ALBERT RK MANN LIBRARY AT CORNELL UNIVERSITY. DATE DUE 1D wov 8) JL \e, ba Ei > te |_Noy 2+ 1999 GAYLORD mann Cornell University Library The original of this book is in the Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000607675 Cambridge Patural Srience Manuals BIOLOGICAL SERIES. GeneraL Epiror:—Artuur E. Suretey, M.A. FELLOW AND TUTOR OF CHRIST'S COLLEGE, CAMBRIDGE, PRACTICAL PHYSIOLOGY OF PLANTS, ZDondon: C. J. CLAY anp SONS, CAMBRIDGE UNIVERSITY PRESS WAREHOUSE, AVE MARIA LANE, AND H. K. LEWIS, 136, GOWER STREET, W.C. Cambritge: DEIGHTON, BELL, AND CO. Leipsig: F. A. BROCKHAUS. Pew Work: MACMILLAN AND CO. PRACTICAL PHYSIOLOGY OF PLANTS BY FRANCIS DARWIN, MA, F.RS., FELLOW OF CHRIST’S COLLEGE, CAMBRIDGE, AND READER IN BOTANY IN THE UNIVERSITY, AND E. HAMILTON - ACTON, M.A, FELLOW AND LECTURER OF sT JOHN’S COLLEGE, CAMBRIDGE WITH ILLUSTRATIONS. CAMBRIDGE : AT THE UNIVERSITY PRESS. 1894 [All Rights reserved. ] Cy, VN Ab Cambringe: PRINTED BY Oe J. CLAY, M.A, AND SONS, AT THE UNIVERSITY PRESS. PREFACE. N 1883 one of us began a course of instruction in the physiology of plants, of which the chief feature was the demonstration of experiments in the lecture-room. Some years later a different arrangement was made, the students were required to perform the experiments for themselves ; and at the same time laboratory work in the chemistry of metabolism was organised by one of us. To enable the students to carry out their work, written instructions were needed, and the present book is the result of an extension and elaboration of what we prepared for our, classes. The book makes no pretence to completeness, it contains merely such a selection of experimental and ana- lytical work as seems to us suitable for botanical students. Part I, which deals with general physiology, is necessarily of a somewhat more elementary character than Part JI, which treats a particular department of physiology in a more special manner, and presupposes a greater amount of knowledge on the part of the student. D. A. b vi PREFACE. The footnotes in Part I (which form an addition to the original draft of the work) merely supply a rough guide to some parts of the literature. Nor is any attempt made to give a full account of the literature of the subjects dealt with in Part II. The papers enumerated at the head of each chapter are merely recommended as being illustrative of the use, in actual research, of the methods. described. We gladly take this opportunity of expressing our thanks to Mr F. F. Blackman, Demonstrator of Botany in the University, for much valuable help in the arrange-’ ment of the experiments in Part I. Also to the Cambridge Scientific Instrument Company for the use of the clichés for Figs. 25 and 26. Botanica, LaBoRAToRY, CAMBRIDGE. August 19, 1894, CONTENTS. PART I. GENERAL PHYSIOLOGY. CHAPTER I. ON SOME OF THE CONDITIONS AFFECTING THE LIFE OF PLANTS. Section A. Respiration. Ei Respiration; accumulation of CO, produced by germinating seeds or buds. 2. Absorption of CO, by- potash. 3. Sachs’ method. 4, 5, 6. Intramolecular respiration. 7, 8,9. Rise of temperature during respiration. 10. Germination of oily seeds. 11. Succulents ‘i . ‘ ‘ 2 pp. 1—10. Section B. The effect of various temperatures: of certain poisons: and of electrical shock. 12. Injurious temperature demonstrated on Oxalis leaf. 13. Do., injected leaf. 14. Do., beetroot. 15. Do., dry and soaked seeds. 16. Circulation of pro- toplasm, Sachs’ Hot-Box. 17. Velten’s method. 18. Circulation of protoplasm, effect of COQ,. 19. Do., chloroform. 20. Oxalis leaf killed by chloroform. 21. Do. by phenol. 22, Do. by in- duced current. 23. Effect of induced current on circulating pro- toplasm . : a A i % i a pp. 10—16. b2 Vili CONTENTS. CHAPTER II. ASSIMILATION OF CARBON. Szcrion A. Formation of starch. 24. Sachs’ Iodine method, 25. Schimper’s method. 26. Variegated leaves. 27. Disappear- ance of starch in darkness. 28. Effect of dull light. 29. Local effect. 30. Gardiner’s experiment. 31. Rays of different refran- gibility. 32. Terrestrial leaves under water. 33. Excess of CO,, 34, Plants deprived of CO,. 35. Temperature and assimilation. 36. ‘Gain in weight. 37. Translocation. 38. Assimilation of sugar. 39. Do., formaldehyde. 40. Leucoplasts. pp. 17—30. Section B. Evolution of oxygen. i. Bubbles of gas. 42. Light of varying intensity. 43. Dependence on presence of CO,, 44, Temperature and gas evolution. 45. Chloroform. 46. Co- loured lights. +47. Collection of gas evolved. 48. Engelmann’s blood method. 49. Phosphorus method. 50. Dehérain’s method. 51. Gas analysis, Pfeffer’s method. 52, Gas analysis, Winkler. Hempel apparatus. 53. Engelmann’s bacterial method. 54. Dif- fusion of gas through cuticle . . 5 . . pp. 30-43, Section C. Reactions of chlorophyll and of some other pig- ments. 55. Separation by benzene, ether, olive oil. 56. Action of light. 57. Aeration and effect of light. 58. Action of acid. 59. Action of copper-salts, 60. Stability of the copper compound. 61. Spectroscopic examination. 62. Red colour of Ricinus, Coleus &e. 63. Floridese. 64. Brown sea-weeds . - pp. 48—46. Section D, Production of chlorophyll, etiolation, sun- and shade-leaves. 65. Appearance of the green colour. 66. Etiolin and light. 67. Pinus. 68. Chlorophyll formation and tempera- ture. 69, 70. Do. and oxygen. 71. Do. and iron salts. 72. Form of etiolated plants. 73. Sun- and shade-leaves. pp. 46—50. CHAPTER III. FURTHER EXPERIMENTS ON NUTRITION. Srection A. Water-culture. 74, Method. 75. Potassium salts necessary. 76. Phosphoric acid necessary. 77. Experi-: ments with Lemna. 78. Calcium oxalate formation. 79. Nitrate reaction . : . : . . . : . : pp. 51—60. CONTENTS. 1x Section B, Nutrition of Fungi and of Drosera. 80. Method. 81. Various cultures. 82. Puccinia. 83. Hanging-drop cultures. 84. Germination of spores. 85. Drosera, digestion of white of egg. 86. Drosera, benefit from feeding 7 7 ‘ , pp. 60—66. _ Secrion C, Functions of roots. 87. De Saussure’s experiment. 88, 89. Root pressure. 90. Moll’s experiment. 91. Absorption by means of dead roots . : ‘ : : - . pp. 66—71. CHAPTER IV. TRANSPIRATION. Secrion A. Absorption of water. 92. Potometer. 93. Kohl’s method. 94, Effect of sunshine. 95, Effect of wind. 96. Effect of light. 97—100. Negative pressure. 101. Permeability of mem- branes. 102, Oozing of water from wood, 103. Permeability of splint-wood. 104. Injection of flaccid shoot. 105. Emulsion ex- periment. 106. Injection with cocoa-butter. 107. Compression. 108. Incisions. 109. Cross-cuts. 110. Do., course shown by eosin. 111. Air-pump and potometer. 112. Strasburger’s air- pump experiment . . : . 7 , . . pp. 72—-88. Section B. Loss of water. 113. Loss of weight during transpi- ration. 114, Transpiration compared with evaporation from water. 115. Loss compared with absorption. 116. Spring balance, pp. 88—93. Section C. Stomata bloom lenticels. 117. Stomatal transpiration. 118. Stipa-hygrometer. 119. Stomata and inter- cellular spaces, 120, Leaf injected with water. 121. Frost effects. 122. Blocking of stomata with water. 123. Movements of stomata. 124. Do. with induced current. 125. Lenticels and intercellular spaces. 126, Bloom as affecting transpiration . pp. 98—100. CHAPTER V. PHYSICAL AND MECHANICAL PROPERTIES. Section A. Imbibition, hygroscopic movements, polariscope, osmosis. 127. Laminaria, microscopic observation. 128. Lami- naria increase not uniform in all direction. 129. Imbibition of seeds, x CONTENTS. temperature effect. 130. Imbibition; salt solution. 131. Stipa, action of. 132, 133. Stipa, temperature, 134, Stipa, salt solu- tion. 185. Stipa, mechanism of movement. 136. Nobbe’s ex- periment. 137. Variability in the swelling of seeds. 138. Rise of temperature. 189. Work done during imbibition. 140. Polari- scope. 141, Polariscope, observations on strained glass rods, 142, Traube’s artificial cells. 143. Slowness of diffusion, 144. Re- lation of membrane to diffusing fluid. 145, Absorption of methylene blue by living cell. é . ‘ ‘ s . pp. 101—113. Szcrron B. Turgor. 146. Plasmolysis, microscopic observations. 147. Recovery after plasmolysis. 148. Osmotic strength of cell-sap in terms of KNO,. 149. Isotonic coefficient. 150. Do., micro- scopic method. 151. Hydrostatic pressure in turgescent tissue, 152. Pfeffer’sgypsum method. . . . #. pp. 118—121, Section C. Tensions of tissues. 153. Longitudinal tensions, 154, Extension of pith in water. 155. Changes in transverse di- mensions of pith. 156. Tangential dimension. 157. Shortening of roots. 158. Imperfect elasticity of tissues. 159. Cyclometer, 160. Hofmeister’s experiment. 161. Loss of rigidity. 162. In- crease in length. 163. Splitting turgescent tissues. 164. Split- ting a root, 165. Splitting a pulvinus ‘ . pp. 121—129. CHAPTER VI. GROWTH. . Section A. Experiments without special apparatus. 166. Method. 167. Free oxygen necessary. 168. Respiration necessary. 169. Full turgescence necessary. 170. Growth at various temperatures, pp. 180—188, Section B. Distribution of growth. 171. Distribution in roots, 172. In air-roots. 173. In stems. 174. Grand period, time observation. 175. Growth and plasmolytic shrinking pp. 138—186. Section C. Auxanometers. 176. Methods. 177. Descent of the weight measured on a scale. 178. Micrometer screw. 179. Are-indicator. 180. Microscope. 181. Self-recording aux- anometer. 182. Do., simple form. 183, 184. Growth and tem- perature, microscopic method. 185. Growth and respiration, micro- CONTENTS, xi scopic method. 186. Growth and temperature, auxanometer. 187. Growth and light, auxanometer. 188. Growth and light, Phycomyces. 189. Growth and light, Sinapis. 190. Periodicity, auxanometer . 2 . F "i . . r pp. 186—149. CHAPTER VII CURVATURES. Szction A. Geotropism, 191. Region of growth and region of curvature, roots. 192. Do., stems. 193. Subsequent change in curvature. 194. Grass-haulms. 195. Noll’s experiment, grass- haulms. 196, 197. Geotropism and respiration. 198. Johnson’s experiment, 199. Pinot’s experiment. 200. Knight’s experiment. 201. Sudden curvature. 202, 203. After effect . pp. 150—159, Section B. CGurvatures due to injury &c. 204. Decapitated roots. 205. Decapitation prevents perception of stimulus. 206. Re- covery after decapitation. 207. Curvature due to injury. 208. Cie- sielski’s experiment. 209. Drooping of leaves in frost pp. 159—164. Section C. Heliotropism. 210. Positive heliotropism. 211. After effect. 212. Light of high refrangibility most effective. 213. Nega- tive heliotropism. 214. eiaes and ie acting together. 215. Trans- mitted stimulus . fi a : pp. 165—168. Section D. Diaheliotropism, diageotropism &c. 216. Diahelio- tropism. 217. Due to specific sensitiveness, klinostat. 2174. EHx- clusion of helio- and geotropism. 218. Rectipetality. 219. Theory of klinostat, grass-haulms. 220. Do., Cucurbita. 221. Diageo- tropism, roots. 222. Growth of secondary roots in light. 223. Dia- geotropism, Narcissus. 224, Horizontal branches. 225. Torsion of internodes. 226. Budsoftheyew. 227. Epinasty. 228. Epinasty and geotropism. 229. Nutation of epicotyls , pp. 168—183. CHAPTER VIII. FURTHER EXPERIMENTS ON MOVEMENT. Section A. Stimulus of contact, chemical agency, moisture, ehanges in illumination and temperature. 230. Tendrils, sensi- tive to contact. 231. Tendrils, De Vries’ injection experiment. xii CONTENTS. 232. Tendrils, Pfeffer’s contact experiment, 233. Mimosa, move- ments produced by stimulation. 284. Mimosa, temperature. 235. Mi- ‘mosa, darkness. 236. Mimosa, continued stimulation. 237. Ovalis acetosella, sensitiveness. 238. Oxalis, Briicke’s experiment. 239. Dro- sera, stimulated by meat and by inorganic matter. 240. Drosera stimulated by dilute solutions. 241. Drosera, inflection indirectly causes. 242. Berberis, irritable stamens. 243. Berberis, effect of chloroform. 244. Stigma of Mimulus. 245. Centaurea, irritable stamens. 246. Phycomyces, curvature towardsiron. 247. Hydro- tropism. 248. Movement of chloroplasts. 249. Chemotaxis, antherozoids. 250. Opening and closing of tulip, temperature. 251. Tulip, sensitive to small change of temperature. 252. Crocus, mechanism of movement. 253. Light and darkness, daisy. 254. Light: and darkness, Trifolium. 255. Nyctitropic movements, Trifolium, 256. Do., Mimosa, self-recorded. 257. Paraheliotropism, Averrhoa. pp. 184-—208. Section B. Autonomous movements. Periodicity. 258. Cir. ecumuutation. 259. Do., twining plants. 260. Autonomous movements, Trifolium. 261. Do., Averrhoa. 262. Do., Desmo- dium. 263. Periodicity, light and darkness, daisy. 264. Perio-., dicity, temperature, daisy. 265. Contrast, daisy pp. 209—216. PART II. CHEMISTRY OF METABOLISM. CHAPTER IX. INTRODUCTION. SOLVENTS. METHODS OF EXTRACTION. GENERAL NOTES ON APPARATUS AND MANIPULATION. Introductory. Preparation of material to be examined. Preparation of extracts: non-nitrogenous plastic substances. Preparation of ex- tracts: nitrogenous plastic substances. Filtration. Evaporation of solutions. Changes occurring in solutions on keeping pp. 221230. CONTENTS, xiii CHAPTER X. PROTEIDS. AMIDES. AMMONIA, NITRATES, &C. Literature. Practical classification of nitrogenous plastic, sub- stances. Qualitative examination for proteids insoluble in ‘water, soluble in dilute alkali—for proteids soluble in water—for peptones and albu- moses—for amides—for ammonia, nitrates, nitrites. Estimation of proteids—of peptones and albumoses. Estimation of amides. Estima- tion of ammonia, nitrates and nitrites. Experiments on nitrogenous metabolism. Qualitative examination of Onobrychis sativa for proteids &c. Comparison of amounts of proteids, peptones, amides in seeds of Onobrychis and in shoots of the same grown under various conditions. Comparison of amounts of ammonia, nitrates, nitrites in shoots of Onobrychis from plants variously treated . : pp. 231—242. CHAPTER XI. OILS AND FATS. GLYCERIN. Literature. Extraction of oils and fats. Qualitative examination of benzene extract. Reactions of glycerin. Quantitative examination. Determination of total oils and fats—of free fatty -acids—of glycerin. Experiments. Determination of oils and fats in seeds of Lepidium— in seedlings of Lepidium, young and old . 