Te inivosi of Tonto bray Wiliam Lash Miller RARDORE A Departnental Library lobeunder scl outinrlatlensfeon the Lbpodiag 3 pthe University dated Marc Af IGIE. are “3 en) aoe Rak DOt BOTANICAL MICRO-CHEMISTRY AN INTRODUCTION TO THE STUDY OF VEGETABLE HISTOLOGY | i / PREPARED FOR THE USE OF STUDENTS 199° per : ‘ VA, POULSEN ut TRANSLATED WITH THE ASSISTANCE OF THE AUTHOR AND CONSIDERABLY ENLARGED BY WILLIAM TRELEASE PROFESSOR IN THE UNIVERSITY OF WISCONSIN Boston S. E. CASSINO AND COMPANY 1884 Se Copyright, Hane « «BY S. E. CASSINO &.CO. © ates . 1883. TO flv Esteemed Teacher, DOCENT EUG. WARMING, Ph.D. AND ; flv Respected Friend, PROFESSOR LEOPOLD KNY, THIS BOOK IS DEDICATED IN THANKFULNESS BY THE AUTHOR, PREFACE TO THE AMERICAN EDITION. ‘Tue translation of the Danish original into German, Italian, and French, and now into Eng-— lish, is an honor not dreamed of when the original . edition was published. I wish to cordially thank all of my collaborators, especially my friends Carl Miiller of Berlin, and Prof. Aser Poli of Melfi, and my honorable col- leagues Dr. Lachmann of Lyon, and Prof. Trelease of Madison, who have not merely enlarged the book, but enriched it with many notes and para- graphs of great value. Once more I wish to express the hope that, in the new shape in whith it appears, my book may - be of use to the students of vegetable histology, for whom it was written. V. A. PouLseEn. ROSENVAENGETS HOVEDVEJ, COPENHAGEN, June 1883. CONTENTS. Pace PREFACE TO AMERICAN EDITION : ‘ : 3 é vii PPeRNSEATORS PRRPACH — ote) eth ee ae ix AUTHOR’S PREFACE : rs é F 2 é > - : x INTRODUCTION ‘ ‘ % z F . e ° . p xi LITERATURE OF BOTANICAL MICRO-CHEMISTRY c ‘. ‘ xv Bart E. MiIcro-CHEMICAL REAGENTS AND THEIR APPLICATION . 2 1 APPENDIX. MOUNTING MEDIA... Sn eats hele Mele? oa Sees seater CEMENTS . Paar eh e Zz : F = - 4 71 Bart IE, VEGETABLE SUBSTANCES AND THE MEANS OF RECOGNIZING THEM . ° ° aeons . ; ‘ . ‘ 75 INDEX . : ° Pherae . . ‘ : . oe = SEO SPECIAL REAGENTS, REACTIONS, AND METHODS. Barcianu’s mucus reaction Barfoed’s glucose test. . . Beale’s carmine . .. . De Bary’s epiplasm reaction Franchimont’s (Unverdorben’s) ESI LOSES M5' a a ese, Ue Grenacher’s alum-carmine . Hanstein’s method of clearing SIBMUIOS oor Sire tee Viet Hanstein’s aniline violet . Hanstein’s metaplasm reaction . Hohnel’s ceric acidtest . . Hohnel’s xylofilin reaction Maupas’ nucleus staining . Millon’s reagent. . . « « PAGE Pringsheim’s hypochlorin re- ACHR N= ec See eae Ranvier’s picro-carmine . . . 53 Raspail’s protoplasm test . . 44 Russow’s potash alcohol. . . 12 Sachs’ reaction for: starch in chlorophyll bodies . . . . 7 Schultze maceration fluid. . . 34 Stromeyer’s starch reaction . . 4 Treub’s method of clearing issues». 56. 04253 es eis Ee Trommer’s sugar test. - » - 35 Unverdorben’s (Franchimont’s) Tesm test <5: 0, Sask eee Wiesner’s lignin test . . . . 46 TRANSLATOR’S PREFACE. WHILE studying in the laboratory of Dr. Far- low, at Harvard University, in 1881, I first be- came acquainted with the German translation of Poulsen’s little treatise, which made its appear- ance in this country about that time. It was proved so valuable by every-day use in the labora- tory, both there and with my classes in the Uni- versity of Wisconsin, that I began a translation of _ the most frequently used parts, having in mind at . first only the convenience of my own students. But several requests having been made that I should complete the translation and publish it, I decided to do so, having received an offer of addi- tional notes from the author. Various causes, how- ever, have delayed my work, so that it is but now, above a year since it was begun, that it is completed. It should be said that the translation is in no sense a literal one. The obvious meaning of the author has been given, but with no effort to pre- serve the idiom or vocabulary, and the usual Eng- lish terminology has commonly been employed. In presenting my translation to American teach- ers of vegetable histology, I feel confident that it will meet the every-day wants of the laboratory. Wma. TRELEASE. MapiIson, Wis., April 12, 1883. AUTHOR’S PREFACE. Tus little attempt at a comprehensive pre- sentation of the more important micro-chemical reagents and methods is the first which has appeared in the Danish language. It has been undertaken on the recommendation of Dr. Eug. Warming, who has often felt the need of a com- pact guide to micro-chemistry in his work with students of vegetable anatomy at the Botanic Garden. To him I am indebted for many valuable suggestions, which I find it a pleasant duty to acknowledge here. I am also indebted to Mr. R. Pedersen, Docent in Vegetable Physiology at the Copenhagen Uni- versity, for many criticisms. Should those inexperienced in microscopy draw even a little useful information from this treatise I shall have accomplished what was aimed at. V. A. PouLsEn. ROSENVAENGET, October 1880. INTRODUCTION. RECENTLY microscopic technology has risen to an importance undreamed of in its early days. The perfection of the microscope, by whose help so many beautiful and important discoveries are made, has given us such an insight into the nat- ural history of the cell as to stimulate the study of elementary organs, so far as possible, with all available aids. The spectroscope, the polar- iscope, and the induction coil, placed in our hands by Bunsen, Huygens, and Faraday, are applicable and must augment the value of the instrument first given us by Hans and Zacharias Janssen; and even photography has of late been employed. Physics has thus striven to bring this instrument to as great a degree of perfection as possible; it remains for chemistry to find means of recog- nizing and rightly understanding the composition of the objects we investigate. In other words, if we employ a thorough system of chemical analysis with the optical apparatus we shall be able to answer all questions lying within the range of pos- sibility.. It is this analysis, applied to objects xi Xil INTRODUCTION. under the microscope, that we designate by the word micro-chemistry. I have endeavored to successively make the reader acquainted with the most valuable reagents used in micro-chemistry, z. ¢. with those sub- stances whose action on the bodies to be studied allows their chemical composition and nature, and sometimes their physical structure, to be recognized. In the first section I have consid- ered the chemicals used in the laboratory; in the second, the vegetable substances to be tested for and the reactions by which they are known. The correctness of the statements which follow rests in part on the long experience of my re- spected teacher, Dr. Warming, and in part on my own. I have also been able to profit by the practice and teaching of Professor Hanstein, of Bonn, made known to me by Dr. Warming. Finally, the scattered experiments and methods, recorded in a large and scattered literature, have been used so far as possible,—far be it from me, however, to suppose that I have exhausted the literature ; nor have I tried to take up all of the chemicals that have been used, my endeavor being to collect only such as are most useful. At the close of the first section I have intro- duced a short chapter on media for the preserva- tion of permanent preparations; to which are added a few words on the cements used in mounting. -_ee ‘ a . oF eres ee ee ee en a ee oe. Us ee INTRODUCTION. xiii Pleasant as it would have been to add a histori- cal outline of the development of micro-chemistry, I have thought best not to do so, partly that the book might be kept within proper limits as to size, partly because that treatment of the subject would more properly find place in a theoretical and comprehensive text-book than in a compact guide for purely practical use. LIST OF THE MORE IMPORTANT PUBLICA- TIONS WHICH HAVE BEEN USED IN THE. PREPARATION OF THIS BOOK. Almquist : Metoder att odla och farga Bakterier. — Hygiea, 1883, Stockholm. Bachmann: Leitfaden zur Anfertigung mikroskopischer Dauerpriparate, Miinchen, 1879. Bary, De: Morphologie und Physiologie der Pilze, Flechten und Myxomy- ceten. — Hofmeister’s Handbuch, Bd. II.,.Abth. 1 ; Vergleichende Anato- mie.— Hofmeister’s Handbuch, Bd. III., 1877. Behrens: Die Nectarien der Bliithen.— Flora, 1879; Hilfsbuch f. mikro- skop. Untersuchungen, 1883. Berg: Zur Kenntniss des in der Cetraria islandica vorkommenden Lichenins und jodblauenden Stoffes, Dorpat, 1872. Bonnier: Les Nectaires.— Ann. Sci. Nat., Bot., 1879, 6 Sér., Tome VIII. Burgerstein: Sitzungsber. wien. Akad., 1874, Bd. LXX., p. 338. Cario: Tristicha hypnoides. — Bot. Zeitung, 1881, p. 31. Dippel: Das Mikroskop, I. and II., 1869; Die neuere Theorie iiber die feinere Struktur der Zellhiille, 1878. Eriksson: Om Meristemet i dicotyla vaxters rétter.— Lunds Universitets arsskrift, 1877, XIII., p. 10. ; Errera: L’épiplasme des Ascomycétes et le glycogéne des Végétaux, Brux- elles, 1882; Glycogéne des Mucorinées. — Bull. Acad. de Belgique, 1882. Flemming: Ueb. das Hermannsche Kernfarbungsverfahren. — Archiv. f. mikr. Anat., 1881, XTX. Fritsche: Ueber den Pollen.— Mem. Acad. des Sci. de St. Pétersbourg, 1837. Hanstein: Die Milchsaftgefiisse und verwandte Organe der Rinde, 1864; Die Scheitelzellgruppe im Vegetationspunkt der Phanerogamen, 1868 ; Organe der Harz-und Schleimabsonderung in den Laubknospen. — Bot. Zeitung, 1868. Hartig: Bot. Zeitung, 1856, p. 262; Entwickelungsgeschichte des Pflan- zenkeimes, Leipzig, 1858; Der Fiillkern, etc. — Karsten’s bot. Unter- suchungen, 1867, I. Xvl LIST OF BOOKS USED. Hegelmaier: Bau und Entwickelung einiger Cuticulargebilde.— Jahrb. wiss. Bot., IX.; Vergl. Untersuch. iiber Entwick. dikot. Keime, 1878, p. If. Higley: Sur acide phosphorique et les phosphates. — Bot. Centralblatt, 1881, No. 27. Hofmeister: Die Pflanzenzelle.— Handbuch der physiol. Bot., 1867, , Bd. I.; Handbuch, Bd. Il. and III. — See De Bary. Hohnel, Von: Ueber Kork und verkorkte Gewebe iiberhaupt. —Sitzungs- ber, wien. Akad., 1877, Bd. LXXVL, Abth. 1. Johow: Zellkerne der héheren Monocotylen., Diss., Bonn, 1880. Kabsch: Chemische Beschaffenheit der Pflanzengewebe.—Jahrb. wiss. Bot., 1863, II., p. 357. Kaiser: Zcitschrift fiir Mikroskopie, 1877-78, Bd. I. Karsten: Gesammelte Beitrage zur Anatomie und Physiologie der Pflan- zen, 1865, L, p. 253. Koch: Verfahren zum Conserviren und Photographiren der Bacterien.— Cohn’s Beitrage zur Biol. der Pflanzen, II., p. 399. Maschke: Pigmentlésung als Reagenz bei mikroskopish-physiol. Unter- suchungen. — Bot. Zeitung, 1859, p. 51. Meyen: Anatom.-physiol. Untersuchungen iiber den Inhalt der Pflanzen- zellen, Berlin, 1828. Mohl, Von: Vermischte Schriften, bot. Inhalts, Tiibingen, 1845; Aufbe- wahrung micr. Praparate. — Bot. Zeitung, 1857. Miiller, N. J. C.: Untersuchungen iiber d. VertheiJung der Harz etc. im Pflanzenkérper. — Jahrb. wiss. Bot., V., p. 397; Handbuch der Botanik, I.: Allgern. Botanik, Erster Theil: Anatomie und Physiol. der Ge- wichse, Heidelberg, 1880. Nageli: Die Starkek6rner, Ziirich, 1858. ; ‘ Nageli and Schwendener: Das Mikroskop, 1877, 2d ed. Planeth: Mikrochemische Analyse der vegetabilischen Zelle, — Promo- tionsschrift an der Univers. Rostock, 1873. Pfeffer: Die Proteinkérner. — Jahrb. wiss. Bot., 1872, VIII. : Pfitzer: Die Bacillariaceen. — Hanstein’s bot. ’Abhandl., 1871, L., Heft 2. Pringsheim: Untersuchungen iiber das Chlorophyll, IV., Das Hypo- chlorin. — Monatsber. ber]. Akad., Nov. 1879. Radlkofer: Krystalle Proteinartige K6rper, 1859. Ranvier: Traité technique d’ histologie, Paris, 1877. Robin: Traité du microscope, Paris, 1877. Russow: Vergl. Unters. iiber Leitbiindel-Kryptogamen. — Mem. Acad. des Sci. de St. Pétersbourg, 1872, 7 Sér., Tome XIX., No. 1; Zur Kennt- niss des Holzes, insonderheit des Coniferenholzes. — Bot. Centralbl., 1883, No. 1. A el 2 ~ ae “ahs “ ere ‘ hy ad ye \ OE a eS ee ee, eee ee ee LIST OF BOOKS USED. xvii Sachs: Keimung der Griaser; Keim der Dattel. —Bot. Zeitung, 1862; Ueber die Stoffe, welche das Material zum Wachsthum der Zellhaut liefern. — Jahrb. fiir wiss. Bot., 1863, III.; Lehrbuch der Botanik, 4th ed,, 1874; Ueber einige seue mikroskopisch-chemische Reaktionsmeth- oden. —Sitzb. wien. Akad., 1859 ; Keimung der Schminkbohne. — Ibid. Sachsse: Chemie und Physiol. der Farbstoffe, Kohlenhydrate und Protein- substanzen, Leipzig, 1877. Sanio- Various papers. — Bot. Zeitung, 1860, 1863; Anatomie der Kiefer. — Jahrb. fir wiss. Bot., 1873-74, IX. Schacht: Die Piiannselle: 1852; Das Mikroskop, 1855} Anatomie oh Phys. der Gewiichse, 1856, I.; Ueber den Bau einiger Pollenkérner. — , Jahrb. fiir wiss. Bot., 1860, II., p. 109. Schimper, A,: Lintinmibetae iiber Protein-krystalloide der Pflanzen, Strassburg, 1879. Schmitz: Becbachtungen iiber die vielkernigen Zellen der Siphonocladia- ceen, Halle, 1879. Strasburger: Befruchtung und Zelltheilung, 1878; > Studien iiber Proto- plasma, 1876; Zellbildung und Zelltheilung, Jena, 1875; 3d ed., 1880. Tangl: Das Protoplasma der Erbse. — Sitzungsber. der wien. Akad. 877- 78, Bd. XXVI. and XXVIII. ; Communication zwischen der Zellen des Endosperms. — Jahrb. wiss. Bot. , 1880, XII.; Beitrage z. Mikrochemie d. Pflanzenzellen. — Sitzb. wien. ’Akad., 1876, Abth. 1 Treub: Méristéme primitif de la racine dans les Monocotylédones, Leide, 1876; Sur le réle du noyau dans la division des cellules, 1878 ; Sur des cellules végétales 4 plusieurs noyaux. — Archives Néerlandaises, T. XV. — Separate, p. 15. Vogl: Anatomie und Histologie der unterirdischen Theile von Convol- vulus arvensis. — Sitzungsber. wien. Akad., 1863, Bd. XIII. p. 257; Bau des Holzes von Ferreira spectabilis.