ALBERT RK MANN LIBRARY AT CORNELL UNIVERSITY. DATE DUE Cambridge Patural Srtence Manuals BIOLOGICAL SERIES. GeneraL Epiror:—Arrtaur E. Suretey, M.A. FELLOW AND TUTOR OF CHRIST'S COLLEGE, CAMBRIDGE. PRACTICAL PHYSIOLOGY OF PLANTS, London: C. J. CLAY anp SONS, CAMBRIDGE UNIVERSITY PRESS WAREHOUSE, AVE MARIA LANE, AND H. K. LEWIS, 136, GOWER STREET, W.C. Cambridge: 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. ] QK Ti D2 Qe, 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. BotanicaL 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. = 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 zi . ‘ ‘ ‘ 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 - ‘ ‘ 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. 81. 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. ii. 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 . si . . 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 . Fi 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. Ssection 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 . . : . . . i . . 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 js . 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 . : ‘ 2 : ‘ . pp. 66—71. CHAPTER IV. TRANSPIRATION. Srcrion 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. 8&—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 stoma'ta. 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. 182, 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 . ‘ ‘ - . 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’ssgypsum 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. 130—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. 188—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 . : . F ‘ 5 . 4 pp. L86—149. CHAPTER VII CURVATURES. Ssction 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. Gurvatures 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. Pe and ee acting together. 215. Trans- mitted stimulus . fi : - pp. 165—168. Section D. Diaheliotropism, diageotropism &c. 216. Diahelio- tropism. 217. Due to specific sensitiveness, klinostat. 2174. Ex- 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 A 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. cumuutation. 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 ei 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 . ‘ c 7 : . 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 dexirins. 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 ‘5 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 ‘ 3 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 . . : F : - : 3 . 5 pp. 296—sS04. APPENDIX II. List of reagents and material required for experiments on meta- bolism ... a2 8 ‘ . . . ‘ pp. 305—308. FIa@, one 5 6 LIST OF ILLUSTRATIONS. Apparatus for demonstrating respiration . Apparatus for estimating CO, produced during ceapieaton Foil clamp for holding cover-slips together under water, for use with Velten’s hot-stage 3 . : Apparatus for the preparation of mpi free from CO, ih not free from oxygen . é . . . . Arrangement for the culture of ae in an atmosphere tae from CO, . : $ : : Apparatus for gananulpeis for use in experiments on assimi- lation . 7, 8,9 Winklestrempal wa panteira for gas- -anelyate 7 10,104 Lemna cultivated in various nutrient solutions . 11 12 13 14 15 16 17 18 19 20 Apparatus for demonstrating root pressure . 5 . Z Another apparatus for the same purpose . The Potometer, for estimating the absorption of wales bya a cut branch . : A é . 2 A modification of Kohl’s aipumtne for ‘tha same purpose . A method of preventing evaporation from the surface of a flower-pot . 7 : . Apparatus for comparing ‘lage ia incieninalion with the swith absorbed . s ‘ A spring-balance for use in eanentcation expetimenta. A hygrometer made of the awn of Stipa for performing Garreau’s experiment . . ‘i ‘ é : . Devaux’s gelatine method of making an air-tight sanstion with the petiole of a leaf . 3 F Apparatus for demonstrating the ligeroasopis merce of the awn of Stipa : ‘ if . 56, 58 PAGE 4 13 24 25 36 38 68 69 33 76 88 90 92 94 96 103 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 39 40 41 42 43 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 Latpenesul ost ise rigidity when bent . ‘i < Micrometer-screw used in growth aupertnsuts Recording auxanometer . Method of using the necdapmiidedayer Tracing illustrating the effect of an increase of teunoentatd on growth Hanging writer for veconting: geonnepie. or thay movements on a revolving drum . Illustrating the curvature of a oot stilehs ha eeeerreradl font the effects of decapitation . Curvature of roots produced mu small planes of ward attadhed to the tips . ‘ : . s 2 Drooping of laurel — in a frost A twig of Veronica salicifolia exposed to oblique hunitantion The klinostat . 7 a 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 noattiod - 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 ‘ * A Apparatus for distillation ides vediased 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 ina 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, Fie, 2, Exp. 3. 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. 