formed. This shows at once the presence of phosphoric acid and magnesium in the globoids. But if the globoids be treated with the ammoniacal am- monium chloride solution before being heated, no formation of ammonio-magnesium phosphate crystals occurs, evidently because the organically combined phosphoric acid behaves differently from the phosphoric acid set free by heating. But the formation of large quantities of crystals of the double salt mentioned takes place when sections, freed as above from fats and proteids, are treated with a mixture of an ammoniacal solution of ammonium chloride and sodium phosphate. I have used in this case, with good results, a reagent containing lo parts of the two salts named and lO parts of the officinal ammonia solution * to lOO parts of water. 391. The presence of calcium was shown by Pfeffer (I, 473) by the addition of an ammoniacal solution of ammonium chloride and ammonium oxalate to the unchanged globoids. There are then gradually formed the characteristic crystals of calcium oxalate. By the addition of sulphuric acid, the formation of the characteristic gypsum needles can be brought about. d. Crystals, 392. The crystals observed within the protein-grains con- sists, like nearly all crystals observed within the vegetable * [This is of 16° Baum6, spec, gravity .960.] 222 BOTA NIC A L MICE O TK CI/N I Q UE. organism, of calcium oxalate. For their reactions §§ 85 \.^ 8 }i: No stratification can be observed in them, even after treatment with caus- J& @"® 6 ^ ^^^ potash or chromic acid. For the examination of the fibrosin bodies Zopf ^from^the cdnfdiTof °Pojn- recommcnds that they be isolated by in different views as indi prcssure on the cover-glass, or that the cated by the dotted lines. , , ^ ^ -.i (xiooo). After Zopf. sporcs be made more transparent with nitric acid or caustic potash. Zopf has determined the following points as^ to their chemical relations : They swell in hot water into roundish bodies ; they are colored neither by iodine-potassium-iodide solution nor by chloro- iodide of zinc ; neither are they dissolved by the latter re- agent. In concentrated sulphuric acid they are soluble with •difficulty ; they are not dissolved by nitric acid, even after 48 hours' exposure. In caustic potash they are insoluble in the cold, but, on warming in it, they swell up into irregu- lar, strongly refractive bodies. They are insoluble in cu- prammonia, alcohol, ether, and chloroform, are not browned by osmic acid, and take up no aniline dye. The fibrosin-bodies therefore agree most closely in their reactions with fungus-cellulose, and are distinguished from the cellulin-grains especially by their insolubility in chloro- iodide of zinc and their difficult solubility in concentrated sulphuric acid. A macrochemical analysis has not yet been carried out, for obvious reasons. 14. The Mucus-globules of the Cyanophyceae. 416. Several authors have observed globular structures in the cells of the CyanophycecE, which always lie in the periph- eral part of the protoplasm (Fig 57, s) and are often es- pecially abundant on both sides of the transverse walls. These may be here termed, with Schmitz, mucus-globules, although no conclusion as to their chemical composition is at present possible. 417. According to the recent investigations of Zacharias SPECIAL METHODS. 233 ^III, p. 12 of separata), these mucus-globules give the follow- ing reactions : They are insoluble in alcohol and in ether, and are un- changed either by boiling in distilled water or by the addition of a i^ solu- tion of soda (NaaCOj). In .3^ hydro- 4:Jiloric acid they swell up and become Fig. 57.-1, living ceii of scyto- . ., 1 1 ,1 1. • nema\ 2, cell of iV<»j/<»c, after invisible, and the same result is pro- staining with acetic carmine. , , , . ^ r , , r •^' mucus-globules. After duced by a mixture ot two volumes of Zacharias. concentrated sulphuric acid and three volumes of water; while they swell even in a mixture of one volume of sul- phuric acid and lOO volumes of water, but remain visible. 3-5^ caustic potash solution causes swelling of the mucus- globules, but in a i^ solution they are unchanged. In a solution of potassium ferrocyanide acidulated with acetic acid they stand out sharply and take a vacuolar structure. In Millon's reagent they remain colorless, and also in iodine- glycerine or in chloroiodide of zinc. lodme and potassium iodide solution and the above-mentioned dilute sulphuric acid cause, on the other hand, a deep brown coloring of the mucus-globules. Acetic carmine colors them deep red, and a deep stain is produced by alum-carmine and by Delafield's haematoxylin, after treatment with alcohol. Their capacity for staining with haematoxylin has been disputed by Biitschli (I, 17). According to his statements, £osin is especially useful for staining them. The mucus- globules may be made visible, even in haematoxylin prepa- rations, by subsequent staining with this agent. [Hierony- mus (II) has studied bodies found in the cells of Cyanophycece, Avhich he terms cyanopJiycin-grains and regards as identical Avith the mucus-globules of Schmitz. According to his statements, the bodies studied by him agree in their reac- tions with those above described, and he gives the following additional facts concerning them : They give no proteid reaction. They are quickly dissolved by nitric acid, by a solution of salt, by eati de Javelle^ chloral hydrate^ and caustic potash solution ; but are insoluble in an artificial gastric juice, in carbon bisulphide, in acetic acid, and in a 234 BO 7'ANICAL MICRO TECHNIQ UE. cold solution of di-sodium phosphate (Na,HPO,); but in a boiling concentrated solution of the last they slowly dis- solve. This author states that these bodies are formed of a knot of thread, and he regards them as probably related ta nuclein (cf. § 236).] 15. Tannin-vesicles. 418. Fig 58.— Cell of Meso- carfus sp. g^ tannin- vesicles;/, pyrenoid; 2, nucleus. In the cells of many ZygneinacecB are found strongly refractive globular structures, which, as Pringsheim (II, 354) recognized, contain large quantities of tannin and are com- monly termed tannin-vesicles. Such tan- nin-vesicles have also been observed in various Phanerogams (cf. af Klercker 1, 15). But, while they are usually present in the Algae m e n - tioned in large numbers, and are of small size (cf. Fig- 58, g\ in the Phanerogams they are usually single or only a few in a cell and are of a ^"^- 59-— Cell from the base of the petiole of Desmanthus ple- relatively large ""^- ^» tannin-vesicle; z, nu- cleus ; c, chloroplasts. size, as in the bases of the petioles of Desmanthus ple7ius (cf. P'ig. 59, g). 419. These tannin-vesicles always arise, as Klercker (I^ 22) has .shown, in the protoplasm, from which they are most probably separated by a true precipitation membrane of albumen tannate. Whether they contain other sub- stances than tannins cannot at present be certainly stated ; but, at all events, they cannot contain dissolved proteids, as Klercker (I, 36) has shown. 420. In the investigation of the tannin-vesicles, all the SPECIAL METHODS. 235 tannin reactions described in §§ 199-208 may, of course, be used. Especially useful is Pfeffer's staining intra vitant with methylene blue (§ 208), which may be used with particular advantage in developmental investigations. As suitable objects for study may be suggested the cells of Zygnema and Mesocarpus (cf. Fig. 58), as well as those from the root-cap of Pistia Stratiotes or from the base of the petiole of DcsniantJius pleniis (cf. Fig. 59). 421. A good fixation of this coloring may be obtained by placing the stained objects in a concentrated aqueous solution of picric acid for 2-24 hours. They are then repeatedly rinsed in pure water, then placed in 10-20^ alcohol, which is gradually replaced by absolute alcohol by means of Schulze's dehydrating vessel (§ 16), then trans- ferred to a mixture of three parts xylol and one part alcohol, finally to xylol, and then mounted in balsam.* In this way I have obtained beautiful permanent preparations of Zygneuia, in which, after six months, the tannin-vesicles alone are stained deep blue ; while threads preserved for the same time in glycerine-gelatine have almost entirely lost their color. I have not experimented as to whether a solution of ammonium picrate, recommended by Dogiel (I) for fixing^ methylene blue stains in animal objects, is to be preferred to the watery solution of picric acid for vegetable prepara- tions also. To obtain permanent preparations of the tannin-vacuoles, af Klercker (III) fixes the objects either with Flemming's chrom-osmic acid, or with a mixture of one volume of Kleinenberg's picro-sulphuric acid and one volume of a 5^ solution of potassium bichromate, or with a mixture of equal parts picro-sulphuric acid and cupric acetate solution. After washing, the objects are imbedded in paraffine in the usual way. Finally, a much darker staining of the tannin * Clove-oil, phenol, or aniline must not be used in the transfer to balsam, as they at once wash out the methylene blue stain. 236 BOTANICAL MICROTECHNIQUE. precipitates may be obtained, by means of a feebly alkaline silver solution, in thin microtome sections. 16. The Reactions of the Various Cell-constituents. 422. In many investigations it is of interest to determine what reaction the various constituents of the cell, especially the cytoplasm and the cell-sap, have in the living cell. The reactions of the sap pressed from large pieces of tissue give very uncertain results in this respect, in view of the extensive division of labor within the cell-organism ; and I therefore refrain from discussing further observations made in this way. 423. So far as the cell-sap is concerned, its reaction may be directly determined in those cases in which it contains a coloring matter in solution which changes its color with the reaction. Such a coloring matter is the so-called antho- cyanin (§ 184), which appears red when the reaction is acid, blue when feebly alkaline, and green to yellow when strongly alkaline. The cell-sap is generally alkaline or neutral in blue parts of flowers, but must be acid in red parts. In the same cell, in the course of its development, a change in reac- tion may occur, as in the flowers of Pulmonaria officinalis, which are first red, and then blue. In the last-named plant, as Pfeffer has shown (V, 140), a blue color of the red parts may be produced at any time by traces of ammonia. 424. In cells with colorless cell-sap, one may reach conclu- sions as to its reaction by the method, proposed by Pfeffer, •of introducing into it an artificial coloring matter which gives different colors, according to the reaction. According to Pfeffer (II, 266), mctJiyl orange is especially suited to this purpose, as its orange-yellow color is not changed by dilute alkalies. Pfeffer used in his experiments a .01^ solution. Pfeffer has (II, 259 and 267) also experimented with cyanin, tropaeolin and corallin ; but these dyes proved less useful. 425. It is, in most cases, more difficult to determine the SPECIAL METHODS. 237 reactions of the protoplasm, which never contains coloring matters that show the reaction directly. In the large plasmodia of ^tJuiliiivi septicuni Reinke (I, 8) was able to determine the alkaline reaction macroscopical- ly ; and he deduced the presence of a volatile alkali from the observation that a bluing of litmus-paper occurred when it was not in direct contact with the plasmodium. But this observation does not in any way exclude the presence of other alkaline substances in the plasmodium ; and it has been shown to be probable, by Schwarz (I, 33), that the alkaline reaction of the protoplasm in the higher plants probably does not depend on the presence of ammonia or ammonium compounds. 426. In these plants Schwarz (I, 20) attempted to deter- mine the reaction of the protoplasm by killing cells that naturally have a colored cell-sap by alcohol, heat, crushing, or electricity, and then noting the color assumed by the dead protoplasm. Again, he treated colorless cells in the same way in a feebly acidified extract of borecole leaves which is yellow-red, purple-red, or red-violet in an acid solution, violet when neutral, and blue, blue-green, grass-green, yellow, or yellowish orange when feebly alkaline. Schwarz found that, after killing by electricity, the protoplasm of a few cells was blue-green ; but in most, it was blue-violet, or red- violet, and he therefore concluded that the reaction of the protoplasm is alkaline. On the other hand, Arthur Meyer (III) observed that the coloring matter of kale is not violet, but blue, when the re- action is neutral, and that, when tin-foil electrodes are used in conducting the current, a violet tin compound of the col- oring matter is formed and taken up by the dead protoplasm, and that various colorations of the extract may be caused near the electrodes by the decomposition of the salts con- tained in it. There can therefore be no doubt that Schwarz's method cannot give trustworthy results. 427. The most certain conclusions may be reached by Pfeffer's methods of introducing artificially certain colored 238 BOTANICAL MICROTECHNIQUE. ^^^^ indicators into the living cell. In fact, Pfeffer has (II, 259 and 266) already shown the alkaline reaction of the cytoplasm in various cells by the aid of cyanin and methyl orange. j 17. Plasmolysis (Plasma-membranes). ^H 428. If living plant-cells are placed in a solution of salt or, the like, the protoplasm withdraws from the wall in the form of a continuous sac, if the solution be above a certain degree of concentration, in consequence of its power of taking up water. This proceeding, now generally known as plasmolysis, offers the best means of showing the continuity of the protoplasmic body in cells poor in protoplasm, and it also plays an important part in various morphological and physiological researches. 429. Concerning the media used in plasmolysis, it is especially important that they shall exert no unfavorable influence on the cells in the degree of concentration in which they are employed. They should also have neither a markedly acid nor an alkaline reaction. It is best, then, to use neutral salts like saltpeter (KNO3) or salt (NaCl), or organic compounds like cane-sugar or glycerine. The latter has the advantage, in some cases, of exerting a clearing effect in consequence of its higher refractive index. No general directions can be given as to the concentration of the solutions to be used, and in the choice of the proper concentration the isotonic coefficient "^ of the compound used should be especially regarded. But, in general, clearly visi- ble plasmolysis may be obtained by using a 4^ solution of saltpeter or a 15^ solution of cane-sugar. 430. If the objects to be plasmolyzed are not prescribed by the nature of the investigation, it is best to choose such objects as are most adapted to the observation of plasmoly- sis, and as have the greatest power of resisting the injurious influences connected with their preparation. Thus the cells of Spirogyra furnish very suitable material for the demon- stration of plasmolysis. Such cells as naturally contain a * [Cf. Zimmermann I, 199-201 : also below, p. 263.] SPECIAL METHODS. 239 colored cell-sap are also very useful, as, for example, the epidermal cells of the red lower surface of the leaf of Tra- descantia discolor. The earliest beginnings of plasmolysis are easily observed in such cells. 