—Jahrb. fiir wiss. Bot., 1873- 74, IX. Vries, De: Keimungsgeschichte des rothen Klees. — Landwirthschaftliche Jahrbiicher, 1877, Bd. VI. Weiss: Die Pflanzenhaare.— Karsten’s bot. Untersuchungen, 1867, Allgemeine Botanik, 1878, Bd. I. Wiesner: Technische Mikroskopie, Wien, 1867; Anatomisches und Histochemisches tiber das Zuckerrohr.—Karsten’s bot. Untersuch., 1867, I.; Das Verhalten des Phloroglucins und eniger verwandter Korper zur Zellmembran.— Sitzungsber. wien. Akad., 1878, Abth. 1. Wigand: Intercellularsubstanz und Cuticula, 1850, Ex XV LIST OF BOOKS USED. LITERATURE OF THE COLORING MATTERS FOUND IN PLANTS. (ExcLUSIVE OF PAPERS ON SPECTROSCOPIC AND OTHER OpTicaAL TESTS.) Askenasy: Bot. Zeitung, 1867, p. 227; 1869, p. 785. Brown, R.: Manual of Botany, p. 589. Cohn: Bot. Zeitung, 1867, p. 38. Frémy: Annales des Sci. Nat., Bot., 1860, T. XIII. Hildebrandt: Die Farben der Bliithen. — Jahrb. fiir wiss. Bot., Ti. ' Kiitzing: Phycologia generalis, p. 17, e¢ seg. Millardet: Comptes rend., 1869. (Cf. Bot. Zeitung, 1869, p. 332.) Miiller, N. J. C.: Handb. d. Bot., Bd. I, Allgem. Bot., Theil 1, 1880, p-. 562. Nageli and Schwendener: Das Mikroskop, 1877, p. 528. Prantl: Bot. Zeitung, 1871, p. 619. Pringsheim: Monatsber. berlin. Akad., October 1874; December 1875. Rosanoff: Mém. de la Soc. de Cherbourg, 1867, XIII. ; met Zeitung, 1866, p. 182. Sachsse: Die Farbstoffe, Kohlenhydrate und Proteinsubstanzen, Leipzig, 1877; Die Chromatophoren der Algen, Bonn, 1882. Timirjaseff : Das Chlorophyll, 1871. Trécul: Annales des Sci. Nat., Bot., 1858, 4 Sér., T. X. Weiss: Allgemeine Bot., 1878, I., p. 106-138. Wiesner: Bot. Zeitung, 1862; Flora, 1874. Besides the works cited here, others, which have been used occasionally, are mentioned in various places in the text. ParT I. MICRO-CHEMICAL REAGENTS AND THEIR APPLICATION. MICRO-CHEMICAL REAGENTS. IODINE. Tuts is one of the most valuable and indis- _pensable substances used in. microscopic botany. It is employed in solution, either in water,’ alcohol, or a solution of potassic iodide in glycerine, chlor- iodide of zinc, or water. The degree of concentra- tion of the solution depends upon the particular case in which it is to be used, the effects of much- diluted solutions being often very evident. For the recognition of starch, iodine is a very con- venient reagent, and the only certain one known. A preparation which contains the minimum of free iodine suffices to reveal this substance, though only in case the starch contains water. With an aque- 1 Nageli: Das Mikroskop, 1877, p. 473. Dippel: Das Mikroskop, 1872, p. 273. Sachs: Jahrbiicher fiir wiss. Bot., 1863, III. Mohl: Ver- mischte Schriften, 1845, p. 335- Hofmeister: Handbuch, I, Pflanzenzelle, 1867, pp. 252, 387. Weiss: Allgemeine Botanik, 1878, I., pp. 18, 77, 159. Schleiden : Wiegmann’s Archiv., 1838, pp. 39, 59. Raspail. Développement et Analyse microscopique dela Fécule.— Ann. Sci Nat, 1 Sér., T. VI, p. 388. Meyen: Untersuchungen iiber den Inhalt der Pflanzenzellen, Ber- lin, 1828, p. 21. Sachs: Lehrbuch der Botanik, 1874, pp 34, 40, 58. - er ee es se BES 4 MICRO-CHEMICAL REAGENTS. ous solution, this condition is fulfilled ; but in case a solution in absolute alcohol is used, water must be added to the preparation. The well-known reaction, discovered by Stro- meyer, is as follows:* According to the strength of the solution and the duration of its action, the starch grains assume a more or less deep-blue color, which vanishes when the preparation is warmed, but reappears when it again becomes cool, and is entirely destroyed by the action of hyposulphite of sodium. Dry starch slowly turns brown when treated with a solution of iodine in chloroform or absolute alcohol, which renders it a good test for the presence of water in alcohol. According to Nageli, the starch grains are com- posed of granulose diffused through a skeleton of starch-cellulose. The former, only, is turned blue by iodine, for after its removal (by digestion with saliva at 45-55° C., or the action of pepsin, dias- tase, organic acids, or the prolonged action of dilute sulphuric or hydrochloric acid) the grains turn yel- low or brown. Most cellulose membranes color yellow or brownish with iodine, especially when a freshly- made solution is employed. An exception, how- ever, is afforded by the paraphyses and asci, —the hymenium of lichens, which often turn blue, like starch. If, however, the cell-wall has been first 1 Nageli: Die Starkekérner, Ziirich, 1858. Wiesner: Technische Mikroskopie, 1857, p. 73- IODINE. 5 treated with strong sulphuric or phosphoric acid, or with an aqueous solution of zinc chloride, it colors blue when iodine is added, these reagents convert- ing the cellulose into amyloid,' a carbo-hydrate related to starch. Hydriodic acid also acts in the same way, and hence old solutions of iodine some- times color the cell-wall blue, when this acid has formed through the action of the water or alcohol — of the solution. Cellulose membranes that have been allowed to dry after treatment with a solu- tion of iodine, and are again moistened with water, often turn blue, the organic matter having in- ‘duced the formatién of hydriodic acid. The epi- dermal cell-walls of many seeds and pericarps that © swell into mucilaginous masses when moistened, as- sume a blue color with iodine-water only after the swelling has reached a certain stage. Not unfre- quently a membrane lying in an iodine solution passes through several shades of color in the course of a longer or shorter time. Most reagents that cause cellulose to turn blue when used in combina- tion with iodine, also cause it to swell, approxi- mately, in the following ascending order: Potassic ‘iodide, iodide of zinc, nitric acid, phosphoric acid, potassic hydrate, hydriodic acid, and sulphuric acid. We have thus an excellent test for pure cellu- lose in iodine and any amyloid-producing reagent. 1 Not to be confounded with the substance known to animal histolo- gists by the same name. 6 MICRO-CHEMICAL REAGENTS, If, however, a membrane does not color blue when treated with sulphuric acid and iodine, it is per- ‘meated by other substances, or is to be regarded as changed chemically ; this is true of wood-cells, - vessels, cork, and, usually, the root-cap. The substances (lignin, suberin) taken up by the walls in these cases must first be removed by the alter- nate use of acids and alkalies, and finally by washing in alcohol, ether, or chloroform, before the cellulose reaction will appear ; but, as yet, with the single exception of the silicified frustules of diatoms, no cell-wall has been found that is not shown by this treatment to contain cellulose.. ~ In the study of lichens, chemical reagents play an important part.t The preparation usually em-, ployed, consists of 5 cg. iodine, 20 cg. potassic iodide, and 15 g. distilled water. In thin sec- tions, the hymenium is usually colored blue by this solution, the medullary layer, occasionally, and the gonidial layer, less commonly. A wine color may also appear under this treatment, which shows that these cell-walls have a composition different from those of other plants. All living protoplasm? is killed by iodine, which is then rapidly imbibed and stains the protoplasm 1 Deichmann-Branth and Rostrup: Lichenes Danie, p. 17. De Bary Morphol. und Physiol. der Pilze, Flechten und Myxomyceten.— Hof- meister’s Handbuch, II., 1866, p. 281. 2 Sachs: Lehrbuch, 1874. Weiss: Allgem. Bot., 1878, I. Tangl: Protoplasma der Erbse.— Sitzungsber. wiener Akad., 1877-8, Bd. LXXVL-LXXVIIL. IODINE. rs brown. This is especially true of the nucleus and chlorophyll bodies, as well as of the nitrogenous fundamental part of the protein grains, for the detection of which a rather concentrated solution should be used. Since a very small quantity of iodine is quickly fatal to protoplasm, besides col- oring it, this reagent is especially useful in study- ing bacteria and other ciliated micro-organisms., In the study of protein grains the glycerine solu- tion is best, because of its clearing action. ; The starch* which occurs in chlorophyll bodies is easily detected by treating very thin sections with ‘alcohol, or potassic hydrate and acetic acid, and afterwards adding a solution of iodine in water which contains potassic iodide, when the swelling starch grains assume the characteristic blue color. The so-called crystalloids consist of protein sub- stances, and consequently turn yellow when treated with iodine. This is also the case with inuline, where, however, the color does not depend upon a real imbibition of the iodine, but only upon a con- | densation of the brown fluid in the fine fissures of ‘the sphaero-crystals. All objects colored with iodine fade in the course of time; and iodine solutions destined for _use should be kept in the dark, to prevent the formation of hydriodic acid, which is greatly, pro- moted by the action of light. When sections or 1 Sachs: Jahrbiicher fiir wiss. Bot., 1863, III., p. 200; Flora, 1862, p. 166. Weiss: Allgem. Bot., 1878, I., p. 111. 8 MICRO-CHEMICAL REAGENTS. other preparations are removed from water into a concentrated alcoholic tincture of iodine, small, black, rhombic crystals of iodine often make their ‘appearance. CHLOR-IODIDE OF ZINC. In the preceding section mention has been made of a solution of iodine in chloride of zinc.t We may now consider this a little more fully. This preparation is made by dissolving zinc in pure hydrochloric acid, evaporating the solution to the density of sulphuric acid, in contact with metallic zinc, and adding as much potassic iodide as the solution will take up. Finally, it is saturated with metallic iodine. The color of the reagent ’ should be reddish-brown; it should have the odor of iodine, and small crystals of pure iodine should precipitate with time. As a precaution against the formation of hydriodic acid, it should be kept in the dark, although this is less important than with the other iodine preparations. Chlor-iodide of zinc is especially useful for the detection of pure cellulose, since the zinc chloride 1 Nageli: Verhalten der Zellhaut zum Jod.— Sitzungsber. d. bayr. Akad. der Wiss., 1863, p. 383; Das Mikroskop, 1877, p. 474. Mohl: Blaue Farbung der Vegetab. Zellmembran durch Jod. — Flora, 1840. See also the works cited above. 2 The directions of Grénland, Cornu and Rivet (Des Préparations Microscopiques, Paris, 1872, p. 75), are incorrect, since the most important element — the iodine— is not present. POTASSIC HYDRATE. 9 converts this substance into amyloid, which is then colored blue or violet by the free iodine. -Cell-walls that have suffered degeneration of the cellulose are not colored blue. Wood-cells, ves- sels, cork-cells, and the cells of the root-cap, as well as the cuticularized layer of the epidermis, the extine of pollen grains and spores, in fine, all lignified or corky membranes, are colored yellow ; the true cuticle, however, is uncolored.. Starch colors blue, but the grains rapidly swell up and undergo disorganization. The walls of fungus hyphz, composed of the so-called fungus-cellulose, ‘remain uncolored to a noticeable degree.*' [They are also usually uncolored by sulphuric acid and iodine. | For the detection of tannin, a very dilute solution of chlor-iodide of zinc is employed, the contents of cells which contain tannin becoming reddish or violet under this treatment.? POTASSIC HYDRATE (Caustic Potash). Next to iodine, caustic potash takes the most important place among micro-chemical reagents.3 1 Schacht: Die Pflanzenzelle, p. 143, e¢ seg. Hofmeister: Handbuch, IL.,. Die Pflanzenzelle, p. 258. 2 Sanio: Bot. Zeit., 1863. Dippel. Das Mikroskop, I., p. 375. 8 Nageli: Das Mikroskop, 1877, pp. 472 and 525. Dippel: Das Mik- roskop, I., p. 278. Wiesner: Technische Mikroskopie, 1867. Sachs: Ueber die Stoffe, welche das Material der Zellhdute liefern.