5 Fi pp. 248—247. CHAPTER XII. TANNINS AND GLUCOSIDES. Literature. Extraction of tannins and glucosides. Many different bodies included under heading tannin and glucoside. Qualitative tests for tannins. Qualitative tests for phloroglucin. Removal of tannins. before examining for sugars. Determination of whether « tannin is a glucoside or not. Glucosides, Identification of salicin. Examination for certain sugars. Experiments. Testing extract of willow-bark for tannin, salicin, sugars. Estimation of certain sugars in young and old fruits of Musa sapientum . ‘ 3 - : . pp. 248—257. XIV CONTENTS. CHAPTER XITI. DEXTRINS AND SUGARS, GLUCOSES, CANE-SUGAR, MALTOSE, &¢, Literature. Soluble carbohydrates. Qualitative test for fermentable sugars. Estimation of fermentable sugars. Removal of dextrins. Tests for glucoses, cane-sugar, maltose, mannite, pentoses. Estimation of glucoses, cane-sugar, maltose. Calculation of results. Experiments on sugars. Testing leaves of Tropgolum majus for various sugars. Estima- tion of fermentable sugars in leaves and roots of Beta vulgaris. Estima. tion of various sugars in leaves of Beta vulgaris under different conditions, pp. 258—271. CHAPTER XIV. STARCH. CELLULOSE. Literature. Estimation of starch and cellulose. Experiments on starch. Estimation of starch in the potato by different processes— in leaves of Acer pseudo-platanus under different conditions. In grains of wheat before and after germination ss 3 fs pp. 272—276, CHAPTER XV. ORGANIC ACIDS AND SALTS. Literature of organic acids. Qualitative examination for organic acids, Determination of ‘acidity’ of extracts. Literature of inorganic salts. Preparation of ash of tissues. The constituents of the ash. Estimation of chlorine—phosphoric acid—alkalies—in ash. Estimation of calcium oxalate in tissues. Experiments on organic acids. Compari- son of acidity of juice from old and young rhubarb petioles—of acidity and amounts of sugars in juice from ripe and unripe apples. Experi- ments on inorganic salts. Weights of ash from normal and etiolated leaves. Estimation of phosphoric acid and alkalies in leaves and grains: of barley—of calcium oxalate in young and old leaves of Sempervivum: tectorum . . . . . . . . . pp. 277 —284, CONTENTS. XV CHAPTER XVI. UNORGANISED FERMENTS. (ENZYMES.) Literature. Extraction of enzymes. Comparison of activity of extracts. Experiments on diastatic ferments. Preparation of solid diastase. Influence of filtration on diastatic power of extracts. Com- parison of diastatic power of malt and ungerminated barley—of leaves of Pisum sativum and Trifolium pratense. Experiments on invertase and glycase. Decomposition of « glucoside (salicin) by an enzyme (synaptase) from another plant ‘ ; 7 ‘ pp. 285— 293. CHAPTER XVII. GENERAL EXPERIMENTS. The increase in weight of growing Spirogyra. The influence of in- organic salts on the formation -of starch, The changes in the reserve materials of an oily seed during germination under different conditions. pp. 294—295. APPENDIX I. Notes on the results likely to be obtained in experiments on metabo- lism . . . ‘ : - : : . 5 pp. 296—S04. APPENDIX II. List of reagents and material required for experiments on meta- bolism ... a2 8 ‘ ‘ . . F pp. 305—308. FIG, 6 LIST OF ILLUSTRATIONS. Apparatus for demonstrating respiration . Apparatus for estimating CO, produced during caapiratton Foil clamp for holding cover-slips together under water, for use with Velten’s hot-stage : : . - Apparatus for the preparation of mien free from CO, it not free from oxygen . é . . . . Arrangement for the culture of a in an atmosphere thee from CO, . . $ : < 3 Apparatus for gananulpes for use in experiments on assimi- lation . 7, 8,9 Winklesttempal apatite for gas- -amelyate F 10,104 Lemna cultivated in various nutrient solutions . 11 12 13 14 15 16 17 18 19 20 Apparatus for demonstrating root pressure . 5 . F Another apparatus for the same purpose . The Potometer, for estimating the absorption of water bya a cut branch . : A é . 2 A modification of Kohl’s aipumtne for ‘th same purpose . A method of preventing evaporation from the surface of a flower-pot . c : . Apparatus for comparing _ ia ieciepinalion with the sehen absorbed . . ‘ A spring-balance for use in eanentcation expetinenta. A hygrometer made of the awn of Stipa for performing Garreau’s experiment . . 5‘ ‘ é r : Devaux’s gelatine method of making an air-tight sanetion with the petiole of a leaf . . : Apparatus for demonstrating the ligeroasopis meeperiee of the awn of Stipa 3 2 ‘ f 103 LIST OF ILLUSTRATIONS. Diagram illustrating the use of turgescent tissue in De Vries’ experiments on isotonic coefficients ., Tracings from split portions of the hypocotyl of Ricinus ea in experiments on isotonic coefficients . 2 : Pfeffer’s gypsum method Arrangement for demonstrating that Lagpeeeu ost ise rigidity when bent . ‘ < Micrometer-screw used in growth aupertneuts Recording auxanometer . Method of using the necdnomiidedayee Tracing illustrating the effect of an increase of teunpoentiatd on growth Hanging writer for veconting: geonnepie. or thay movements on a revolving drum . Illustrating the curvature of a oot whieh — weeerreradl front the effects of decapitation . Curvature of roots produced i small planes of easd attadhed to the tips . ‘ . s 3 Drooping of laurel leaves in a frost A twig of Veronica salicifolia exposed to oblique flhunitantion The klinostat . 4 2 Section showing part of the mechan of he idingetat A seedling Cucurbita which has germinated on the klinostat ; showing the frill-like growth of the heel or peg Diageotropism of the flower of Narcissus poeticus The twisted internodes of Lonicera : Leaves of clover in the day and night nositiod - The sleep-movements of Mimosa recorded on a drum by means of a hanging writer Paraheliotropic movement in Averrhoa Dilimbi ‘ Diagram representing the circumnutation of a cabbage- seedling i ‘ * 6 Apparatus for distillation ides vediaged pressure . Xvii PAGE 115 116 120 127 138 141 145 147 158 160 162 164 169 171 172 177 179 181 204 206 207 211 228 ADDENDA AND CORRIGENDA. Page 1, line 4 for ‘‘ grow-” read ‘‘ growth-’’- » 68, line 14 for “developement” read “ development.” » 120, Fig. 23 is copied from Pfeffer’s paper in Abhandl, k. Stichs Ges. Band xx. 1893. » 121, line 8 for ‘‘silk-paper” read ‘“‘ tissue-paper ”. » 188, 141, Figs. 25 and 26 are from the Catalogue of the Cam- bridge Scientific Instrument Company. PART I. GENERAL PHYSIOLOGY. CHAPTER I. ON SOME OF THE CONDITIONS AFFECTING THE LIFE OF PLANTS. Section A. Respiration. Suction B. Temperature— Poisons — Electricity. Section A. Respiration. The presence of free oxygen is a necessary condition of the life of all the higher plants. This fact will be more conveniently demonstrated in the chapters on growth and grow-curvatures. The present section is intended as an introduction to the study of the facts without special reference to the importance of respiration. (1) Take a stoppered jar of about 500 c.c. capacity, fill it to one-third of its. height with horse-chestnut buds (in spring) or with beans (in winter) which have been soaked in water for 12 hours and have been afterwards placed in damp cocoa-fibre for 12 hours. Place the jar in a warm, dark room, and after 12 hours cautiously open the jar and lower a lighted taper which will be extinguished as it enters the CO, produced. D. A. 2 RESPIRATION. [cH. 1 (2) Take a filtering flask of 400 or 500 cc. capacity, having a lateral opening as shown in fig. 1 to which a glass Fie. 1. Exp. 2. tube, A, (4 or 5mm. bore) is attached by thick rubber tubing and wire ties. The end of A dips into the mercury in the beaker Hg. The flask contains enough CH. I] RESPIRATION. 3 germinating barley to cover a piece of wet filter paper at the bottom of the flask. Barley germinates well in winter: it should be soaked in water for 24 hours and kept in damp air for 24 hours before use. A test-tube T half full of strong KHO is introduced into the flask, which is then closed by a sound tightly fitting rubber cork, As the CO,, produced by respiration, is absorbed by the KHO, the mercury in the beaker Hg is sucked up the tube A. In starting the experiment it is necessary to warm the air in the flask before the end of A is forced into the Hg, so that as the air cools again the mercury may be sucked a little way up the tube to a point which will then serve as zero for subsequent observations. The warming may be done by immersing the flask in water at 40° for a few minutes; or it may be warmed by the hands. (3) Sachs’ method?. Place 10 germinating beans in a jar A, fig. 2, closed by an india-rubber cork pierced by two holes and fitted with glass tubes. One tube is connected with an aspi- rator so that a current of air is drawn through the vessel and keeps up continuous normal respiration. The other tube serves to admit to the flask air free from CO,; for this purpose it is connected with a filtering bottle F containing a few sticks of KHO. The air is admitted to F through a tube 7 filled with soda-lime. In order that it may be certain that no extraneous CO, enters the 1 Physiologie Végétale (French Translation), 1868, p. 295, fig. 35. Also Pfeffer’s Physiologie, 1. p. 349, fig. 38. 1—2 4 | RESPIRATION. [cH. 1 flask, another washing bottle B containing baryta water is fitted between F and the experimental flask, A. The drop-aspirator figured by Detmer’ answers very well, it is made from a distillation tube and is attached to a tap through which a current of water in detached drops passes, and produces a correspondingly slow suction- current of air at the side tube (c in Detmer’s figure); The outflow tube. should be about 2 feet in length to insure that the suction is strong enough. In the absence of a drop-aspirator the current may be moderated as Sachs recommends by allowing the air to enter the first washing bottle through a fine capillary tube. If the sink in the laboratory is inconveniently placed. the 1 Praktikum, p. 179, fig. 76. CH. I] RESPIRATION. 5 air suction may be carried to any part of the room by means of fine lead-tubing. Between the flask and the aspirator two washing bottles P, C, containing baryta water are fitted in which the CO, produced by respiration of the plants is caught and precipitated as BaCO,. The amount of the precipitate may be estimated by titration, for which see Sutton, Volumetric Analysis, 5th Ed. pp. 80—89. (4) Intramolecular respiration. To demonstrate the fact, the following simple form of experiment may be tried. Soak 6 peas in water for 12 hours, when the seed- coats can easily be removed without injury to the embryo; the removal of the testa is necessary to avoid introducing air with the peas, the object of the experiment being to show that CO, is produced in the absence of free oxygen. Fill a test-tube with mercury and invert it in a mercury trough which should stand in a strong wooden tray. This precaution is advisable in all experiments involving the use of mercury, so that if any accident occurs the mercury may not escape and get into the cracks of the floor. It is desirable to use clean mercury which has been redistilled, but the experiment will succeed perfectly without any special care being taken in its treatment. Pass the peeled peas one at a time under the rim of the test-tube so that they float up into the mercury, and occupy the upper end of the test-tube. On the following day it will be found that the test-tube is half full of gas, 6 RESPIRATION. [CH. I and the peas are therefore clearly visible, instead of being partly hidden by mercury. A few drops of water are now passed in under the test- tube rim with a bent pipette, and a fragment of caustic potash added from below, in this way a strong solution of KHO is supplied, by which the CO, is absorbed. (5) Another method. The Torricellian vacuum was used by Wortmann in his work on intramolecular respiration’. A tube closed at one end, and of greater length than the height of the barometric column, is filled with, and inverted over mercury. Three or four peas are floated into the vacuum at the top of the tube. After 24 hours a depression of several cm. will be observed in the height of the column, which will rise to nearly its original level on the addition of KHO. (6) Pfeffer’s method. ‘To get -accurate results another method must be followed ; the following is taken from Pfeffer’s paper in his Tiibingen Untersuchungen, Vol. 1. p. 637. The principle is that already described for estimating ordinary respiration, but instead of air, a current of hydrogen is drawn through the vessel in which the plants are contained. It is necessary to prevent the entrance of extraneous CO, and to make sure that the hydrogen has no admixture of oxygen. 1 Sachs’ Arbeiten, u. p. 500. CH. I] RISE OF TEMPERATURE. 7 (7) Rise of Temperature. The spadix of an Arum is the classical material for demonstrating the heat produced by respiration in plants. We have used the British Arum maculatum, but for some reason the experiment has not always succeeded. Remove the spathe from 4 or 5 spadices (of which the spathes are just beginning to expand) and fix their stalks in wet sand, so that the specimens stand vertically in a ring. Suspend a delicate thermometer in such a way that the bulb falls among the Arums, and wrap them round with a flock of cotton wool so that they may touch the bulb. Kraus+ has shown that the respiration of Arums is easily affected by any injury to the surface of the spadix, the specimens must therefore be delicately handled. Hang a second thermometer (which must be previously compared with the first) close to the Arums, and cover all with a bell-jar standing in water. The bell-jar is necessary to prevent currents of air. (8) Rise of temperature. The following is Sachs’ arrangement for showing the rise of temperature in germinating peas”. We have found flowers, such as those of dandelions, treated in the same way to answer well. Gather a large handful of dandelion flowers* cutting the stalks just below the head, place them in a large funnel supported in a beaker half-filled with KHO. Hang a thermometer so that the bulb is 1G, Kraus, Naturforsch. Ges. Halle, xv1. 1884. 2 Sachs’ Text-book, Hd. u. p. 724. Fig. 472. 3 Or in winter of young flowers and buds of a small-flowered Chrysan- themum. 