431. In many cases the plasmolysis may be made plainer by an artificial coloring of the cell-sap. In cells containing tannic acid this may be accomplished by exposing the ob- jects on the slide, in a drop of the plasmolyzing solution, to the vapor of osinic acid for a time, according to the method described in § 308. The protoplasts are then completely fixed in their original position and are much easier to ob- serve on account of the browning or blackening of the cell- sap. Plasmolysis may also often be made plainer by pre- liminary staining intra vitam. Finally, by adding an indifferent pigment, like eosin, to the plasmolyzing fluid, a difference in color between it and the cell-sap may be produced. To test for the presence of a living plasma-body within the vessels, Th. Lange (I, 404) injected large pieces of tissue with a 5^ solution of saltpeter under the air-pump, and then added a dilute solution of picric acid to fix the tissues. After washing in water and dehydrating in alcohol, he placed them in clove-oil. Very good thin sections were prepared from the material so treated, and in these the pro- toplasm was stained with borax-carmine or eosin. Abnormal Plasmolysis. 432. As was shown by H. de Vries (I), many solutions cause the complete killing of the protoplasm and its inclu- sions, with the exception of the inner plasma-membrane bordering on the cell-sap, which is not changed in its osmotic relations, so that it still completely excludes the cell-sap (cf. Fig. 60). If this proceeding, which de Vries calls abnormal plasmolysis, does not justify such far-reaching conclusions as he has drawn from it (cf. on this point Pfeffer VIII, 240), yet abnormal plasmolysis may do good service in many investigations. 240 BO TA NICA L MICRO TECHNIQ UE. of 433. To produce it, one may use a 10% solution ot sai peter to which a httle eosin h; been added. This pigment ha< the double advantage of at onc< staining the killed part of the pro- toplasm and of causing a sharpei definition of the uncolored cell- '"„°o,^arp';l'm.?W»-'r»TSV.oi"sat sap. Pretty large Spirogyra-ctWi S::"h/°mo°?soiateTvi"Soiir'"; furnish excellent objects for stud remains of the dead chloroplasts. / r p«* fir>\ 434. For fixing the isolated vacuolar membranes Went recommends (I, 314) ^-i^ chromic acid, which he allows to act one or two days. He then washes the objects in running water six hours and transfers them, in the usual way, to parafifine, for the preparation of microtome sections \ 18. Methods of determining whether certain Bodies lie In the Cyt plasm or in the Cell-sap. 435. This question often cannot be answered by direct microscopical examination, and already various methods have been devised which make a certain decision possible even in diflficult cases. Wakker (I) observed, with micro- scope tipped down, what motions the bodies in question un- derwent with the slide in a vertical position. If they simply fall downward in the cells in a vertical line in consequence of their greater specific gravity, it is very probable that the bodies lie in the cell-sap. But, since the starch-bearing chro- matophores of the starch-sheath, which undoubtedly lie in the cytoplasm, sink down rather rapidly in the cells, as was recognized by Dehnecke (I, 9) and Heine (I, 190), it has seemed useful to me to modify this method by reducing the ready displaceableness of the protoplasm by killing the cells, which may be easily done with an iodine and potas- sium-iodide solution. The movement of the chromato- phores in the starch-sheath was at once stopped by iodine solution, while the protein crystalloids in the epidermis of the leaf of Polypodium irreoides, which undoubtedly lie in the SPECIAL METHODS. 24! cell-sap, continued the motions due to their weight after the same treatment (cf. Zimmermann II, 68). 436. Wakker (I) has also used abnormal plasmolysis with a loio solution of saltpeter, containing eosin (cf. §§ 432 and 433), for the same purpose. It is often to be determined with certainty by direct observation whether the bodies in question lie in the vacuoles isolated by this method. Be- sides, a further confirmation of the conclusions reached by direct observation may be obtained from the movements taking place in such preparations when the slide is placed vertically. 19. Aggregation. 437. Complicated changes of arrangement take place within the cells of the glandular hairs of Drosera rotundi- folia in consequence of chemical stimulation, which consist essentially in the origin of rapid circulation-currents in the protoplasm and the breaking up of the large central vacuole into a large number of small vacuoles which gradually con- tract more and more. This process, discovered by Darwin and studied in detail especially by H. de Vries (II), will be termed exclusively, in the following account, aggregation, in agreement with de Vries, though Darwin and various other authors use the same term also for the artificial pre- cipitation which accompanies the process, but also occurs in very many plants. 438. The tentacles of Drosera are especially adapted to the observation of aggregation, since their vacuoles stand out very sharply on account of their red cell-sap. Aggre- gation may be produced in them by bringing a leaf of the plant in contact with an insect, a bit of cooked albumen, or the like. It may also be observed in isolated tentacles which have been placed in a i^ solution of ammonium car- bonate. Aggregation then begins at the bases and at the tips of the tentacles, and may be especially well followed in its separate stages at their middles (cf. Fig. 61). 439. According to recent investigations of Bokorny (IV), ■42 B O TA XICA L MICR O TE CHNIQ UE. lo observed aggregation in other plants also, it is produced I J J J jj by the most various <^^j/r'substances ; but if they have highly poisonous properties, as caustic potash or am- monia, they must be used in very dilute form. This author observed a very far-reaching aggregation on leaving superficial sections from peri- anth-leaves of Ttilipa suaveolens for tv^o or three days in a .i^ coffein so- lution. 440. A somewhat different appear- FiG. 61.— Cell from a marginal ten- , . , . . , ,1 lacie of the leaf of Dresera ro- ance, whicli IS Certainly Very closely tundifolin in three different , -^ ^ stages of aggregation, caused related to acfgrecration, was observed by a .ijf solution of ammonium '^^ ° carbonate. The interval be- by Bokomy (I V, 45 I ) On placing^ SCC- tween I and II is six minutes, '' j \ ^ -tj j r & and that between II and III, tions of the maig^in of the stigma of two minutes. After H. de o t> vries. Ctocus vcrmis in a .i^ solution of coffein. The vacuolar wall did not draw together as in normal aggregation, but the whole protoplasmic mass con- tracted, so that there arose between it and the cell-mem- brane a space filled with water. 20. Artificial Precipitates. 441. Artificial precipitates may be produced in the cyto- plasm and in the cell-sap without injury to the vitality of the cell, by the most various reagents. Such excretions may often arise in the cell-sap during plasmolysis. The chemical composition of these bodies has as yet been determined in but few cases ; but it may be regarded as very probable that protein-\\}^^ compounds, especially, are widely distributed in these precipitates with tannins and related substances. 442. Precipitates which consist of relatively pure tannic acid are produced in a great variety of cells by alkaline car- bonates (cf. § 206). That these usually globular bodies con- tain no important amount of proteid substances was recog- nized by Klercker (I). Similar precipitates were produced by Bokorny (V and IV) in various plant-cells by the most various basic com- SPECIAL METHODS. 243 pounds, as, for example, by a .1^ solution of coffein. These may lie partly in the cytoplasm and partly in the cell-sap, and, since they also occur in cells free from tannin, cannot consist of tannin in all cases, at least. But to what extent proteid substances or "■ active albumen " (of. § 445), which forms their chief constituent, according to Bokorny, occur in them cannot be certainly determined from our present Icnowledge. It is not impossible that we have here to do -with very various bodies. 443. Precipitates occurring in plasmolysis were first observed by PfefTer (II, 245) in the root-hairs of Azolla, and they occurred in the same manner whether the plasmolysis was accomplished by means of sugar, saltpeter, or calcium <:hloride. These precipitates agree essentially with those produced by ammonium carbonate, and consist chiefly of tannin, in most cases. But, as af Klercker (I, 29) has shown, other substances must be present in the cell-sap "which remain dissolved in the vacuolar fluid during the ■excretion, and prevent the partial re-solution of the. tannin. It is also noteworthy that Klercker (I, 43) has observed similar precipitates in artificial cells of tannate of glue, on plasmo- lyzing them with a solution of saltpeter, while plasmolysis with sugar contracted the whole cell into a glassy mass. 444. Plasmolytic precipitates which are not due to the presence of tannins may be very easily ob- served in the epidermal cells of the red under surface of the leaves of Tradesca?itia discolor. If tangential sections of these cells be placed in a. small dish with a 10^ solution of saltpeter, it may be seen in ten minutes that strong plasmo- lysis has taken place in all the cells, and pretty strongly refractive, deeply colored spheres (cf. p,G. 62.-Epider- Fig. 62) occur in most cells, while the cell-sap Sl^unTJr^ide"" has in some cases become markedly clearer. ^^scantiadiscoiTr If water is afterwards added, these precipitates J ^^^ ^s^aitpeter ,. , , «T • •. , 1 1 solution, show- are redissolved. No precipitates are produced ing piasmoiytic in these cells by ammonium carbonate, and no p^'^'^'P''-^ ^^• blackening occurs with osmic acid. 244 BOTANIC A. 21. The Loew-Bokorny Reagent for "Active Albumen." 445. Loew and Bokorny (cf. I and II) have, in various papers, maintained the view that living and dead proto- plasm differ from each other in that only the former h:ij the power to precipitate silver from an alkaline silver- solution. These authors conclude from this that livinj albumen contains aldehyde groups which at once underg( a breaking up, at its death. [They have observed a pre- cipitation, in living cells treated with a .1^ solution oi coffein, of abundant small granules which they believe to^| consist of '* active albumen," and term '*proteosomes."j 446. It has, however, been shown by various authors (cf.j especially Pfeffer VII) that the observations of Loew and Bokorny are partly incorrect and that the bodies called by them " active albumen " certainly consist in part of tannin and similar substances. But, since the Loew-Bokorny " Life-reagent " has already been used by other investigators for the recognition of living albumen, and may perhaps be capable of furnishing a basis for some conclusions concern- ing the contents of the cell, when more critically employed^ the methods used by these investigators may be briefly out- lined here. 447. The silver reagent called by Loew and Bokorny " Solution A " is prepared by mixing 13 ccm. of caustic potash solution of specific gravity 1.33 (containing 33 J^ of KOH) and 10 ccm. of aqua ammonia of specific gravity .96 (containing 9^ of NH3) and diluting the mixture to icx> ccm. For use, i ccm. of this solution is mixed with i ccm. of a i^ silver nitrate solution, and the mixture is diluted to one liter (1000 ccm.). 448. The " Solution B " is prepared by adding 5 to 10 ccm, of a saturated solution of lime to a liter of. a j^Vo^ solution of silver nitrate. Both solutions must be used in large quantities on account of their extreme dilution, and but a small number of the objects used should be placed in them. The deposition of silver usually begins only after some hours, and it is gen- SPECIAL METHODS. 245; erally necessary to leave the objects from 5 to 24 hours- in the solutions. A more rapid reaction is obtained with a more concen-^ trated solution containing i gram AgNO, and .3 gram NH, to a liter of water. This can be used, however, only with resistant objects and with such as contain neither sugar nor tannin (cf. Bokorny VI, 195). 22. Protoplasmic Connections. 449. Proof has been furnished by the investigations of Tangl, Gardiner, Russow, and others that, besides the ele- ments of the sieve-tubes, the protoplasts of the separate: cells of various other tissue-systems are in direct connection, with each other through perforations of their cell-walls.. Kienitz-Gerloff (I, 22) has recently concluded from his re- searches that all the living elements of the entire body of the higher plants are united by protoplasmic threads. The- threads which accomplish this union, the so-called proto- plasmic connections, are, however, in most cases so fine that nothing can be seen of them within the living cell ; and rather complicated preparation-methods are necessary to- their recognition. 450. If one has to do with relatively tJiick protoplasmic connections, like those of many sieve-tubes, they may be made visible, in many cases, simply by treatment with chloroiodide of zinc or with iodine and sulphuric acid- Gardiner (II, 55, note 4) recommended for this purpose a_ solution of Hofmann's violet in concentrated sulphuric acid. This solution has a brownish color and does not stain strongly sections placed in it. But if the acid is washed out with a large quantity of water, after acting for about half a minute,, the sections take at first a green, then a bluish, and finally a violet color, and the protoplasts are almost exclusively colored. I have obtained in this way very instructive preparations of alcoholic material, adding to pretty thin sections on the slide, after drying them externally with filter- paper, a drop of the sulphuric acid containing the staining- 246 BOTANICAL MICROTECHNIQUE. material, and at once dropping on a cover-glass to prevent too great warping of the sections. After a short time the whole slide is plunged into a large vessel of v^^ater in which it remains until the sections have beconne clear violet. Al- though the cover-glass usually separates from the slide, some sections generally remain attached to it, and these can, naturally, best be used for study. 451. A very deep staining of the protoplasmic connections contained in the sieve-pores may be obtained in microtome sections of stems of Cuairbitacece by the use of Altmann's acid f uchsin method (cf. § 345). This brings out the proto- plasmic threads without the previous use of any swelling media. In many cases the double staining, described in § 290, Avith aniline blue and eosin gives very fine preparations of the sieve-plates with the protoplasmic connections passing through them. 452. Delicate protoplasmic connections, on the other hand, have as yet been made visible only by the successive action of swelling and staining media. For swelHng, chloroTodide of zinc and sulphuric acid have been chiefly used. ChloroTodide of zinc was recommended especially by Oardiner (II). He treated sections of fresh material first with a solution of iodine and potassium iodide, then added chloroTodide of zinc and let it act a longer or shorter time, according to the capacity of the membranes for swelling, commonly about 12 hours. Before staining, the chloroTodide was washed out with water or, where the membranes were much swollen, with alcohol. 