— Jahrb. fiir. wiss. Bot., 1863, III.; Keimung von Allium Cepa. — Bot. Zeitung, 1863, Nos. 8-9; Zur Keimungsgeschichte der Grdser, Keimung der Dattel. — Bot. Zeitung, 1862. Ny 10 MICRO-CHEMICAL REAGENTS. The commercial potassic hydrate is used in an aqueous or alcoholic solution, whose concentra- tion depends upon the particular purpose for which it is to be employed. In general, a moderately concentrated solution is preferable, since it may be diluted when this is necessary, and a very strong solution is seldom to be recommended, since, although its action is more rapid, it destroys the tissues too quickly. In making an aqueous solution, care should be taken to add the alkali in small quantities, to avoid undue heating. The alcoholic solution is made as follows:* 85-90 per cent. alcohol is mixed with a concentrated aqueous solution of caustic potash until a precipitate is formed. After being repeatedly shaken it is set aside for twenty-four hours, at the end of which time the clear, pale-yellow fluid is decanted, and, after dilution with distilled water, is ready for use. Russow recommends the employment of solutions of. two densities, one consisting of one part of dis- tilled water and two parts of the saturated solu- tion; the other, of one part of water to three of the original solution. : Since potash solutions readily take up carbonic acid from the air, and further tend to crystallize out in the neck of the bottle in which they are kept, it is necessary to keep them in bottles with glass stoppers, which must be frequently loosened. 1 Russow: Mém. Acad. St. Pétérsbourg, Sér. 7, Tome 19, No. 1, p. 15, note. POTASSIC HYDRATE. It [A slight coating of paraffine will prevent the stopper from sticking.] In microscopic manipulations the value of potash depends upon its solvent and softening actions, those which accompany the absorption of water. It dissolves many of the fine granules in proto- plasm, bleaches various coloring matters, forms soluble soaps with fats, and effects the swelling of the starch which often renders tissues opaque. It thus destroys the protoplasmic structure of cells and makes these clearer and more transparent, thus, when dilute, playing a very important part as a clearing medium in the study of otherwise » opaque sections ; while it permits the examination of thick masses of tissue, or even of entire organs, ~ as embryos, trichomes, sections of the punctum vegetationis, or whole stems and leaves, as in the study of the course of fibro-vascular bundles [and laticiferous tissue]. This method was first proposed by Hanstein. The sections are treated with a solution of potash, washed, and neutralized with hydrochloric or acetic acid. If this renders them too opaque they may be cleared by washing first in pure water, then in ammonia water. If, on the other hand, they are too transparent, they may be improved by washing 1 Hanstein: Die Scheitelzellgruppe im Vegetationspunkt der Phanero- gamen, 1868; Entwickelung des Keimes, Botan. Abhandl,, I., Heft 1, p. 5. Koch: Cuscuta, in Hanstein’s Bot. Abhandl., 1., Heft 3, p. 25. Oels: Anat. d. Droseraceen., Diss., Breslau, 1879, p. 13. _ 12 MICRO-CHEMICAL REAGENTS. in alum water. Sometimes the process has to be repeated several times before the desired effect is obtained. After washing in distilled water, prep- ‘ arations made in this way can be kept for a long time in glycerine, which clears them still more. _ Russow’s alcoholic potash is used for the same purpose as the aqueous solution, and in most cases is preferable, since the presence of alcohol pre- vents the excessive swelling of the cell-wall, that/ often occurs when the Hanstein process is em- ployed. With this reagent acetic acid may be used for neutralization, and the preparations keep well in glycerine. Starch swells and is destroyed in potash, which is therefore useful for clearing up tissues, like the albumen of. seeds, that contain much starch. The aqueous solution of potash, causing swelling of the cell-wall, often facilitates the study of its striation and stratification; this is especially the case in collenchyma. A warm, solution of potash, being a solvent for the so-called intercellular substance, is sometimes employed for isolating cells by maceration. Potash is useful as a test for suberin.t When thin sections of corky tissue are well boiled in this reagent the suberin is extracted from the cell- wall, appearing as yellow drops, that soon run together. 1 Hohnel: Kork u. verkorkte Gewebe. —Sitzber. wien. Akad,, 1877, Abth. L, p. 16, el NT: POTASSIC HYDRATE. 13 Cell-walls (wood-cells, ducts, etc.), that do not immediately give the cellulose reaction with iodine and sulphuric acid, because of the presence of so-called incrustation matters, give this reaction promptly after treatment with boiling potash* — often used in connection with nitric or other acids — since this removes the foreign sub- stances. Sometimes tannin may be recognized by the use of potash, as cells which contain it —and which color green with salts of iron—assume a yellow color with potash. Cells containing chrysophanic acid? become pur- ple-red with the same fluid. Sachs has employed potash as an analytic re- agent in various histologo-physiological studies, obtaining good reactions when using it with cupric sulphate in testing for different sugars, protein matters, and carbo-hydrates.3 When protoplasm is first treated with nitric acid, and afterwards with dilute potash or ammonia, it assumes a beautiful yellow color from the forma- tion of potassic or ammonic xanthoproteate.* The so-called crystalloids swell and change their angles in potash, thus showing their organic nature. 1 Hofmeister: Handbuch, I., several places. 2 Borscow: Bot. Zeit., 1874, p.20. Weiss: Allg. Bot., 1878, I., p. 288. 8 See cupric sulphate, below. The reader is referred to the preceding papers, and to H. de Vries’ Keimung des rothen Klees, — Landwirthsch. Jahrb., 1877, VI., p. 468. 4 Dippel ; Das Mikroskop, II., pp. 10, 18. 14 MICRO-CHEMICAL REAGENTS. AMMONIA. ‘A concentrated solution of ammonia in water is often used instead of potash, where the latter would act too powerfully. We have also men- -tioned its use in clearing tissues by Hanstein’s method. As a test for protein combinations, including crystalloids, it is employed with nitric acid to in- tensify the color of the xantho-protein reaction. When athin section of a tissue composed of cells with thickened walls is successively treated with nitric acid and ammonia, the middle lamella (inter- cellular substance) is colored yellow.’ Finally, it is employed ‘in the preparation of © cupro-sulphate and carminate of ammonia.3 Ammonia is also valuable for restoring the form of herbarium specimens of phzenogams, algze, and ~ mosses, as well as spores, pollen: grains, etc., that are to be examined microscopically. CUPRAMMONIA (Cuoxam, Cramer; Cupridiamin), This important reagent must be freshly made when needed for use, as it deteriorates with age. When it is desirable to keep it for a time it should be set in the dark. 1 Dippel: Das Mikroskop, IL, p. 279. Weiss: Allgem. Bot., 1878, PP- 77; 144- 2 Dippel: Das Mikroskop, II., p. 100. 3 See the section on staining agents. CUPRAMMONIA. 15 To prepare it an aqueous solution of sodic hydrate is slowly added to a solution of cupric sul- phate, until a precipitate of cupric hydrate forms. The precipitate is collected on a filter, transferred to a test tube, washed, and dissolved in strong ammonia. The solution, which is of a beautiful dark-blue color, is at once ready for use. It may also be prepared by allowing 16 per cent. ammonia to stand upon copper turnings in an open flask. However prepared, it should be used only so long as it has the power of quickly dissolving cotton fibers. Pure cellulose swells much and is dissolved with- out conversion into amyloid. Cell-walls incrusted with lignin, suberin, etc., are only dissolved after these substances have been removed by Schultze’s maceration. - ; In general, neither the cuticle nor the so-called middle lamella or intercellular substance dis- solves. According to Kabsch, cuprammonia is a test for pectose. When a tissue containing this substance is treated with the reagent a fine skeleton of cupric pectate is left behind.' | 1 Schweitzer: Vierteljahrschrift, nat. Ges., Ziirich, 1857, II. Kabsch: Jahrb. wiss. Bot., 1863, p. 357. Hofmeister: Handbuch, I., p. 240. Dippel: Das Mikroskop, I., p. 280. Wiesner: Technische Mikroskopie, 1867, p. 38, note 2. Nageli: Das Mikroskop, 1877, p. 474. Frémy: Mém. del’ Acad., Paris, 1859; Journ. de Pharm. et Chim., XXXVI. Ep- stein: De Conjunctione cellulose cum cupro oxydato. Dissertatio, Bres- lau, 1860 (Cf Bot. Zeit., 1860, p. 234). 16 MICRO-CHEMICAL REAGENTS. MINERAL ACIDS! SULPHURIC ACID (Oil of Vitriol). Sulphuric acid is used in a concentrated or dilute form according to circumstances. The most use- ful proportion is obtained by diluting one volume of strong acid with three of water.? Dilute sulphuric acid causes starch grains to swell, and similarly affects cellulose, especially in collenchyma, at the same time transforming it into one of its isomers, amyloid, which differs from cellulose in assuming a blue color when treated with iodine. Hence, to determine whether a cell- wall consists of pure cellulose, it is only necessary ‘to treat it first with a tincture of iodine and then with sulphuric acid, when it turns blue, if unin- crusted. Concentrated sulphuric acid dissolves both cell- wall and starch grains, greatly swelling all parts it 1 In using any of these acids a large cover-glass should be employed to prevent injury to the objective. 2 Dippel: Das Mikroskop, I., p. 276. Nageli: Das Mikroskop, 1877, p- 474. Weiss: Allgem. Bot., 1878, I., p. 62, etc. Hofmeister: Hand- buch, L., p. 252, ete. 8 [M. Vetillart advises the use of the following mixture, in place of pure acid, in the cellulose test. Three volumes of sulphuric acid (spec. grav. 1.84), one of water, and two of glycerine, are slowly mixed to avoid heat- ing. This does not destroy the tissue. — Christy’s Fibres, p.17. W. T.] MINERAL ACIDS. 17 comes in contact with. Starch is thus converted into dextrin. The cuticularized parts of the cell-wall (cork, cuticle,t extine of pollen, exospore, root-cap, etc.) resist the action of this reagent, as does the pre- viously mentioned middle lamella. Protoplasm is destroyed after a time, while young protoplasmic bodies are often colored rose-red under its action,? the reaction being rendered more certain by the addition of a solution of cane-sugar. Fat-bodies occurring in the protoplasm are not dissolved, but run together, forming small refractive drops. Oil which previously existed, diffused through the pro- toplasmic mass, manifests itself similarly.s NITRIC ACID (Aqua fortis). This is employed as a macerating reagent in combination with potassium chlorate, g. v. ; When the contents of a cell are treated with nitric acid alone, or with this reagent followed by ammonia, they assume a bright yellow color when -protein matters are present, through the formation of xantho-protein acid. According to Hohnel, nitric acid, either alone or 1 DeBary: Hofmeister’s Handbuch, II., 1878, pp. 84,°131. Bron- gniart: Ann. Sci. Nat., 1830, 1 Sér., T. XXI., p. 427. 2 Sachs: Bot. Zeitung, 1862, p. 242. 8 Sachs: Bot. Zeitung, 1862, p. 146. 4 Dippel: Das Mikroskop, I., p. 275. _Nageli: Das Mikroskop, 1877, p- 474. Sanio: Bot. Zeit., 1863, p. 362, note. Bs agar MICRO-CHEMICAL REAGENTS. with potassic chlorate, is a good test for suberin.* To obtain this so-called ceric’ reaction, thin sec- tions of the tissue are treated with the reagent. If suberin is present, after the solution of the other parts of the cell-wall, yellow or spherical masses remain, which are at’ first granular but later become homogeneous. They consist of ceric | acid, and are soluble in alcohol, ether, benzol, and chloroform. Warm nitric acid, followed by ammonia, colors the middle lamella (or intercellular substance) yellow.? Nitric acid dissolves starch grains after causing them to swell greatly; hence it may often be used in a dilute condition for clearing tissues which contain much starch. ~‘ CHROMIC ACID. This must be free from sulphuric acid. It is employed concentrated or dilute, according to cir- cumstances.3 Since it tends to form crystals in the neck of the bottle in which it is kept, the stopper should be coated with vaseline, or fre- quently loosened by turning. The cell-wall swells and finally dissolves in chro- 1 Hohnel: Sitzber. wien. Akad., 1877, Abth. I. 2 Dippel: Das Mikroskop, II., p. 100. 