8 OILY SEEDS. [CH. I covered by the flowers, and let the control thermometer be supported in a funnel containing coarse sawdust slightly moistened and loosely packed. This arrangement is meant to equalise the conditions of the two thermometers, and to prevent the bulb among the flowers acting as a wet- bulb. We find, with the control thermometer hanging simply in the air, that the flowers keep about 2° C. above the control temperature, As before, the whole must be covered with a bell-jar. Sachs uses a tubulated bell of which the opening is plugged with cotton-wool. (9) Oxygen necessary. Several of the earlier observers have shown that when the air is replaced by indifferent gas the tempera-_ ture falls. Pfeffer! recommends that the germinating seeds or other material should be placed in a glass balloon having three apertures—one of which serves for a thermo- meter. When the temperature of the respiring material has been proved to be steadily above that of the surround- ing air, the atmosphere in the balloon is replaced by hydrogen, for which purpose the two lateral apertures will serve, The readings of the two thermometers should now become practically equal, and it should be possible to re-establish the difference by readmitting air. (10) Germination of oily seeds, Repeat experiment 2, using oily seeds for the ger- minating material, and omitting the test-tube of KHO. Hemp seeds will serve the purpose: they should not be 1 See Pfeffer, Physiologie, u.p, 408. Fig. 40. CH. I] SUCCULENTS. 9 soaked in water before they are used, but placed air-dry on wet filter paper in the flask. The mercury rises in the tube in spite of the absence of KHO. As a control experiment an equal weight of barley should be placed in precisely similar conditions. In our experiments the column of mercury was depressed in the case of the barley. The two flasks should be kept as nearly as possible at a constant temperature; for an accurate determination of the results, barometer readings must be taken at the beginning and end of the experiment, and corresponding corrections must be made. But any external influences will affect the experimental and the control flask equally, so that the difference between their readings must depend on the different behaviour of the seeds in germination. (11) Succulents}. In certain succulents an increase of the acidity of the cell sap is accompanied by a fixation of oxygen. There- fore, when the plant is placed in the apparatus used in experiment 10, a rise of the mercury takes place. The following is taken from Detmer, p. 224, He recommends Rochea falcata as especially good for the experiment. A leaf is taken from the plant at the close of a hot summer day, cut into pieces and introduced into the eudiometer (Detmer, fig. 14). The lower end of the graduated tube is placed in water and the apparatus kept in the dark until the following morning when a considerable rise in the water column is visible. As a 1 See De Saussure, Recherches Chimique. (An. xii=1804), p. 65. 10 INJURIOUS TEMPERATURES. (cH. I control a similar eudiometer is fitted up with non-succu- lents, such as pieces of young sunflower’. Section B. The effect of various temperatures : of certain poisons: and of electrical shock. (12) Temperature. To get a rough idea of the upper limit of temperature which ordinary plants can endure, it is best to make a few simple experiments with plants in which the moment of death is marked by some obvious change, e.g. in colour. Ozxalis acetosella is useful for this purpose, because death is indicated by a dingy yellow colour due to the action of the acid cell-sap on the chlorophyll. Fill a beaker with water at 25°C., and suspend in it a thermometer, to the bulb of which a leaf of Oxalis is attached. Heat the water by means of a gas flame, and note the temperature at which the leaf loses its fresh green tint. (13) Temperature. Ifthe Oxalis leaf is injected with water before the experi- ment, it changes colour at a temperature several degrees. lower than in (12). This is a simple way of demonstrating | the fact given by Sachs (Physiologie Végétale, p. 71) that plants in air endure a temperature which they cannot bear in water. The cells of the injected Oxalis leaf acquire the tem- 1 To complete the experiment, the relative acidity of the Rochea in the evening and next morning, should be compared, See Part 1, CH. I] INJURIOUS TEMPERATURES. 11 perature of the water more quickly than those of the uninjected leaf, and this is probably the explanation of the difference. (14) Temperature. When a turgid cell is killed the cell sap escapes through the dead protoplasmic wall, and if the cell sap is coloured, the escape will be a marked occurrence. The Beet-root may be used in this way as a rough indicator of the fatal temperature at which the protoplasm is killed. Cut a slice of beet-root, wash it to free it from any cell sap adhering to the cut surfaces, and suspend it with a thermometer in a beaker of water at about 25° C., which is to be heated as in experiment 12. A similar experiment may be more accurately made under the microscope, using one of the methods described below, by which a microscopic object can be subjected to a given temperature. (15) Dry and soaked seeds’. The effect of a high temperature depends, among other things, on the condition of the subject of the experiment. Thus, dry seeds can endure a temperature which is fatal to seeds which have been soaked. Take 20 peas, half of which (a) are to be left in water for 12 hours, or until they are thoroughly soaked, while the other 10 (5) are reserved for comparison. The dry seeds (b) are placed in a dry test-tube, while the imbibed seeds (a) are placed in a test-tube half full of water: both 1 Sachs’ Physiologie (French Tr.), p. 72. Fig. 8. 12 PROTOPLASMIC CIRCULATION. (cH. 1 test-tubes are corked and are immersed in a beaker of water kept by means of thermostat at 60°C. for 2 hours. Both sets of seeds should now be sown in damp saw-dust, —the lot (b) having been previously soaked in cold water for twelve hours: it will be found that lot (6) germinate, while (a) do not do so, and show other obvious signs of being dead. (16) Circulation of Protoplasm—Sachs’ Hot-Boa. Any parts of plants, in which circulating protoplasm can be observed, serve as material for studying the effects of temperature. The staminal hairs of Tradescantia, or other plant-hairs are convenient, or the tentacles of Drosera may be used. But the leaves of Elodea are perhaps most easily obtainable throughout the year. Mount a leaf of Elodea upside down in a drop of water under a large cover-glass; look for circulating protoplasm near the mid-rib}, and subject it to gradually increasing temperature by means of any of the recognised “hot- stages,” e.g. with Sachs’ Hot-box. The arrangement is described and figured in Sachs’ Text Book, English Trans- lation, p. 736. It consists of a hollow-walled metal-box, into which the microscope is placed so that by filling the walls with warm water the object under observation can be subjected to the desired temperature. A window admits light, and a hole in the moveable lid allows the microscope-tube and fine adjustment to project. The felt lining of the lid should be wetted and a little water 1 The leaves should be cut off an hour before they are wanted, because, in winter at any rate, circulation is not visible until some time after the leaves have been cut. CH. I] EFFECT OF HEAT. 13 should be spilt on the floor of the box, so that the atmo- sphere surrounding the object may be damp. A thermo- meter passes through a hole in the lid or, as we find more convenient, through a cork fitting one of the lateral openings. The glass slip on which the object is mounted should be separated from the stage of the microscope by a perforated plate of cork, so that the object may assume the temperature of the air, rather than that of the micro- scope,—although these two temperatures will after a time be nearly identical. The hot-box may be conveniently supported on wooden blocks and heated by a gas flame. As the warming of a considerable mass of water is a slow process it is advisable to fill the box with water 10°C. above the room tempera- ture. Notice the accelerating effect of warmth, and record the temperature at which the circulation (1) becomes slower (about 42°C.); (2) stops altogether (about 46° C.). (17) Velten’s method. A simpler and quicker plan is that of Velten’, which, Fic. 3. Exp. 17. 1 Flora, 1876, p. 177, also F. Darwin, Q. Journal Microscopical Science, N.S. Vol. xvit. p. 245. Cf. Pfeffer, Zeitschr. fiir wiss. Mikro- skopie, 1890. 14 CARBON DIOXIDE. (cH. I however, should not be used with a valuable microscope. The objective and the preparation are immersed in water contained in a glass dish standing on the stage of the microscope. A siphon, provided with a tap, allows warm water to run into the dish, while a second siphon and tap provides for the overflow. The object is mounted between two cover-slips, which are gently clamped together by a bit of tinfoil of the form shown in fig. 3, the flaps being bent up at 45° along the dotted lines. Unless some such plan is adopted, the upper cover-slip is liable to be washed off by currents in the water. (17a) The same method may be used to subject cir- culating protoplasm to a low temperature. (18) Effect of CO,, To observe the effect of gases on circulating protoplasm, the Elodea leaf is mounted in a small drop of water, on the under surface of a cover-slip forming the roof of a gas chamber: if the cover-slip projects fairly well beyond the edges of the hole on which it lies, the apparatus can be made sufficiently gas-tight by painting the edges of the cover-slip with olive oil; or the slip may be fixed with putty. Having under observation a circulating cell, attach the tube of the gas chamber to the CO,-generating apparatus’, and observe that the protoplasm comes to rest: by disconnecting and allowing air to pass, the circulation can be renewed. 1 The CO, must be made to bubble through water before it reaches the gas chamber. CH. I] POISONS. 15 In this experiment the CO, acts, not by preventing access of oxygen, but as a narcotic. This can be shown by connecting with a hydrogen-generator, the rapid retardation previously observed will be absent. (19) Chloroform. The same apparatus serves to demonstrate the effect of chloroform and other hurtful vapours. Shake up one per cent. of chloroform in a bottle of water, through which (by means of an aspirator) a current of air is made to bubble. The air, thus charged with chloroform is allowed to pass through the gas-chamber. The circulation can be stopped without killing the leaf. (20) Chloroform. The effects of poisons may also be conveniently demon- strated on the leaf of Owxalis acetosella, using the colour test already described. Shake up 1 cc. chloroform in 200 e@c. water in a stoppered bottle and add an Oxalis leaf cut into small pieces. Note the time required for the discoloration to occur. (21) Carbolic acid. (Phenol.) Make the same experiment, substituting 0°5 per cent. carbolic acid for chloroform-water. (22) Induced current. If an Oxalis leaf is impaled on a pair of needles (in an insulated handle) connected with the induction coil, the region between the punctures is killed and becomes 16 INDUCED CURRENT. [cH. 1 discoloured when the current passes: the needle points should not be more than 2—3 mm. apart. (28) Induced current. Two triangles of platinum foil are sealing-waxed on to a glass-slip, the points being about 1 mm. apart. To make the platinum adhere well it is necessary to heat the glass over a flame until the wax between the glass and the metal is thoroughly soft, and then to apply pressure. An Elodea leaf is mounted in water so that a cell showing circulation lies between the points, and by connecting the foil triangles with an induction coil, the effect of the current can be observed. The wires from the coil are most conveniently connected by means of the insu- lated screw-binders, obtainable from instrument makers; in the absence of screw-binders the following arrangement will be found to answer quite well. A cork ring is sealing-waxed on to each foil-triangle near its base, and into the little vessels so made, mercury is poured, into which the connecting wires are placed. To get a rough idea of the current needed, it is advisable to note the position of the coil when the current is just bearable on the tongue, and compare it with position of the coil when the protoplasmic circulation has been stopped. CHAPTER II. ASSIMILATION OF CARBON. Section A. Formation of Starch. Section B, Evolution of Oxygen. Szorion C. Reactions of Chlorophyil. Szction D. Conditions of chlorophyll formation: Etio- lation: sun and shade leaves. SeEcTion A. Formation of Starch. (24) Sachs’ [odine-method* (Iod-Probe). This is a macroscopic method well adapted for many experiments. Almost any leaves will serve as material for the demonstration of the method, but since in research it is of importance to employ material which allows of rapid work, the choice of plants is a point to be considered. Submerged water-plants are useful, and among land plants, Tropzeolum and clover are especially valuable. The leaves to be tested are to be boiled for about one minute in water’, when they should be flaccid and free 1 Sachs’ Arbeiten, 11. p. 1. 2 Sachs allows a longer period, viz. 10 minutes, he states also that the addition of a few drops of strong KHO to the boiling water hastens the process. D. A. 2 18 ASSIMILATION. [CH. II from intercellular air. They are then placed in methyl- ated spirit warmed to 50°-60°C.: cold spirit will remove the chlorophyll equally well but not so quickly: if the specimens are not wanted at once the best results will be obtained by putting them in the sun for a few hours. The preliminary boiling in water must on no account be omitted, it shortens the process of decolorising in the most remarkable manner; of this it is easy to convince oneself by trying, for instance, to decolorise an Entero- morpha without the hot-water treatment. To produce the iodine reaction place the decolorised leaves in alcoholic tincture of iodine diluted with water’ to the colour of dark beer. In a few minutes they will be stained, and after washing in fresh water, they should be spread out on a white plate so that their tint—by which the amount of starch is roughly gauged—may be well seen. When full of starch they are almost black, and ‘with less amounts of starch the colour sinks through purple, grey, and greenish grey to the yellow tint of starchless leaves. (25) Schimper’s method’. In some cases it is necessary to use the microscope, this is especially necessary: when the amount of starch present is small, or where, as in Schimper’s researches, the distribution of starch in the leaf is particularly studied. Prepare a strong solution of chloral hydrate by dis- solving the crystals in as much distilled water as will just 1 Spring water answers perfectly well. 2 Bot. Zeitung, 1885. CH. I] IODINE METHOD. 