453. When sulphuric acid is used, the sections are also usually first fixed with iodine and potassium iodide solution. Kienitz-Gerloff (I, 8) used for this purpose a solution con- taining .05 gram of iodine and .20 gram of potassium iodide in 15 grams of water. He recommends, especially for juicy tissues, the method used by A. Fischer (III) with the best results in the study of the contents of the sieve-tubes (cf. § 455). This author scalded large plants or large parts of SPECIAL METHODS. 247 plants in boiling water as quickly as possible and hardened them, before cutting, in absolute alcohol. Concerning the strength of the sulphuric acid used for swelling it may be remarked that it has been used partly in concentrated form, partly after dilution with one fourth its volume of water. With membranes which swell strongly,, the dilute acid is, of course, to be preferred. The time for it to act depends chiefly on the character of the membranes. But usually a few seconds is sufficient for concentrated acid. The acid must, of course, be removed by careful washing in water, before staining. 453. The following dyes have been used for staining sec- tions treated in this way. 1. Hofmanns blue (same as aniline blue). Gardiner (II) recommends a solution of this dye in 50^ alcohol saturated with picric acid. The solution is washed out with water^ and the preparation is then either mounted in glycerine, or is gradually transferred through dilute to strong alcohol, cleared with clove-oil, and mounted in Canada balsam. In this way very permanent preparations of the protoplasmic connections may be obtained. Gardiner also used a solution of Hofmann's blue in 50^ alcohol acidified with a few drops of acetic acid. The sub- sequent treatment is the same as in the previous case. Terletzki (I, 455) stains simply with a strong aqueous solution of aniline blue and examines the preparation in water, after washing. 2. Hofmamis violet is used by Gardiner, simply in an aqueous solution. This at first stains wall and protoplasm about equally ; but, after lying for a long time in glycerine, in many cases several days, the color is removed from the walls, while the protoplasts and protoplasmic connections remain strongly stained. 3. Methyl violet is recommended by Kienitz-Gerloff (I, 9) for such cells, like those of hairs, etc., as do not permit the penetration of Hofmann's blue, on account of cuticulariza- tion of the walls. He uses it in a concentrated aqueous solution. 248 BO TA NIC A L MICRO TECHNIQ UE. It may be observed that Gardiner finds it useful to brush over the sections with a camel's-hair brush after swelling ^nd staining. 454. A method differing essentially from previous ones has lately been used by Kohl (I, 12) for demonstrating the proto- plasmic connections. It agrees in essence with Loefifler's method for staining cilia (§ 476). After preliminary mor- •danting with tannin, Kohl stained with nietJiyle7ie blue or Bismarck brown ; and if the staining of cell-walls or gelatin- ous sheaths, due to the presence of pectic substances, inter- fered with observation, these substances were removed by an acid. This author has not given more exact details of iiis method. 23. Contents of Sieve-tubes. 455. Since the contents of the sieve-tubes, which com- municate by relatively large openings, are partly pressed out on cutting the tissues, and undergo the most various changes, A. Fischer (III) has devised a method of fixing the r<~i^ J yy Fig. 63.--Parts of sieve-tubes of Cucurbita Pefio. a, from a plant cut and then scalded: X, ' Schlauchkopf . b, scalded in an uninjured condition (X 675). After Fischer. -contents of the sieve-tubes in the uninjured tissues. For this purpose he plunges carefully unpotted plants or pieces of an uninjured plant, such as branch-tips, into boiling water and leaves them in it until the contents of the sieve-tubes are coagulated, for which two to five minutes' exposure is usually quite sufificient. He was able to show that the accumulations of strongly refractive and readily staining substance observed at one side of the sieve-plate in Cucur- SPECIAL METHODS. 249 bita and many other plants when the ordinary methods are employed, the so-called " Schlauchkopfe " (Fig. 63, a^ j), represent artificial products and originate only when the sieve-tube system is cut. They were entirely wanting in material scalded as above (Fig. 63, U)y but were not changed by boiling water in tissues where they had arisen from cut- ting before the scalding. 456. According to investigations of this scalded material by A. Fischer (VI), we may distinguish three sorts of sieve- tubes, according to the organization of their contents, as follows : 1. Sieve-tubes with coagulable contents (in the Cucurbi- taced) have a thin protoplasmic wall-layer and a clear sap coagulable by heat. 2. Sieve-tubes with mucus (as in Humulus) contain a delicate wall-layer filled with large and small slime-masses and a clear, non-coagulable, watery fluid. 3. Sieve-tubes with starch-grains (as in Coleus) contain a delicate wall-layer carrying small masses of mucus and a clear, non-coagulable fluid with small starch-grains. METHODS OF INVESTIGATION FOR BACTERIA. 457. Since the methods employed in Bacteriology differ in many respects from those used in the study of other plants, I have preferred to collect the former into a special chapter. In the following pages I have not attempted to give an even approximately exhaustive compilation of bac- teriological methods. I have rather chosen to bring together a number of trustworthy methods of preparation which should be quite sufficient for most cases in the study of Bacteria. I must refer persons who wish to devote them- selves especially to Bacteria to the special works on the subject, particularly those of Giinther (I) and Hueppe (I). I. The Observation of Living Bacteria. 458. The observation of living Bacteria may be generally conducted like that of other lower organisms. If it is to be continued for a long time, the Bacteria may best be placed in a hanging drop (cf. § 2). Under some circumstances, a frequent renewal of the culture-fluid is necessary. In case of rapidly motile Bacteria, they may be brought to rest by proper fixing media, like the fumes of iodine or of osmic acid. Finally, I may remark that, in some cases, the dark-field illumination of the Abb^ condenser may be used with suc- cess in the examination of Bacteria. 250 BACTERIA. 2c;i II. Fixing Methods. 1. Cover-glass Preparations. a. Fixing by Dry Heat. 459. For fixing Bacteria from fluids containing them, from gelatine cultures, or the like, dry heat is almost exclusively used at present, and commonly, according to R. Koch's method, as follows : A small drop of fluid containing Bacteria is transferred, by means of a platinum wire sterilized by heat, to a carefully cleaned cover-glass,* and spread out over it as evenly as possible. If the Bacteria are taken from a solid substratum, a drop of water is first placed upon the cover and a very small particle of the material is rubbed up in it as completely as possible with a platinum wire. The fluid is now allowed to evaporate from the cover- glasses thus smeared with Bacteria, at the ordinary tempera- ture, until the preparation is "air-dry." The Bacteria are then fixed by passing the cover-glasses,, with their Bacteria-sides upward, three times through the non-luminous flame of a Bunsen burner.f Johne's rule may- give an idea of the rapidity with which this should be done. According to this, the hand should describe an horizontar circle a foot in diameter in a second, moving at an equals rate throughout the course, and passing through the flame: at one part of it. 460. In this way the Bacteria are so fastened to the cover- glass that they may be treated with staining-fluids and other media without fear of separation. It is thus possible to stain or restain, at any time, preparations which have been mounted for a long time in Canada balsam. For this pur- *The cleaning of cover-glasses may be accomplished by the method pro- posed by Giinther (I, 40, note), which consists in heating the glasses, after cleaning with alcohol, in the non-luminous flame of a Bunsen burner. f In these and the following manipulations the so-called Kuehne forceps are very convenient. Their arms are bent at about i^ cm. from their tips^ and end in broad surfaces. . 252 BOTANICAL MICROTECHNIQUE. pose one need only gently warm the preparation until the balsam becomes fluid, remove the balsam from the raised cover-glass with xylol, and finally wash off the xylol with alcohol. It may be remarked, finally, that Bacteria fixed in the above manner may be preserved in this condition for an indefinite time without harm, if they are protected from dust and moisture by being wrapped, for example, in filter- paper. 461. To remove the strongly staining substance from the red blood-corpuscles, in preparations from the blood, Gun- ther (I, 63) recommends rinsing the objects, fixed in the usual way, in 1-5^ acetic acid. The stainable haemoglo- bin is thus extracted from the red corpuscles, and a large part of the blood-plasma is washed out of the preparations, leaving the Bacteria unchanged. Preparations which give no satisfactory results with this method, on account of hav- ing been kept dry for a long time, have been treated by Gunther with a 2-yf> solution of pepsin, with the best re- sults. b. Other Fixing Methods. 462. Since certain inequalities can hardly be avoided with the fixing methods described above, H. Moeller (II, 274) has proposed fixing the air-dry preparations with absolute alcohol, instead of heating them ; he leaves them in this fluid two minutes. 463. A. Fischer (II) demonstrated the noteworthy fact that artificial appearances often arise, especially when prepa- rations are allowed to dry, which are chiefly the result of plasmolysis of the bacterial cells (cf. § 428). As Fischer has shown, the Bacteria are plasmolyzed by solutions of pretty slight concentration. In general a i^ salt solution is suffi- cient to produce plasmolysis in most Bacteria. Fischer (II, 73) recommends the use of a loj^ solution of lactic acid for fixing Bacteria, which does not prevent subsequent staining with alcoholic solutions of aniline dyes. 464. Besides, the fixing methods used for higher plants BACTERIA. 253 may certainly be used with some success in the investiga- tion of the minute structure of the bacterial cell. Their use may generally be successfully carried out by Overton's method. But it should be noted that the membranes of the Bacteria are characterized by relatively great impermeabil- ity. It has been shown by A. Fischer (II, 72) that i^ osmic acid and a i^ solution of corrosive sublimate, in particular, cause only an incomplete fixation of bacterial cells. 2. Sections. 465. Absolute alcohol is commonly used for fixing Bac- teria within infected organisms, pieces of the tissue being placed directly in it. The microtome should be used for cutting sections from these, after imbedding in paraffine (§ 43) or celloidin (§ 49a). III. Staining Methods. 466. Under this head a number of methods will be de- scribed which can be successfully used, in most cases, for recognizing the presence of Bacteria in a fluid or in a dis- eased organism. Then follows a special description of stain- ing methods for tubercle Bacilli, the spores, and the cilia of Bacteria. I. Staining with Loeffler's Methylene Blue. 467. Loefifler's methylene blue consists of 30 ccm. of a concentrated alcoholic solution of methylene blue and 100 ccm. of an aqueous .01^ solution of caustic potash. It keeps indefinitely. With cover-glass preparations, it may be used by care- fully warming the preparation with a few drops of the stain until steam is seen to rise, then washing off the stain with water and drying in the air, without heat. A drop of Cana- da balsam is then placed on a slide, and the cover-glass is placed on it. This method does not give a very deep stain, but often brings out delicate differentiations sharply. 254 BOTANICAL MICROTECHNIQUE. For staining sections which would be injured by warnning^ the methylene blue may be allowed to act for a longer time. The staining fluid is then washed off with water, and the preparation is transferred to balsam, either by being first allowed to dry (§ 23), or by the use of aniline between water and xylol (§ 24). Many Bacteria, such as the anthrax Ba- cillus, endure treatment with alcohol very well, and may therefore be transferred to balsam in the ordinary way. 2. Ziel's Carbol-fuchsin. 468. Ziel's solution of carbol-fuchsin is prepared by rub- bing up one gram of fuchsin with 100 ccm. of a 5^ aqueous solution of carbolic acid, with the gradual addition of 10 ccm. of alcohol. It is very stable. With cover-glass preparations, it is allowed to act only about a minute. Under some circumstances its action may be hastened by warming. The preparations may be washed in water and mounted, after drying, in Canada balsam. But strongly stained objects will endure longer washing with alcohol, and may be transferred to xylol, and then to Canada balsam. Carbol-fuchsin seems less adapted to use with sections^ 3. Ehrlich's Aniline-water Solutions. 469. These solutions are prepared by adding 1 1 ccm. of a concentrated alcoholic solution of fuchsin, gentian violet, or methyl violet to 100 ccm. of aniline-water. Turbidity arises at first in this mixture, which prevents its immediate use. But it may be used for staining in 24 hours, after previous filtering. It remains fit for use but a few weeks. It is sufficient to let these solutions act for a minute^ while heated, on cover-glass preparations. The dye is then washed off with water, and the preparation is dried and mounted in balsam. Betteir staining of the Bacteria contained in sections is obtained by Gram's method, described in the next paragraph. BACTERIA. 255 4. Gram's Method. 470. The so-called Gram's method is adapted especially for sections, because it stains the Bacteria in them deeply without staining the nuclei at the same time. But it may also be used with cover-glass preparations, especially if they contain many other stainable bodies besides Bacteria. According to Gram's original account, this method con- sisted in placing the sections first for several minutes in Ehrlich's aniline-water-gentian-violet solution (§ 469), and then transferring them to a solution containing one part of iodine and two parts of potassium iodide in 300 parts of water. After a few minutes in this, they are washed with alcohol until no more color comes off, then transferred to clove-oil, which removes more of the dye, and finally mounted in balsam. 471. But, according to Giinther (I, 89), it is better, in most cases, to treat the sections, after removal from the iodine solution, for half a minute with alcohol, then/?/5/ ten seconds* with 3^ hydrochloric acid-alcohol, and then at once with pure alcohol until they are completely decolor- ized. For transferring them from alcohol to balsam, this author recommends xylol, instead of clove-oil. According to the method described by Weigert, aniline is gradually dropped upon the sections, differentiating and dehydrating them, and they are then passed through xylol into balsam. 472. A sharp double staining, by which the nuclei are differently stained from the Bacteria, may be obtained by preliminary staining with picro-carmine (§ 318). This solu- tion is allowed to act one or two minutes on the sections, which are then carefully washed with water, placed in alcohol, and finally stained again according to the Gram or the Gram-Giinther method. * For pretty thin paraffine sections this time is certainly too long. 256 BOTANICAL MICROTECHNIQUE. 