8 Dippel: Struktur der Zellhiille, 1878. Sanio: Bot. Zeitung, 1860- 1863. Kabsch: Jahrb. wiss. Bot., III., p. 387. Nageli: Das Mikroskop, 1877, P- 475- MINERAL ACIDS. 19 mic acid, and, as the swelling proceeds slowly, the dilute acid is useful for showing the stratification of the wall. Only silicified and corky* layers resist its action. Lignified cells are entirely dissolved. Those containing suberin become very transparent and nearly invisible; but after the acid has been removed by washing they usually reappear, though the prolonged action of the reagent dissolves them. In general, chromic acid is useful in the study of the stratification of the cell-wall, starch-grains, etc. It is also sometimes applicable to the fixation of protoplasm [e. g. the plasmodia of Myxomy- cetes], but must be used in a very dilute form for this purpose. PEROSMIC ACID. This poisonous and ill-smelling reagent,? which is usually kept in the crystalline form in hermet- ically-sealed tubes, and dissolved in water when needed for use, has of late years been much em- ployed in the investigation of the minute structure 1 Hohnel: Ueber Kork.— Sitzungsber. wiener Akad., 1871, Abth., I. HGhnel differs here from Pollender: Chromsdure als Losungsmittel fiir Pollenin und Cutin. — Bot. Zeitung, 1862, p. 385. 2 Dippel: Das Mikroskop, I., p. 375. Strasburger: Befruchtung und Zelltheilung, 1878; Studien iiber Protoplasma, 1876. Pfitzer: Die Bacil- lariaceen, — Hanstein’s Bot. Abhandl., I., Heft 2, 1871, p. 33. Nageli: Das Mikroskop, 1877, p. 476. Ranvier: Histologie, 1875, p. 55. Robin: Microscope, 1877, p. 220. 20 MICRO-CHEMICAL REAGENTS. of protoplasm. As its vapor attacks the mucous membrane of the eyes and air passages, the use of the osmiamid has been recommended, instead of the acid, which it replaces well in all reactions. Oil and fats reduce osmic acid, precipitating me- tallic osmium, which colors the oil drops brown or. even black. Tannin is recognizable by the same reaction. ‘ A one per cent. solution is especially valuable for the instantaneous hardening of living protoplasm. The stages in the division of the nucleus of cells, and other structural peculiarities of protoplasmic bodies are thus fixed in a few minutes, and, after washing, may be preserved for future study in a dilute solution of glycerine, which, however, has the disadvantage of rendering the preparations very transparent. Recently perosmic acid has been used in the study of young meristem tissues.‘ The organs to be studied were laid in a very dilute aqueous solu- tion (.I-1 per cent.) of the acid until they black- ened, after which they were treated with alcohol, cleared with clove oil, and imbedded for sectioning in cocoa-butter (butyrum cacao of pharmacists). The sections were mounted in Canada balsam. A mixture of nine parts of .25 per cent. chro- mic acid and one part I per cent. perosmic acid 1 Parker: Journ. Roy. Microsc. Soc., 1879, IL, p. 382. I have not tested the method. MINERAL ACIDS. 21 also gave good results. Staining and hardening were thus effected simultaneously, the terminal buds of Chara serving as material for the study. PHOSPHORIC ACID. This has a limited usefulness through inducing the imbibition of water. It causes crystalloids to swell. [Its occasional use in the cellulose test has already been mentioned ¢ p. 5.] HYDROCHLORIC ACID (Muriatic Acid). Like other powerful acids this? induces the swelling of starch, and young cell-walls, especially when it is concentrated. Its employment in*‘Han- stein’s method of clearing tissues has been already mentioned (cf. p. 11). Kabsch? has used it with concentrated sulphuric acid and potash to isolate the tertiary lamella of wood-cells, the sections be- ing successively treated with the separate reagents, and washed with water after each has acted a suf- ficient length of time. After lying in the acid for a long time nitrogen- ous substances (protein matters) assume a violet 1 Hofmeister: Handbuch, I., several places. Kraus: Jahrb. wiss. Bot., VIII., Crystalloids in the Epidermis. 2 Dippel: Das Mikroskop, I., p. 276; II, p. 41. Hohnel: Sitzber. wien. Akad., 1877, Abth., I., p. 21. 8 Jahrb. wiss. Bot., 1863, III. 22 MICRO-CHEMICAL REAGENTS, color. Hydrochloric acid is further valuable for showing the nucleus of diatoms, etc.’ ‘Crystals based on carboni¢ acid emit bubbles of this gas, when treated with hydrochloric acid, the carbonates being converted into chlorides. On the other hand, crystals based on oxalic acid dis- solve without effervescence. Recently, Pringsheim? has employed hydro- chloric acid as a reagent for hypochlorin, one of the components of chlorophyll bodies, g. v. Newly- cut sections are allowed to lie in the acid for sev- eral-hours, when the hypochlorin separates as small semi-fluid exudation-masses, brownish or red in color, at first nearly spherical, but afterward forming needle-shaped crystals. ORGANIC ACIDS. ACETIC ACID. This acid 3 is used, in the form in which it is kept by pharmacists, in various micro-chemical investi- gations; ¢.g. it may replace hydrochloric acid in Hanstein’s method for clearing opaque meristem (see the section on potassic hydrate, Cf p. 11). The sections, first rinsed in water, are placed in a drop 1 Pfitzer: Bacillariaceen.— Hanstein’s Bot. Abhandl., L., Heft 2, p. 31. _ 2 Monatsber. berlin. Akad., Nov. 1879. 8 Dippel: Das Mikroskop, I., p. 277; IL., p. 10. Nageli: Das Mikro- skop, 1877, p. 476. Hanstein: Scheitelzellgruppe, 1868; Entwickelung des Keimes.— Bot. Abh., Bd. I., Heft 1, p. 5. Strasburger: Studien iiber Protoplasma, 1876, p. 5. ORGANIC ACIDS, Sy ae of acetic acid on the slide. With the neutraliza- tion of the alkali they often become somewhat opaque, but may be re-cleared, often to great trans- parency, by laying them in glycerine. In the study of crystals, those composed of an oxalate may be distinguished by being insoluble in acetic acid, but soluble in hydrochloric acid, (p. 22); while salts of carbonic acid, occuring as crystals or as incrusting components of the cell- wall, are soluble with effervescence in either. - Acetic acid sharply differentiates the nucleus, and is often a valuable medium in the study of the intimate structure of protoplasm. Stras- burger" employs a one per cent. aqueous solution for fixing the nucleus when staining the latter with methyl-green; at the same time it often clears up protoplasmic structures, and swells the condensed ectoplasm. Acetic acid has also found. application in coch- ineal solution, and in glycerine in which prep- arations stained with carmine are to be preserved. OXALIC ACID. The aqueous solution? is employed with certain coloring matters in staining tissues. The alcoholic 1 Zellbild.u. Zelltheil., 1880, 3 ed. 2 Dippel: Das Mikroskop, I., p. 285. Frey: Das Mikroskop, p. 77. Bachmann: Dauerpraparate, p. 27. Wiesner: Technische Mikroskopie, ~ Pp. 247, 258. 24 MICRO-CHEMICAL REAGENTS. solution is useful for removing some of the color from too deeply stained sections. A concentrated aqueous solution is used as a test for pectose, which it dissolves after previous treatment of the section with potash. CARBOLIC ACID (Phenol). This has a very limited application in micro- chemistry.‘ Cell-walls that, after treatment with carbolic acid, assume a greenish-yellow or bluish- green color, when moistened with hydrochloric acid, are considered to be lignified, and phenol thus becomes a reagent for lignin (?). Leitgeb? has employed a solution of carbolic acid in alcohol asa clearing medium in studying the histogeny of mosses. Phenol should be added in small quantity to glycerine-jelly to prevent molding, and when dilute it may be used as a preservative for bacteria (Warming), which remain sharply defined, but be- come clear and homogeneous internally. 1 Bot. Zeitung, 1877, p. 786. 2 Nageli: Das Mikroskop, 1877, p. 476. ALCOHOLS. ALCOHOLS. ALCOHOL (Ethyl Alcohol). The known disinfecting value of spirits of wine, or alcohol,‘ depends upon its fatal action on all protoplasm ; hence its application as a preservative for animal and vegetable preparations. Absolute alcohol has the property, in common with peros- mic acid, of rendering protoplasm *rigid,-and it is thus applicable in studying the more intimate structure of protoplasmic bodies, the division of the nucleus, etc. Its avidity for water causes the protoplasm to contract from the cell-wall, so that the ectoplasm becomes visible; and the same peculiarity is taken advantage of when we employ t alcohol for hardening tissues that are too soft for . section-cutting when fresh. It may also be used advantageously for removing the air from intercel- lular spaces, etc., in preparations, since it pene- trates into capillary cavities much more readily than water does. In difficult cases warming the sections often helps this action, alcohol being added from time to time to replace that lost by 1 Dippel: Das Mikroskop, I., p- 282. DeBary: Vergl. Anat., p. 86. Nageli: Das Mikroskop, 1877, p. 476. Sachs: Bot. Zeit., 1864, Nos. 12- 13. Weiss: Allg. Botanik, 1878, pp. 182,185. Tangl: Protoplasma der Erbse.- Sitzber. wien. Akad., Abth. 1, 1877-8. Strasburger: Zellbildung und Zelltheilung, 1875, p. 2; Befruchtung und Zelltheilung, 1878, p. 38. 26 MICRO-CHEMICAL REAGENTS. evaporation. When this expedient does not pro- duce the desired effect, recourse should be had to the air-pump. Alcohol is a solvent for volatile oils and resins, while fatty oils and vegetable wax are insoluble in cold alcohol, though it causes the oil globules to become confluent. In tissues, ¢. g. nectaries, which contain much cane sugar, this may be forced by the use of abso- lute alcohol to separate in small stellate crystals soluble in water.? In all tissues containing inulin the prolonged action of alcohol effects its precipitation within the cell in the form of sphaero-crystals; ¢. g. in Inula, Helianthus, Dahlia, etc. Other sphaero- crystal-forming substances, such as_hesperidin,? crystallize under the same treatment. (See Inulin and Hesperidin.) Asparagin may also be detected by the use of absolute alcohol, the sections that are tested being alternately moistened with the reagent and allowed to dry, when the asparagin 3 crystallizes out — often with other substances. It is recognized by its in- 1 Bonnier: Les Nectaires. — Ann. Sc. Nat., 1879, T. 8, pl. 8, figs. 124, 126. 2 Prantl: Das Inulin, 1870. _Rosenvinge: Sferokrystaller hos Mesem- brianthemum.— Nat. Foren. vidsk. Meddelelser, 1877-78, p. 305, with table (with literature of the subject). Pfeffer: Sachs’ Lehrbuch, 1874, p. 65.~ Russow: Leitbiindelkryptogamen, 1872, p. 110. 8 Hartig: Entwickelung des Pflanzenkeimes, 1858. Pfeffer: Ann. Sci. Nat., Bot., 5 Sér., T. XIX., p. 391. Sachs: Lehrbuch der Botanik, 1874, - p. 689. Borodin: Bot. Zeitung, 1878, p. 803. Detmer: Vergl. Physiol. d. Keimungsprocesses, 1880, p. 171. ALCOHOLS. 27 solubility in a warm solution of asparagin. The same treatment may, perhaps, be used with effect in testing for other substances; it has already been found satisfactory for tyrosin. Alcohol is also employed as a solvent for a part of the reagents used in micro-chemical tests ; e.g. anilin dyes, corrosive sublimate, phloroglucin, iodine, etc., as well as in the capacity of an anhy- drating medium for preparations that are to be mounted in volatile oils or Canada balsam. GLYCERINE. This fluid,* especially useful as a preservative for permanent preparations, for which use I pre- fer the nearly anhydrous form known as _ glyce- rinum Wilsoni, is also employed for many other purposes. According to circumstances it is diluted with alcohol or water, or with both. Preparatory to final mounting it is often well to place prepara- tions temporarily in a mixture of equal volumes of glycerine, distilled water, and absolute alcohol. It is used like alcohol as an anhydrating medium in the study of protoplasm. It can be employed very successfully as a clearing medium in many cases ; ¢. g. in studying the histology of the fibro- vascular bundles, and as a preservative or final 1 Kraus: Bot. Zeitung, 1877, p. 329. Sachs: Bot. Zeitung, 1864, Nos. 12,13. Nageli: Das Mikroskop, 1877, p. 475. Hegelmaier: Entwick- elung dicotyledoner Keime, p. 11. 