19 cover them! The solution is now coloured by the addition of a little tincture of iodine, and is ready for use. Delicate leaves, such as those of submerged water-plants, when placed in Schimper’s solution, are rendered so trans- parent that every detail of starch-distribution can be studied under the microscope in the leaf examined as a transparent object. (26) Variegated leaves. Test Sachs’ method on a variegated leaf such as that of the ivy or of Arundo donaxz. In the case of the ivy a rough plan of the green and white parts of the leaf must be traced on paper placed under the leaf, which may best be done by a broken line made with a blunt instru- ment dotted along the lines separating the chlorotic from the green parts of the leaf. The iodine-stained leaf is then compared with the plan. With Arundo no such process is necessary, the chlorotic regions are in longi- tudinal stripes, and it is only necessary to cut out of the leaf a short piece, which, after staining in iodine, can be replaced between the base and apex of the leaf to which it. belonged: the colourless stripes in the fresh part corre- spond to yellow stripes in the stained part, and the purple to the green. Twelve hours is necessary for extracting the chlorophyll, and an hour for iodine staining. (27) Disappearance of starch in darkness. Either of the methods may be tried on submerged water-plants (e.g. Elodea, Potamogeton) which have been 1 Chloral hydrate 8 parts, water 5 parts. a) 20 ASSIMILATION. [CH, iI placed in the dark room for about four days. The control-plants must be grown either out of doors or in a greenhouse. (28) Effect of dull light. Sachs’ method may be used to demonstrate a fact, the knowledge of which is of practical value to the physiologist?, namely, that plants in a laboratory suffer from want of light far more than would be readily supposed—and that accordingly experimental plants can- not be too carefully kept in the best light available. Choose two equally vigorous pots of clover, let one remain in bright diffused light out of doors, and place the other on a table in the middle of the laboratory. The plant in the laboratory must be under a bell-jar on account of the dryness of the air, and therefore to make the control experiment fair the plant out of doors should also be under a bell. After two days compare the amounts of starch in the two plants. . (29) Local effect. Various means may be used to convince oneself that assimilation is confined to the illuminated regions of a leaf. Part of a leaf may be darkened, while still attached to the plant, by bending it down and burying the apical half in a flower-pot of finely sifted dry earth. The leaf should be buried one day and examined in the afternoon of the following day, taking care before the leaf is un- covered to mark on it the depth to which it was buried. 1 See Detlefsen. Sachs’ Arbeiten, 111. p. 88. CH. II] PHOTOGRAPHIC METHOD. 21 (30) Gardiner’s experiment’. A plant growing in a flower-pot (for convenience of moving) is placed in the dark for 24 hours, or until the leaves are found to be free from starch. One of the leaves is now covered with a photographic negative and left exposed to bright light out of doors, or in a greenhouse, until the evening, when the leaf is tested for starch. It will be found that an accurate copy of the photograph has been printed in starch. (31) Effect of rays of different refrangibility. The effect of the different parts of the spectrum may be demonstrated by a similar method as has been done by Timiriazeff* In the absence of the necessary appa- ratus we may compare the effects of light transmitted through coloured fluids. Fill a couple of double-walled bell-jars, (1) with potassium bichromate solution, (2) with ammoniacal CuSO, solution. Under each bell place a young Tropeolum or Clover plant in a small pot, ora seedling plant of any kind dug up and placed with its roots ina bottle of water. The bell-jars should stand in saucers of dry earth or sawdust, so as to ensure the exclusion of colourless light. They must be exposed to diffused light—in sunshine the temperatures are not the same in the two bell-jars. The experiment may be started in the afternoon and the leaves tested on the following evening. 1 W. Gardiner, Annals of Botany, tv. p. 163. 2 Timiriazeff, Comptes rendus, T. ex. p. 1346. 22 ASSIMILATION. [cH. 1 (32) Terrestrial leaves under water. To show that the leaves of land-plants do not form starch as those of aquatic plants do under water+, it is only necessary to tie a leaf so that it is partly immersed in a beaker of water. The experiment may be started in the morning and concluded on the afternoon of the follow- ing day. (33) Effect of excess of CO). | To show that excess of CO, diminishes assimilation? floating water-plants are convenient. We use Callitriche, and possibly Lemna might be used, but these must be kept a long time in the dark before they are de- starched. Two graduated jars of 200 c.c. capacity are filled with and inverted over water, and plants of Callitriche, which have been previously deprived of starch, are passed under the edge and allowed to float up. Into one jar equal quantities of air and CO,, while into the other 12 volumes of air to one of CO, are passed. The propor- tion of CO, in the atmospheres so prepared does not of course remain constant, since the water absorbs the gas. But if the experiment is started in the evening and concluded in the evening of the next day, one jar will certainly contain far more than the optimum of CO,, while the other will not fall much below the optimum. A still simpler plan is to use beakers of about 800cc. capacity inverted in saucers of water. The beakers are 1 Nagamatz (Sachs’ Arbeiten, 111.) shows that leaves covered with bloom can assimilate under water. 2 Godlewski. Sachs’ Arbeiten, 1. p. 343, CH. I] CULTURE WITHOUT CO, 23 graduated as follows: into one 550c.c. of water is poured and the level marked with a diamond, a second mark being made after the addition of 50c.c. The other beaker is marked at 300 and 600cc. The beakers are filled with water and inverted in saucers, and the rosettes of Callitriche floated up under the rims of the beaker. Three hundred cc. of air are now introduced into one beaker and 550 c.c. into the other, using a finger bellows for the purpose ; afterwards CO, is added until each beaker contains 600 c.c. of mixed gas, one containing 50 p.c., the other 8p.c. of CO,. In our experiments the Callitriche exposed to 50c.c. CO, showed hardly any starch, while the control-plants were black with it. The experiment may be more accurately performed with a pair of graduated tubes inverted over mercury (covered with a few drops of water) and containing leaves of land-plants. (84) Plants deprived of CO,. To show that the formation of starch depends on the presence of CO, it is necessary to cultivate plants in such a way that they have access to oxygen but not to CO, Water-plants. Water which has been boiled and allowed to cool in a closed flask will be free from both O and CO,. But if the flask is in connection with an arrangement for preventing 1 Godlewski, Flora, 1873, p. 378. 24 ASSIMILATION. [cH. 0 the access of CO, while allowing other gases to pass in, the boiled water will after a time become oxygenated. A convenient method is the following. A flask A (fig. 4) is filled with spring water which has been freshly Fic. 4, Exp. 34. boiled, and filtered from precipitated calcium carbonate; it is connected with the bottle B, half filled with strong KHO solution. The water in A is boiled 20 minutes, with the stop-cock C left open. The flame is now removed and C is closed. As the flask A cools, air is sucked in by D, and in passing through the KHO in the bottle B, is freed from CO,. The water so prepared is now used for the culture fluid: the vessel containing CH. IT] CULTURE WITHOUT CO, 25 the plants must be closed by a rubber cork through which passes a tube of soda-lime like the one shown in fig. 5. A similar flask filled with spring water (to which a little extra CO, may be added by blowing air from the lungs through it) and closed by a U tube containing coarse sand, will serve for a control. The CO, may also according to Pfeffer: be removed by careful treatment with lime water. Land-plants. Seedlings with their roots in water, or plants of Fic. 5. Exp. 34. 1 Pfeffer, Physiologie, 1. p. 111. 26 ASSIMILATION. [cH. 11 Tropzolum or Clover in small pots, are to be used. The pot is supported in a crystallising glass (G, fig. 5) half filled with soda-lime, which rests on a ground glass plate, and is covered by a tubulated bell-jar, the lower edge of which is ground, but need not be welted. The ground edge is smeared with wax-mixture!, and the junction with the glass plate is made secure by a little embankment of wax-mixture melted into the angle with a hot wire. The aperture of the bell is closed by a rubber cork pierced for the tube 7, which contains soda-lime. The apparatus should be placed out of doors or in a brightly lighted greenhouse. A control-plant must be fitted up in a similar way except that G may be dispensed with and that 7 must be filled with sawdust or some indifferent coarsely grained powder. We find that ex- posure from 10a.m..until the afternoon of the next day gives good results. (35) Temperature. Elodea or Potamogeton should be deprived of starch by darkness. One portion of the plants should be placed in a glass jar containing about 2000c.c. of spring water chilled to a temperature of about 5°C. by lumps of ice, the rest in a similar jar kept by meatis of gas regulator at 25°—80°. The ice will want renewing occasionally during the course of the experiment, which should take place in a bright light and should be continued from 1 Wax-mixture consists of resin 15 parts, bees-wax 35 parts, vaseline 50 parts. The wax and the vaseline are melted together, the resin is powdered, gradually added and stirred. CH. IT] GAIN IN WEIGHT. 27 10 a.m. to 5 or 6 p.m., when the amounts of starch are to be compared. (36) Gain in Weight. Sachs! has shown that a given area of leaf is heavier in the evening than in the morning, owing to the accumu- lated products of assimilation. The following are Sachs’ instructions for performing the experiment. Out of a board 3mm. in thickness cut out a square of 10 cm. to the side and another rectangular piece of 10 x 5 cm.: these are to be used as templates by which to cut out areas of 100sq. cm. and 50sq. cm. respectively. The plants used must be large leaved kinds, eg. Helianthus, Cucurbita, Rheum. The experiment must be begun soon after sunrise. Having selected 5 or 6 healthy leaves of, say Helianthus, each must be cut longitudinally close to one side of the midrib, the part which is thus freed from the plant is to be investigated at once, while the other half remains on the plant till the evening. Each half-leaf is treated in the following way. It is laid on a flat board, the lower side of the leaf being upwards, so that the projecting veins may be easily seen. The templates are now fitted in between the larger veins so as to get as many areas as possible consisting of mesophyll without large veins, The rectangular pieces of leaf so obtained are quickly killed by steam, After being allowed to become air dry, they are powdered, dried, and weighed. 1 Arbeiten, 111. p. 19. 2 Unless the plant is placed in a dark room on the previous evening, in which case the operator chooses his own time in the morning. 28 TRANSLOCATION. [cH. 11 In the evening a similar process is gone through with the control halves. The following is the result of one of Sachs’ experiments. A hundred sq. cm. were cut out of the halves of 7 leaves of Helianthus annuus; the dry weight of the 700 sq. cm. was :— 5 a.m. 3054 grams. 3 p.m. 3693 639, This equals 0°9 grams per sq. meter of leaf surface, per hour. Mutatis mutandis the weighing method is used by Sachs for showing the loss by translocation in the night. (37) Translocation. Sachs’ iodine method is also useful for studying the translocation of carbohydrates, ic. that the products of assimilation wander from the leaf to the body of the plant. In the evening remove the halves of several leaves and having tested small pieces of each (which should be preserved for further comparison) place the freed halves on wet filter-paper under a bell-jar in a dark room; the plant must also be placed under a bell in the same room. In the morning the half-leaves attached to the plant — will have lost more starch than the free halves. (38) Assimilation of Sugar. Water- plants, such as Elodea or Potamogeton, are placed in vessels of 400 or 500 cc. capacity, containing spring water, to one of which (A), 3°/, cane sugar has 1 More accurate methods are described in Part 1. Chaps. xiii. and xiv. CH. I] SUGAR CULTURE. 