1. 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, 403. 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, 4 — 7 cy 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. 1 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. i 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 Owalis acetosella, using the colour test already described. Shake up 1 cc. chloroform in 200 ec. 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. Szotion C. Reactions of Chlorophyill. Section 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, Tropzolum 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. 11 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. II] 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 in a 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 800 cc. 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. It] 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 50.c.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. (87) 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. 380 FORMALDEHYDE. [cH. IL. posed into formaldehyde and NaHSO,) can be used in culture fluids in the proportion of 01 per cent. without injury to Spirogyra. Bokorny (Joc. 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 illumination 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 em. 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 bition 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. IT] 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}. aan (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 ¢°. See Bunsen and Roscoe, Gasometric Analysis. 1 38 GAS ANALYSIS. [CH. 1 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. II] .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. Exp. 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 LZ 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 V du Fig. 9. Exp. 52. graduations on Gt, 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. LZ 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. CH. 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. 1 (56) Action of light. Fill three test-tubes with alcoholic leaf-extract, cork them and place A in sunlight, B in diffused light, 0 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°/, CuS,? solution a 1 Or of strong solution of copper acetate and strong HCl. CH. I] 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 T 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. 47 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) ttolin 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. LEtiolated 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 variety 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—6?. 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 ‘3 a Magnesium sulphate (crystals) 1 ef Pe 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 tobe 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 Ny 2 i Pwehyo ¢-# wae 84ee Worn Os "eh «4 ddctoZo &O feoveret rover re ee ee oe ee ee eh ee @ Orie N RON mMaynu-Be-e | ar) + a . i) an oe * e 4 a €¢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, sculus 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. 111 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. UT] 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 cc. 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. 2 H. 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, &. 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. 011] 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., 1e, 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 0:26 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. II] 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 03 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. CH. IV] POTOMETER. Fic. 13. Exp. 92. 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 A, fill it to the brim with water. Support the potometer on a firm retort-stand and fix the plant to the same stand to avoid any possible movement between the plant and the in- strument. The end of the capillary tube dips into a small vessel of water W supported on two blocks, of which the upper one is small enough to be conveniently seized in one hand, and of such a height that when it is removed the thermometer tube no longer dips in the water. When this is done, (if the plant is absorbing vigorously) a column of air will be sucked? in at the lower end of the 1 If herbaceous plants, or woody plants with delicate leaves, are used for transpiration experiments it is necessary to cut them from the parent plant under water, to prevent the entrance of air, which rushes in to satisfy the negative pressure. It is generally possible to force a branch below the surface of a basin held by an assistant, and divide it with a strong pair of gardeners’ shears, In the case of herbaceous plants the process is made easier if a couple of sharp bends are made in the stem, a proceeding which need not admit air, and has no effect in the transpira- tion current in the plant. For all research-work connected with the transmission of water the specimens should be thus treated, but for the following experiments, in which laurel or Portugal laurel are used, cutting under water is not necessary. 