28 MICRO-CHEMICAL REAGENTS. clearing fluid in the Hanstein and Russow meth- ods of clearing tissues. A mixture of dilute potash - and glycerine has been employed by Hegelmaier in the study of the embryo. Kraus has employed glycerine as a reagent for sugar and inulin. When sections that contain these substances in solution are placed in glyce- rine, strongly refractive rounded drops appear in the cells. If inulin is present these drops change to the characteristic spaero-crystals, and remain ; but if only sugar is present in the tissue they rapidly dissolve again. The sections should not be laid in water, but must be placed directly in the glycerine. The reliability of this reaction is cer- tainly worthy of further tests. With larger masses of tissue, glycerine can also be used for the sepa- ration of inulin, and it is a good preservative medium for inulin preparations, Iodin-glycerine is useful in the study of protein grains. It is prepared by dissolving a little iodine in glycerine, to which a small quantity of iodide of potassium has previously been added. More spe- cific directions as to the relative quantities are un- necessary. Warm glycerine is also used for the same pur- pose. Init the protein grains, which under natural © conditions are uniformly refractive, become differ- 1 [For the use of glycerine in the cellulose test, see sulphuric acid, p. 16. For its employment instead of potassium tartrate in Fehling’s sugar test, see cupric sulphate, p. 36. W. T.] { / ETHERS, ETC. 29 entiated so that globoids and crystalloids are dis- tinguishable. Since the evaporation of glycerine is siete imperceptible, it is one of the best preservatives for permanent preparations; but as it absorbs the moisture of the air when concentrated, the cover- glass should be sealed with some air-tight cement. ETHERS, ETC. ETHER, BENZOL, CHLOROFORM, BISULPHIDE OF CARBON, ETHEREAL-OILS.' As reagents for determining to what extent a substance is a fat, a volatile oil, a resin, etc., sub- stances have been employed which, from a chemi- cal standpoint, are often quite different. They allow us to recognize these substances because of their solvent powers. For this purpose ether, chloroform, alcohol, benzol, oil of turpentine, and carbon bisulphide are used, 1 Weiss: Allgemeine Bot., 1878, p. 178. DeBary: Vergl. Anatomie, p. 86. H. de Vries: Keimung des rothen Klees. — Landwirthschaftliche Jahrb., 1877, Bd. VI., p. 468. Nageli: Das Mikroskop, 1877, p. 476. -Dippel: Das Mikroskop, I., p. 374. Wiesner: Technische Mikroskopie, p. 81. For the use of carbon bisulphide as a eet for sulphur, see this substance in Part II. 30 MICRO-CHEMICAL REAGENTS. Resins are soluble in ether, cold absolute alco- hol, carbon bisulphide, and oil of turpentine. Fatty oils are soluble in carbon bisulphide, ethe- real oils, hot alcohol, and ether. When treated with concentrated potassic hydrate they form soaps, which are soluble in water. Ethereal otls are easily soluble in oil of turpen- - tine and cold absolute alcohol. Most of them are also dissolved by ether and carbon bisulphide.* It will be seen that their behavior, when treated — with alcohol, is distinctive for fatty and volatile oils; but for resins other reactions must be relied upon, which will be spoken of later. The substances which dissolve fatty oils may also be used as reagents for wax. ; With respect to the application of these reagents, we can only say that since carbon bisulphide, ether, oil of turpentine, and benzol are insoluble in water, the sections should be placed immediately in them to secure the best results. They cannot be pre- pared in water which is replaced by allowing the reagent to penetrate under the cover-glass, as with so many other reagents. The most convenient | plan is to treat the sections in a watch-glass, with a considerable quantity of the reagent. 1 Dippel (Mikr., 1, p. 374) states that they are insoluble in ether, which I do not understand. INORGANIC SALTS. 31 ETHEREAL OILS. Several volatile oils beside turpentine, which has already been mentioned, find use in microscopy. Oil of cloves and lemon oil are especially useful as clearing fluids in the study of pollen. They are also good preservative media for objects which can- not be studied in water, but require a fluid of some other refractive index. Since they decrease the re- fraction they are very useful in the study of many strongly refractive substances, by the aid of polar- ized light. Preparations which have been in oil must. be washed with ether or chloroform and afterward with alcohol before they can be ix in water or glycerine. INORGANIC SALTS. CHLORIDE OF SODIUM (Table Salt). A dilute aqueous solution' is used as a morpho- logical reagent for the contraction of protoplasmic bodies, a phenomenon which is to be attributed to its avidity for water. Many other salt-solutions have this property. A dilute solution of table salt ! Dippel: Das Mikroskop, I., p. 279. 32 MICRO-CHEMICAL REAGENTS. has the power of dissolving crystalloids, at least in the embryo of Bertholletia.* CHLORIDE OF CALCIUM. In an aqueous solution (two to three parts of water to one of the salt) this substance is some- times used as a mounting medium for permanent preparations excepting those which contain amy- lum, although for this purpose glycerine has largely replaced it. Recently it has found application for clearing tissues. The section which is to be treated is placed in a few drops of water and sprinkled with the dry pulverized salt. It is then warmed over a gentle flame until nearly dry, and again moistened with a few drops of water, after which it is laid in glycerine, where, in the course of a few hours, it acquires a very satisfactory degree of transparency. (Treub’s method.’) CHLORIDE OF MERCURY (Corrosive Sublimate). A very dilute aqueous solution (1:100) is used to make the finest protoplasmic currents evident.’ 1 Weyl: Zeitschr. fiir phys. Chemie, Bd. I., p. go. 2 Treub: Méristéme primitif ‘de la racine. — Musée de Leyde, 1876, Tome IIL, p.9. Eriksson: Meristemet i dicotyla vaxters rotter, p. 10. Flahault: Accroissement terminale de la racine. — Ann. des Sc. Nat., Bot., . 1878, p. 24. 8 Dippel: Das Mikroskop L, p. 281. INORGANIC SALTS. 33 Pfeffer’ has used it in a two per cent. alcoholic tincture in the study of protein grains. It unites with these albuminoids, forming a compound insol- uble in water; but to secure satisfactory results the preparation must lie at least twelve hours in the fluid. CHLORIDE OF IRON. An aqueous solution may be employed as reagent for tannin? when this is not present in too small quantity. The cells to be examined, when placed immediately in the reagent without the previous contact of water, which easily removes the tannin, - assume a dark green or bluish-black color, accord- ing to the nature of the tannin compound. The green cells color yellow if potash is added. The solution should not be too concentrated, as the tannate of iron which is formed is soluble in an excess of this compound, and its prompt solu- tion renders the test less evident. For this reason acetate and sulphate of iron’ have largely replaced the chloride. Being more certain in their action, they are to be preferred. . 1 Pfeffer: Jahrb. fiir wiss. Bot., 1872, VIII, p. 491. Weiss: Allg. Bot., p- 140, note. Sachs: Lehrb., 1874, 'p. 55. Duchartre: Eléments de Bot., 1877, p. 102. 2 Karsten: Gesammelte Beitr. zur Anat. u. Phys. d. Pflanzen, I., 1865, p-253- Dippel: Das Mikroskop, I., p. 375. Wiesner: Technische Mikro- skopie, p. 83. Weiss: Allg. Botanik., I., p. 181; Die Pflanzenhaare, Kar- stens Bot. Unters., I. Ndageli: Das Mikroskop, 1877, p. 475. 8 Cf. Link: Grundlehrend. Anat., 1807, p. 80, where, to my knowledge, it is mentioned for the first time as a micro-chemical reagent. 34 MICRO-CHEMICAL REAGENTS. CHLORATE OF POTASSIUM. This reagent is used with nitric acid, either in a concentrated aqueous solution, or, better, in the crystalline form, for the destruction of the “ middle lamella” and the consequent isolation of cells, especially in investigations of wood.* The mix- ture, the so-called Schultze maceration-medium, is boiled for a few minutes in contact with pieces of the tissue to be studied. After careful washing in alcohol, these macerated preparations may be pre- served in glycerine. This process must be carried on at a distance from microscopes and other appar- atus that can be injured by the gases developed. The Schultze mixture has also been found useful as a reagent for suberin. Thin sections are boiled for a long time in it; all parts of the cell-wall soon become clear, but those which contain suberin possess dark and sharp contours and resist the action longer than the others, though finally they become distorted, abruptly swell, and melt to form rounded drops of ceric acid, which are soluble in ether, benzol, chloroform, caustic potash, and boil- ing alcohol. In the process a part of the suberin is dissolved by the reagent, only a part being changed into ceric acid. Membranes which are 1 Sanio: Bot. Zeitung, 1863, p. 362, note; Anatomie der Kiefer.— Jahrb. fiir wiss. Bot., IX. Héhnel: Ueber Kork.— Berichte der wiener Akad., 1877, Abth. I. Schacht: Das Mikroskop, 1855, p. 27; Anatom. ue Physiol. der Gew., L., p.14. Dippel: Das Mikroskop IL, p.1or. Nageli: - Das Mikroskop, 1877, p. 474. INORGANIC SALTS. 35 only slightly suberiferous will, therefore, hardly show this reaction; and to detect their suberin, the sections are placed in the cold fluid a few mo- ments, and then removed to a solution of potash. The walls, which after the first treatment stand out sharply, assume an ochre-yellow color under the action of the potash —jin all cases after being slightly warmed. CUPRIC SULPHATE (Blue Vitriol). This substance has a very extensive application in micro-chemistry.‘ It is always used in a tolera- bly concentrated aqueous solution, and must be chemically pure. It is used in the following (Trommer’s) test for sugar. A moderately thick section of the tissue which is to be studied is allowed to lie from two to ten minutes in a concentrated solution of the salt. The surface is then rapidly rinsed with distilled water, and the section transferred to a boiling mixture of equal parts by weight of water and potassic hydrate. Cells which contain cane sugar (saccharose) assume a bright blue color, while those 1 Nageli: Das Mikroskop, 1877, pp. 475, 525. Dippel: Das Mikro- skop, I., p.372; IL, p.20. Weiss: Allgem. Botanik, 1878, I., pp. 77, 171,174. Sachs: Flora, 1862, p. 289; Jahrbiicher fiir wiss. Bot., III., p. 187. De Vries : Keimung des rothen Klees. — Landwirths. Jahrb., 1877, VL, p. 468. Wiesner: Technische Mikroskopie, p. 79. Fresenius: Quantitative chem- ische Analyse, Braunschweig, 1853, p. 496. Fehling: Ann. der Chemie und Pharmacie, Bd. LXXIL., p. 106. 36 MICRO-CHEMICAL REAGENTS. which contain grape sugar (g/ucose) become clouded by the deposition of a finely granular or flocculent orange precipitate of reduced oxide of copper. By this test we are thus able to determine which kind of sugar was present in the tissue. If the blue solution of cane sugar is boiled with dilute sulphuric or nitric acid it is changed into grape sugar, which then gives the red reaction. Trommer’s test also serves for the detection of dextrine. The contents of cells containing this sugar assume a vermilion color, while small gran- ules in the precipitate exhibit the Brownian movement. If the dextrine is mixed with pro- tein compounds, however, the precipitate is yel- lowish. If in manipulation too much of the cupric sul- phate has penetrated the cells the reaction is often masked bya precipitate of cupric hydrate. To avoid this difficulty Fehling’s fluid, which gives the same reaction, may be used. It is prepared in the following manner. Dissolve 4 gm. of cupric sul- phate in 16 gm. distilled water; and 16 gm. of potassium tartrate in the minimum of water. The two solutions are to be mixed. The reagent should be kept in the dark and needs frequent renewal. [Prof. W. S. Haines has found in glycerine a very desirable substitute for the tartrate in Feh- ling’s test. The proportions employed by him for qualitative examinations are: Cupric sulphate, 2 Se INORGANIC SALTS, 37 gm.; potassic hydrate, 6 gm. ; pure glycerine, 7.5 cc.; distilled water,.178 cc. — W. T.] Arabin (arabate of potassium) Cerasin (meta- gummate of potassium) and Bassoriz do not reduce the Trommer reagent ; a dark blue precipitate only is formed, the flocks of which unite into balls when heated. Protein compounds are sometimes recognized by the same tests, the contents of young cells assuming a beautiful violet color, though older cells fail to show the reaction. Cell-walls which are not lignified are colored faintly blue by long soaking in an aqueous solution of cupric sulphate. POTASH ALUM (Alum).2 An aqueous solution? is employed as a mordant, in various staining processes, e. g. in Frey’s haem- atoxylin and Grenacher’s alum-carmine; as an anhydrating medium ; or, finally, as an aid in the Hanstein method of clearing tissues, g. v. POTASSIC NITRATE (Niter, Saltpeter). This has been used in a one-fourth per cent. aqueous solution as a culture-fluid for the living 1 Wheeler: Organic Chemistry, p. 187. 2 The ordinary commercial alum, which is ammonium-alum, is quite as useful. 