5. Staining Tubercle-Bacilli. 473. The Bacilli of tuberculosis and of leprosy are acterized by a peculiar behavior with staining media which' makes possible the staining of them alone in a mixture ofj Bacteria, and therefore their certain distinction from othei species of Bacteria. Of the numerous methods recom- mended for staining tubercle Bacilli, only the following, due to Czaplewski (I), need be here referred to ; and I hav< obtained excellent results with it. Cover-glass preparations are treated, after fixing, first foi a minute with carbol-fuchsin (§ 468) heated to boiling. The] are then washed with the so-called Ebner's fluid * until hardly a trace of color can be seen, are then repeatedly rinsed with pure alcohol, and stained again with a mixture of three parts water and one part concentrated alcoholic solution of methylene blue. This is then rinsed off with water, and the preparation is dried and mounted, in the usual way, in balsam. If a mixture of tubercle Bacilli and other Bacteria, for example, which can easily be prepared by mixing pure cul- tures, be treated in this way, it will be found that, with the exception of the very rare \Gipr3.-Bacilli, only the tubercle- Bacilli are colored red, while all other Bacteria are blue. 474. The staining of tubercle Bacilli in sections can be accomplished by essentially the same methods. If one has parafiine sections attached to the slide with albumen (§ 52), they may be heated in carbol-fuchsin, whose action for a few minutes is sufficient. But if the sections will not endure heating, the fuchsin solution must be allowed to act for a longer time (about 24 hours). It is also better to wash the methylene blue from sections with alcohol, and to transfer them to balsam through xylol. In preparations treated in this way, only any tubercle Bacilli that may be present are stained red ; all other Bac- teria, and the nuclei of the tissue, are blue. * This consists of 20 parts water, 100 parts alcohol, .5 part hydrochloric acid, and .5 part sodium chloride. BACTERIA. 257 6. Staining the Spores of Bacteria. 475. A well-differentiated staining of the spores of Bac- teria may be obtained, according to the method proposed by H. Moeller (II), by plunging cover-glass preparations fixed by heat or by alcohol in 5^ chromic acid "^ for from five seconds to ten minutes, then thoroughly rinsing in water, adding carbol-fuchsin in drops, and warming the whole in the flame for 60 seconds, allowing it to boil up once. The carbol-fuchsin is then poured off, the cover-glass is plunged in 5^ sulphuric acid until it is decolorized, and again thor- oughly washed with water. An aqueous solution of methyU ene blue or malachite green is then allowed to act for 30- seconds, and is rinsed off with water. The preparation is allowed to dry and mounted in balsam. In good prepara- tions, the spores are to be seen as bright red spots within the blue or green bodies of the Bacteria. Zinc chloride or chloroi'odide may be used as a mordant,, instead of chromic acid, but these commonly require a longer time of action than the latter. 7. Staining the Cilia of Bacteria. 476. For staining the cilia of Bacteria, Trenkmann (I) and Loeffler (I) have proposed different methods. According to Loeffler's latest publication, the following method is best adapted for the purpose. The Bacteria are first spread upon the carefully cleaned cover-glass (§ 459, note), and fixed by being passed three times through the flame, too strong heating being carefully avoided. The mordant is then placed on the warm cover-glass. This is best prepared * In general, the action of chromic acid for about 30 seconds is sufficient ; but different species of Bacteria show great differences in this respect. Ac- cording to Moeller, the most favorable duration of its action is : for the brown \^oy.2Xo- Bacillus, 30 sec; for the yellow one, 2 min.; for the while one, 10 min.; for Bacillus cyanogenus, 30 sec; for the a.nihra.x Bacillus, 2 min. ; for the tetanus Bacillus, 2 min. I obtained beautiful staining of the spores of Bacillus subtilis on allowing the chromic acid to act 30 sec. on cover preparations fixed by heat, ^58 BOTANICAL MICROTECHNIQUE. "by mixing lO ccm. of a 20^ aqueous tannin solution, 5 ccm. of a cold saturated solution of ferrous sulphate, and i ccm. of an aqueous solution of fuchsin. According to the char- acter of the Bacteria, a few drops of sulphuric acid or of caustic soda* must be added to this mixture. The cover- glass holding it is now warmed over the flame until steam is formed and, after half a minute to a minute, the mordant is rinsed off with distilled water, and the cover-glass is dried las usual. Then the staining fluid is dropped on until the Cover-glass is wholly covered by it, the whole is again warmed for a minute until steam forms, and is finally rinsed in a stream of water, dried, and mounted in balsam in the usual way. Loefi^er recommends as a staining-fluid, a solu- tion of fuchsin in aniline-water, or a mixture of 100 ccm. of aniline-water, i ccm. of a ij^ soda solution, and solid fuchsin in excess. * For staining the cilia of typhus Bacilli, 22 drops of a \% aqueous solu- tion of sodium hydrate should be added to 16 ccm. of the above mordant ; for Bacillus sublilis, 28 to 30 drops. On the other hand, the cholera Bacilli require the addition of ^ to i drop of a sulphuric acid that will just neutral- ize the same volume of 1% caustic soda ; Bacillus pyocyaneus, the addition of 5 to 6 drops of the above acid ; Spirillum rubrum, 9 drops of the same. But the above mordant has just the right reaction for Spirillum concentri- cum. According to the writer's experiments, it is also well suited to Spiril ium Undula without further addition. TABLES FOR REFERENCE. Weights, Measures, and Temperature. The metric system is based on the 7neter, which was in- tended to be one ten-millionth of a meridian quadrant of the earth in length. The unit of capacity is the liter^ whose volume is that of one cubic decimeter or looo ccm. The unit of weight is the gram, which is the weight of one cubic centimeter of water at 4° C. The centigrade or Celsius' thermometer has for its zero the freezing-point of water, and for its 100° point the boiling- point of water. One degree of the scale is -^\-^ of this in- terval. Comparison of Measures of Length. English and U. S. Metric. I foot = .3048 meter = 30.48 cm. I inch = .0254 " = 25.4 mm. i " = 3.175 mm. TOTT = .254 " = 254. ^ TTHRT = .0254 " = 25.4-" 39.37 in. = I meter ^ .3937 in. = I cm. .0394 in. = I mm. Comparison of Measures of Capacity. English. U.S. Metnc. 1 quart, Imp. = 1.2 qt., wine = 1.135 liter. .833 qt., " = I qt., = .9463 '* 1 fluid ounce = 28.38 ccm. I " dram = 3-55 " .8811 quart, Im] p. = I. 0567 qt., wine = i liter .0352 fl. oz., or .2817 fl .dr. = I ccm. 259 26o TABLES FOR REFERENCE. Comparison of Weights. Avoirdupois, Apothec. Metric. I grain = .0G48 gram I dram = .4558 dr. = 1. 7718 " 2.194 dr. = I dram = 3.888 " I ounce = .9115 oz. = 28.3495 '• 1.0971 oz. = I ounce = 31-1035 " lib. = 1. 215 lb. = 453-59 .8229 lb. = I lb. = 373.242 " .5643 dr. = 15-432 gr. = I gram. Comparison of Thermometer Scales. r c. = \(t - 32) " F. /' F. = \e c. + 32" •F. •c. •F. "C. »F. »C. °C. p "C. OF. »c. °F. 400 204.5 130 54-4 45 7.2 300 572 no 230 35 95 350 176.7 120 48.9 40 4-4 280 536 100 212 30 86 300 148.9 IIO 43-3 35 1-7 260 500 95 203 25 77 280 137-8 100 37-8 30 — I.I 240 464 90 194 20 68 260 126.6 95 350 25 - 3.9 220 428 85 185 15 59 240 "5-5 90 32.2 20 - 6.7 200 392 80 176 10 50 220 104.4 85 29.4 15 - 9-4 190 374 75 167 5 41 200 93-3 80 26.7 10 — 12.2 180 356 70 158 32 190 87.8 75 23.9 5 - 15-0 170 338 65 149 - 5 23 180 82.2 70 21. 1 - 17.8 160 320 60 140 - 10 14 170 76.7 65 18.3 - 5 — 20.5 150 302 55 131 -15 5 160 71. 1 60 15-5 — 10 - 23-3 140 284 50 122 — 20 - 4 150 65-5 55 12.8 - 15 — 26.1 130 266 45 113 - 25 - 13 140 6o.O| 50 lO.O — 20 — 28.9 120 248 40 104 - 30 — 22 Specific Gravity and Percentage Composition of Solutions. The following tables are based on Balances JiydrometerSy a set (two) of which is assumed to be available. The scale of the hydrometer /^r liquids lighter than water has its zero-point at the bottom. This is the point to which the instrument sinks in a 10^ solution of common salt. The 10° point is that to which it sinks in pure water. One de- gree of the scale at any part is one tenth of this interval. The hydrometer /^r liquids heavier than water has its zero point at the top of the scale. This is the point to which it sinks in pure water ; and the 10° point is that to which it TABLES FOR REFERENCE. 26 r sinks in a 10^ salt solution. One degree of the scale is one tenth of this interval. A solution of a given percentage strength is prepared by adding to a number of parts of the substance to be dissolved equal to the required percentage, a sufficient quantity of the solvent to make 100 parts in all. Thus, a 10^ salt solution consists of 10 parts of salt in 90 parts of water. For practical purposes, one cubic centimeter of water or alcohol may be considered as one gram. The figures in the following tables refer to parts by volume at about 60° F. Intermediate values may be obtained from those given above with sufficient accuracy by interpolation. a Heavier than Water. Lighter than Water. Specific % ^HCl. % % Specific % % e Gravity. KOH. 22« B. H,S04. HN03. Gravity. Alcohol. NH3. "jfl . Italics 3 5 8 1.022 2.6 12.6 3.8 4.0 4J OJ = % 1.037 1.060 4.5 7.4 20.4 33-6 5.8 8.8 6.3 10.2 is NH4OH 26° B. 10 1.075 9.2 42.0 10.8 12.7 1. 000 0. 0. 12 1. 091 10.9 50.7 I3-0 15.3 .986 10. 3.3 28.0 15 I.II6 13.8 64.7 16.2 19.4 .967 28. 8.0 3Q-Q 17 1. 134 15.7 74.5 18.5 22.2 •954 38. II. 4 S8.a 20 1. 162 18.6 89.6 22.2 26.3 .936 49. 16.6 71 S 22 1. 180 20.5 1 00.0 24.5 29.2 .924 55. 20.4 q2.0' 25 1. 210 23.3 119.0 28.4 33.8 .907 62. 26.3 27 1. 231 25.1 31.0 37.0 .896 67. 30.7 30 1.263 28.0 34.7 41.5 .879 74. 32 1.285 29.8 37. 45.0 .869 78. 35 1.320 32.7 41. c 50.7 .854 83. 37 1.345 34.9 44.4 55.0 .844 87. 40 1.383 37.8 48.3 61.7 .829 91. 42 1. 410 39-9 51.2 67.5 .820 94. 45 1.453 43.4 55.4 78.4 .807 97. 47 1.483 45.8 58.3 87.1 .798 99. 50 1.530 49.4 62.5 lOO.O 66 1.842 lOO.O 262 TABLES FOR REFERENCE. Table for Acetic Acid. The difference between the specific gravities of water and of glacial acetic acid is small. The mixture of the two sub- stances has the peculiarity that its specific gravity increases up to a certain point with the addition of acid, and then "decreases to that of the pure acid. If the specific gravity is above 1.055, ^"<^ ^^ increased by the addition of water, the acid is above 78^ ; if it is decreased by the addition of water, the acid is below 78^. •B. Sp.gr. % HC,H,0,. »B. Sp. gr. j«HC,H,Oa. I 2 3 5 1.0068 I. 0138 1.0208 1.0280 I.0353 5.0 — 9.7 — 14.6 — 19.7 — 25.2 — 6 7 8 9 10 1.0426 1. 0501 1.0576 1.0653 I.0731 1.0748 31.2 — 37.9 — 45.6 or 99.1 55-0 or 95.4 69.5 or 87.0 77 to 80 Table for Diluting Alcohol. 100 volumes of alcohol of % strength of 90 85 80 75 70 65 60 55 50 Alcohol. require addition of vols, water 85 6.6 • 80 13.8 6.8 75 21.9 14.5 7.2 70 31. 1 23.1 15.4 7.6 $5 41.5 33.0 24.7 16.4 8.2 60 53.7 44-5 35.4 26.5 17.6 8.8 55 67.9 57-9 48.1 38.3 28.6 19.0 9.5 50 84.7 73.9 63.0 52.4 41.7 31.3 20.5 10.4 45 105.3 93.3 81.4 69.5 57.8 46.1 34.5 22.9 11.4 40 130.8 117.3 104.0 90.8 77.6 64.5 51.4 38.5 25.6 35 163.3 148.0 132.9 117.8 102.8 87.9 73.1 58.3 43.6 30 206.2 188.6 171.1 153-6 136.4 118.9 101.7 84.5 67.5 25 266.1 245.2 224.3 203.5 182.8 162.2 141. 7 121. 2 100.7 20 355.8 329.8 304.0 278.3 252.6 227.0 201.4 176.0 150.6 15 505.3 471.0 436.9 402.8 268.8 334.9 301. 1 267.3 233.5 zo 804.5 753.7 702.9 652.2 601.6 551. 1 500.6 450.2 399.9 TABLES FOR REFERENCE, 263 Crystal Systems. Name of Principal Axes. System, No. Rel. Length. Rel. Positions. Cubic or monometric. 3 all equal. at right-angles. Tetragonal or dimetric. 3 two equal, third variable. at right-angles. Hexagonal 4 three equal, fourth variable. three at 60" with each other; fourth at 90° with these. Rhombic or trimelric. 3 of different lengths. at right-angles. Monoclinic or oblique. 3 of different lengths. two at right-angles, third oblique. Triclinic or asymmetric. 3 of different lengths. all oblique to each other. Isotonic Coefficient. Two solutions of equal power to take up water are said by De Vries to be in isotonic concentration. He terms a num- ber showing the water-absorbing power of a given solution, as compared with that of a solution of saltpeter of equal strength taken as a standard, its isotonic coefficient. For con- venience, the coefificient assigned to saltpeter is 3. De Vries finds that compounds fall into six groups whose coefficients are approximately whole numbers, as follows : I. Organic compounds not containing metals, and free acids ; e.g., Cane-sugar, tartaric acid, citric acid, etc. 2. earths with one acid group in the Magnesium sulphate and malate. 2. earths with two acid groups in the II. Salts of alkaline molecule ; e.g. III. Salts of alkaline molecule ; e.g. cium chloride. Magnesium chloride and citrate, cal- 264 TABLES FOR REFERENCE, IV. Salts of alkali-metals with one atom of alkali in the molecule ; e.g., Potassium or sodium nitrate, chloride, acetate, etc. 3. V. Salts of alkali-metals with two atoms of alkali in the molecule ; e.g., Potassium sulphate, oxalate, tartrate, malate, etc. 4. VI. Salts of alkali-metals with three atoms of alkali in the molecule ; e.g.. Potassium citrate. 5. Thus a solution of cane-sugar has f the water-attracting power of one of saltpeter of the same strength ; and a solu- tion of sugar must be | as strong as one of saltpeter to pro- duce the same osmotic effects. LITERATURE. Allen, Edwin West.— I. Untersuchungen uber Holzgummi, Xylose und Xylonsauren. Inaug.-Diss. Gottingen, 1890. Altmann.— I. Die Elementarorganismen und ihre Beziehungen zu den Zellen. Leipzig, 1890. II. Ueber Nucleinsauren. Archiv f. Anatomie u. Physiologic, Physiol. Abt. 1889. p. 524. III. Die Struktur des Zellkernes. lb. Anatom. Abt. 1889. p. 409. Ambronn, H. — I. Ueber das optische Verhalten der Cuticula und der verkorkten Membranen. Ber. d. D. bot. Ges. 1888. p. 226. Arnaud, a. — I. Recherches sur la composition de la carotine, sa fonction chimique et sa formule. Comptes rendus. 1886. T. 102. p. 1 1 19. II. Reclierches sur les matieres colorantes des feuilles; identite de la matiere rouge orange avec la carotine, C18H34O. lb. 1885. T. 100. p. 751. u. Pade, L. — I. Recherche chimique de Tacide nitrique, des nitrates dans les tissus vegetaux. Comptes rendus. T. 98. p. 1488. ASKENASY, E. — I. Beitrage zur Kenntniss des Chlorophylls und einiger dasselbe begleitenden Farbstoffe. Bot. Zeitg. 1867. p. 225. Bachmann, E. — I. Emodin in Nephroma lusitanica. Ber. d. D. bot. Ges. 1887. p. 192. II. Spektroskopische Untersuchungen iiber Pilzfarbstoffe. Progr. d. Gymnasiums zu Plauen. Ostern 1886. III. Mikrochemische Reaktionen auf Flechtenstoffe als Hilfs- mittel zum Bestimmen der Flechten. Zeitschr. f. w. Mikrosk. Bd. III. p. 216. IV. Ueber nicht krystallisierte Flechten farbstoffe. Prings- heim's Jahrbucher. Bd. XXI. p. i. V. Mikrochemische Reaktionen auf Flechten farbstoffe. Flora. 1887. p. 291. VI. Referat iiber Solla (II). Zeitschrift f. w. Mikrosk. Bd. 2. p. 260. 265 266 LITERATURE. DE Bary. — I. Ueber die Wachsiibcrziige der Epidermis. Bot. Zeitg. 