29 been added, to (B), 5°/, glycerine, while to (C) nothing has been added. It is of importance that specimens similar in size and in general vigour shall be selected, and that the specimens should be small in comparison with the volume of water in the beaker. Leave the vessels in the dark room for 5 or 6 days, when the plants in (A), (B) and (C) are to be compared as to condition, growth, and especially as to the contained starch. The chief difficulty experienced is the growth of moulds in the solution. Something may be done by washing the vessels with 4 p.c. corrosive sublimate and then in boiled distilled water; the culture fluids should be boiled and allowed to cool in vessels closed with cotton-wool plugs. [See Chap. ii.] Chlorophyll is not necessary for this form of assimila- tion, colourless parts of plants form starch vigorously. The white flowers of Phlox paniculata are especially useful for this experiment. They are simply floated in the above described solutions of sugar or glycerine, control specimens being placed in water. In a few days they become rich in starch, while the control flowers are starchless. The employment of colourless objects, such as white flowers, is especially convenient, since the use of alcohol as a decoloriser is avoided. The flowers must, however, be boiled before being placed in the iodine fluid. (39) Formaldehyde. _ Loew’ and Bokorny? have shown that although form- aldehyde is poisonous even in very dilute solutions, yet that oxymethyl natrium sulfonate (which is easily decom- 1 Botan, Centralblatt, xurv. p. 315. 2 Berichte d. D. Bot. Ges. 1x. p. 103. 30 FORMALDEHYDE. [cH. IL. posed into formaldehyde and NaHSO,) can be used in culture fluids in the proportion of 0°1 per cent. without injury to Spirogyra. Bokorny (loc. cit.) has shown if Spirogyra is cultivated in the light in a nutrient solution containing 0-1 per cent. oxymethyl natrium sulfonate that the starch in the plant increases considerably,—a result which we have confirmed, The access of CO, must of course be prevented; and for this reason the culture fluids should be examined for moulds, bacteria, &c., which might serve as a source of CO, to the alge. The nutrient solution must contain 0'1 per cent. dinatrium phosphate to counteract the evil effect of the NaHSO, set free. After four or five days the plants must be compared with control specimens which have not been supplied with oxymethy] natrium sulfonate, but have been in otherwise identical conditions. (40) Starch-formers (leucoplasts). These may be examined in the tubers of Phajus grandifohius, according to the method given by Stras- burger. The sections are to be placed in alcoholic tincture of iodine diluted with half its volume of distilled water. The relative positions of starch-former and starch- grain and the elongated crystalloid are well shown in Strasburger’s figure 29. The leucoplasts in the rhizome of Iris germanica are given in his fig. 30. Section B. The Evolution of Oxygen. (41) Bubbles of gas given off. Place a branch or two of a submerged water- plant, such as Hottonia, Potamogeton crispus, or Elodea, ' Practicum, pp. 67, 68. CH. It] ; GAS EVOLVED. 31 in a beaker of spring water. The cut ends of the plants must be upwards; and must be below the surface, to effect which it may be necessary to tie the specimens to a glass rod (see Pfeffer, Physiologie, I. fig. 17, and Detmer, fig. 12). The beaker is to be -placed in sunlight, and evolution of gas from the cut ends of the specimens to be observed. To obtain a convenient series of small bubbles Pfeffer recommends varnishing the cut end of the shoot and pricking a fine hole in the membrane so produced. Select a branch which seems to be yielding a satisfactory ‘amount of gas, and record, with a stop-watch, the time which elapses while 10 or 20 bubbles are given off. The observation must be repeated until the rate of bubbling is fairly constant. [It is important to know that the evolution of bubbles of gas may be produced by other causes than illumination. Thus a plant which is exposed to feeble ilhumination and is not giving off bubbles may be made to do so by being transferred to a beaker containing soda-water freshly drawn from a “syphon.” Devaux! has shown that this depends on the internal atmosphere rapidly assuming the gas-pressure of the water, by the diffusion of CO, from outside into the intercellular spaces.] , (42) Light of different intensities. Now move the beaker into the shade, or cover it with a sheet of white paper, and take a fresh series of readings, and finally replace it in sunshine and record the rate once more. In the absence of sunshine, an incan- 1 Ann. Se, Nat. 1889. 32 GAS EVOLVED. [cH. II descent electric light of 2 or 3 candle power may be used, the intensity of illumination being easily varied by placing the light at various distances from the plant. (43) Dependence on CO,. Transfer the plant to a beaker filled with water which has been boiled in the apparatus shown in fig. 4. After a time the water may be supplied with CO, by blowing vigorously into it through a glass tube. Repeat the observation with the stop-watch. (44) Temperature. Provide two beakers of water, one at a temperature of 24°—26°C., the other at 4°—5°C. Place a specimen in the warmer of the two and when the readings are constant transfer it to the cold water. During the experiment take note of any changes in the brightness of the sky; if this precaution is forgotten it is easy to be deceived by a passing cloud causing an alteration in the rate of assimilation. (45) Chloroform. Repeat experiment 41 and add a small quantity of chloroform-water, that is, of water in which not more than 1 per cent. of chloroform has been shaken. If the experiment is cautiously performed it should be possible to seriously diminish the rate of gas-discharge without killing the plant. (46) Coloured light. Proceed as in experiment 41, and when constant CH. IT] GAS EVOLVED. 33 readings are obtained, cover the beaker with a double bell-jar containing ammoniacal copper-sulphate solution and note the result. After an interval of ten minutes, when the readings should be approaching constancy, replace the blue jar by another containing potassium bichromate solution, and take a series of readings. It will probably be necessary to alternate the blue and orange light several times before a definite result is obtained. (47) Collection of the gas. Place a quantity of any of the above-named water- plants in a glass jar of about 12—14cm. diameter. Press the plants down into the water with an inverted funnel, which should be a large one, and should fit easily inside the jar; its neck should be cut short, so that the opening may be easily submerged. The gas given off by the plants will be guided by the funnel and may be collected in an inverted test-tube filled with water and placed over the opening. If the neck of the funnel is covered with 4 inch of india-rubber tubing, and if a test-tube be selected which fits tightly over the tube, no other support for the test-tube is needed. The funnel may be kept in its place by 3 bent glass rods hooked over the rim of the jar, and ending in glass rings by which they are tied to the neck of the funnel. When the test-tube is nearly full, the gas may be shown to be oxygen by the glowing of a splinter of deal which has been lighted, and is blown out just before it is thrust into the gas. The test-tube should be of such a size that it can be easily covered with the thumb. D. A. 3 34 PHOSPHORUS METHOD. [cH. II (48) Engelmann’s blood-method’. Pass a stream of CO,, or of hydrogen, through some defibrinated bullock’s blood? so that it may take on a dark venous colour. A filament of Spirogyra about lcm. in length is mounted in a drop of the blood under a cover slip. The preparation is now placed in a bright diffused light, and in about 15 minutes a stripe of scarlet, due to arterial blood, is seen to border the alga. In sunlight the scarlet tint appears more quickly. According to Engelmann the most delicate method of showing the evolution of the oxygen is by means of the spectroscope, the spectrum of the blood changing as the oxyhemoglobin appears. (49) Boussingault’s phosphorus method’, Fill a bell-jar over water with hydrogen and add a small proportion of CO,, ie. not more than 8 per cent. of the volume. Introduce a stick of phosphorus and a leafy branch. The oxygen in the intercellular spaces of the plant will attack the phosphorus, and the bell-jar will be filled with white fumes. The bell-jar must there- fore be placed in the dark for two or three hours, or until the white vapour is dissolved in the water, and the contents of the jar are clear and transparent. The bell- jar Is now exposed to the sun when in a few minutes it becomes clouded with white fumes. We find that, when replaced in the dark, a quarter of an hour is sufficient for the absorption of the fumes. 1 Pfliiger’s Archiv, Vol. xutt. 2 According to Engelmann the blood may be slightly diluted. 3 See Deherain, Chimie Agricole, p. 82. CH. Ir] GAS ANALYSIS. 85 (50) A demonstration method recommended by Deherain' is the following. A current of air is drawn by means of an aspirator through a glass tube, which is carefully lined (paved as it were) with leaves, and exposed to bright light. The air, after slowly traversing the tube, is made to bubble through baryta water. If the current of air is kept slow the baryta water is said to remain clear while a current of the same rapidity which has passed through an empty tube clouds the solution. (51) Pfeffer’s method’. A leaf is exposed to light in a calibrated tube con- taining a known volume of CO,: after a certain number of hours the amount of CO, decomposed is estimated by absorbing what remains with KHO. The tube is almost 36 cm. in length, of which 26cm. is a calibrated tube of 14-15 mm. in diameter, 7' (fig. 6); above this part the tube is blown into a balloon and ends above in a narrow tube B with flat ground edges. The whole tube contains about 120c.c. The leaf to be experimented on is rolled into a cylinder and gently pushed up the tube with a wooden rod until it reaches the wide part of the tube where it unfolds of itself, After the experiment is over, the leaf is to be removed by means of a piece of thin iron wire, W, attached to the stalk before the leaf was inserted. The wire should be attached outside the tube by an elastic band #. The tube is fixed vertically in a glass beaker, H, having upright sides and containing 1 Chimie Agricole, p. 75. 2 Sachs’ Arbeiten, 1. p. 15. See also Pfeffer’s Physiologie, 1. p. 188. 38—2 36 GAS ANALYSIS. [cH. II mercury, and a drop or two (0'2—03c.c.) of water is placed above the mercury column in the calibrated tube to protect the leaf from mercury fumes. By applying suction at B the mercury column is raised: to a desired height. The suction is best ap- plied through a washing bottle contain- ing water, so that the breath of the operator may not come directly in com- munication with the air in the gas-tube. An india-rubber tube fitting over B serves to connect with the washing bottle, and also to close the tube when desired. T When the mercury column is at a suffi- cient height, the tube is temporarily closed with a clip and afterwards more securely by a-bit of glass rod R, whose lower surface is ground flat and greased, so that when pushed home it fits close | w against the ground surface of B. E The volume of the air contained in the tube is now read off on the calibrated tube, and at the same time the height of the little column of water above it is re- Onna eo ition corded, Readings of the barometer and loc. cit. thermometer are also taken. From 8 to 10cc.-of CO, which has been washed in NaHCO, to free it from HCl is now passed into the tube, and the readings are again taken. Before introducing the CO, its purity should be tested by ascertaining that it is entirely absorbed over R +B CH. I] GAS ANALYSIS, 37 KHO. The apparatus is now exposed to bright diffused light for 5 or 6 hours, or it may be exposed to sunlight. When the exposure to light is complete the leaf must be pulled out by the wire, and when the apparatus has cooled, readings are again taken. In order to estimate the quantity of CO, which has been decomposed, about 0:2 or 0'3c.c. of concentrated KHO is injected into the gas-tube; this Pfeffer recom- mends to be done by the heat of the hand acting on a closed pipette. After 2 hours the CO, may be assumed to be all absorbed, when readings are again to be taken. The volume of the leaf is also to be ascertained by sinking it in a narrow measuring glass and reading off the altered position of the level; the fluid may be a mixture of alcohol and water which prevents adhesion of bubbles to the leaf. The volume of the leaf being known it must be applied as a correction to the readings of the gas-volume. To obtain the result it is necessary to reduce the readings of the calibrated tube (before and after the injection of KHO) to 0°C. and to 1 meter mercury pressure, and to make allowance for water vapour tension, etc. This is to be done according to the formula of Bunsen}. Pe aie (0 — by — by) 1+0:00366¢° Where v,=the reduced volume of gas. v =the observed volume, m=the correction for meniscus. b =the barometric reading, b, =the mercury. pressure in the eudiometer. b,=the water-vapour tension at the temperature 1°. See Bunsen and Roscoe, Gasometric Analysis. 1 38 GAS ANALYSIS. [CH. 11 It is by no means necessary to employ a tube of the above described form. We often employ tubes of test-tube form of 2 cm. internal diameter and containing 100 cc. Oleander leaves (which are especially good material in the winter months) fit these tubes well. The mercury is raised to the desired height by a thick-walled india-rubber tube pushed up.into the cavity, and connected with a water air-pump. The rubber tube is then closed between the fingers and drawn out: if in this process a few drops of mercury are drawn into the tube, they may be sucked (by turning on the pump) into a bottle fitted like a washing bottle, which serves as a trap between the pump and any vessel to which suction is to be applied. (52) Winkler-Hempel apparatus. For demonstration purposes, where it is desirable to avoid barometer readings, calculations, &c., fair results may be obtained with the Winkler-Hempel apparatus. A jar, J, fig. 7, containing leaves is filled with air con- taining about 8°/, of CO,: the exact proportion is of no importance, but it must be accurately determined at the beginning of the experiment. The bent tube ¢ serves to draw off a sample of the gas in the jar J, and as it is drawn off, the water flows through the tube J from the beaker o outside, into the second vessel inside 7. The tubes ¢ and / are now clamped, and the apparatus exposed to bright light for 4 or 5 hours when a fresh sample of gas is drawn off and analysed. The water introduced absorbs some of the CO, and causes an error, which however is not so serious as to interfere with the results for demonstra- CH. It] .GAS ANALYSIS, 39 tion purposes. The analysis is made in the following manner :—A strong KHO solution (1 in 2) is introduced into B (fig. 8) until its level reaches A, and then by blow- Fig. 7. Exp. 52. Fig. 8. Hxp. 52. ing down B the KHO is forced up the fine tube # and into a thick-walled india-rubber tube connected with it. As soon as the solution appears at the open end of the tube, the clamp C is closed. The tubes G and F (fig. 9) of the measuring burette are then a little over 4 filled with water, care being taken that no air bubbles remain in the connecting india-rubber tube. F is then raised till water flows out of H; then the stop-cock L is closed and H is connected by tubing with the vessel J in fig. 7 con- taining the gas to be analysed. ’, now nearly empty, is lowered and Z opened, so that a sample of gas is drawn 40 GAS ANALYSIS. [cH. II ‘into the burette. Z is closed and H disconnected, The volume drawn in is then measured by means of the H y/ a Fig. 9. Exp. 52. graduations on G, after bringing the water in the two tubes to one level. To absorb the CO, A is connected with the india-rubber tubing C of the absorption pipette (fig. 8). Fis raised, and L and the clamp C opened. The gas is thus forced over into D where it is retained for a CH. 11] BACTERIAL METHOD. 