8 Frey: Das Mikroskop, p. 93. Bachmann: Dauerpraparate, p. 28. _ Hanstein : Scheitelzellgruppe, 1868. 38 MICRO-CHEMICAL REAGENTS. | tissues of higher plants during observations on the division of the nucleus." MERCURIC NITRATE (Millon’s reagent).? This is made by dissolving mercury in its weight of concentrated nitric acid, and diluting with an equal volume of distilled water. It should be freshly prepared when required for use. It causes the cell-wall to swell, and so renders | its striation more evident, but its most important use is for the detection of protein compounds. These, after lying in it for some time, and espe- cially after slight warming, assume a very red color. The surface membrane (Hautschicht) of proto- plasm is slightly colored if at all. It should, how- ever, be added that the reagent is not very sensitive, and the reaction is not always obtained (Nageli). CHLORIDE OF GOLD. A one-half per cent. solution has recently been used in America for coloring the tissues of fungi. It requires from one to six hours to produce the 1 Treub: Quelques recherches sur le réle du noyau dans la division des cellules végétales, 1878, p. 9-— Naturk. Verh. d. koning]. Akad., Vol. XIX., Amsterdam. 2 Dippel: Das Mikroskop, I., p. 281; II., p. 18. Niageli: Das Mikroskop, 1877, pp. 475, 527. Weiss: Allgem. Bot., I., pp. 77, 144- Millon: Ann, de Chim, et de Phys., 3 Sér., Tome 29, p. 507. Radlkofer: Ueber krystal. proteinart. Kérper, Leipzig, 1859. INORGANIC SALTS. 39 proper effect. Preparations stained with it may be mounted in dilute glycerine." NITRO-PRUSSIATE OF SODIUM. This is useful in its aqueous solution for the detection of free sulphur.?. It should be prepared when required for use. The crystals need to be kept from the air, from which they very readily take up water. The preparation to be tested is heated with potassic hydrate, after which treat- ment the granules of sulphur unite to form larger yellow masses, which are colored violet by the nitro-prussiate. POTASSIC FERRO-CYANIDE (Yellow Prussiate). An aqueous solution precipitates ferric salts, thus causing a blue color. This reaction has been taken advantage of for the detection of fer- ric hydrate in the cell-wall, ¢. g. in Crenothrix.$ Cells, the brown color of whose walls causes a suspicion of an encrusting compound of iron, are treated with a mixture of hydrochloric acid and the ferro-cyanide. If the beautiful color of Prus- sian blue appears the suspicion caused by the brown color is confirmed. 1 W. Hassloch: New York Med. Journ., Nov. 1878,—also Journal Royal Microscop. Soc., 1879, Vol. II., p. 170. Ihave not tested this my- self. 2 Cohn: Untersuchungen iiber Bacterien, II.— Beitr. zur Biol. d. Pfl., Bd. I., Heft 3, p. 175. 8 Cohn: Ueber den Brunnenfaden. — Beitr. zur Biol. d. Pfl, Bd. IL, Heft 1, p. 119. 40 MICRO-CHEMICAL REAGENTS, SULPHO-CYANATE OF POTASSIUM. An alcoholic solution is employed, sometimes in combination with hydrochloric acid, for the detec-- tion of iron in the cell-wall. (See the second sec- tion, Iron.) POTASSIUM BICHROMATE. An aqueous solution is used for the detection of tannic acid.* Masses of tissue of considerable size are left in the reagent for sometime. Cells which contain tannin assume a reddish-brown color. The iron-reactions, however, are preferable. It is also used for hardening resin masses. NITRATE OF SILVER. A very dilute alkaline solution in water has been employed for testing the living condition of proto- plasmic bodies which, in this condition, contain aldehyde. - According to Loew and Bokorny,’ the discoverers. of this reaction, the reagent is pre- pared as follows: 1. A one per cent. solution of nitrate of silver is made in distilled water. 2. A mixture of 13 cc. potassic hydrate solution (s. g. 1.333), IO cc. ammonia (s. g. .964), and 77 cc. dis- 1 Dippel: Das Mikroskop, L., p. 280. Sanio: Bot. Zeitung, 1863, p. 17. Hanstein: Organe der Harz und Schleimabsonderung. — Bot. Zeitung, 1868, p. 702. Nageli: Das Mikroskop, 1877, p. 475. Weiss: Allgem. Bot., L, p. 187, note. 2 Pflugers Archiv., 1881, XXV.; Jahrb. wiss. Bot., 1882. 5 a F = . a = 4 4 "INORGANIC SALTS. - 4 tilled water is made. Before using these solutions they are mixed, 1 cc. of each being taken, and the mixture diluted so as to makea liter. The mixture can be made only at the moment when it is to be employed ; otherwise metallic silver is precipitated by the light. This reagent colors living protoplasm black, while dead protoplasm remains uncolored. The reaction may be obtained with a solution diluted very greatly (even I : 1,000,000).* Tannic acid also gives a reaction, but this is effected only with a less dilute solution (I : 10,000). Glucose gives a reaction with a solution of I : 100,000, the cells coloring brown, and not black. In this proportion, therefore, nitrate of silver is an excellent reagent for this sugar. 1 These very dilute solutions cannot be employed in as small quantity as micro-chemical reagents usually are. To obtain good results it is neces- sary, according to Bokorny, to immerse a few cells (e. g. of Spirogyra) in a large quantity (.5—1 liter) of the reagent for six to twelve hours.— French Translator. 42 MICRO-CHEMICAL REAGENTS, ORGANIC SALTS. ACETATE OF IRON Is used in an aqueous solution, as has been de- scribed for the chloride. ACETATE OF COPPER (Verdigris) Is used as a means of recognizing resins. Masses of tissue are soaked five or six days in an aqueous solution of the salt, after which treatraent the resinous masses assume an emerald green color.' SULPHATE OF ANILIN. An aqueous solution of this substance, the so- called Wiesner anilin reagent, is used for the detec- tion of lignin, the constituent of wood.2? The sections are first placed in a dilute solution of the sulphate until they are well saturated with it ; even with no other treatment, lignified membranes often assume a faint yellow color, which, however, is 1 Franchimont: Origine et constitution chimique des résines de ter- pénes. — Cf Archives Néerlandaises, 1871, T. VL, p. 427, with PI. 8. 2 Wiesner: Technische Mikroskopie, 1867, p. 64, Karsten: Bot. Un- tersuchungen, I., p. 120, note. Burgerstein: Sitzungsber. der wiener Akad., 1874, Bd. LXX., Abth. 1, p. 338. Hohnel: Ueber Kork, etc.— Sitzungsber. der wiener Akad., 1877, Bd. LXXVI., Abth. 1, p. 21. ORGANIC SALTS. 43 intensified by transferring the section to dilute sulphuric acid. If it is desired, the reagents may be mixed before using. Since this anilin salt is found in the market only in a very impure and barely soluble form, it is bet- ter to replace it by the next. CHLORIDE OF ANILIN Is used in aqueous solution for the same purposes as sulphate of anilin, and in the same manner, except that the acid used must be hydrochloric." An alcoholic tincture of either anilin salt may replace the aqueous solution, and the colors pro- — duced are then more intense. CHLORAL. This has been recently introduced into micro- chemistry by Meyer.? It is employed in aqueous ‘solution: five parts of chloral to two parts of ° water; and should be:used at a temperature of 15° C., as crystals are precipitated at lower tempera- tures. Its effect upon fats and volatile oils is similar to that of alcohol. . It dissolves the. same saccharine and amylaceous matters (Kohlenhy- drate) as water, and causes the swelling of starch grains. It swells or dissolves protein matters, and ° is, therefore, frequently useful in clearing tissues. 1 Hohnel: Ueber Kork, etc., p. 21. 2 Arthur Meyer: Das Chlorophyllkorn, Leipzig, 1883. 44. MICRO-CHEMICAL REAGENTS, OTHER ORGANIC COMPOUNDS. SOLUTION OF CANE SUGAR (Syrup). Vegetable cells containing much sugar often assume a beautiful red color on the addition of sulphuric acid. ‘This fact has been utilized in ob- taining a reaction for protoplasmic substances. The tissues to be tested are first saturated with a solution of cane sugar and water. On the addition of sulphuric acid the red color ‘manifests itself. q This reaction (Raspail’s)' is, however, often diffiewlt a to obtain, and not very good. The sections have’ . to be left in the acid for some time before the color appears. Syrups of different degrees of concentration also find application as anhydrating media.? A three per cent. solution had been used as a fluid of prep- aration in the study of transparent ovules, e. g. embryo sac of Monotropa and Orchis; and a five per cent. solution has been recommended for pollen-cultures under the microscope. 1 Raspail: Chimie organique, 1839, II., p.139. M. Schultze: ‘Ann, der Chem. u. Pharm., Bd. LXXI., p. 270. (Second discovery of the reaction.) Schacht: Mikroskop, p. 27; Pflanzenzelle, pp. 27, 28; Anat. u. Physiol. ‘ der Gewichse, I., pp. 46, 61. Dippel: Mikroskop, IL, p. 283. Ndageli: Mikroskop, 1877, pp. 476, 526. Frey: Mikroskop, p. 73. Hofmeister: Pflanzenzelle. — Handb. der phys. Bot., I., p. 2. Weiss: Allg. Bot., L., p. 77. Duchartre: Eléments de Bot., 2 ed., p. 25. 2 Strasburger : Befruchtung u. Zelltheilung, pp. 16, 29, 52, etc. OTHER ORGANIC COMPOUNDS. 45 ASPARAGIN. Borodin * has recommended a tepid concentrated aqueous solution for the detection of asparagin, which has been precipitated in the tissue by alcohol (p. 27), on the principle that a crystallized sub- stance is insoluble in a saturated solution of the same substance. _ Asparagin is prepared by evaporating the well- boiled and filtered sap of young seedlings of legu- minosze (especially lupines) which have germinated in the dark, or by the evaporation of the dialyzed aqueous decoction of althza root; when it sepa- rates as crystals. DIPHENYLAMIN. This officinal substance has of late been used as a reagent for nitrates and nitrites? The sections to be tested are allowed to dry on the slide and afterwards moistened with a solution of .ol—.1 gm. of diphenylamin in 10 cc. pure sulphuric acid. Even very small quantities of compounds contain- ing nitric acid are indicated by the appearance of a dark blue color in the cells; the reaction is ve distinct. 1 Borodin: Bot. Zeitung, 1878, p. 804, e¢ seg. Detmer: Physiol. u. Keimung d. Samen, 1880, p. 171, e¢ seg. 2 Molisch: Ueber den Mikrochemischen Nachweis von Nitraten und Nitriten etc. — Ber. d. deutschen bot. Ges., 1883, I., p. 150. 46 MICRO-CHEMICAL REAGENTS. BRUCIN Has been introduced into micro-chemistry for the same purpose as diphenylamin, but it does not serve so well for the detection of small quantities of nitrates and nitrites. Molisch* recommends a solution of .2 gm. brucin in 10 cc. pure sulphuric acid. A dry section of a tissue which contains N, O; assumes a bright red or reddish-yellow tint when placed in a drop of the solution. INDOL. This substance has been employed quite recently by Niggl as a reagent for lignin, or lignified mem- branes.?_ The discovery of this reaction is due to Professor Baeyer, of Munich.3 Several crystals of indol are dissolved in a sufficient quantity of warm distilled water. The sections to be tested are placed in a drop of the reagent for several minutes, after which they are washed in dilute sulphuric acid (I pt.:4 pts. water). Lignified cell-walls assume a very intense red color. : PHLOROGLUCIN. One of the prettiest and best reactions of micro- chemistry has recently been discovered by Wiesner ; 1 Molisch: Ber. deutsch. bot. Ges., 1883, L, p. 150. 2 Niggl: Flora, 1881, No. 35, p. 545- 8 Ann. d. Chem. u. Pharm., Bd. CXL. OTHER ORGANIC COMPOUNDS, 47 viz. the phloroglucin test, for lignin. An aqueous solution or, better, an alcoholic tincture of the reagent is employed, and even in excessively small quantity induces the reaction.t The section to be studied is treated with hydrochloric acid, and then placed in a drop of phloroglucin on the slide. The parts containing lignin assume a beautiful and in- tense rose-red color witha rapidity depending upon the concentration of the solution. The prepara- tions may be kept for a considerable time. If difficulty is experienced in getting the reagent —and at present it is quite expensive and hard to obtain — an extract of cherry wood diluted with water may replace it. This contains the substance in question among others, but gives a more violet reaction than the pure reagent.” ROSOLIC ACID Is recommended by Janczewski as “ the best of the reagents which color the callosities of sieve tubes.” A little ammonia or sodic carbonate should be added to it. 1 Wiesner: Sitzungsber. der wiener Akad., Bd. LXXVII., Abth. 1., Januarheft. 2 This has been called the xylofilin reaction by its discoverer, Héhnel. — Sitzungsanzeiger d. wiener Akad., 1877, No. 23, pp. 228, 229. 