1 87 1, p. 129 und 566. II. Morphologic u. Biologic der Pilzc. II. Aufl. Leipzig, 1884. Behrens, J. — Ueber cinige atherisches Oel secernierende Haut- driisen. Ber. d. D. bot. Ges. 1886. p. 400. W. — I. Leitfaden der botanischen Mikroskopic. Braunschweig, 1890. II. Tabcllen zum Gebrauch bei mikroskopischen Arbeiten^ lb. 1887. III. HilfsbuchzurAusfiJhrungmikroskopischer Untersuchungen^ lb. 1883. Beilstein. — I-III. Handbuch derorganischenChemie. II. Auflage. Leipzig, 1 886-1 890. Bd. I-III. Belzung. — I. Recherchcs morphol. et physiolog. sur Tamidon ct les grains de chlorophylle. Ann. d. so. nat., Bot. Ser. VII. T. 5. p. 179- II. Sur divers principes issus de la germination et leur cristal- lisation intracellulaire. Journ. de Botanique. 1892. p. 49. et Poirault. — I. Sur les sels de I'Angiopteris evecta et en parti- culier le malate neutre de calcium. Journ. de Botanique. 1892. p. 286. Berthold. — L Studien uber Protoplasmamechanik. Leipzig, 1886. II. Beitrage zur Morphologic und Physiologic der Meeresalgen. Pringsheim's Jahrbiicher. Bd. XIII. p. 569. BOKORNY, Th. — L Eine bemerkenswerte Wirkung oxydicrtcr Eiscn- vitriollosungen auf Icbcndc Pflanzenzellen. Ber. d. D. bot. Ges. 1889. p. 274. II. Ueber den Nachweis von Wasserstoflfsupcroxyd in lebenden Pflanzenzellen. lb. p. 275. III. Das Wasserstoflfsupcroxyd und die Silbcrabscheidung durch aktives Albumin. Pringheim's Jahrb. Bd. 17. p. 347. IV. Ueber Aggregation. lb. Bd. 20. p. 427. V. Ueber die Einwirkung basischer Stoffe auf das lebende Protoplasma. lb. Bd. 19. p. 206. VI. Neue Untcrsuchungen iiber den Vorgang der Silbcrab- scheidung durch aktives Albumin, lb. Bd. 18. p. 194. Borodin. — I. Ueber die mikrochcmischc Nachweisung und die Verbrcitung des Dulcits im Pflanzenreich. Revue d. sc. n. ^ p. p. 1. Soc. d. Nat. d. St. Peters b. 1890. N. i. p. 26. (Ref. : Bot. Centralbl. 1890. Bd. 43. p. 175.) n. Ueber die physiologische Rollc und die Verbrcitung des Asparagins im Pflanzenrcichc. Bot. Zeitung. 1878. p. 801. III. Ueber cinige bei Bearbeitung von Pflanzenschnitten mit Alkohol c«tstehende Niederschlagc. lb. 1882. p. 589. LITER A TURE. 267 Borodin.— IV. Ueber Sphaerokrystalle aus Paspalum elegans und iiberdie mikrochemische Nachweisung von Leucin. Arbeiten d. St. Petersb. Naturf. Ges. Bd. XIII. p. 47. (Ref. : Bot. Centralbl. 1884. Bd. XVII. p. 102.) BORSCOW, El. —I. Beitrage zur Histochemie der Pflanzen. Bot. Zeitg. 1874. p. 17' Braemer, L.— Un nouveau reactiv histo-chimique des tannins. Bull. Soc. d'hist. nat. de Toulouse. Janv. 1889. (Ref. : Zeitschr, f. w. Mikroskopie. Bd. VI. p. 114.) Bredow, Hans. — I. Beitrage zur Kenntniss der Chromatophoren. Pringsheim's Jahrb. f. w. Bot, Bd. 22. p. 349. BUESGEN. — I. Art und Bedcutung des Tierfanges bei Utricularia vulgaris. Ber. d. D. bot. Ges. 1888. p. LV. BuETSCHLi, O. — I. Ueber den Bau der Bakterien und verwandter Organismen. Leipzig, 1890. BussE, W. — I. Photoxylin als Einbettungsmittel fiir pflanzliche Objecte. Zeitsch. f. wiss. Micros. Bd. IX. p. 47. 1892. II. Nachtriigliche Notiz zur Celloidin-Einbettung. lb. Bd. IX. p. 49. 1892. Campbell, Douglas H. — I. The Staining of living Nuclei. Unter- such. a. d. botan. Institut zu Tiibingen. Bd. II. p. 569. Clautriau, G. — I. Recherches microchimiques sur la localisation des alcaloides dans le Papaver soniniferum. Ann. de la Soc. beige de Microsc. T. XII. 1889. p. 67. (Ref.: Zeitschr. f. w. Mikrosk. Bd. VI. p. 243.) CoHN, Ferdinand. — I. Untersuchungen iiber Bakterien. II. Cohn's Beitr. z. Biol. d. Pflanzen. Bd. I. Heft III. p. 141. Correns, Carl Erich — I. Ueber Dickenwachstum durch Intus- susception bei einigen Algenmembranen. Munchener Inaug.- Diss. 1889, u. Flora 1889. II. Zur Anatomie und Entwicklungsgeschichte der extranup- tialen Nectarien von Dioscorea. Sitzungsber. d. k. Akad. d. W. in Wien. Mathem.-naturw. CI. Bd. XCVII. Abt. I. 1888. p. 652. III. Zur Kenntniss der inneren Struktur der vegetabilischen Zellmembranen. Pringsheim's Jahrb. f. w. Botan. Bd. XXIII. p. 254. CouRCHET.— I. Recherches sur les chromoleucites. Annales d. sc. nat., Bot. Sen VII. T. VII. 1888. p. 263. Crato, E.— I. Die Physode, ein Organ des Zellenleibes. Ber. der D. bot. Ges. Bd. X. p. 295. 1892. CZAPLEWSKI, Eugen.— I. Die Untersuchung des Auswurfs auf Tuberkelbacillen. Jena, 1891. Dehnecke.— I. Ueber nicht assimilierende Chlorophyllkorper. I naug.- Dissert. Bonn, 1880. 268 ^^^B^ LITER A TUKE. Detmer, W.— I. Das pflanzenphysiologische Praktikum. 1888. DiPPEL.— I. Kalium-Quecksilberjodid als Quellungsmittel. Zeit- schrift f. w. Mikroskopie. Bd. I. p. 251. II. Handbuch der allgemeinen Mikroskopie. II. Aufl. Braunschweig, 1882. DOGIEL, A. S. — I. Ein Beitrag zur Farbenfixierung von mit Methy- lenblau tingierten Praparaten. Zeitschr. f. w. Mikrosk. Bd. VIII. p. 15. Dragendorff, Georg.— I, Die qual. und quant. Analyse von Pflanzen und Pflanzenteilen. Gottingen, 1882. DUFOUR, Jean. — I. Notices microchimiques sur le tissu epidermique des vegetaux. Bull, de la Soc. vaud. d. Sc. nat. T. XXII. Nr. 94. II. Recherches sur I'amidon soluble, lb. T. XXI. Nr. 93. Engelmann, Th. W.— I. Neue Methode zur Untersuchung der Sauerstoflfausscheidung pflanzlicher und tierischer Organis- men. Bot. Zeitung. 1881. p. 44i- Errera, Leo. — I. Ueber den Nachweis des Glycogens bei Pilzen. Bot. Zeitung. 1886. p. 316. II. Sur le glycogene chez les Basidiomycetes. Mem. de 1' Acad. d. Belgique. T. 37. (Ref.: Zeitschr. f. w. Mikrosk. Bd. III. p. 277.) III. Sur I'emploi de I'encre de Chine en microscopic. Bull. d. 1. Soc. beige de Microscopic. T. X. p. 478. (Ref. : Zeitschr. f. w. Mikrosk. Bd. II. p. 84.) IV. Canarine. lb. p. 183. (Ref. : Botan. Jahresber. 1884. p. 1 93-) V. Sur la distinction microchimique des alcaloides et des matieres proteiques. Annales de la Soc. beige de Microscopic. Memoires. Tome XIII. (Ref.: Bot. Centralbl. 1891. Bd. 46. p. 225.) Maistriau et Clautriau. — I. Premieres recherches sur la localisa- tion et la signification des alcaloides dans les plantes. Ann. de la Soc. beige de Microscopic. T. XII. 1889. p. i, (Ref. : Zeitschr. f. w. Mikrosk. Bd. VI. p. 389.) Eternod, a. — I. Instruments destines a la microscopic. Zeitschr. f. w. Mikrosk. Bd. IV. p. 39. Eyclesheimer, a. C. — I. Celloidin imbedding in plant histology. Bot. Gazette. Vol. XV. p. 272. 1890. Fischer, Alfred. — I. Ueber das Vorkommen von Gypskrystallen bei den Desmidiaceen. Pringsheim's Jahrb. Bd. XIV. p. 133. ^— II. Die Plasmolyse der Bakterien. Berichte der K. Sachs. Ges. d. W. Math.-phys. Kl. 1891. p. 52. LITERA TURE. 269 Fischer, Alfred. — III. Ueber den Inhalt der Siebrohren in der unverletzten Pflanze. Ber. d. D. bot. Ges. 1885. p. 230. IV. Neue Beobachtungen iiber Starke in den GefUssen. lb. 1886. p. XCVII. V. Beitrage zur Physiologie der Holzgewachse. Pringsheim's Jahrb. Band XXII. p. 73. VI. Neue Beitrage zur Kenntniss der Siebrohren. Ber. d. math.-phys. Klasse der K. Sachs. Ges. d. Wiss. 1886. Emil. — I. Synthesen in der Zuckergruppe. Bericht d. D. chem. Ges. 1890. Bd. 23. p. 21 14. Flemming, W. — I. Ueber Teilung und Kernformen bei Leukocyten und iiber deren Attraktionsspharen. Archiv f. niikr. Anatom. Bd. 37. p. 249. II. Weiteres iiber die Entfarbung osmierten Fettes in Terpen- tin und anderen Substanzen. Zeitschrift f. w. Mikrosk. Bd. VI. p. 178. — III. Neue Beitrage zur Kenntniss der Zelle. II. Teil. Archiv f. niikr. Anatomic. Bd. 37. p. 685. FORSSELL, K. B. J. — I. Beitrage zur Mikrochemie der Flechten. Sitzungsber. d. K. Akad. der W. z. Wien. Mathem.-naturw. Kl. 1886. Bd. 93. Abt. I. p. 219. Frank, B. — I. Untersuchungen iiber die Erniihrung der Pflanze mit Stickstoff. Landwirtsch. Jahrb. 1888. p. 421. II. Bemerkungen zu vorstehendem Artikel (Kreusler I). lb. p. 723- Gardiner, Walter. — I. On the Phenomena accompanying Stimula- tion of the Gland-Cells in the Tentacles of Drosera dichotoma. Proceedings of the R. Soc. of London. V. XXXIX. p. 229. II. On the continuity of the protoplasm through the walls of vegetable cells. Arb. d. bot. Instit. in Wiirzb. Bd. III. p. 52. III. The determination of Tannin in vegetable cells. The Pharm. Journ. and Transact. 1884. N. 709. p. 588. (Ref. : Zeitschr. f. w. Mikroskop. Bd. I. p. 464. Geofkroy.— I. Journal de Botanique. 1893. p. 55. GiBELLi, Giuseppe. — I. Nuovi studi sulla malattia del Castagno detta deir inchiostro. Bologna. 1883. (Ref.: Zeitschr. f. w. Mi- krosk. Bd. I. p. 137.) Gierke, Hans. — I. Farberei zu mikroskopischen Zwecken. Braun- schweig, 1885. GiLSON, Eugene. — I. La suberine et les cellules du liege. La Cellule etc. p. p. Carnoy. T. VI. 1890. p. 63. GiLTAY, E.— I. Ueber das Verhalten von Haematoxylin gegen Pflanzenzellmembranen. Sitzungsbericht der K. Akadem. d. Wiss. zu Amsterdam. 27. Oktober 1883. p. 2. Gravis, A.— I. L' Agar-Agar comme fixatif des coupes microto- 270 LITERA TURK. miques. Bull. d. 1. Soc. beige de Microscopic. 1889. p. 72. (Ref. : Bot. Centralblatt. 1890. Bd. 41. p. 13.) Green, J. R. — I. On the germination of the tuber of the Jerusalem Artichoke. Annals of Botany. 1889. Vol.1, p. 223. (Ref.: Zeitschr. f. vv. Mikrosk. 1889. Bd. 6. p. 244.) GUENTHER, Carl.— I. Einfiihrung in das Studium der Bakteriologie. Leipzig, 1 891. GUIGNARD, Leon. — I. Developpement et constitution des anthero- zoides. Revue gen. de Botanique. Bd. L p. 11. IL Sur la localisation des principes qui fournissent les essences sulfurees des Cruciferes. Comptes rend. 1890. T. iii. p. 249. in. Sur la localisation, dans les plantes, des principes qui four- nissent I'acide cyanhydrique. lb. 1890. T. no. p. 477- IV. Nouvelles 6tudes sur la fecondation. Annales d. sc. nat., Bot. Ser. VII. T. XIV. p. 163. Haberlandt, G. — I. Das reizleitende Gewebesystem der Sinnprlanze. Leipzig, 1890. Hanausek, T. F. — I. Zur histochemischen CofTeinreaktion. Zeit- schr. d. AUg. Oesterreich. Apotheker-Vereins. 1891. p. 606. (Ref. : Bot. Centralbl. 1891. Bd. 48. p. 284.) Hansen, A. — I. Ueber Sphaerokrystalle. Arb. d. botan. Instituts in Wiirzburg. Bd. III. p. 92. II. Das Chlorophyllgriin der Fucaceen. lb. p. 288. III. Die FarbstofTe der Bliiten und Friichte. Verb. d. Physik.- Med. Gesellschaft zu Wurzburg. N. F. Bd. XVIII. N. 7. Hanstein, J.— I. Ueber eine Conferve, welche die Eigentiimlich- keit hat, sich mit Giirteln von Eisenoxydhydrat zu um- kleiden. Sitzungsber. d. niederrh. Ges. zu Bonn. 1878. p. 73. Harz, C. O. — I. Ueber Physomyces heterosporus n. sp. Botan. Centralblatt. 1890. Bd. 41. p. 405. II. Ueber die Entstehung und Eigenschaften des Spergulins, eines neuen Fluorescenten. Botan. Zeitg. 1877. p. 489. Haug, R. — I. Winke zur Darstellung von Praparaten von intra vitam mit Anilinfarben injizierten Geschwulstpartien. Zeitschr. f. w. Mikrosk. Bd. VIII. p. 11. Hauptfleisch, Paul.— I. Zellmembran und Hiillgallerte der Des- midiaceen. Inaug.-Dissert. Greifswald, 1888. Haushofer, H. — I. Mikroskopische Reaktionen. Braunschweig, 1885. Hegler, Robert. — I. Histochemische Untersuchungen verholzter Zellmembranen. Flora. 1890. p. 31. Heine, H. — I. Ueber die physiologische Funktion der Starkescheide. Ber. d. D. bot. Ges. 1885. p. 189. Heinricher, E.— I. Verwendbarkeit der Eau de Javelle zum Nach- LITERATURE. 2/1 weis kleiner Starkemengen. Zeitschr. f. w. Mikrosk. Bd. III. p, 213. Henking. — I. Ein einfaches Mikrotommesser. Zeitschr. f, w. Mikroskopie. 1885. Bd. II. p. 509. Hermann, F. — I. Beitrage zur Histologic des Hodens. Archiv f. mikrosk. Anatomic. Bd. 34. p. 58. II. Beitrage zur Lehrc von derEntstehung der karyokinetischcn Spindel. lb. Bd. 37. p. 569. Herrmann, Ottoman — I. Nachweis ciniger organischer Verbind- ungen in den vegetabilischen Geweben. Inaug.-Diss. Leip- zig, 1876. Hertwig, O.— I. Die Zelle und die Gewebe. Jena, 1892. Hieronymus, G. — I. Ueber Dicranochaete reniformis Hieron., cine neue Protococcacea des Slisswassers. Cohn's Beitrage z. Bio- log, der Pflanzen. Bd. V. p. 351. II. Beitrage zur Morphologic und Biologic der Algen. II. Cohn's Beitrage zur Biol. d. Pfl. Bd. V. p. 461. 1892. V. HoHNEL. — I. Ueber den Kork und verkorktc Gewebe iiberhaupt. Sitzungsber. d. Akad. der Wiss. zu Wien. Bd. 76, I. p. 507, II. Histochemische Untersuchungen iiber das Xylophilin und das Coniferin. lb. Band 67, I. p. 663. HOFFMEISTER, W. — I. Die Rohfaser und einige Formen der Cellu- lose. Landwirtschaftl. Jahrblicher. 1888. p. 239, II. Die Cellulose und ihre Formen. lb. 1889. p. 767. HOLZNER, Georg. — I. Ueber Krystalle in den Pflanzenzellen. Inaug.- Dissert. und Flora. 1864. HuEPPE, Ferdinand. — I. Die Methoden der Bakterien-Forschung. V. Auflage. Wiesbaden, 1891. HUSEMANN, A. und A. Hilger und Th. Husemann, — I. Die Pflan- zenstoffe. II. Aufl. 1882-84.. Ihl, Anton. — I. Einwirkung der Phenole auf Cinnamaldehyd. Zimmtaldehyd, ein wahrscheinlicher Bestandteil der Holz- substanz. Chemikcrzeitung. 1889. p. 560. (Ref. : Bot. Cen- tralblatt. 1889. Bd. 39. p. 184.) II. Ueber neue empfindliche Holzstoff- und Cellulose-Reagen- tien. lb. 1885. p. 266. (Ref. : Zeitschr. fur w. Mikrosk. Bd. II. p. 259.) Immendorff, H. — I. Das Carotin im Pfianzenkorper und Einiges iiber den griinen FarbstofI des Chlorophyllkorns. Landwirt- schaftl. Jahrblicher. 1889. p. 507. JoNSCON, B, — I. Entstehung schwefelhaltiger Oelkorper in den Mycelfaden von Penicilliumglaucum. Botan. Centralbl. 1889. Bd. 37. p. 201. KaerNER, W.— I. Ueber den Abbruch und Abfall pflanzlicher Be- haarung und den Nachweis von Kieselsaure in Pflanzenhaaren. 272 LITER A TURE. Nova Acta d. Ksl. Leop. -Carol. D. Acad. d. Naturf. 1889. Bd. 54. N. 3. p. 219. Kienitz-Gerloff. — I. Die Protoplasmaverbindungen zvvischen benachbarten Gewebselementen in der Pflanze. Botan. Zeitg. 1891. p. I. KiRCHNER.— I. Die mikroskopische Pflanzenwelt des Siisswassers. Braunschweig, 1885. Klebahn.— I. Studien iiberZygoten I. Pringsheim's Jahrb. Bd. 22. p. 415. Klebs.— I. Ueber die Organisation einiger Flagellatengruppen. Un- tersuch. a. d. bot. Institut zu Tiibingen. Bd. I. p. 233. II. Ueber die Organisation der Gallerte bei einigen Algen und Flagellaten. lb. Bd. II. p. 333. III. Beitrage zur Physiologic der Pflanzenzelle. lb. p. 489. IV. Einige Bemerkungen zu der Arbeit von Krasser " Untersuch- ungen iiber das Vorkommen von Eiweiss jn der pflanz- lichen Zellhaut, etc." Botan. Zeitg. 1887. p. 697. V. Ein kleiner Beitrag zur Kenntniss der Peridineen. lb. 1884^ p. 721. Klein, J. — I.. Die Krystalloide der Meeresalgen. Pringsheim's Jahrb. 6d. XIII. p. 23. Klercker, John af. — I. Studien iiber die Gerbstoffvakuolen. Tiib- inger Inaugur.-Dissert. 1888. II. Ueber das Kultivieren lebender Organismen unter dem Mi- kroskop. Zeitschrift f. w. Mikrosk. Bd. VI. p. 145. III. Ueber Dauerpraparate gerbstoffhaltiger Objecte. Verb. d. biol. Vereins in Stockholm. Bd, VI. No. 3. 1891. Koch, L.— I. Microtechnische Mittheilungen. Pringsheim's Jahr- biicher. Bd. XXIV. p. i. 1892. Kohl, F. G.— I. Protoplasmaverbindungen bei Algen. Bericht d. D. botan. Ges. 1891. p. 9. II. Anatomisch-physiologische Untersuchung der Kalksalze und Kieselsiiure in der Pflanze. Marburg, 1889. Krabbe, G. — I. Untersuchungen iiber das Diastaseferment unter spezieller Beriicksichtigung seiner Wirkung auf Starkekorner innerhalb der Pflanze. Pringsheim's Jahrb. f. w. Bot. Bd. 21. p. 520. Krasser, Fridolin. — I. Untersuchungen iiber das Vorkommen von Eiweiss in der pflanzlichen Zellhaut, nebst Bemerkungen iiber den mikrochemischen Nachweis der EiweisskSrper. Sitzungs- bericht der K. Akad. der Wiss. zu Wien. 1886. Bd. 94. Abt. I. p. ir8. - II. Ueber eine Conservirungsfliissigkeit und die fixirende Eigenschaft des Salicylaldehyds. Bot. Centralbl. Bd. LIL p. 4. 1892. LITER A TURE. 2/3 Krasser Fridolin.— III. UeberneueMethodenzurdauerhaften Pra- paration des Aleuron und seiner Einschliisse. Sitzungsber. d. zool.