41 minute or so and gently shaken in contact with the KHO, the clamp C and stop-cock ZL being closed meanwhile. When absorption should be complete the gas is sucked back into G (fig. 9) by lowering F, with C and Z open. L is then closed and G and F brought to a level so that the diminished volume of gas can be again read off. The difference gives the amount of CO, originally present. To make sure of complete absorption the gas may be again passed into D, shaken and returned, when it should show no further reduction in volume. When any potash is sucked back into @ along with the gas the tubes must be carefully washed clean before being used for another sample of gas. (53) Engelmann’s bacterial method. This depends on the extreme sensitiveness of certain bacteria to the presence or absence of free oxygen. One of the difficulties connected with the experiment is the providing a sufficiently sensitive bacterium. Pfeffer recommends that a pea having been killed by boiling shall be allowed to putrefy in 200 cc. water; according to Detmer a pure culture should be made of the bacteria so obtained. It is best to begin with a study of the behaviour of bacteria mounted simply under a cover slip. They will be found to swarm round any air bubbles which may be included in the fluid under the cover slip; and to collect round the edges of the preparation, and in fact to seek out sources of free oxygen. Ifthe preparations are sealed by a coating of olive oil painted round the edge of the cover 42 . DIFFUSION. [CH. II slip, the bacteria ultimately become sluggish and come to rest. It is of this fact that Engelmann’s method takes advantage. If a filament of Spirogyra or the leaf of a submerged plant be included with the sealed bacteria we have it in our power, by the exposure of the preparation to light,—to produce free oxygen. Thus all that is necessary is to place the preparation in the dark until the bacteria are at rest, then to expose it to light, and to watch the swarming of the bacteria round the green plant. By means of Engelmann’s Micro-spectral Objective it is possible to cast a spectrum on the filament of Spirogyra and to observe the distribution of the swarming bacteria in the different colours. We do not propose to enter into Engelmann’s method of “successive observa- tions” for which the student may consult Engelmann’s papers in the Botanische Zeitung from 1881 onwards. (54) Diffusion. In connection with assimilation the diffusion of gas through the cuticularised epidermis should be studied. Detmer’s method}, may be used. A piérced rubber cork is fitted over a glass tube (3 cm. diameter) so that the surface of the cork is flush with the upper rim of the tube. On the aperture in the cork a piece of fine wire gauze is laid and on this a leaf (e.g. that of Platanus) is placed with the stomatal surface uppermost, and firmly cemented with wax-mixture to the cork. The tube is filled with CO,, and its lower end plunged into mercury. As the CO, diffuses out through 1 Praktikum, p. 107. OH. 11] CHLOROPHYLL. 43 the leaf, the mercury rises in the tube. The wire gauze serves to prevent the leaf bulging inwards into the tube. The best method of filling the tube is by displace- ment of the air, which is allowed to leave the tube by a small gap purposely left uncemented between the leaf and the cork, and which can be closed when the air has been replaced by CO.. Section C. Reactions of chlorophyll and of some other pigments. To study the simpler reactions of chlorophyll we extract the green colour of leaves by means of alcohol. The leaves! are boiled for a few minutes in water, roughly dried with filter paper and placed in alcohol. The ex- traction must go on in the dark, because light has a destructive action on the colouring matters. (55) Separation by Benzol, etc. Place some of the alcoholic extract in a test-tube, dilute it with a few drops of distilled water; add benzol, shake the mixture, and allow. it to settle. The benzol which floats above the alcohol is of a bright greenish blue (cyanophyll) while the alcohol dissolves the yellow pigment (xanthophyll) which forms part of the alcoholic leaf-extract. A-similar separation may be effected by adding to the alcoholic extract :-— (a) Ether. (6) Olive oil. 1 Almost any leaves will serve the purpose: grass answers well. 44 CHLOROPHYLL. [cH. 11 (56) Action of light. Fill three test-tubes with alcoholic leaf-extract, cork them and place A in sunlight, B in diffused light, C in the dark. After a few hours note the changes in colour. The solution which has been exposed to sunlight rapidly becomes brown or yellowish brown, while C is unchanged and B is intermediate in tint. In the absence of sunlight the effect may be shown by placing A close to the window, B in a dull corner, and C in the dark. Exposure for 24 hours is necessary. Chlorophyll solution may be compared with an alcoholic extract of etiolin which is far more stable in light. (57) Aeration in connection with the action of light. Boil some of the alcoholic solution in a test-tube, sO as to remove the air, cork it and allow it to cool. Place ‘it with an unboiled sample in bright diffused light, and note that the absence of oxygen delays the light effect. If the extract is boiled for too long a period it becomes more concentrated and therefore of a darker tint than the unboiled sample, this may be rectified by dilution with boiled alcohol before exposure to light. (58) Action of acid. Add a few drops of HCl to the alcoholic extract and note the appearance of a brownish tint (phyllocyanin) ; with excess of acid a muddy blue is produced. (59) Action of copper salts. By the addition of a little 10°/, Cu8,? solution a 1 Or of strong solution of copper acetate and strong HCl. CH, II] CHLOROPHYLL. 45 copper compound with phyllocyanin is produced, which has the general appearance of chlorophyll, but differs notably in not being tiuorescent. To observe this point, compare it with unaltered chlorophyll extract; fluores- cence is most easily visible with a strong solution in a narrow test-tube. (60) Stabelity of the copper compound. Fill two test-tubes A, B, with the copper compound and two others C, D, with unaltered leaf-extract: place A and @ in sunlight, B and D in the dark. After some hours note by comparison with B and D, that the copper compound is not destroyed while C is affected. (61) Spectroscopic examination. To see the characteristic chlorophyll band I in the red, a small direct-vision spectroscope may be used: the solution may be in a test-tube, and ordinary daylight will suffice. In Detmer’s Praktikwm, p. 17, a convenient holder for test-tubes is figured and described. For the other bands direct sunlight is needed, the solution which must be a weak one, should be placed in a parallel-sided vessel, and a more elaborate spectroscope should be used. (62) Other pigments. The red varieties of Ricinus, Coleus and Amaranthus may be used. In the last named the red colour can be obtained by boiling a leaf in water, which takes out the coloured cell sap, and leaves the leaf green. In the case of Ricinus and Coleus the red colour is destroyed by boiling. If these leaves are partly immersed in boiling 46 PRODUCTION OF CHLOROPHYLL. [cH. n water, the parts which have been heated reveal, almost at once, the chlorophyll. To obtain a solution of the red colour the leaves must be killed by ether vapour, cut up and placed in distilled water. Even in cold water the red of Ricinus is soon decomposed. (63) Floridee. In some species at any rate, the colouring matter reddens cold fresh water in which the sea-weeds are placed, but the colour is destroyed by boiling. In Polysiphonia it is not destroyed. (64) Brown sea-weeds. A portion of Fucus or Laminaria yields a brown colour to water in which it is boiled—while the boiled thallus shows a greenish colour and yields a green alcoholic extract. But it is impossible as far as we have seen to extract the whole of the colouring matters. Section D. Conditions necessary for production of chlorophyll. Etiolation. Sun and shade leaves, (65) Formation of chlorophyll. Seedlings of any sort, e.g. cereals or cress (Lepi- dium) or mustard, or V. faba, are grown in the dark and are then placed in the morning in a good light close to the window and the time necessary for the production of a distinct green colour is noted». 1 Etiolation proper can only be observed in parts of plants which have developed in the dark. The already formed chlorophyll may become discoloured by starvation, but this is not etiolation. Many leaves retain their green colour for a long time in darkness, CH. IT] ETIOLATION. AT Place similar etiolated plants in the darkest corner of the laboratory and when chlorophyll has been developed show, by an examination of the leaves with Sachs’ test, that light too weak for assimilation is strong enough for chlorophyll-formation. (66) tiolin and light. The following point is of less importance. Compare the colour of etiolated seedlings, which have been exposed to light for one or two hours but have not developed chlo- rophyll, with control specimens left in the dark. They will be found to be of a darker yellow or orange colour. In this way Elfving? showed that light increases the forma- tion of etiolin. (67). Pinus. Light is not necessary for chlorophyll formation in certain Gymnosperms. The seeds of various species of Pinus should be sown 3 weeks or a month before they are needed for demonstration. Let them be kept in the dark continuously and at a temperature of at least 15°C. Peas, or beans should be grown with them to prove by their appearance that the cupboard is dark enough to etiolate ordinary plants. (68) Temperature. Sink an empty beaker into a larger one half filled with water, and keep the water at 30° or 31°C. by means of a thermostat. Etiolated plants such as seedling cereals, or the epicotyls of beans are placed in the inner beaker 1 Sach’s Arbeiten, 11. p. 495. 48 CHLOROSIS. [cH. 1 which is covered by a glass plate. A similar vessel contains control plants and is allowed to remain at the room temperature of about 15°C. After 2 or 3 hours a distinct difference in the greenness of the plants at 31°C. as compared with the control plants is perceptible. (69) Oaygen necessary for chlorophyll-formation. Germinate mustard in the dark and when the coty- ledons are free from the seed coat pass 2 or 3 plants under the rim of an inverted test-tube filled with water. They float up to the top of the tube and are thus fully exposed to light, but they do not become green; while control plants placed on wet filter paper under a bell-jar soon develope chlorophyll. It is not necessary to use boiled water, the amount of air in ordinary spring water being insufficient for the respiration of land-plants. (70) Seedlings in hydrogen. To demonstrate the fact in another way mustard seed- lings may be placed in hydrogen. We use the L shaped vessels recommended by Detmer’*. The difference between the experimental seedlings and the control in air is clear after 24 hours. The vessel may be filled with hydrogen by displacement of water., (71) Tron. The effect of iron salts in restoring a green colour to chlorotic? leaves, may be occasionally demonstrated on chance specimens. Professor Elfving of Helsingfors, 1 Praktikum, p. 26. 2 See Sachs’ Arbeiten, 11. p. 483. CH. II] ETIOLATION. 49 when a student at Wiirzburg, restored a healthy green to a chlorotic branch of Robinia by screwing a funnel into the tree close to the base of the branch, and pouring into it a solution of an iron salt}. In the absence of chance material, chlorotic plants must be produced by growing them, by the water culture method, without iron. It is probably best to grow some 5 or 6 iron-starved plants so as to have control plants and to make sure of material for several experiments, Add a few drops of iron chloride solution to one culture jar, and use another for Gris’ experiment, which consists in painting a leaf with very dilute ferric chloride solution. (72) Form of etiolated plants. For a thorough study of the changes of form and structure which accompany etiolation it would be neces- sary to grow a great vartety of plants. The best for the purpose are plants produced from tubers or bulbs, or from large seeds full of reserve material, since here the effects of darkness in producing starvation do not complicate the result. Among Dicotyledons Dahlia, Helianthus tuberosus, Hop, and Beans (Faba and Phaseolus) may be grown. Among Monocotyledons any of the cereals and Narcissus. In each case control plants of the same species must be grown in light. Compare the two sets as to develop- 1 I cannot be sure of the details, but I remember the fact. [F. D.] ° For an account of the experiments of Gris see Sachs, Physiologie Végétale (French Trans.), p. 159. Also Sachs, Arbeiten, m1. p. 433, for Chlorosis. D. A. : 4 50 SUN AND SHADE-LEAVES. (cH. 11 ment of leaf, measured in length and breadth; length and diameter of stem, and length of internode. (73) Sun and shade-leaves. To see the remarkable structural characters described by Stahl’, the leaves of the beech will serve. Transverse sections must be cut from leaves which have grown (1) in the fullest sunshine, and (2) in deep shade. The chief point to note is the difference in the palisade tissue. 