3 Etudes comparées sur les tubes cribreux. — Mém. de la Soc. des Se. nat. de Cherbourg, 1881, XXIII, p. 350. The discovery is due to Szyszyloviez, , 48 - -‘MICRO-CHEMICAL REAGENTS. COLORING AGENTS. Lately various dyestuffs have been employed for the differentiation of the tissue systems, as well as for the recognition of some of the cell contents; of these only the most important will be mentioned. TINCTURE OF ALCANNA. A red dye, extracted by alcohol from the root of Alcanna tinctoria, is employed for coloring resins, which have a special avidity for this substance. Protoplasm is also colored pale red by it.!' Prepa- — rations colored by alcanna do not endure drying. COCHINEAL. An aqueous extract of cochineal is employed with acetic acid or alum, for staining the prosen- chyma (bast cells) of the phloem in fibro-vascular © bundles. After lying in the dye for some time, these cells assume an intense red color, while the other elements are not affected, or but slightly colored. Yet there are certain kinds of wood which imbibe the cochineal extract, but after-treat- 1 N. J.C. Miiller: Untersuchungen tiber die Vertheilung der Harze. — Jahrbiicher fiir wiss. Bot., V., p. 387. Hanstein: Organe der Harz- und - Schleimabsonderung. — Bot. Zeitung, 1869, pp. 707, 708. are COLORING AGENTS. 49 -ment with hydrochloric or sulphuric acid removes the color from everything except the bast cells, in which it becomes more intense. As a coloring agent in the investigation of pro- tein bodies, this substance has also found use.* CARMINE. _ A solution of carmine in dilute potash, such as is sold by dealers, is used for staining the nuclei of cells. The solution, which should contain very little undissolved carmine, is filtered and then mixed with alcohol or glycerine in various propor- tions. The object requires to lie in the solution - for some time. Only the nucleus (and protein grains) imbibe the color. The carminate of ammonium (ammonia carmine) is, however, more commonly used.’ It is prepared in the following manner, suggested by Hartig. Carmine powder is dissolved in a strong solution of ammonia until this is saturated; the solution is evaporated to dryness over a water bath, and the carminate thus formed is prepared for use by solution in water. 1 Wigand: Bot. Zeitung, 1862, pp. 129, 139. Maschke: Bot. Zeitung, 1859, p. 22. Vogl; Anat. u. Histol. der unterirdischen Theile von Convol- vulus arvensis. — Sitzungsber. d. wiener Akad., 1863, XIII. 2 Hartig: Der Fiillkern, etc.— Karsten’s botan. Untersuchungen, I., p. 282, note. 8 Dippel: Das Mikroskop, I., p. 184, Frey: Mikroskop, 1873, pp. 87, 88, go. Bachmann: Dauerpraparate, p. 26. Tangl: Protoplasma der Erbse. — Sitzungsber. der wiener Akad., Bd, XXVI.-XXVIII. 50 MICRO-CHEMICAL REAGENTS. Thiersch gives the following formula for its pre- paration. - One part of carmine is dissolved in one part (by weight) of concentrated ammonia and three parts of distilled water. This solution is mixed with eight times its volume of dilute oxalic acid (1:22). Twelve volumes of absolute alcohol are then added, and the whole is filtered. The filtrate may be rendered more orange in color by the addition of oxalic acid, or more violet by the addition of ammonia. If oxalate of ammonium is precipitated, it can be removed by filtration or redis- solved by the addition of a few drops of ammonia. Grenacher’s alum-carmine, recently introduced into vegetable histology by Tangl,' is prepared by him in the following manner. A saturated aqueous solution of alum is made. The desired quantity of carmine is dissolved in it, the solution is boiled for about ten minutes, allowed to cool, and filtered. Walls consisting of cellulose are colored bright-red by this preparation, while those containing su- berin or lignin remain unstained. Protoplasmic bodies and the nuclei of cells are also-stained with _ difficulty, and but slightly. It is recommended that parts of plants from which sections are to be cut, shall be hardened in absolute alcohol, as this increases the power of the membranes to take up the coloring matter. 1 Tangl. Ueber offene Communication zwischen den Zellen des Endo- sperms. — Jahrb. fiir wiss. Bot., 1880, XII., p.170. Grenacher: Archiv. f. ~ mikr. Anatomie, 1879, p. 465; Zeitschrift f. Mikroskopie, 1879, Jahrg. IL, p. 55- COLORING AGENTS. 51 If the preparation should be too deeply stained by this carmine fluid, it can be bleached in an alcoholic solution of oxalic acid. Carmine stains all protoplasm if allowed to act a sufficient length of time. The nuclei of cells are most deeply colored. Living protoplasm does not imbibe the coloring matter. This occurs only after it has been killed by the addition of the reagent. In general it may be said that it colors most vegetable albuminoids, while starch and cellulose take it up in much smaller quantity or not at all. [The double-staining of clear sections, usyally bleached by the action of Labarraque’s solution, or — some similar fluid, is capable of yielding very good results where convenience of demonstration in the class-room is concerned. As good direc- tions as any are those of Dr. Rothrock,’ who uses Woodward’s ammonia carmine and the aniline color known as iodine-green. The section is placed in alcohol faintly colored by the addition of a few drops of a concentrated tincture of the green, where it is allowed to remain from twelve to twenty-four hours, according to circumstances. It is then successively passed through a series of fluids in the following order: one, water ; two, carmine ; three to five, alcohol; six, absolute alcohol ; seven, oil of cloves : being merely 1 Rothrock : Staining and double-staining of vegetable tissues. — Bot. Gazette, Sept. 1879, Vol. IV., pp. 201-6. §2 MICRO-CHEMICAL REAGENTS. dipped into the water, remaining from twenty sec- onds to a minute in the carmine, being well rinsed in the first watch-glass of alcohol, and staying ten to twenty minutes in each of the others. It is left in the oil of cloves until cleared up, when it is ready for mounting in balsam. The gen- eral experience of teachers seems to be, how- ever, that more time is consumed in making the few successful preparations than they are worth. —W. T.] Beale’s carmine, which is especially useful in dif- ferentiating the nucleus, is prepared by dissolving .6 gm. carmine in 2 gm. boiling ammonia water. The solution is set aside for an hour to allow some of the ammonia to escape. Sixty gm. dis- tilled water, 60 gm. glycerine, and 15 gm. absolute alcohol are then added. After standing for some time, the fluid is filtered and ready for use." Strasburger, in the study of the embryo sac, stains the protoplasm with a boracic solution of carmine, prepared as follows: Four parts borax are dissolved in fifty-six parts distilled water. To this one part of carmine is added. One volume of this solution is diluted with two volumes abso- lute alcohol and filtered. By the use of this dye the study of the forms of the nucleus is greatly facilitated. The preparations may be preserved in glycerine or glycerine jelly.?. 1 Frey: Mikroskop, p. go. 2 Strasburger: Zellbild. u. Zelltheilg., 1880, p. 9. - COLORING AGENTS, . 53 Czokor recommends the following coloring fluid: -7 gm, cochineal are pulverized with an equal quan- tity of calcined alum. This is dissolved in 700 gm. distilled water, and the whole is boiled down to about 400 gm. After it is cool a drop of car- bolic acid is added, and the fluid is filtered. It should last at least six months without deteriora- tion. At the end of this time it should be re- filtered after the addition of another drop of car- bolic acid.? PICROCARMINATE OF AMMONIUM (Picrocarmine). This staining agent, which is much used by students of animal histology, is employed in botani- cal micro-chemistry, chiefly for differentiating the nucleus.? It is prepared by adding a strong solu- tion of ammonium-carminate to a concentrated aqueous solution of picric acid, until this is neu- tralized. After evaporating it to four-fifths its original volume, it is set aside for a time, and then filtered, when the dark orange fluid is ready for use.3 Another method has been recommended by Gage. Equal parts by weight of picric acid and 1 Czokor: Die Cochenille Carminlosung. — Arch. f. mikr. Anat., 1880, XVIII, p. 412 e¢ seg. — See also Bot. Centralblatt, 1880, p. 1280. 2 Treub: Actes du congrés international 4 Amsterdam, 1877, Leyden, 1879, p. 146. 8 Frey: Mikroskop, p. 91. Bachmann: Dauerpraparate, p. 27. Treub: Réle du noyau dans la division des cell., 1878, p. 23. Pelletan: Le Microscope, 1870, p. 207. Gage: American Monthly Micr. Journ., 1880, p. 22; Journal of the Royal Micr. Soc., 1880, Vol. III., p. sor. . 54 MICRO-CHEMICAL REAGENTS. carmine are dissolved ; the former in one hundred parts. of water, the latter in fifty parts of concen- trated ammonia. The solutions are then mixed and filtered, evaporated to dryness, and the residue is. redissolved in a hundred times its weight of water. - ‘ Protoplasm is colored a yellowish-red by it. The nucleus quickly assumes a deeper color, especially after very short action of the coloring matter. The best degree of concentration is a one per cent. solution. Maupas recommends the use of alcohol, picro-carmine, and glacial acetic acid for staining the nucleus." [Mayer’s picro-carmine? is prepared as follows :— “To a mixture of powdered carmine (2 g.) with water (25 cc.), while heating over a water-bath, add sufficient ammonia to dissolve the carmine. The solution may then be left open for a few weeks in order that the ammonia may evaporate ; or the evaporation may be accelerated by heating (Hoyer). So long as any ammonia remains large bubbles will form while boiling, but as soon as the free ammonia has been expelled the bubbles will be small, and the color of the fluid begin to be a' ‘ little lighter. It is then allowed to cool, and fil- tered. To the filtered solution is added a concen- trated aqueous solution of picric acid (about four 1 Maupas: Comptes rendus, July 1879, No. 4, p. 250. 2 Mayer: M T. Zool. Stat. Neapel, 1880, Il., pp. 1-27. Journ. Roy. Mic. Soc., Dec. 1882, Ser. 2, Vol. IL, pp. 876-7. COLORING AGENTS. 55 volumes of the acid to one of the carmine solu- tion). The addition of the acid should cease before a precipitate begins to form. In order to protect this fluid against changes attributed to bacteria by Hoyer,’ Dr. Mayer places a small crystal of thymol in the containing bottle ; Hoyer uses chloral-hydrate (I per cent. or more) for the same purpose.” Weigert* prepares the reagent by the following process: “ Over 2 gm. of carmine are poured 4 gm. common ammonia, and the whole left twenty-four _ hours in a place protected against evaporation ; 200 gm. of a concentrated picric acid solution are then poured in; the mixture is left twenty-four hours, until all soluble matters are dissolved. Very small quantities of acetic acid are then added, until a slight precipitate comes down even after stirring; a rather copious precipitate is usually thrown down in the course of the next twenty-four hours; it should be removed by filtration. A picro-carmine which does not stain readily may be improved by the addition of acetic acid.”"—- W. T.] HAMATOXYLIN.? This substance is the active principle in the ex- tract of logwood, but.is not found in great quantity in the ¢énctura lignt campeschiani. It may be 1 Hoyer: Beitr. z. histol. Technik. — Biol. Centralblatt, 1882, II., pp. 17-19. 2 Arch. pathol. Anat. (Virchow), 1881, Vol. 84, pp. 275-294. — Journ. Roy. Micr. Soc., Feb. 1883, Ser. 2, Vol. III., p. 139. 8 Frey: Mikroskop, p. 91. Pelletan: Le Microscope, p. 209. Ranvier : 56 MICRO-CHEMICAL REAGENTS. obtained in the market ready prepared. The stain- ing fluid is made by dissolving .35 gm. hamatoxy- lin in 10 gm. water. To this are added a few drops of an alum solution (which acts as a mordant in fixing the color), made by dissolving 3 gm. alum in 30 gm. water. This makes a beautiful violet fluid, which colors the nucleus deep blue. It is the best staining fluid that is now known for the nucleus." Preparations need to lie in it for some time. They may be preserved in glycerine. I have used with success the method of staining bacteria first published by Koch. The dried preparation is treated with a concentrated extract of Campeachy-wood in water. After removing the superfluous dye with distilled water, the color is fixed by the use of dilute chromic-acid. The prep- aration can be preserved, after drying, in glycerine or Canada balsam. Cilia and the bodies of the cells are sharply differentiated by this method.? Koch has later recommended coloring with hazm- atoxylin. - Staff-shaped bacteria, however, do not color by this substance, according to him.3 I have ~ however used the hematoxylin tincture with suc- cess even on certain staff-shaped bacteria. After rinsing, my preparations are preserved dry. Histologie, p. 103. Robin: Microscope, 1877, p. 250. Bachmann: Dauer- praparate, p. 28. Schmitz: Niederrhein. Gesellsch., Nov. 1879. 