-bot. Ges. zu Wien. Bd. XLI. 1891. Kreusler. — I. Zum Nachweis von Nitraten im Erdboden, etc. Land- wirtsch. Jahrbiicher. 1888. p. 721. Kugler, Karl. — I. Ueber das Suberin. Strassburger Inaug.-Dissert. 1884. Lagerheim, G. — I. Ueber die Anwendung von Milchsaure bei der Untersuchungtrockener Algen. Hedwigia. 1888. p. 58. (Ref.: Ztschr. f. w. Mikr. Bd. V. p. 552.) II. L'acide lactique, excellent agent pour 1 etude des champig- nons sees. Revue mycologique. T. XI. 1889. p. 95. (Ref.: lb. Bd. VI. p. 380.) Lange, Gerhard. — I. Zur Kenntniss des Lignins. I. Zeitschrift fiir physiologische Chemie. Bd. XIV. p. 15. II. Id. II. Mitteilung. lb. p. 217. — — Theodor. — I. Beitrage zur Kenntniss der Entwicklung der Gefasse und Tracheiden. Flora. 1891. p. 393. Lecomte, Henri. — I. Contribution a I'etude du liber des Angiosper- mes. Ann. des so. nat., Bot. Ser. 7. T. 10. p. 193. Leitgeb, H. — I. Ueber die durch Alkohol in Dahliaknollen hervor- gerufenen Ausscheidungen. Botan. Zeitung. 1887. p. 129. II. Der Gehalt der Dahliaknollen an Asparagin und Tyrosin. Mitteilungen a. d. Botan. Instit. zu Graz. Heft II. p. 215. LiNDT, O. — I. Ueber den Nachweis von Phloroglucin. Zeitschr. f. w. Mikroskop. Bd. 2. p. 495. II. Ueber den mikrochemischen Nachweis von Brucin und Strychnin. lb. Bd. I. p. 237. LOEFFLER. — I. Weitere Untersuchungen iiber die Beizung und Farb- ung der Geisseln bei den Bakterien. Centralbl. fiir Bakteriol. u. Parasit. 1890. Bd. VII. p. 625. LoEW, O. — I. Noch einmal iiber das Protoplasma. Botan. Zeitg. 1884. p. 113. ^ II. Ueber den mikrochemischen Nachweis von Eiweissstoflfen. lb. p. 273. und Bokorny. — I. Ueber das Verhalten der Pflanzenzellen zu stark verdiinnteralkalischerSilberlosung. II, Botan. Central- blatt. 1889. Bd. 39. p. 369. II. Die chemische Kraftquelle im lebenden Protoplasma. Miin- chen, 1882. LUDTKE, Franz. — I. Beitrage zur Kenntniss der Aleuronkorner. Pringsheim's Jahrb. Bd. 21. p. 62. LuNDSTRoM, Axel N. — I. Ueber farblose Oelplastiden und die biolo- gischeBedeutung der Oeltropfen gewisser Potamogeton-Arten. Botan. Centralblatt. Bd. 35. p. 177. 2/4 ^■■r LITERATURE. ^^^^^^^ Malfatti, H. — I. Zur Chemie des Zellkerns. Ber. der naturw.- med. Vereins in Innsbruck. XX. Jahrg. 1891-2. (Ref. Bot. Centralbl. LV. 152.) II. Beitriige zur Kenntniss der Nucleine. Zeitsch. fiir physiol Chemie. Bd. XVI. p. 68. 1892. (Ref. : Bot. Centralbl LV. 154.) Mangin, Louis. — I. Observations sur la membrane du grain de pol len mur. Bull. d. 1. soc. bot. de France. T. 36. 1889. p 274. II. Sur lacallose, nouvelle substance fondamentale existant dans la membrane. Comptes rendus. T. 1 10. 1890. p. 644. III. Sur la structure des Peronospor6es. lb. T. in. 1890. p. 923. IV. Sur la presence des composes pectiques dans les vegetaux. lb. T. 109. 1889. p. 579. V. Sur les reactifs colorants des substances fondamentales de la membrane. lb. 1890. T. iii. p. 120. (Ref.: Zeitschr. f. w. Mikroskopie. Bd. VII. p. 409.) VI. Sur la substance intercellulaire. lb. T. no. p. 295. (Ref.: lb. p. 545.) VII. Sur les reactifs jodes de la cellulose. Bull. d. 1. soc. bot. d. France. T. 35. 1888. p. 421. (Ref. : lb. Bd. VI. p. 242.) VIII. Observations sur la membrane cellulosique. Comptes rendus. T. CXIII. p. 1069. 1891. IX. Sur la constitution des cystoliths et des membranes in- crustees de carbonate de chaux. lb. T. CXV. p. 260. 1892. Mattirolo, O. — I. Skatol e Carbazol, due nuovi reagenti per le membrane lignificate. Zeitschr. f. w. Mikrosk. Bd. II. p. 354. Mayer, P. — I. Aus der Mikrotechnik. Internat. Monatschr. f. Ana- torn, u. Physiol. Bd. IV. 1887. (Ref. : Zeitschr. f. w. Mikrosk. Bd. IV. p. 76.) II. Einfache Methodezum Aufkleben mikroskopischerSchnitte. Mitt. a. d. Zool. Stat. Neapel. Bd. IV. 1883. p. 521. (Ref. : lb. Bd. II. p. 225.) III. Ueber das Farben mit Carmin, Cochenille und Haematein Thonerde. lb. Bd. X. p. 480. 1892. Melnikoff.— I. Untersuchungen iiber das Vorkommen des kohlen- sauren Kalkes in Pflanzen. Inaug.-Diss. Bonn, 1877. Mesnard, E.— I. Recherches sur la mode de production de parfum dans les fieurs. Comptes rendus. T. CXV. p. 892. 1892. (Ref.: Bot. Zeitung. LI. 185.) Meyer, Arthur.— I. Ueber Starkekorner. welche sich mit Jod rot farben. Berichte d. D. bot. Ges. 1886. p. 337. LITERATURE, 2/5 Meyer, Arthur.— II. Das Chlorophyllkorn. Leipzig. A. Felix, 1883. (Ref. : Zeitschr. f. w. Mikrosk. Bd. I. p. 302.) III. Kritik der Ansichten von Frank Schwarz iiber die alkali- sche Reaktion des Protoplasmas. Bot. Zeitg. 1890. p. 234. IV. Mikrocheniische Reaktion zum Nachweis der reduzierenden Zuckerarten. Ber. d. D. botan. Ges. 1885. p. 332. V. Chloralkarmin zur Farbung der Zellkerne der Pollenkorner. lb. Bd. X. p. 363. 1892. MiGULA. — I. Beitrage zur Kenntniss des Gonium pectorale. Botan. Centralbl. 1890. Bd. 44. p. 72. jMiliarakis.— I. Die Verkieselung lebender Elementarorgane bei den Pflanzen. Wiirzburg 1884. Inaug.-Diss. MOELLER, Hermann. — I. Anatomische Untersuchungen iiber das Vor- kommen der Gerbsaure. Ber. d. D. bot. Ges. 1888. p. LXVI. II. Ueber eine neue Methode der Sporenfarbung. Centralblatt f. Bakteriologie und Parasitenkunde. 1891. Bd. X. p. 273. MOLISCH. — I. Grundriss einer Histochemie der pflanzlichen Genuss- mittel. Jena, 1891. II. Ueber merkwiirdig geformte Proteinkorper in den Zweigen von Epiphyllum. Ber. d. D. botan. Gesellsch. 1885. p. 195. III. Ein neues Coniferinreagenz. lb. 1886. p. 301. IV. Zur Kenntniss der Thyllen, nebst Beobachtungen iiber Wundheilung in der Pflanze. Sitzungsber. d. K. Akad. d. W. in Wien. Math.-nat. Kl. Bd. 97. Abt. I. p. 264. V. Zwei neue Zuckerreaktionen. lb. 1886. Bd. 93. Abt. II. p. 912. VI. Ueber den mikrochemischen Nachweis von Nitraten und Nitriten in der Pflanze mittelst Diphenylamin oder Brucin. Bericht der D. botan. Gesellsch. 1883. p. 150. VII. Die Pflanze in ihren Beziehungen zum Eisen. Jena, 1892. VIII. Bemerkungen iiber den Nachweis von maskirten Eisen. Ber. der D. bot. Ges. Bd. XI. p. 73. 1893. Moll, J. W. — I. Eene nieuwe mikrochemische looizuurreactie. Maandblad voor Natuurwetenschappen. 1884. (Ref. : Bot. Centralbl. 1885. Bd. 24. p. 250.) MONTEVERDE. — I. Ueber die Ablagerung von Calcium- und Magne- sium-Oxalat in der Pflanze. Petersburg, 1889. (Ref. : Bot. Centralbl. 1890. Bd. 43. p. 327.) II. Ueber Krystallablagerungen bei den Marattiaceen. Arb. d. St. Petersb. Naturf. Ges. 1886. Bd. 17. p. 33. (Ref.: lb. Bd. 39. p. 358. Mueller, Carl Oscar.— I. Ein Beitragzur Kenntniss der Eiweissbild- ung in der Pflanze. Leipziger Inaug.-Diss. 1886. (Sep.-Abdr. aus Landw. Versuchsstat.) 276 ^^^r LITERATURE. Mueller, C. O.— II. Kritische Untersuchungen uber den Nachweis maskirten Eisens, etc. Ber. der D. bot. Gesell. Bd. XI. p. 252. 1893. N. J. C— I. Spectralanalyse der Blutenfarben. Pringsheim's Jahrbiicher. Bd. XX. p. 78. Nadelmann, Hugo.— I. Ueber die Schleimendosperme der Legu- minosen. Pringsheim's Jahrbiicher. Bd. 21. p. 609. Naegeli, C. und S. Schvvendener.— I. Das Mikroskop. II. Aufl. 1877. W. I. Beitrage zur niiheren Kenntniss der Stiirkegruppe. Miin- chener Inaug.-Dissert. Leipzig, 1874. Nebelung, Hans.— I. Spektroskopische Untersuchungen der Farb- stoffe einiger Siisswasseralgen. Bot. Zeitg. 1878. p. 369. Nickel, Emil.— I. Die Farbenreaktionen der Kohlenstoffverbindung- en. II. Aufl. Berlin, 1890. JI. Bemerkungen iiber die Farbenreaktionen und die Alde- hydnatur des Holzes. Botan. Centralblatt. 1889. Bd. 38. p. 753. NiGGL. — I. Das Indol ein Reagenz auf verholzte Membranen. Flora 1881. p. 545. NOBBE, Hanlein und Councler. — I. Vorl. Notiz betr. d. Vorkommen von phosphorsaurem Kalk in der lebenden Pflanzenzelle. Landw. Versuchss^at. 1879. Bd. 23. p. 471. Noll, Fritz. — I. Experimentelle Untersuchungen iiber das Wachstum der Zellmembranen. Wiirzburger Habilitationsschrift u. Abhandl. der Senckenberg. naturf. Gesellsch. Bd. XV. 1887. p. loi. Obersteiner, H.— I. Ein Schnittsucher. Zeitschr. f. w. Mikrosk. Bd. III. p. 55. Overton. — I. Mikrotechnische Mitteilungen. Zeitschrift f. \v. Mikroskopie. 1890. Bd. VII. p. 9. II. Beitrage zur Histologic und Physiologie der Characeen. Botan. Centralbl. 1890. Bd. 44. p. i. Palla, Ed. — I. Beobachtungen iiber Zellhautbildung an des Zell- kernes beraubten Protoplasten. Flora. 1890. p. 314. Pfeffer. — I. Untersuchungen iiber die Proteinkorner und die Bedeutung des Asparagins beim Keimen der Samen. Prings- heim's Jahrb. Bd. VIII. p. 429. II. Ueber Aufnahme von Anilinfarben in lebenden Zellen. Untersuchungen a. d. botan. Instit. zu Tiibingen. Bd. II. p. 179. III. Hesperidin, ein Bestandteil einiger Hesperideen. Botan. Zeitung. 1874. p. 529. IV. Beitrage zur Kenntniss der Oxydationsvorgange in lebenden Zellen. Abhandlungen der mathem.-phys. Kl. d. K. Sachs. Gesellsch. d. W. Bd. XV. p. 375. LITERA TURE. 277 Pfeffer. — V. Osmotische Untersuchungen. Leipzig, 1877. VI. Die Oelkorper der Lebermoose, Flora. 1874. p. 2. Vli. Low und Bokorny's Silberreduktion in Pflanzenzellen. lb. 1889. p. 46. — - VIIL Zur Kenntniss der Plasmahaut und der Vakuolen. AbhandL d. math.-phys. KI, der Kgl. Sachs. Ges. d. Wiss. Bd. XVL p. 187. IX. Studien zur Energetik der Pflanzen. lb. Bd. XVIIL p. 151. 1892. Pfeiffer, Ferdinand, R. v. Wellheim. — I. Mitteilungen iiber die Anwendbarkeit des venetianischen Terpentins bei botanischen Dauerpraparaten. Zeitschrift i. w. Mikrosk. Bd. 8. p. 29. Pfitzer, E. — I. Ueber ein Hartung und Farbung vereinigendes Ver- fahren fiir die Untersuchung des plasmatischen ZelUeibes. Berichte d. D. botan. Gesellscli. 1883. p. 44. Plugge. — I, Salpetrige Saure-haltiges Quecksilbernitrat als Reagenz auf aromatische Korper mit einerOruppe OH am Benzolkern. Archiv der Piiarmacie. 1890. Bd. 228. p. 9. POULSEN. — I. Botanische Mikrochemie. Uebersetzt von C. Miiller. Cassel, 1 88 1. Engl, trans, by Trelease. Boston, 1882. II. Note sur la preparation des grains d'aleurone. Revue gen. d. Botan. 1890. p. 547. Prael, Edmund. — I. Vergleichende Untersuchungen iiber Schutz- und Kern-holz der Baume. Pringsheim's Jahrb. Bd. XIX. p. I. Pringsheim. — I. Ueber Cellulinkorner. Ber. d. D. botan. Ges. 1883. p. 288. II. Ueber Lichtwirkung und Chlorophyllfunktion. Prings- heim's Jahrbucher. Bd. 12. p. 288. Rabl. — I. Ueber Zellteilung. Morphologisches Jahrbuch. Bd. X. 1885. p. 214. Radlkofer, L. — I, Zur Klarung von Theophrasta und der Theo- phrasteen. Sitzungsbericht der math.-physik. Kl. d. k. B. Akad. d. Wiss. zu Miinchen. 1889. Bd. 19. p. 221. II. Ueber die Gliederung der Familie der Sapindaceen. lb. 1890. Bd. 20. p. 105. Ranvier. — I. Technisches Lehrbuch der Histologic. Uebersetzt v. Nicati und von Wyss. Leipzig, 1888. Reichl, C. und C. Mikosch.— I. Ueber Eiweissreaktionen und deren mikrochemische Anwendung. Jahresbericht der K. K. Ober- realschule in d. II. Bezirke von Wien. Wien, 1890. Reinitzer, Friedrich.— I. Bemerkungen zur Physiologic des Gerb- stofTs. Ber. d. D. botan. Ges. 1889. p. 187. II. Ueber die wahre Natur des Gummifermentes. Zeitschrift f, physiol. Chemie. Bd. 14. p. 453. 2/8 LITERA TURE. Reinke, Friedrich. — I. Untersuchungen iiber das Verhaltniss der von Arnold beschriebenen Kernformen zur Mitose u. Amitose. Inaug.-Diss. Kiel, 1891. J. — I. Die chemische Zusammensetzung des Protoplasma von Aethalium septicum. Untersuch. a. d. botan. Labor, d. Univ. Gottingen. Heft II. p. i. 11. Beitrag zur Kenntniss des Phycoxanthins. Pringsheim's Jahrbiicher. Bd. X. p. 399. Reiss. — I. Ueber die Natur der Reservecellulose und iiber ihre Aufl5sungsweise bei der Keimung der Samen. Landwirt- schaftliche Jahrbiicher. Bd. XVIII. 1889. p. 711. Rhumbler, L. — I. Die verschiedenen Cystenbildungen und die Entwicklungsgeschichte der holotrichen Infusoriengattung Colpoda. Zeitschr. f. wiss. Zoologie. Bd. 46. 1888. p. 549. (Ref.: Zeitschr. f. w. Mikrosk. Bd. VI. p. 50.) RiCHTER, K. — I. Beitrage zur genaueren Kenntniss der chemischen Beschafifenheit der Zellmembran bei den Pilzen. Sitzungsber. d. Akadem. d. Wiss. zu Wien. Bd. 83. I. p. 494. Rosen, F. — I. Beitrage zur Kenntniss der Pflanzenzellen. Cohn's Beitr. zur Biol. d. Pfl. Bd. V. p. 443. 1892. ROSOLL, Alexander. — I. Ueber den mikrochemischen Nachweis der Glykoside und Alkaloide in den vegetabilischen Geweben. 25. Jahresber. des Landes-Realgymnasiums zu Stockerau. 1889-90. II. Beitriige zur Histochemie der Pflanzen. Sitzungsbericht d. Akad. d. Wiss. zu Wien. 1884. Bd. 89. Abt. I. Mathem.- naturw. Kl. ROSTAFINSKI, J. — Ueber den roten Farbstoff einiger Chlorophyceen, sein sonstiges Vorkommen und seine Verwandtschaft zum Chlorophyll. Botan. Zeitung. 1881. p. 461. RUSSOW. — I. Ueber die Verbreitung der Callusplatten bei den Gefasspflanzen. Sitzungsbericht d. naturf. Gesellsch. d. Univ. Dorpat. Bd. 6. p. 63. Sachs, Julius v. — I. Ueber die Stoffe, welche das Material zum Wachstum der Zellliaute liefern. Pringsheim's Jahrb. Bd. III. p. 183. Sanio, Carl. — I. Ueber die in der Rinde dicot. Holzgewachse vor kommenden krystallinischen Niederschlage und deren ana- tomische Verbreitung. Monatsber. d. Berl. Akad. 1857. p. 252. II. Einige Bemerkungen iiber den Bau des Holzes. Botan. Zeitung. i860, p. 193, SCHENCK. H. — I. Ueber Konservierung von Kernteilungsfiguren. Inaug.-Diss. Bonn, 1890. (Ref. : Zeitschr. f. w. Mikrosk. Bd. VII. p. 38.) LITER A TURK. 279 SCHIMPER. — I. Ueberdie Krystallisation der eiweissartigen Substan- zen. Zeitschrift f. Krystallogr. u. Mineral. Bd. V. 1881. p. 131. II. Zur Frage der Assimilation der Mineralsalze durch die griine Pflanze. Flora. 1890. p. 207-261. III. Untersuchungen iiber die Chlorophyllkorper und die ihnen homologen Gebilde. Pringsheiin's Jahrb. Bd. 16. p. i. ScHoNFELD, Selmar. — I. Modification of Pagan's growing slide. Journ. R. Microsc. Soc. 1888. pt. 6. p. 1028. (Ref.: Zeitschr. f. w. Mikroskopie. Bd. VI. p. 51). SCHOTTLANDER, P. — I. Beitrage zur Kenntniss des Zellkerns und der Sexualzellen bei Kryptogamen. Cohn's Beitrage zur Biol. d. Pfl. Bd. VI. p. 267. 1892. SCHMITZ. — I. Die Chromatophoren der Algen. Bonn, 1882. II. Beitrage zur Kenntniss der Chromatophoren. Pringsheim's Jahrbiicher f. w. Bot. Bd. XV. p. i. SCHUTT, Franz. — I. Ueber Peridineenfarbstoffe. Ber. d. D. bot, Ges. 1890. p. 9. II. Ueber das Phycoerythrin. lb. 1888. p. 36. III. Ueber das Phycophaein. lb. 1887. p. 259. IV. Weitere Beitrage zur Kenntniss des Phycoerythrins. lb, 1888. p. 305. SCHULZE, E. — I. Ueber die stickstofffreien Reservestoffe einiger Leguminosensamen. Ber. d. D. botan. Ges. 1889. p. 355. II. Zur Chemie der pflanzlichen Zellmembranen. II. Abhand- lung. Zeitsch. fiir physiol. Chemie. Bd. XVI. p. 387. 1892. (Ref. : Bot. Centralbl. LV. 157.) E., E. Steiger und W. Maxwell. — I. Zur Chemie der Pflanzen- zellmembranen. I. Abhandlung. Zeitschrift f. physiologische Chemie. Bd. 14. 1890. p. 227. Franz Eilhard. — I. Ein Entwasserungsapparat. Archiv f. mikrosk. Anatomic. Bd. 26. p. 539. SCHWARZ, Frank. — I. Die morphologische und chemische Zusam- mensetzungdes Protoplasmas. Breslau, 1887. Cohn's Beitrage zur Biologic der Pflanzen. Bd. V. H. i. 11. Chemisch-botanische Studien uber die in den Flechten vor- kommenden Flechtensauren. lb. Bd. III. p. 249. Shimoyama. — I. Beitrage zur Kenntniss des japanischen Klebreisses. Inaug.-Diss. Strassburg, 1886. Singer. — I. Beitrage zur naheren Kenntniss der Holzsubstanz und der verholzten Gewebe. Sitzungsber. d. Wfener Akad. d. W. Bd. 85. Abt. I. p. 345- SOLEREDER, H.— I. Studicn uber die Tribus der Gaertnereen Bcnth.- Hook. Ber. d. D. bot. Gesellsch. 1890. p. (71). SOLLA, R. F.— I. Zur naheren Kenntniss der chemischen und physi- 28o LITERA TURE. kalischen Beschaffenheit der Intercellularsubstanz. Oesterr. bot. Zeitschr. 1879, November. (Ref.: Bot. Jahresber. 