1 Bot. Zeitung, 1880. CHAPTER III. FURTHER EXPERIMENTS ON NUTRITION. Section A. Water-culture. Section B. Experiments on Fungi and on Drosera. Section C. Absorption and other functions of the root. Section A. Water-culture. (74) Method. To show what elements are necessary for the development of a green plant and the relative proportions in which they are absorbed by its roots, the method of water-culture should be used, either alone or in com- bination with studies on the ash obtained by incinerating the plants cultivated. Full directions for conducting water-culture experi- ments are given in Sachs’ Lectures, Eng. Ed. Lect. XVIL p. 283, and by Detmer, Ch. 1. pp. 1—61. Although we have never succeeded in preventing the failure of a small proportion of such experiments, the liability to failure may be much diminished by careful 1 Compare also Acton, Proc. Royal Soc. Vol. xuvit. (1889), pp. 152-157. 4—2 52 WATER-CULTURE. (CH. III attention to the following precautions. The cylinders used should not contain less than 500 cc. of the solution? in an experiment and should therefore be of at least 700 c.c. capacity. Every cylinder used should be carefully cleaned just before setting up the experiment. For this purpose the cylinders are thoroughly washed and then rinsed out with strong commercial nitric acid which is removed by distilled water. They are then again rinsed out with a strong aqueous solution of mercuric chloride and lastly with distilled water, which has been boiled for some time immediately before use, till portions of the wash-water give no trace of turbidity with a solution of silver nitrate. The culture solution should be boiled rapidly for at least half-an-hour, the water which evaporates off being replaced from time to time with pure distilled water, and transferred to the cylinder as soon as it has cooled. Two holes should be cut in the cork, one for the plant 1 Sachs recommends the following: Potassium nitrate 1:0 gram Sodium chloride 05 Calcium sulphate 0°5 Magnesium sulphate 0°5 Caleic phosphate 0-5 Water 1000 c.c. Pfeffer, Physiologie, Vol. 1. p. 253 quotes from Knop the following : Calcium nitrate 4 parts by weight Potassium nitrate 1 rs a Magnesium sulphate (crystals) 1 i i Potassium phosphate 1 5 ‘5 One part of the mixture of salts is dissolved in 50 parts of water: for use it is diluted to 2 or 3 per mille. A drop or two of iron chloride must be added to it as in the case of all normal nutrient solutions, Schimper (Flora, 1890, p. 220) gives a variety of useful formule. CH. 111] WATER-CULTURE. 53 and one for a tube to admit air to the interior of the cylinder. For the latter purpose a short glass tube is inserted through the hole in the cork’ so that the ends project about 5 cm. beyond the upper and under surface ; the upper open end is attached to a small bulb tube loosely packed with recently ignited asbestos which will exclude dust etc. but allow a circulation of gases. This tube is also useful for introducing fresh water, when required, without touching the plant, as it is only necessary to remove the bulb tube and afterwards replace it. To fix the plant in position in the cork soft asbestos, which has been recently heated, is preferable to cotton- wool, and the material should not project beyond the lower surface of the cork, as it is desirable to keep it as dry as possible, since ‘damping off’ at the ‘collar, from the attacks of Pythium, is the commonest cause of failure in culture experiments. For the same reason, only those plants should be selected for use which are uninjured at the ‘collar, and great care taken that no injury is inflicted at this part when fixing in position. When changing the plants into fresh cylinders the whole cork should be taken out and put into the new cylinder, but if for any reason the asbestos around the collars should get damp it is better to take a fresh cork and to fix the plant again with dry material. At the end of each week the plants should be changed 1 Out of fifty-six unsuccessful experiments where plants died within three weeks, more than thirty were attacked in this way; the plants were seedlings of Epilobium hirsutum and Cheiranthus cheiri. 54 WATER-CULTURE. [CH. 111 into cylinders containing only pure distilled water and left in the same for three or four days, when they may be again placed in the culture solution, using for this purpose a fresh 500 cc. of the solution put into the vessels with the same precautions as at first. The longer such cultures are continued, if the plants keep healthy, the more striking will be the results, but three weeks, during average summer weather, will be sufficient to demonstrate the facts illustrated in the selected experiments, Pure chemicals should be used in making up culture solutions; the solutions do not keep well even in the dark and should be freshly made for each set of experiments. A useful rough rule for making up such solutions is to dissolve twice the weights of the solids, given in grams per liter, in an ordinary blue glass Winchester quart bottle, containing roughly 2 liters. Water-plants cannot generally be recommended for accurate experiments extending over any considerable time, as we have found it much more difficult to grow them satisfactorily in culture solutions than to grow ordinary plants with the roots immersed. Strong seedlings of any common green plants may be ‘used; of the plants used by Acton (loc. cit.) the best were found to be Epilobium hirsutum and Cheiranthus cheiri. In experiments where the time required is not very long, shoots of plants with the cut end in the solution may be used ; shoots of Alisma plantago and Scrophularia aguatica are good for this purpose, and when it is . convenient to have a woody stem, branches of Acer pseudoplatanus or Tilia europea answer well. CH. IIT] WATER-CULTURE. 55 (75) Potassium salts necessary. Take three plants A, B, C, as nearly as possible of equal weight and equally developed. Dry A at 100° and determine its dry weight. Grow B in normal culture solution and C in a fluid containing the same salts as the normal solution but with an equivalent weight of sodium —instead of the potassium—salt. Continue the cultures for about three weeks, then take out the plants B and C, dry them at 100° and determine their dry weights. B should be considerably heavier than C. To confirm the fact that the greater increase in weight shown by B is associated with the actual ab- sorption of the potassium, B and C should be incinerated after weighing and the absolute amounts of K,O in the whole ash of each determined. Instructions for obtaining the ash and making an accurate estimation of the K,O are given in part II. (76) Phosphoric acid necessary. The same method is used as in the last experiment but a somewhat longer time will be required for satis- factory results. The solution which contains no salt of: phosphoric acid may have the usual calcium phosphate replaced by an equivalent quantity of calcium nitrate. Instructions for determining P,O, in the ash are given in part IT. In this as the preceding experiment it need scarcely be pointed out that it is much better to start five or six separate cultures under each set of conditions than to rely on one only. If all develope well, the mean result of the best three may be taken in each case. 56 LEMNA. {cH. 111 (77) Experiments with Lemna. Though as above stated water-plants are not generally to’be recommended, yet we have found Lemna ‘useful for purposes of demonstration. They grow rapidly and their increase being principally in one plane is easily noticed at a glance. Moreover a rough numerical estimate of the K Ww Fic. 10. Exp. 77. CH, 111] LEMNA. 57 amount of increase in a given time can be made by counting the fronds; thus in fig. 10 the culture S which has about 21 fronds consisted originally of six separate fronds, as shown in culture W. We grow the Lemna in narrow cylinders containing 300 ec. of fluid; if the cylinders are darkened by black card- board covers the cultures keep reasonably free from alge. Fig. 10 gives the result of an experiment carried on in a greenhouse in the winter. Three jars S, K, W, were prepared, in each of which six fronds were placed. S contained 0°25 °/, Sachs’ culture fluid: K contained 0:25°/, potassium nitrate, while W contained only distilled water, a drop of dialysed iron being added to each culture. The amount of increase is shown in the figure, the difference in root production as well as in the amount of frond is noticeable. In this and similar experiments the Lemna died in a short time in distilled water; whether this is due simply to starvation we have not ascertained. Fie. 10 A. Exp. 77. 58 LEMNA, [CH. II X, ©eeh yw @ HAR O A Pwehsyeo f-# wat 82ee WernrOs "eh 4 dbcteZo B&O feoveret rover ee oe t Past rehe ge ee he ee © Cries ROAM mMayru-Be-e < # > 4 ts» -% S oe * e 4 eo ¢ €¢9 Fic, 104. Exp. 77, Fig. 10 A gives the comparative result of culture in Sachs’ fluid (S) and in the same without phosphates (P). Four or five weeks (in May) are necessary to give the result. Owing to an accident the figures do not show the strong growth of roots in (8) (78) Calcium oxalate formation. The leaves of the Horse-chestnut, dsculus hippo- castanum, of Acer negundo, Ulmus campestris, and Humulus lupulus are according to Schimper? useful to demonstrate the fact that calcium oxalate accumulates in leaves with age. To make this out, young and old leaves of some of these species should be compared. The method described in Chapter v. of detecting small amounts 1 In both cultures there were originally 6 plants each with 3 fronds. 2 Botan, Zeitung, 1888, p. 83. 7 CH, 111] CALCIUM OXALATE. 59 of calcium oxalate with the polariscope may be used, but will probably not be needed. The appearance of the oxalate is connected with illumination: Schimper states that in the Horse Chestnut this is especially noticeable, leaves which have grown in full sunshine having far more crystals than older leaves developed in the shade. The formation is also connected with the presence of chlorophyll. The comparison of a pure green and a white leaflet of a leaf of Acer negundo is, as Schimper states, especially instructive. In the white leaflet only a small amount of minute crystals occur. The variegated Pelargonium may also be used. (79) Nitrate reaction. Schimper has shown that the appearance of calcium oxalate is connected with the decomposition of calcium nitrate in the leaf. The calcium being deposited as an oxalate while the nitrate is assimilated. The disappear- ance of nitrate out of leaves shows therefore the same relation to light and to the presence or absence of chloro- phyll that he has shown to exist for the oxalate formation. The presence of nitrates is to be tested by the diphenyl- amin-sulphate test?; a not too thin section of a leaf or leaf-stalk is placed on a glass-slide and a drop of diphenyl- amin sulphate added; if nitrate is present a deep blue colour appears. Schimper recommends the leaves of the Elder, Sambucus nigra, adding that the large leaves de- veloped on the long spring shoots should be avoided, and 1 Molisch, Deutsch. Bot. Gesellech. 1883. For the precautions necessary in drawing conclusions from observations based on this test see Zimmerman, Botanische Mikrotechnik, 1892, p. 49. 60 FUNGI. (CH. II that leaves developed in shade on short twigs should be employed. The cut leaves having been tested and found to contain nitrate are placed with their stalks in water and exposed to light. He describes an experiment in which the leaves lost the greater part of the nitrate in four or five days under these circumstances. When a varie- gated Elder is used for the experiment, the diminution of nitrate takes place in the green, not in the chlorotic parts. The importance of light was also shown in the case of Taraxacum dens leonis, Aristolochia sipho? and some other plants by observing that after some weeks of sunny weather the sun-leaves gave no nitrate reaction while the shade-leaves showed a moderate or even strong reaction. Pelargonium zonale is also especially useful according to Schimper, the nitrate reaction in this plant varies with the weather: in bright sunny periods there is no reaction, after dull weather it appears again. Section B. Nutrition of Fungi® and of Drosera. (80) Method. Make the following‘ nutritive solution (N). Dextrose 5 to 10:0 grams Peptone lto 20 Ammonium nitrate 10 1 loc. cit. p. 132. 2 loc. cit. p. 188. 3 For the form of the instructions here given we are indebted to Professor Marshall Ward. 4 Or any of the solutions given on p. 172 of Zopf, Die Pilze. Solution N is compiled from Elfving, Studien iiber die Einwirkung des Lichts auf die Pilze, 1890, p. 30. CH. IT] FUNGI. 61 Potassium nitrate 0°5 Magnesium sulphate crystals 0°25 Potassium monophosphate 0°25 Calcium chloride 001 Pure water 100°0 Take 1000 cc. of solution N and add to it 100 grams of pure gelatine (Coignet’s gold label); sterilise in a flask plugged with cotton-wool, filter while hot and distribute into sterile plugged tubes: sterilise and preserve for use. Expose a saucer of solution N to the air, until it is infected with one of the blue moulds:—Penicillium or Aspergillus. With a sterilised needle remove spores of the mould selected and shake up in a small flask of pure water: rapidly filter through a sterile funnel, plugged with cotton-wool to make the spores separate from one another. Add one drop, or more, according to the quantity of spores in the water, to a tube of the gelatine just liquefied, and pour it into a sterile glass dish. When set, put it aside at 20°C. in the dark; after 48 hours or so there will probably be isolated pure cultures of the mould. Take spores from these with a sterile needle, and touch the nutrient gelatine of a series of the prepared tubes: this gives pure cultures of fungus for stock. (81) Various cultures. Prepare a series of small flasks (200 c.c.), plugged with cotton-wool and sterilised. To the flasks (A to E) add 50 c.c. of the following liquids: A. Pure distilled water. B. Solution N minus the dextrose. 