1 Johow: Zellkerne d. héheren Monocotylen., Diss., Bonn, 1880, p. 9, note. 2 Koch: Conserviren und Photographiren der Bacterien.— Cohn’s Beitr. z. Biol. d. Pfl., IL, p. 421. 3 Wundinfectionskrankheiten, 1878, p. 30. COLORING AGENTS. 57 NIGROSIN. [Errera recommends* this tar derivative for staining the nucleus. It is soluble in water, but insoluble in alcohol and ether. After remaining a short time in a solution of nigrosin, the section is transferred to water, where it remains until no more of the color is removed, when it may be - mounted in glycerine or glycerine jelly, or trans- ferred to alcohol, and afterward cleared in oil of cloves and mounted in balsam. The latter me- dium serves best for specimens intended to show the chromatin; while the former are preferable for those intended to show the achromatin of Flemming. — W. T.] EOSIN. This beautiful rose-colored derivative of phthalic acid has a pronounced green fluorescence. It is employed in aqueous [or alcoholic] solution. Even a very small quantity has great coloring capacity.2, It has been used for staining Sarcina and Sarcinoglobulus. It does not appear adapted _ for use on bacteria (Bacillus, Bacterium, etc.). [I have succeeded in obtaining fair preparations of Bacillus subtilis by staining in alcohol-eosin, and 1 Procés-verbal de la séance mensuelle du 25 Juin, 1881, Soc. belge de Microscopie, p. CXXXIV. 2 Poulsen: Om nogle mikroskopiske Planteorganismer. — Nat. Foren. vidsk. Meddel., 1879-80, p. 235. Preparations stained with eosin may be preserved in glycerine. = 58 MICRO-CHEMICAL REAGENTS, mounting in balsam.— W. T.] In the tissues of higher plants, where its effect has not yet been fully studied, it stains dead protoplasm a beautiful rose color. Eosin is especially useful for coloring the plasma of sieve tubes and the nuclei of cells. It has also been used as the first dye in double- staining preparations, which are afterwards treated with Nicholson’s blue, fixed with absolute alcohol and mounted in dammar.* ANILINE COLORS.-? . Within a few years the beautiful aniline colors have been generally applied in many histological researches. In none, however, are they more useful than in the preparation of bacteria, not only in sections of animal tissues, but also in films ob- tained by drying (and hardening through a flame) a thin layer of fluid containing these organisms on the cover-glass. The discovery, by Weigert, in 1871, of the:power of carmine to color bacteria 3 led him to try the effect of the aniline salts, in which a very energetic coloring matter —almost a reagent for bacteria—was found. The names 1 Journ. of Roy. Micr. Soc., 1880, III., p. 693. 2 Wigand: Bot. Zeitung, 1862, p. 17. Hanstein: Organe et Harz- und Schleimabsonderung in den Laubknospen. — Bot. Zeitung, 1868, p. 708. 8 The “sulphate of rosanilin” has been strongly recommended by Salo- monsen for staining bacteria in putrid blood. It is used in a concentrated aqueous solution prepared by heating, and filtered after it has become cold. — Cf. Studier over Blodets Forraadnelse, Copenhagen, 1877, p. 15. COLORING AGENTS. 59 of Ehrlich and Koch are also well-known in con- nection with the further improvement of this method of research. In the following paragraphs only some of the most- useful aniline dyes will be mentioned ; their number is daily increasing. In general it may be stated, that all preparations -stained by aniline colors should be carefully washed before being mounted; they must also be kept in the dark, as most of* these colors soon fade in the light. - FUCHSIN, In.an alcoholic tincture, colors especially well thick- ened cell-walls ; the different layers often with dif- ferent intensity. Sections which are to be stained should be free from potash, which destroys the color, and they must be treated in alcohol, the addi- tion of water precipitating the dye. Stained prepa- rations remain unchanged only for a limited time. Fuchsin has recently been applied by Ehrlich* similarly to vesuvin, with methylene blue, for the staining of the Bacillus of tuberculosis. HANSTEIN’S ANILINE VIOLET Is prepared by mixing about equal parts of methyl’ violet and fuchsin, and dissolving them in alcohol. Its action depends upon the different avidity of 1 Ehrlich: Zeitschr. f. klin. Medicin, 1882, II. 60 MICRO-CHEMICAL REAGENTS. ' the various substances which are found in the tissues or cells for the mixed colors. _ Protoplasm is stained violet-blue. Amyloid sub- ~ stances, the nucleus and gums assume various shades of red; the membrane of the nucleus stains bluish ; resins, pure blue (the cuticle also colors blue in many colleters). Tannin assumes a foxy color; the cell-wall stains pale violet, deeper if it contains lignin, reddish if it is more gelatinous. Bast cells are stained a deep red; sieve tubes and the soft bast do not assume any intense color, which is of advantage in the study of the fibro- vascular bundles of endogens. METHYL VIOLET Has been recommended as a staining agent for bacteria by Koch,’ whose methods we give. A few drops of a concentrated tincture of me- thyl violet are added to 15 to 20 gm. of distilled water, so that this is deeply colored. With a small pipette a couple of drops of this are placed upon the film of bacteria to be colored, where the ~ fluid is allowed to flow back and forth until it is thought that the specimen is sufficiently stained. After a little practice it is easy to determine the proper concentration for the fluid, and the length of time it requires to act. If it is too weak the bacterian film loosens from the glass, while if it 1 Koch: Cohn’s Beitr. z. Biol. der PAl., IL, p. 406. COLORING AGENTS. 61 contains too much of the coloring matter the mass in which the bacteria lie stains too deeply. After the process of staining is finished, the preparation is rinsed with distilled water, or with a ten per cent. solution of potassic acetate. After lying for half an hour exposed to the air, the slide is ready for mounting in balsam. Gly- cerine cannot be used, as it removes the color. Preparations which are to be photographed should be mounted in a fifty per cent. solution of potassic acetate and sealed air-tight. The coloring matter is so quickly taken up by the bacteria that we have in it a useful reagent for these organisms, which might be easily con- founded with small oil globules or other very minute rounded bodies. ANILINE BLUE. Wilhelm* and Russow? have recommended an aqueous solution of this dye for staining the cal- ‘lous-plates of sieve-tubes. After the sections have been submitted to the action of the dye they are rinsed in water. The protoplasm colors violet- blue; the nuclei, deep indigo. Cellulose mem- branes assume a blue color, while the callous- plates become azure. Preparations mounted in glycerine change in a few days, so that only the 1 Wilhelm: Siebréhren, 1880, p. 36. 2 Russow: Sitzber. d. naturf. Ges., Dorpat, 1881, p. 63. 62 MICRO-CHEMICAL REAGENTS. nuclei and callous-plates remain colored. The latter are very evident, because of their considera- ble refractive power. Such preparations remain unchanged for several months. I can recommend this blue for giving a very intense color to bacteria. It is used precisely like methyl violet. The preparations should be mounted in pure Canada balsam, as the chloroform solution removes the coloring matter. 4 [More recently methyl blue has been used for ; detecting the Bacillus of consumption. The tu- berculous matter is dried in a thin film on a cover- glass in the usual way, and then floated for twenty- four hours on a fluid composed of 1 part saturated methylene blue in alcohol, 2 parts 10 per cent. potash, 200 parts distilled water. After rinsing it is submitted to the short action of a few drops of vesuvin. Only the bacteria retain the blue color. — W. T.] ; ANILINE BROWN Is used in the same way as methyl violet.’ It is better than the latter for specimens which are to be photographed; but the preparations must be mounted in glycerine, since the color is removed by potassic acetate. A concentrated solution in equal parts of glycerine and distilled water is rec- ommended as a staining fluid, the superfluous color being washed away with glycerine. 1 Koch: Cohn’s Beitr., IL, p. 406. COLORING AGENTS. 63 ANILINE GREEN (Methyl Green) Has been recommended by Hanstein for staining chlorophyll grains, to which it imparts a deep green color, different from their natural tint. Treub* recommends it for staining the nucleus. Nuclei which are not in process of division are colored pale green. In those which are dividing the so-called nuclear-plates assume an _ evident green color. Strasburger? has used it for the same purpose in combination with 1 per cent. acetic acid, [For the use of iodine-green in double staining cell-walls for convenience in class demonstrations, © see Ammonia Carmine. ] “MAGENTA. [Dr. Gibbs recommends magenta and chrysoidin for staining consumptive sputum. Three fluids | are required: A— Magenta crystals, 2 gm.; pure anilin, 3 gm.; alcohol (s.g. 830), 20 cc.; distilled water, 20 cc.; B—A saturated solution of chrys- oidin in distilled water, to which a crystal of thymol has been added to prevent deterioration ; C — Dilute nitric acid (1:2). The sputum, after being thoroughly dried on the cover-glass, is floated on A for 15-20 minutes, then washed in 1 Treub: Sur des cellules végétales 4 plusieurs noyaux. — Archives Néerlandaises, 1880, T. XV.; pp. 7, 17 of the Separate, 2 Strasburger: Zellbild. und Zelltheil., 3d ed., 1880. 64 MICRO-CHEMICAL REAGENTS, C till the color disappears, rinsed in pure water, which restores a little of the color, floated on B a few moments, transferred to absolute alcohol, and finally dried and mounted in Canada balsam. The Bacillus of consumption is stained by the magenta, which does not color putrefactive bac- teria, and so differentiated in the brown sputum.' —W. T.] The above coloring matters are those which are most commonly employed in micro-chemistry, and which give the best results. There are some others which are used for various purposes, but they may be omitted here. : 1 The Lancet, Aug. 5, 1882. APPENDIX TO PART I. MOUNTING MEDIA. Before this section is closed the most important substances used in mounting botanical specimens - should be noticed. In many cases the student must learn by trial the best medium for a prepara- tion. Still there are a number of substances which are known to be so well adapted for the preservation of very different objects that they should be tried first. GLYCERINE is excellent for nearly all botanical preparations. The Florideae and Diatoms, how- ever, usually require preservation in other media; the former since their cell-walls often swell greatly in this fluid, especially if they have not been pre- viously anhydrated with absolute alcohol; the latter because their structure does not appear in it with the required degree of distinctness. Bac- teria, also, become so transparent in glycerine, and in particular if they have not been stained, as to be almost indistinguishable. [FARRANT’S SOLUTION, which is employed some- what in animal histology, is a good substitute for glycerine in mounting many vegetable tissues. Frey’s formula is: Equal parts of gum arabic, glycerine, and a saturated solution of arsenious acid. If the slide is allowed to lie a day or two 65 66 APPENDIX TO PART L before being sealed, the gum hardens at the edge of the cover, and so aids in fastening it. This medium does not render sections so transparent as pure glycerine. GLYCERINE AND AcETIc AcID in equal parts _make a convenient preservative for many fungi and other preparations. The fluid should be boiled and filtered to remove mold spores and other impurities. Like other fluid media, this requires the employ- ment of cells of some sort,—the usual ring of asphalt or the wax cell, if firmly fixed, answering very well. In covering such a cell it is best to lower the cover gradually from one side, so that “any superfluous fluid which is forced out shall penetrate between the cell and cover-glass only at one side, where it must be carefully wiped off with blotting-paper before the cell is sealed. _ A neat and useful cell may be made by placing three small balls of white wax on the slide, at equal distances apart, just within the line where the edge of the cover is to come, and flattening them to the requisite thickness by pressing the slide against any flat body covered by a piece of muslin or paper. = Ae cA <0 =. 7" a i] eS | *8sBeTez +I8q Ty OSETA ‘usstnog