2 To hasten the entrance of the air column, it is best to absorb, with piece of filter-paper, the water hanging at the end of the tube. CH. IV] POTOMETER, 75 tube. As soon as this appears the small block is replaced, the tube once more dips into the water, and a bubble of air included in it travels up, and serves as an index of the rate of absorption. The bubble must be of uniform size in successive readings, because other things being equal a long and short bubble travel at different rates. To insure this, mark the tube with a file at 5 or 6mm. from its end, and replace the vessel W when the air column has reached the file-mark. The movement of the air-bubble is timed from a mark on the tube: the upper limit of its course is the upper end of the capillary tube, the moment of its impinging against the column of water in the potometer being easily visible. It is for this reason that the tube projects above the cork, for otherwise a convenient place of ending for the course of the bubble would not be visible. The starting point of the course should be at least 2 inches above the file-mark, so that the bubble may settle down to a uniform pace before the course begins; and because time is needed for the observer to put down the block and the small vessel of water, and take up the stop-watch. _ In this way numerous readings can be taken in a short time; the air admitted collects at A, and can be removed after a time; it is obvious if the branch were placed in A and the cork in C, that the admitted air might diminish the surface of contact between the water and the absorbing surface of the branch. It is well to make graphic representations of one or more of the following experiments, using the reciprocals of the stop-watch readings, which will be proportional to 76 KOHL’S METHOD. [CH. IV the rates of absorption!. The actual volume of water absorbed per unit of time may be obtained by calculation from the length and diameter of the tube 7. Or by re- placing the plant by a siphon delivering a known weight of water per hour’. (98) Kohl’s method [slightly modified]. This apparatus, like the potometer, is a modification of Sachs’ instrument. Here the index is not a bubble of air, but the column of air which travels onwards as the water is absorbed. We use a potometer fitted up as Fie. 14. Exp. 93. shown in fig. 14. The end, c¢, as before, takes the branch br; a takes a tube connected by a rubber tube with a funnel f, and closed by a clamp; 6 takes a glass tube connected with a horizontal tube h, which is coarser 1 Reciprocals may be got from published mathematical tables. 2 See Darwin and Phillips, loc. cit. CH, Iv] SUN AND WIND. 77 than that used in exp. 92, and also longer. When no readings are being taken the bent free end of h dips into the vessel of water v; when v is removed a column of air enters and travels along h, its rate of movement being noted by timing it over intervals of 5 or 10mm. For this purpose h may be graduated, or a millimeter scale may be set up behind it. When the column of air has nearly reached b it can be brought to the zero of the scale by opening the clip and allowing the water from the funnel f to drive it back along h. (94) Sunshine. Take a branch of Portugal laurel? which has been cut and placed in water for at least 6 hours. This pre- caution is necessary to satisfy the negative pressure in the branch, if this is not effected variations in the rate of absorption will by no means represent variations in rate of transpiration, Fit up the branch in the potometer and take readings until the rate of absorption is fairly constant. Then place the plant in sunshine and observe the increased rate of absorption, and finally replace it in shade. (95) Wind. When the rate is once more steady, open a door and a window so that the plant is exposed to a draught. 1 The bore of the tube must be varied according to the size and absorbing power of the specimen. In the figure h is represented shorter than it actually is. 2 Kohl’s apparatus will answer for any of the experiments for which the potometer is recommended, and vice versa. 78 LIGHT. [CH. IV Compare the effect on the plant with the readings of a wet and dry bulb thermometer. (96) Light and darkness. Select a rounded bushy branch of a tree, and if neces- sary tie in the branches to make the specimen compact. Fit the branch air-tight into a hole in a glass plate resting on a large tripod, the branch will thus be above the plate and may be covered with a tubulated bell-jar, while the apparatus is below and free to be manipulated. Into the tubulure fit a cork through which two glass tubes pass, one of which is connected with an aspirator so that a current of air is drawn through the bell-jar. The other tube is connected with chloride of calcium tubes so that the air in the jar may be dry. Readings of the potometer and of the hygrometer inside the bell-jar should now be taken in varying conditions of the air. The current aspired