1880. p. 8.) SOLLA. R. F. — II. Ueber zwei wahrscheinliche mikrochemische Reaktionen auf Schwefelcyanallyl. Botan. Centralbl. 1884. Bd. XX. p. 342. Spatzier, W.— I. Pringsh. Jahrbiicher. Bd. XXV. p. 39. 1893. SteinaCH, Eugen.— I. Siebdosen, eine Vorrichtung zur Behandlung mikroskopischer Praparate. Zeitschrift f. vv. Mikrosk. Bd. IV. p. 433- Strasburger. — I. Das botanische Praktikum. II. Auflage. 1887. Streng, a. — I. Ueber eine neue mikroskopisch-chemische Reaktion auf Natrium. 24 Ber. d. Oberh. Gesellsch. f. Nat. u. Heilk. Giessen, 1885. p. 56. (Ref. : Zeitschr. f. w. Mikrosk. Bd. III. p. 129.) II. Ueber einige mikroskopisch-chemische Reaktionen. lb. p. 54. (Ref. : lb. Bd. II. p. 429) III. Ueber einige mikroskopisch-chemische Reaktionen. Neues lahrbuch fur Mineralogie. 1888. Bd. II. p. 142. (Ref. : Zeitschr. f. w. Mikroskopie. Bd. V. p. 554.) SUCHANNEK. — I. Technische Notiz betrefTend die Verwendung des Anilinols in der Mikroskopie sovvie einige Bemerkungen zur Paraffineinbettung. Zeitschrift f. w. Mikrosk. Bd. VII. p. 156. Temme.— I. Ueber Schutz-und Kernholz, seine Bildung und physio- logische Bedeutung. Landwirtsch. Jahrb. 1883. p. 173. Terletzki, p.— I. Anatomic der Vegetationsorgane von Struthiop- teris germanica Willd und Pteris aquilina L. Pringsheim's Jahrb. Bd. 15. p. 452. Thorner, W. — I. Ueber den im Agaricus atrotomentosus vorkom- menden chinonartigen Korper. Bericht d. D. chemisch. Ge- sellsch. 1878. p. 533 und 1879. p. 1630. VAN TiEGHEM. — I. Sur les globules amylaces des Floridees. Comptes rendus. T. 61. 1865. p. 804. et Douliot. — I. Recherches comparatives sur I'origine des mem- bres endogenes. Ann. des sc. nat., Bot. Ser. VII. T. 8. Trenkmann. — I. Die Farbung der Geisseln von Spirillen und Bacil- len. Centralblatt f. Bacteriol. und Parasitenk. 1889. Bd. VI. P- 433. Treub. — I. Quelques Recherches sur le role du noyau dans la division des cellules vegetales. Naturk. Verb. d. K. Akad. Vol. XIX. Amsterdam, 1878. ViNASSA, E. — I-III. Beitriige zur pharmakognostischen Mikroskopie. Zeitschrift f. w. Mikroskopie. Bd. II. p. 309. Bd. IV. p. 295. u. Bd. VIII. p. 34. LITERATURE, 28 1 ViRCHOW, H.— Ueber die Einwirkung des Lichtes auf Gemische von chromsauren Salzen (resp. Chromsaure), Alkohol und extra hierten organischen Substanzen. Archiv f. mikrosk. Anato- mic. 1885. Bd. XXIV. p. 117. (Ref. : Zeitschr. f. vv. Mi- krosk. Bd. II. p. 272.) VoiGT, A.— I. Lokalisierung des atherischen Oeles in deii Geweben der AUium-Arten. Jahrbuch der Hamburgischen wissen- schaftliciien Anstalten, VI. 1889. (Ref.: Bot. Centralbl, 1890. Bd. 41. p. 292.) VOSSELER.— I. Einige Winke fur die Herstellung von Dauerprapara- ten. Zeitschr. f. w. Mikrosk. 1890. Bd. VII. p. 457. Venetianisches Terpentin als Einschlussinittel fiir Dauerprapa- rate. lb. Bd. VI. p. 292. DE Vries,— I. Plasmolytische Studien iiber die Wand der Vakuolen. Pringsheim's Jahrb. Bd. 16. p. 465. — — II. Ueber die Aggregation im Protoplasma von Drosera rotun- difolia. Botan. Zeitung. 1886. p. i. Waage, Th. — I. Ueber das Vorkommen und die Rolle des Phloro- glucins in der Pflanze. Ber. d. D. botan. Ges. 1890. p. 250. Wakker, J. H.— I. Studien iiber die Inhaltskorper der Pflanzenzel- len. Pringsheim's Jahrbiicher. Bd. 19. p. 423. Weber van Bosse, A.— I. fitudes sur les Algues de I'Archipel Malaisien. II. Annales Jard. Bot. de Buitenzorg. T. VIII. p. 165. 1892. Wehmer. — I. Das Calciuraoxalat der oberirdischen Teile von Cratae- gus Oxyacantha L. im Herbst und im Friihjahr. Ber. d. D. botan. Gesellsch. 1889. p. 216. Weiss, Adolf J. und Julius Wiesner. — I. Vorlaufige Notiz iiber die direkte Nachweisung des Eisens in den Zellen der Pflanzen. Sitzungsber. d. Wiener Ak. der Wiss. Math.-naturw. Kl, i860. Bd. XL. p, 276. Went, F. A. F. C. — I. Die Vermehrung der Vakuolen durch Teilung. Pringsheim's Jahrbiicher. Bd. XIX. p. 295. DE Wevre, A. — I. Localisation de I'atropine. Bull. Soc. beige de Microsc. T. XIII. 1887. p. 19. (Ref.: Zeitschr. f. w. Mi- krosk. Bd. V. p. 119.) Wiesner. — I. Beobachtungen iiber die Wachsiiberziige der Epider- mis. Botan. Zeitung. 1871. p. 769. II. Ueber die krystallinische Beschaflfenheit der geformten Wachsiiberziige pflanzlicher Oberhaute. lb. 1876. p. 225. III. Note iiber das Verhalten des Phloroglucins und einigerver- wandter Korper zur verholzten Zellmembran. Sitzungsber. d. Akad. d. W. zu Wien. Math.-naturw. Kl. Bd. -]-]. Abt. l. 1878. p. 60. — — IV. Ueber das Gummiferment. eln neues diastatisches Enzym, 282 LITER A TURK. welches die Gummi- und Schleimmetamorphose in der Pflanze bedingt. lb. 1885. Bd. 92. Abt. I. p. 41- WiESNER.— V. Untersuchungen iiber die Organisation der vege- tabilischen Zellhaut. lb. 1886. Bd. 93. Abt. I. p. 17. WiNOGRADSKY, Sergius.— I. Ueber Schwefelbakterien. Bot. Zeitg. 1887. p. 489- II. Ueber Eisenbakterien. lb. 1888. p. 261. WiNTERSTEiN, E.— I. Ueber das pflanzliche Amyloid. Zeitsch. fur physiol. Chemie. Bd. XVII. p. 353. 1892. (Ref. : Bot. Centralbl. LV. 149.) VAN WiSSELlNGH, C. — I. Sur la lamelle subereuse et la suberine. Archiv neerland. T. XXVI. p. 305. 1893. (Ref.: Bot. Centralbl. LV. 109.) WOTHTSCHALL, E.— Ueber die mikrochemischen Reaktionen des Solanin. Zeitschr. f. w. Mikrosk. Bd. V. p. 19. ZaCHARIAS, E. — I. Ueber Eiweiss, Nuklein und Plastin. Botan. Zeitung. 1883. p. 209. II. Beitrage zur Kenntniss des Zellkernes und der Sexnalzellen. lb. 1887. Nr. 18 bis 24. III. Ueber die Zellen der Cyanophyceen. lb. 1890. Nr, 1-5. IV. Ueber das Wachstum der Zellhaut bei Wurzelhaaren. Flora^ 1891. p. 467. V. Ueber die chemische Beschaffenheit von Cytoplasma und Zellkern. -.Bee- der D. bot. Gesell. Bd. XI. p. 293. 1893. ZiMMERMANN.— I. Die Morphologie und Physiologie der Pflanzen- zelle. Breslau, 1887. II. Beitrage zur Morphologie und Physiologie der Pflanzenzelle^ Heft I. Tubingen. 1890. III. Idem. Heft II. 1891. VI. Idem. Heft III. 1893. IV. Eine einfache Methode zur Sichtbarmachung des Torus der Hoftiipfel. Zeitschrift f. w. Mikroskopie. Bd. IV. p. 216. V. Botanische Tinktionsmethoden. Zeitschr. f. w. Mikroskopie^ Bd. VII. p. I. VII. Microchemische Reactionen von Kork und Cuticula. lb. Bd. IX. p. 58. 1892. VIII. Ueber die Fixirung der Plasmolyse. lb. Bd. IX. p. 184. 1892. ZOPF. — I. Die Pilztiere Oder Schleimpilze. Schenk's Handbuch. Bd. III. Halfte 2. p. I. II. Die Pilze. Ibid. Bd. IV. p. 217. III. Ueber einen neuen Inhaltsk(5rper in pflanzlichen Z*»llen. Ber. d. D. bot. Gesellschaft. 1887. p. 275. IV. Ueber das mikrochemische Verhalten von Fettfarbstoffen LITEKATURE. 283 und Fettfarbstoff-haltigen Organen. Zeitschr. f. w. Mikrosk. Bd. VI. p. 172. ZOPF.— V. Ueber Pilzfarbstoffe. Bot. Zeitg. 1889. p. 53. VI. Zur physiologischen Deutung der Fumariaceen-Behalter. Ber. d. D. bot. Gesellsch, 1891. p. 107. ZWAARDEMACKER, H. — I. Flemiiiing's Safraninfiirbung unter Hinzu- ziehung einer Beize. Zeitschrift f. w. Mikroskopie. Bd. IV. p. 212. INDEX. Abnormal plasmolysis, 239, 241. Abrus precatorius, 109. Absorption spectrum, loi, 103, 104, 105, 106. Acanthospheres, 224. Acetic acid, as reagent, 60, 63, 71, 90, 94, 99, 113, 188, 220, 252. for maceration, 6. Specific gravity, 262. Achromatic figures, 194. Achyranthes Verschaffelti, 205. Acids, 70, 85. Acid alcohol, 181. for maceration, 6. Acidfuchsin, 148, 191, 192, 195, 196, 197, 202, 205, 207, 213, 218, 246. Aconitine, 120, Active albumen, 243, 244, ALthalium septicum, 134, 237. Agar-agar for attachment, 39. Agaricus armillatus. Pigment of, 113. Agave, 210. Agave atnericana, 72, 150, 152, 208. Aggregation, 241. Albumen for attachment, 40. Alcanna tinctoria, 71. Alcannin, as reagent, 71, 74, 89, 90, 91, 95, 209, 210, 211. Alcohol, as reagent, 51, 69, 82, 124, 125. for dehydrating, 12, 32. for fixing, 176, 252, 253. Specific gravity, 261. Table for diluting, 262. Alcohols, 69. Aldehydes, 86. as reagents, 131. Aleurone, 215. Algae, Study of, t, 5. Alkaloids, 119. Alkyl thiocarbimides, 81. Alliutn, 81, 88. Alloxan, as reagent, 131. Allyl sulphide, 81. Allyl sulphocyanate, 81. Aloe, Pigment of flowers, 103. Aloe verrucosa, 75. Altmann's acid-fuchsin staining, 195. Alum, 181. Alum carmine, 233. Alum cochineal, 184. Amido-caproic acid, 82. Amido-compounds, 82. Ammonia, as reagent, 87, 94, 96, 113, 122, 123. Ammonia-fuchsin, 153. Ammonia, Specific gravity, 261. Ammonio-magnesium phosphate, 53. Ammonium, 57. carbonate, as reagent, 67, 88, 117. carminate, 182. chloride, as reagent, 52, 65, 118, 221. molybdate, as reagent, 52, 65, 117. oxalate, 6r. as reagent, 66, 167. sulphide, as reagent, 121. vanadate, as reagent, 97. 28s 286 INDEX. Amorphous starch, 229. Ampelopsis, 63. Amphipyrenin, 135, 136. Amygdalin, 136. Amylodextrine, 229. Amyloid, 156. Anqiopteris evecia, 64. Anhydrite, 66. Aniline for dehydrating, 17, 29. Aniline blue, 142, 153, 165, 210, 246, 247. chloride, as reagent, 145. sulphate, as reagent, 86, 145, 147, 157. water, 185. solutions, 254. Anisic aldehyde, as reagent, 131. Anthoceros, 206. Anlhochlorin, 107, 108. Anihocyanin, 107, 109, 236. Antimonic oxide, as reagent, 70. Araban, 163. Arabanoxylan, 163. Arthonia gregaria, 1 1 2. Arthonia-violet, 112. Artificial cells. Precipitates in, 243. Artificial precipitates, 242. Asaron, 85. Asarum europaum, 85 Ash-skeletons, 168. Asparagin, 51, 82. Astrosphere, 19S Atropine, 120. Attaching sections to slide, 37. Attractive spheres, 198. A vena orientalis, 21 1. Azolla, 243. Bacteria, Fixing Methods for, 251. by alcohol, 252. by heat, 251. by lactic acid, 252. Membranes of. 161. Observation of living, 250. Staining, 253. spores, 257. cilia, 257. Bacteria, reagent for oxygen, 44. Bacterio-purpurin, 106. Bacterium termo, 44. Balsam glass, 16. Balsam Tolu, for mounting, 224. Barium chloride, as reagent, 49, 59, 62, 70. Baryta-water, as reagent, 87, 88, 93, 112. Beale's carmine, 182. Beggiatoa, 47. Benzol, as reagent, 124, 127. Berberin, 120. Berberis vulgaris, 120. Berlin blue, as reagent, 132, 168, 172, 190. as stain, 142. Bertholletia excelsa, 74, 219 Beta, 225. Betula alba, 70. Betuloretic acid, 70. Bitter principles, 99. Bohmer's haematoxylin, i8l. Boletus edulis, 161. Borecole, 237. Borodin's method, 49. Borraginacea, 164. Bottles for reagents, 15. Brandt's reaction, 97. Bromine for fixing, 176. Brucine, 122. as reagent, 51. Caffeine (see Cofifein), 127. Calcium, 57, 66. carbonate, 60. chloride, 112. as reagent, 70. malate, 64. nitrate, as reagent, 70. oxalate, 57, 61, 222. phosphate, 64. sulphate, 62. tartrate, 63. Callose, 163, 164. Slain for, 165. Callus, 163. INDEX. 287 Calycin, 99. Calycium chrysocepkalum, 99. Campanula tracheliu?n, 195. Canada balsam, for mounting, 11, 16, ^o, for sealing, 43. Canarin for staining, 10. Candollea adnata, 194. Cane-sugar, 78, as reagent, 130. Canna, 225. Canna Warszewiczii^ 205. Carbazol, as reagent, 145. Carbohydrates, 75. Carbol-fuchsin, 215, 254, 256, 257. Carbon bisulphide, as reagent, 71, 102. Carbonization, 174, Carmalum, 183. Carmine, 182. Beale's, 182. P. Mayer's, 183. Carminic acid, 183. Carotin, loi, 106, 204, 209. Caustic potash for clearing, 9. for maceration, 7, Caustic potash, for reagent, 59, 63, 73, 77, 78. 84, 87, 88, 92, 94, 95, 103, no, 112, 113, 114, 125, 130, 137, 151, 209, 217, 233. for swelling, 8. Specific gravity, 261. soda, as reagent, 139, 141, 164. Caulerpa, 168. Celloidin blocks, Attaching, 36 Cutting, 36. Hardening, 37. for attachment, 28, 38. for imbedding, 36. Cell-sap, Bodies in, 240. Reactions of, 236. Cellulin grains, 231. Cellulose, 138, 139, 149- bodies, 231. Stains for, 142. Cell-wall, 138. Development, 168. Cell-wall, Minute Structure, 170. Centrosome, 198. Centrospheres, 198. Stain for, 199, Cerasus lusitanica, 137 Ceric acid, 151. sulphate, 126. ChcBtopteris, 214. Chara, 225. Characece, 224. Chemical differences in walls, 173, Chlamydomonas, 215. Chloral carmine, 184. Chloral hydrate, for clearing, 9, 10, 60. for reagent, 71, 90, 193, 227, 233. gelatine, 42. Chlorine for fixing, 176. Chloroform, as reagent, 71, 102, 150, 151. Chlorolodide of zinc, as reagent, no, 139, 140, 143, 155, 166, 245, 246. Chlorophyll, as reagent, 151. Chlorophyll-grains, 136. green, loi. yellow, loi. Chloroplastin, 135, 136. Chloroplasts, 201. Chlororufin, 106. Chromatic figures, 193. Chromatin, 134, 136. spheres, 191. Chromatophores, 201. Inclusions of, 204. Minute Structure, 203. Methods of study, 5, 201. Pigments of, 100. Chrome-yellow, 159. Chrom-formic acid, 178. Chromic acid for fixing, 177, 240. for maceration, 7, for reagent, 54, 113, 116, 125, 257, for swelling, 8. Chromic-acid-platinum-chloride. 180. 288 INDEX. Chromoplasis, 201. Chrom-osmic-acetic acid, 178. Chrom-osmic acid, 235. Chrysophanic acid, 88. Chylocladia, 212. Cicer arietinum, 128. Cilia, Stains for, 214. of Bacteria, Stains for, 257. Cinchonacece, 210. Cinchonamin, as reagent, 51. Cinnamic aldehyde, 146. as reagent, 131. Citrus Auraniiutn, 94. medica, 58. vulgaris, 58. Cladophora, 176. Cladothrix, 68, Clearing media. Chemical 9 Physical, 11. Clearing sections in celloidin, 38. Clivia nobilis, 152. Closterium, 62, 68. Clove-oil for clearing, 14, 15, 33. Cloves, 84. Cochineal, Czokor's, 184. Cocoa-bean, 72, 126. Coffee-bean, 73, 94. Coffee-tannin, 94. Coffein, as reagent, 242, 243. Colchicine, 122. Colchicum officinale, 122. CoUus, 249. Collodion, for attachment, 37, 39. Coloring matters, 100. Combretacea, 210. Comparison of therixLometers, 260. of weights and. measures, 259. Concentration of a'.cohol, 13, 28. Copt/ervacea, 68. Congo red, 143, 169. Coniferce, 92, 142. Coniferin, 92, 144 146. Conjugate, Sheaths of, 157. Constitution of resting nucleus, 191. Convolvulus tricolor, 205. Copper sulphate, as reagent, 130, 137. Corallin, as reagent, 155, 104. Cordiacece, 2 ID. Cork, 149, 152. Corrosive sublimate for fixing, 179^ 213, 218. Corydalin, 122. Cosmarium, 206. Cover-glass preparations, 251. Creosote for clearing, 29. Crocin, 96. Crocus vernus, 242. Cruciferce, 95, 137. Crystalloids, Staining, 195. Crystals, Observation of, 43. Crystals of ammonio- magnesium phosphate, 53. asparagin, 51, 69, 83 berberin, 121. calcium oxalate, 58, 60. sulphate, 66. tartrate, 63, 7 dulcite, 69. gypsum, 62. hesperidin, 94. piperine, 125. protein-grains, 221. saltpeter, 51, 69, 83. silver chloride, 48. sulphur, 47. , Preparation of, 43. Crystal Systems, 263. Cucurbit acea:, 246, 249. Cucurbita Pepo, 168, 248. Culture slide for Algae, 3. Cuprammonia, as reagent, 139, 140,. 155, 157, 162. Cuprammonia for swelling, 8. Cupric acetate, as reagent, 90, 115. sulphate, as reagent, 77, 78. Curcuma a mat a, 107. Curcumin, 107. Cuticle, 148, 150, 152. Cyan in, 46, 72, 90, 152, 154, 209, 211, 214, 238. Cyanophilous nuclei, 191. Cyanophycece, 109, 232. Pigments of, 104. INDEX. 