62 FUNGI. (CH. III C. Solution N minus the peptone and nitrates. D. A 10°, solution of dextrose only. E. Solution N. [N.B. These experiments need the greatest possible care to avoid any trace of impurity in the salts, water etc.] . Add to each flask one drop of pure water in which spores have been shaken, and separated by filtering through cotton-wool as described above, taking care that the drop contains only a few spores. If properly done each drop should contain about a dozen spores. Place the flasks in a temperature of 20° to 25°C., and compare the growths, which will be as follows :— A.' No perceptible growth’. B. Fair growth at first which soon, however, comes to an end. . C. ‘Hardly perceptible growth which soon stops. D. Fair growth at first, ceasing soon. E. Standard growth, rapid and large. If sufficient care is taken as to absolute purity (a difficult bit of manipulation’), it is possible to show, by leaving one out at a time, that each of the salts mentioned is necessary. Also to show that, with Penicillium, magnesium sul- 1 The microscope shows that the spores germinate, but the mycelium does not continue its growth. 2 Owing to the cotton-wool, dust, glass, water &c. rather than the chemicals themselves. CH. II] FUNGI. 63 phate can be replaced by magnesium sulphite or hyposul- phite, but not by some other sulphur compounds. K by rubidium or cesium, but not by Na, Li, Ba, Sr, Ca, Mg. Ca by Mg, Ba, or Sr, but not by K or Na}. (82) Puccinia. Obtain teleutospores of Puccinia graminis which have wintered on the straw of wheat or Triticum repens, and sow in February—April in, (A) water, (B) nutritive solu- tion, and keep at 10—15°C. in the dark. Both will germinate, and even proceed to develope the “gporidia,” but these die off eventually. Their further developement can only be got by infecting young leaves of the Barberry. The same thing is true of other Uredinee. (83) Hanging-drop cultures. A damp-culture cell is to be prepared as follows’. A deep glass ring is placed on a broad glass slide and a drop of previously sterilised olive oil allowed to run in, or melted paraffin may be run in, in the same way while the slide and ring are hot; this cements the ring to the slide, while a cover-slip placed on the ring like a roof supports the hanging drop. Or a chamber may be made as described and figured by Marshall Ward, loc. cit. p. 131, which is especially useful where it is desired to control the nature of the atmosphere to which the drop is exposed. 1 See Nageli, Hrndhrung der Niederen Pilze. 2H. Marshall Ward, Philosophical Transactions, 1892, n, p. 130. 64 FUNGI. (CH. III Everything being sterilised and the cell ready, take a clean cover-slip, heat it between two sheets of tale over a flame, and allow it to cool. Then, with forceps, place the cover-slip.on any convenient support, and with a platinum needle place a drop on the centre. The drop is got thus :— Infect a tube (gelatine or fluid medium) with a drop of water containing: spores, and shake thoroughly. Hold a platinum needle in a flame, and let it cool; dip it into the infected medium and place the drop on the cover-slip. Then rapidly invert the latter, and cement it to the cell with gelatine, or with oil, or paraffin. The drop should contain one spore, and trials have to be made to insure this. In gelatine media, the student can work with two to five spores if well isolated. All the foregoing experiments can be repeated with - drop-cultures. (84) Germination. Place a culture containing one spore in focus under the microscope. Record the temperature, and fix the spore under the eye-piece micrometer, and cover the whole with a darkened bell-jar. Examine the preparation from time to time, and note the stages of germination. Measure the germinal filaments, mycelial branches &ec., and plot out the rate of growth on sectional paper. (85) Drosera: digestion of white of egg’. Drosera may be grown in wet moss in soup-plates: the moss should be running with water which may advanta- 1 ©. Darwin, Insectivorous Plants, p. 93. CH. III] DROSERA. 65 geously be changed every few days. Drosera cannot be successfully cultivated in large towns. For the experi- ments fresh young leaves having good drops of secretion on their tentacles should be selected. From the white of a hard-boiled egg cut cubes of which the side measures about a millimeter in length: place two of such cubes on each of several leaves, and at the same time put other cubes on the wet moss to serve as a control. They should be examined in 24 hours and again after a further interval of 24 hours. It will be seen that the egg on the Drosera shows a distinct rounding at the angles of the cubes, which are afterwards converted into spheres surrounded by zones of transparent fluid. Still later the spheres generally disappear and nothing but a small quantity of viscid fluid is left. (86) Drosera: benefit derived by feeding}. The plants are, as in exp. 85, to be grown in soup- plates, each of which holds from 20 to 30 plants. Each plate must be divided in two by a thin wooden partition, this serves to mark off those plants which are to be fed from those which are to receive no food. Roast meat is cut across the grain into thin slices and the fibre teazed and cut into fragments so small that 15 together weigh 2 centigrams. A given leaf should not receive more than two of these particles at a time; they may be placed on the glands of separate tentacles: the feeding may be repeated every four or five days. The plants should be 1 F, Darwin, Linnean Society’s Journal, vol. xvii. For references to- other similar experiments see Insectivorous Plants, 2nd Edit. 1888, p. 15. D, A. 5 a 66 ROOTS. [CH. III grown under wooden frames covered with fine netting (mesh 1:5 mm.) to exclude insects. The fed plants soon begin to look clearly greener and more vigorous than the unfed ones. To get a good result the experiment should be begun in May or June and continued to the middle of August. The number and height of the flower scapes, the number and weight of capsules, the number of seeds per capsule, &c. should be compared. Or the plants may be carefully washed and dissected out of the moss and the dry weight per plant of the fed and starved specimens compared. SEcTIoN C. Roots. (87) De Saussure’s experiment’. When plants are placed in solutions of various salts they do not, except under certain conditions, absorb the water and salt in the same proportion. De Saussure, using solutions that were not very dilute, found that the plant absorbed a relatively less salt than might have been expected. This condition of things is sometimes spoken of as absorption according to De Saussure’s law, and although it is well known to be only a special case, the fact itself is worth confirming. In our experiments we proceeded as follows. , A bunch of rooted water-cresses (Nasturtium officinale) was taken up, washed and placed in distilled water for three days to allow the roots to recover from their in- juries. They were then placed in a beaker containing 1 De Saussure,’ Recherches chimiques, 1804, p. 247. CH. IT] ROOTS. 67 700 cc. of a solution made by dissolving 1 part of potassium chloride in 1000 parts of water. They were left in the fluid for 8 days, by which time only 260 cc. of solution were left in the beaker. This was ana- lysed volumetrically, by titrating with decinormal silver nitrate, using potassium chromate as indidator. If the salt and the water had been absorbed in the same proportion the remaining solution should have still con- tained 01 p.c., ie, 0°26 grams; in other words, the plant should have absorbed 0°44 grams. It was found however that less than this had been taken up, and that $, ie. 0'5 grams, of the original potassium chloride instead of 026 grams were still present. Other salts give various different coefficients for this same strength of solution. If sufficiently dilute solutions be made use of, it has been found that, in contrast, relatively more salt than water is absorbed and the remaining portion of the liquid contains less than the due proportion of the original salt. (88) Root pressure. Root pressure can be easily observed in young plants of Phaseolus. An indiarubber tube 7 (Fig. 11) is tied on the cut stump, S, of the plant and is filled with water: a capil- lary glass tube G is tied into the tube, leaving about six centimeters of rubber tube full of water between the stump and the bottom of the glass tube. The glass tube is now fixed in a clip and after a time drops of water fall from the end £. To get an idea of the rate of flow it is only necessary to gently pinch the rubber tube so as to press the fluid out, and to absorb it with filter-paper held 5—2 68 ROOT PRESSURE. [CH, III at E. When the rubber tube is released a column of air is drawn into the tube and serves as an index of the Fie. 11. Exp. 88. rate of flow as it travels up the tube, which should be graduated. By watering the earth with warm water a greatly accelerated rate of flow is obtained, but whether it is due to increased root pressure or to the expansion of air in the tissues is not easy to say. (89) Root pressure. To demonstrate the force of root pressure a striking method is that used by Mr Gardiner in his lectures. He uses a plant of Sparmannia growing in a large pot. The CH. 111] ROOT PRESSURE. 69 stump is attached by rubber tubing to a potometer tube! filled with a solution of nigrosin in water; to one arm of the potometer a vertical glass tube, a few mm. in diameter and several feet in length, is attached; the other arm of the potometer is closed with a cork. The nigrosin seems to have no bad effect on the plant and makes the rising column of fluid easily visible. If the tube is supported against a wall it can be elongated by fresh lengths of glass tubing and thus a column of 8 or 10 feet can easily be shown. (89. A) Root pressure. The classical method of observing root pressure is that described and figured by Sachs in his Physiologie Végétale (Fr. Trans.), p. 223, of which the following (Fig. 12) Fic, 12. Exp. 89 A. is a modification. A T tube (7) having one arm B bent 1 The arrangement is similar to that figured in Sachs’ Vorlesungen, p. 328, fig. 211. 70 WATER EXUDED. [ox. 11 so as to be parallel to the two others, is tied into a piece of pressure tube which is also tied to the plant. The arm B passes through a rubber cork firmly tied into a wide- mouthed (stoppered) bottle, in the bottom of which is half an inch of mercury, Hg: the tube M, which serves for manometer readings, fits tightly into a hole in the cork and reaches the bottom of the bottle. Water W is now poured in at C' so that the bottle and the arm 7 are filled. At first the plant will usually absorb water, so that C should be left open until the rise begins, when it may be filled up and closed by means of a clamp. The mercury will rise to a considerable height and will show diurnal variations about its mean position which should be carefully noted. (90) Moll’s Eaperiment. Various kinds of plants, when placed under a bell- jar standing in a dish of water, will give evidence of root pressure by the drops of water exuding from the leaves. Root pressure may as Moll has shown! be replaced by that of a column of mercury. The branch or leaf-stalk, as the case may be, is fixed air-tight into the short arm of a U tube filled with water, and mercury is then poured into the long arm until about 20 cm. pressure is obtained. The whole is then covered with a bell-jar standing in water, and after a time drops of fluid are found hanging to the leaves. We found that with 25 cm. of mercury the drops appear very rapidly on the leaves of the 1 Bot. Zeitung, 1880, p. 49. References are given to Sachs’ Lehrbuch, 1874, p. 660, and de Bary, Bot, Zeitung, 1869, p. 888, for similar results. CH, III] DEAD ROOTS. 71 Balsam (Impatiens balsamina). Moll also recommends Begonia and Phaseolus: in the last named the fluid is, as Moll says, found on the lower surface of the leaf. (91) Absorption by means of dead roots. Several observers’ have shown that transpiring plants can absorb water from the soil even after the roots are dead. We have confirmed the fact on pot-plants of Helianthus tuberosus. A thermometer having been forced into the earth, the flower-pot is immersed in water so hot that the soil is kept at a temperature of 60°—65° C. for two hours. In spite of this violent treatment the leaves remain turgescent for several days, whereas control- plants shaken out of their pots and freed from soil rapidly wither. 1 Strasburger, Leitungsbahnen, 1891, p. 849, where references to earlier experiments are given. CHAPTER IV. TRANSPIRATION. Section A. Absorption of water by transpiring planta. Section B. Loss of weight due to transpiration. Section C. Stomata, Bloom, Lenticels. Section A. Absorption. (92) Potometer'. In the first series of experiments (Section A) the rate of absorption of water by transpiring plants under varying circumstances is to be observed. This may be done with potometer as shown in fig. 13. Of the three openings of the potometer, A and B are closed by rubber corks; that in B is perforated by a thermometer-tube of about 0'3 mm. bore: the tube should just project beyond the cork on the inside and should have a total length of 7 or 8 inches. The end 4 is closed by an unperforated cork, while to C is fitted about 4 inches of rubber tubing, of which 2 inches project beyond the end of the tube. The cork B should first be fitted in, then fill the potometer with water and 1 Darwin and Phillips, Cambridge Philosoph. Society, Vol. v. 1886. 74 POTOMETER. {CH, IV force the branch! into the rubber tube C, as far as it will go. The joint between the rubber tube and the branch must be secured by tying; for this it is best to use strong uncovered elastic thread, which must be stretched while it is being wrapped round the tube, and can be secured by a simple tie, a knot being unnecessary. The rubber tube may be secured to the glass tube with wire. Turn the potometer upside down so that any air in C may rise and collect at A, and before corking