2% Cyanophycin-grains, 233. Cycas circinalis, 58. Cynoglossum, 164. Cystoliths, 61. Cytisine, 123, Cytisus Laburnum, 123. Cytoplasm, Bodies in, 240. Cytoplastin, 135, 136. Czaplewski's stain for tubercle-Ba- cilli, 256. Dahlia, stain, 189. Dahlia variabilis, 79, 82, 83, 86. Dammar lac for mounting, 18 Datisca cannabina, 93. Datiscin, 93. Dauctis Carofa, loi, 203. Dehydrating vessel, Schulze's, 12. Dehydration, 11. by alcohol, 12. by drying, 17. Klercker's method, 12. Overton's method, 13. Delafield's haematoxylin, 180. Delicate objects, To mount, 15, 18. Dermatosomes, 174. DesmantJiMS plenus, 234. Desmidiacea, 17, 157, 160. Development of cell-wall, 168. Dextrine, 80, Dextrose, 77. Diastase, 229. DiatomacecEy Pigments of, 105. Diatomin, 105. Dicranochcele reniformis, 207. Digestive fluids for chromatin, 192. Dioncca, 222,. Diphenylamine as reagent, 50, 69, 83. Discocrystals, 229. Dishes for staining, 24. Double staining, 147. Draccena, 210. Draining boxes for washing, 22. Drosera dichotoma, 223. rotundifolia, 241. Dulcite, 69. Eau de Javelle, for bleaching, 6. for clearing, 10. for reagent, no, 145, 152, 228 233. Ehrlich's solutions, 254. Elaioplasts, 209. Ellagic acid, 86. Ely??t us g iga n teus, 211. Emodin, 87. Emulsin, 136. Eosin, 153, 154, 165, 185, 199, 216, 218, 233, 246. Eosin-haematoxylin, 199. Epiphylluni, 222. Equisettwi arvense, 204. hiemale, 54. Erysipkece, 231. Erythrophilous nuclei, 191. Eternod's apparatus, 25. Ethereal oils, 89. Eugenol, 84. Euglena acus, 230. Ehrenbergii, 230. Spirogyra, 230. Euphorbia, 225. caput-m^dusce, 64. Evonynius japonicus, 69. Exclusion of Bacteria from cult- ure, 4. Eye-spot, 209. Fats and fatty oils, 71. Fehling's solution, 77, 78, 92. Ferments, 136. Ferric acetate, as reagent, 115. chloride, as reagent, 91, 93, 94, 95, 98, 115, 132, 143. Ferrous sulphate, as reagent, 45, 95, 98, 115, 2ig. Fibrosin-bodies, 231. Ficus elastica, 61, 62. Fixing-fiuids, Removal of, 22. Fixing, Methods for, 20, 21, 27. Fixing-methods for cell-contents, 176. Flemming's fixing-fluid, 178. FloridecE, 230 290 INDEX. ./'loridece, Pigments of, 103. Floridean starch, 230. fluids for study of living cells, 4. Fceniculum officitiale, 162. Frangulin, 93. Fuchsin, 147, 188, 191, 203, 204, 258. Fucus, 176. Fundamental mass of protein-grains, 216. Fungus-cellulose, 160, 232. Fungus-gamboge, 91. Fungi, Study of, i, 5. Funkia, 210. Gaertneracea, 210. Galactose, 163. Garlic oil, 81. Gelatinized walls, 154. Gelatinous sheaths of Conjugatce, 157. Gentian violet, 148, 153, 185, 186, 255. Globoids, 219. Glceocapsa, I ID. Gloeocapsin, no. Glucose, 77. Glucosides, 92. Glycerine for clearing, 11. for dehydrating, 13. for mounting, 41. Glycerine and chrome alum for mounting, 41. Glycerine-gelatine for mounting, 41, 42. Glycogen, 80. Gold-chloride, as reagent, 81, 122, 126, 127. Gold-size for sealing, 43. Gonium pectorale 214. Graminea:, 54, 210. Gram-GUnther method, 255, Gram's method for staining, 185, 255. Grana of chromatophores, 204. Granula, 213. Gratiola officinalis, 222. Grenacher's borax-carmine, 182. haematoxylin, 180, Growth of cell-wall, 168. Guiacum officinale, 5 J Gums, 154. Gypsum, 59, 62, 63, 64, 65, 66. Haematein, 181. Haematochrome, 106. Hcematococcus, 106. Haematoxylin, 142, 153, 180, 197^ 208. Hanging drop culture, 2. Hebeclinium 7nacrophyllum, 63. Hedera, 205. Helianthus annuus, 74. Helichrysin, 109. Helichrysiim, 109. Hemicelluloses, 161. HepaticcB, 210. Hesperidin, 93. Higher plants. Study of, 5. Hoffmann's reagent, 130. Hofmann's blue, 224, 247. violet, 245, 247. Humulus, 2^9. Hydrocarbons, 88. Hydrocellulose, 142, Hydrochloric acid, 48. as reagent, 58, 65," 67, 84, 86, 90, no, 113, 121, 126, 127, 137, 194. Specific gravity, 261. Hydrofluoric acid, as reagent, 53, 55. Hydrogen peroxide, 45, 117, 178. Hydrolysis, 139, 162, 163. Hydroxylamine, as reagent, 147, Imbedded objects. Attachment to carrier, 35. Imbedding in celloidin, 35. paraffine, 31. Impatiens Balsamina, 156. parvijlora, 148. Inclusions of chromatophores, 204. of nucleus, 194. India-ink, Use of, 158. Indol, as reagent, 145. Inorganic Compounds, 44. Intercellular substance, 166. INDEX. 291 Inulin, 78. Invert-sugar, 78. Iodine, as reagent, 155, 227. for fixing, 27, 176. for removing sublimate, 179. Iodine in sea-water for fixing, 212. Iodine and potassium iodide for fix- ing, 214, 224. as reagent, 80, 102, 103, 106, 120, 122, 123, 128, 129, 185, 186, 233. and sulphuric acid, as reagent, no, 139, 140, 143. Iodine-calcium chloride, as reagent, 141. Iodine-green, 188. 202. Iodine-phosphoric acid, as reagent, 141. Iridescent plates of Algae, 212. Iridous chloride, as reagent, 124. Iron, 68. Isotonic coefficient, 238, 263. Javelle water, see Eau de Javelle. Juglans regia, 87. Juglon, 87. Karyokinetic figures, 185, 187, 192, Kinoplasm, 194. Lactic acid for dried plants, 5. for fixing, 252. Lamellation of wall, 170, Lathrea squamaria, 225. Lead acetate as reagent, 93, 94, 98, 107, 109. LeguminoscE , 162. Lenzites sepiaria, 92. Lepidium, 169. Lepra- Bacillus, 256. Leptoniihts lacteus, 23 1. Leptophrys vorax, 230. Leptothrix ochracea, 69. Leucin, 82. Leucoplasts, 201. Leucosomes, 205. Lichen-pigments, no, in. Life reagent, 244. Lignic acids, 144. Lignified walls, 143. Rea(!tions of, 145. Lignin, 143. Liliutn Martagon, ig8, 199. Lime water, as reagent, 87, 88, 93, 96, 113. Linin, 135, 136. Lipochromes, 106. Lipocyanin, 106. Lithospermum, 164. Live-staining, 119, 189, 224, 235. Living Bacteria, Study of, 250. Living tissues, Staining, 19, 119. Loeffler's blue stain, 253. Loevv-Bokorny reagent, 244. Lophospermum scandenSy 195. Lupinus luteus, 162. Maceration, 6, Madder dye, 95. Magnesium, 67, oxalate, 67. phosphate, 67. sulphate, 52, 65. Mandelin's reaction, 97. Mannose, 162. Marattiacece, 63. Masked iron, 68. Maskenlack for sealing, 43. Mass-staining, 182. Measures of capacity, 259. length, 259. Melampyrite, 69. Melampyrum arvense, 194. Membranes of Bacteria, 161. Mercuric chloride, as reagent, 122, 128. iodide, 57. for swelling, 8. nitrate, as reagent, 129. Mesocarpus, 234. Metadiamidobenzol, as reagent, 86, 157. Metaxin, 135, 136. Methylal, as reagent, 124. 29^ INDEX. Methyl alchol, 171. blue, as stain, 142, 153, 188, 190. as reagent, 1 19. Methylene blue, 166, 168, 173, 191, 192, 224, 235, 248, 256. Loeffler's, 253. Methyl green, 188, 190. orange, as reagent, 236, 238. Methyl violet, 1S9, 247, 254. Micrasterias rotata, 62. Microcosmic salt, as reagent, 67. Microsomes, 212. Microtome knife, 31. Microtomes, 29, 30. Microtome technique, 29. Middle lamella, 6, 167. Millon's reagent, 85, 129, 137. Mimosa pudica, Glucoside from, 98. Mimulus Tillingi, 195. Minute structure of cell-wall, 170. Moist chamber, 2. Mordant for cilia of Bacteria, 258. spores of Bacteria, 257. Morphine, 123. Mounting delicate objects, 15. in air, 43. in balsam, 16. with alcohol, 12. drying, 17. phenol, 17. Mucus-globules, 232. Musa paradisiaca, 58. Mustard oils, 81. Myrosin, 81, 137. a-Naphtol, as reagent, 76, 79, 145. Narcelne, 124. Narcotine, 124. Neottia nidus-avis^ 204. Nephroma lusitanica, 87. Nerium, 171, 172, 173. Nessler's reagent, 57. Nickel sulphate, as reagent, 50. Nicotine, 128. Nigrosin, 189. Nitella, 190, 224, 225. Nitric acid, 50. for fixing, 213. for maceration,6. for reagent, 52, 54, 65, 85, 86, 96, 103, 112, 113, 121, 122, 129, 156. Specific gravity, 261. Nostoc, 233. Nucin, 87. Nuclear divisions, Fluid for, 4. Nuclear membrane, 192, Nucleic acids, 133. Nuclein, 234. — '- Artificial, 134. Nucleins, 133. Nucleolus, 191. Nucleus, Constituents of, 175. Inclusions of, 194. Oil-bodies, 210. Oil-drops, 208. Oil-formers, 2og, Oil of bergamot for clearing, 39. Oils, Ethereal, 89. To distinguish, 90. Fatty, 71. for clearing, 14. Ononis spiuosa, 90. Opium alkaloids, 123. Oplismenus imbecillus, 21 1. Orange, stain, 186. Orcin, as reagent, 79, 84, 86, 136, 137. 145, 157- Organic compounds, 69. Ornithogalum, 2 ID. Orseillin, 199. Oscillatoria, 104. Osmic acid, as reagent, 27, 72, 90, 117, 152, 178, 211, 212, 213, 2I4» 215, 219. 239. Overstaining, 26. Oxalic acid, 62, 70. Oxalic acid for maceration, 6. Oxygen, 44. Oxynaphthoquinone, 87. INDEX, ^93 Pceonia, 156, 217, 219. Fallacious chloride, as reagent, 124. nitrate, as reagent, 81. Pancreatin, as reagent, 133. Pandorina, 215. PanicecBy 67. Papaver somniferum, 123. Paracarmine, 183. Paraffine blocks, To attach, 35. To preserve, 35. for imbedding, 32. oven, 34. Paragalactan, 162. Paragalactan-like substances, 161. Paralinin, 135, 136. Paramylum, 230. Paris quadrifolia, 162. Paspalum elegans, 82. Passijlora ccerulea, ill. Paxilhis atrotomentosus, Pigment of, 113- Pectic acid, 167. substances, 166. Pellicle of protein grains, 217. Penicillium, 48. Pepsin, as reagent, 133, 134, 252. Peridinecc, Pigment of, 105. Peridinin, 105. Permanent preparations, 40. PeronosporecE, 164. Peziza, 187. Phacus parvula, 230. PhcEophycecB, 2y:i. Pigments of, 104. Phaeophycean starch, 230. Phellonic acid, 149. Phenol, for clearing, 10, 60. for dehydrating, 17. for reagent, 71, i45i 146. Phenols, 84. Phenosafranin, 166, 168. Phloicnic acid, 149. Phloridzin, 95. Phloroglucin, 84, 119. as reagent, 80, 86, 144, 145. I47» 157. Phoenix dactylifera, 162. Phosphoric acid. 52. Phospho-molybdic acid, as reagent^ 119, 120, 123. -tungstic acid, as reagent, 128. Photophore, 25, Photoxylin for imbedding, 37. Phycocyanin, 105. Phycoerythrin, 104. Phycophsein, 104. Phycopyrrin, 105. Pkyllosiphonacea, 231. Physcia parietina, 88. Physodes, 214. Phytelephas, 162, 174. Phytophysa Treubii, 231. Picric acid, for differentiating, i82> 188. for fixing, 177, 189, 207» 210, 216, 235. for reagent, 123. Picrocarmine, 183, 255. Picro-nigrosin, 189, 216. Picro-sulphuric acid, 177, 235. Pigments, 100. dissolved in cell-sap, 107. in oils, 107. Fatty, 106. in the cell-wall, 108, 109. of Aloe flowers, 103. Chromatophores, 100. CyanophycecE, 104. DiatomacecE y 105. FloridecE, 103. lichens, no, in. Peridinece, 105. • Pha:ophyce(E, 104. on the cell-wall, 112. Piperacea, 124. Piperine, 124. Pirus Mains, 95, Pistia Stratiotcs, 235. Plant-mucilages, 154. Plasma-membranes, 238. Plasmolysis, 238, 243. Plastin, 134. Plastoids, 223, Platinum chloride, for fixing, 180. 294 INDEX. Platinum chloride, for reagent, 56, 81, 128. -osmic-acetic acid, for fix- ing, 180. Pleochroism, 204. Pleurotccnium Trahecula, 159. Plugge's reagent, 130. Podocarpus elongatus, 173. Podosphara Oxyacanthce, 231, Polarized light, 60, 65, 73, 88, 220, 226. Polygonacea, 88. Polypodiiim irreoides, 222, 240. Polyporus hispidus, 91. Potamogeton, ll\. Potassic-mercuric chloride, as re- agent, 122. iodide, as reagent, 122, 123, 12S. Potassium, 56. acetate, as reagent, 70. bichromate for antiseptic, 4. for differentiating, 1S2, 192, 197. for reagent, 116, 123, 219. -bismuth iodide, as reagent, 123. -calcium iodide, as reagent, 123. carbonate, as reagent, 86. caryophyllate, 84. chlorate for maceration, 6. chromate, 4, 122. ferrocyanide, as reagent 68, 132, 135, 172, igo, 192. hydrate. See Caustic potash. iodide, 45, 57. myronate, 81, 95. nitrate, 51. as reagent. See Salt- peter. oxalate. Acid, as reagent, 70. permanganate for differentiat- ing, 200. platinum chloride, 56. sulphate, 49, 50. sulphocyanide, as reagent, 68, 123. Pritnulacea, 156, Protelds, 128. Proteids, Reactions of, 129. Protein crystalloids, 194, 205, 217, 222. grains, 215. Proteosomes, 244. j Protoplasm and cell-sap, 174. Protoplasm, Reactions of, 237. Protoplasmic Connections, 245. Prunus Lauro-cerasus, 136. Pulmonaria officinalis, 236. Pulverization methods, 174. Pyrenin, 135, 136. Pyrenoids, 206. Pyroligneous acid, 180. Quercus Suber, 149. Quinones, 87. Raphides, 58, 60. Raspail's reagent, 138, 224. Reactions of cell-sap, 236. of protoplasm, 237 Reagent-bottles, 1=;. Peniijia purdieana, 51. Removing air from tissues, 5. Replacement of alcohol, 14. Resedacecc, 137. Reserve-cellulose, 162. Resin, 211. Resins, 90. Resorcin, as reagent, 86, 145. Resting nucleus. Recognition of, 190. Retinic acids, 91, 92 Rhabdoids, 223. Rhamnus frangula, 87, 93. Rhodospermin, 223. Riciitus, 117, 118, 130, 216, 218, 219. Rochelle salt, as reagent, 77. Ruberythric acid, 95. Ruhiacea, 210, Rubia iinciorutn, 95. Rutin, 96. Saccharose, 78. Saffron-yellow, 96. Safranin, 148, 152, 185, 186, 207. Salicin, 96. Salicylic aldehyde, 146. INDEX. 29s Salicylic aldehyde for fixing, 202. for reagent, 131, 132. Salt, 238. Saltpeter, 5, 240, 263. Sapindacece, 210. Saponaria officinalis, 230. Saponification of fats, 73. Saponin, 96. SapotacecE, 210. Sap rolegn ia cecE, 231. Schulze's dehydrating vessel, 12. macerating mixture, 6, 151. settling cylinder, 16, Schweizer's reagent, 140. Sculpturing of wall, 170. Scytonefna, no, 233. Scytonemin, no. Sealing media, 43. Sec ale cereale, 148. Section-finder, 25. Selenic acid, as reagent, 122. Seminin, 162. Seminose, 162. Setaria viridis, 67. Settling cylinder, Schulze's, 16. Sieve-plates, 246, 248. Sieve-tubes, Callus of, 163, 165. Contents of, 248. Silica skeletons, 54, 168. Silicic acid, 53. Silvering, 173, 226. Silver nitrate, as reagent, 48, 81, 85, 173. Silver-solution, 244. Sinapine, 125. Skatol, as reagent, 145. Small objects. Fixing and Staining,27. Smilax, 225. Soda solution, 165. Sodium, 56. carbonate, as reagent, 113. carminate, 183. hydrate, see Caustic soda. metatungstate, as reagent, 122. phosphate, as reagent, 67, 217, 218, 220. selenate, as reagent, 97, 124. 235. Sodium silico-fluoride, 55. tungstate, as reagent, 117. uranyl acetate, 56. Solanin, 97. Solanum tuberosuju, 202, 222. Soluble blue extra 6B, 165. Soluble starch, 229. Solutions, Percentage Composition^ 260. Preparation of, 261. Solvents for fats, 71. Sorbus aucupaHa, 204. Specific gravity of solutions, 260. Spergula vulgaris, 99. Spergulin, 99. Sphaerocrystals, 63, 64, 65, 73, 74,. 79, 82, 85, 93. Spirogyra, 18, 45, 115. 118, 131, 132,. 206, 238, 240. Spores of Bacteria, Staining, 257. Staining attached sections, 38. in mass, 24, 182. intra vitam, 119, 189, 224, living tissues, 19. Methods for, 20, 24, 27. methods for cell-contents, sections, 25. Stains for cellulose, ^42, 143. cuticle and cork, 152, lignified walls, 147, 148. Stapelia picta, 52, 53, 64. Starch-grains, 225. Medium for, 19. Starch, Recognition of, 227. Starch skeletons, 229. Staurastrum bicorne, 159. Stender dishes, 24. Stratification of cell-wall, 170. of starch-grains, 226. Striation of cell-wall, 170. Strontium nitrate, as reagent. 49. Strychnine, 125. Strychnos nux-vomica, 122, 126. Suberic acid, 149. Suberin, 148, 150. Suberized membranes, 75, 148, 150, 152. [80. 154. 296 INDEX. Sulphur, 47. compounds, 81. Sulphuric acid, 49. for maceration, 7. for reagent, 54, 59, 62, 64, 65, 66, 67, 68, 70, 79, 81, 84, 86, 88, 91, 92, 96, 97, 98, 99, 102, 103, 105, io6, 107, 112, 113, 120, 122, 124, 127, 130, 140, 164, 246. for swelling, 8. Specific gravity, 261. Sulphurous acid for washing, 22, 177. Swelling, 8. Syringa vulgaris, 98. Syringin, 98. Tables for reference, 259. ' Tannic acid, 242. Tannin, as reagent, 45, 143. Tannins, 114, 242. Tannin-vesicles, 234. Tartaric acid, as reagent, 70, 119. Terpenes, 90. Thallin sulphate, as reagent, 49, 145, 146. Theine, 127. Thelephora, 91, 112. Thelephoric acid, 112. Theobromine, 126. Thermometer scales, 260. Tholuidendiamine, as reagent, 145. Thymol, as reagent, 76, 79, 86, 145, 157. Titanic acid, as reagent, 124, Tradescantia, 200. albijlora, 214. discolor, 58, 205, 206, 239, 243. virginica, 46, 189. Trametes cinnabarina, 91, 113. Trianea bogotensis, 46. Tropaolacea, 137, Tropceolitm ma jus, 156. Trypsin, 192. Tubercle-bacilli, Staining, 256. Tulipa suaveolens, 242. TurnbuU's blue 169. Tyrosin, 85. Unequal water-content of cell-walls, 171. Unverdorben - Franchimont reac- tion, 90. Uranyl-acetate, as reagent, 56, 67, 70. Uranyl-magnesium acetate, as re- agent, 56. Urceolaria ocellata, 112. Urceolaria-red, 112. Urticales, 164. Vanda furva, 222. Vanilla, 86. planifolia, 209. Vanillin, 86, 144, 146. as reagent, 84, 131. Venetian turpentine for mounting, 18. Veratrine, 127. Veratrum album, 127. Vessel for staining sections, 26. Vesuvin, 165. Vicia Faba, 46, 187. Vinca, 172, 173. ViolacecE, 137. Vitis Labrusca, 63. vinifera, 220, 222. Washing, 22, 27. apparatus, 23, 27. Wax, 74. Wax feet for cover-glass, i. Weights, Table of, 260. Willows, 96. Wood-gum, 144. Wound-gum, 157. Xanthein, 107. Xanthin, 103, 106, 209. Xanthine, 128. Xantho-proteic acid, 129. Xanthotrametin, 113, Xylol for clearing, 14. for imbedding, 33. Zinc chlorine, as reagent, 231. sulphate, as mordant, 199, Zygnema, 115, 176, 206, 235. Zygnemacece, 157, 158, 234. 152808 /^