must not be too rapid, or the air pressure inside the jar will be less than atmospheric pressure; a small mercury manometer fitted into the cork of the tubulure will show this difference if it arises. If the air inside can be kept at a constant hygrometric condition the effect of alternate light and darkness may be shown as in Kohl’s research: the bell-jar is darkened by a cover, which can be removed when darkness is exchanged for light. Kohl’s method is an excellent one for getting rid of the difficulty which meets the experimenter in all researches involving 1 If it is merely desired to show the effect of dry and damp air ina rough way, a simple bell-jar may be used which may either cover the plant completely or be removed, or may be propped up to produce an intermediate hygrometric condition. CH. IV] NEGATIVE PRESSURE. 79 changes from light to darkness, and vice versa, namely, that the hygrometric state of the air does not remain the same when the change in illumination takes place. For some reason we have not found it easy to keep this condition uniform even with Kohl’s apparatus, but it is clear from his excellent results that it is to be done. Kohl performed the experiment with rooted plants, which is in every way preferable. His method will be under- stood from his illustrations, fig. 2: and his results from the curves on page 63+. (97) Negative pressure. The object of this experiment is to prove the need of the precaution mentioned under experiment 94, viz. leaving the cut end of the branch under water for some hours before using it. Cut a branch from a Portugal laurel, fit it at once to Kohl’s apparatus, and take a series of readings. The absorption will be found to be very quick at first, and then to become slower. (98) Negative pressure. Fit up a laurel branch in a potometer and allow it to remain for a day or so. Having taken a series of readings take out the branch and shave a few millimeters off the eut end, which will have become dirty: the partial stoppage of the vessels, which gives the dark tint, produces a slowing of the current, and when it is removed the readings of the stop-watch show an immediate increase in rate. When the readings are fairly steady 1. Kohl, Die Transpiraticn der Pflanzen, 1886. 80 NEGATIVE PRESSURE. [CH. IV again, repeat the removal of a shaving of wood to prove that this per se has no special effect. (99) Negative pressure. A similar result may be got more quickly by filling the potometer with an emulsion of skim-milk diluted with three times its volume of water. The slowing of the current, and the recovery when the blocked ends of the vessels are cut off, may thus be seen within a short space of time. (100) Negative pressure. The fact of the existence of negative pressure may best be demonstrated by von Hohnel’s method’ If a strongly transpiring stem or branch be cut under a watery solution of eosin, immediately removed and examined, the red colour will be found to have rushed into the vessels to a considerable height. We find that “Solomon’s seal” (Polygonatum multiflorum) answers well. Or plants of Helianthus tuberosus grown in pots and left without water until they are on the point of withering. In winter, seedlings of Vicia faba pulled out of the loose sawdust in which they have grown, and allowed to lie on the table until nearly withered, show good injection. (101) Permeability of cell membranes. Negative pressure depends on the fact that dry cell membranes are far more easily permeable to air than are wet membranes. (i) Take a cylinder 2 inches long by 4 inch diameter, turned from the splint wood of a fresh branch of Yew or of Pinus, and attach it by. a strong 1 Pringsheim’s Jahrbiicher, xii. 1879, p. 47. cH. IV] FILTRATION EXPERIMENT. 81 rubber tube to the short arm of a U tube. By pouring mercury into the long arm it will be found possible to force air through; to make this obvious paint the upper surface of the wood with olive oil in which the escaping bubbles are visible. (ii) A similar cylinder which has been thoroughly soaked in water is fitted into the U tube: the wet cell- membranes will be found to be extremely, though not absolutely, impermeable to air. (iii) If the U tube is now filled with water it will be found that a very slight pressure of water forces water through. (102) Oozing of water from the lower end of wood. The experience gained in experiment 101 enables us to understand the following experiment’. A yew-branch (two inches in length) is placed in water until thoroughly soaked. When removed from the water and held with its axis vertical no water escapes from the lower surface (although water is contained in the tracheids) because if water is to escape air must enter, and air does not easily pass wet membranes. If however a drop of water is added (e.g. with a wet paint brush) to the upper surface, water immediately oozes out below. (103) Permeability of splint wood. A modification of the experiment may be used to illustrate the fact that the water travels in the splint-- 1 See Sachs in his Arbeiten, 1. p. 296. Also Godlewski, Pringsheim’s Jahrbiicher, xv. D, A. 6 82 FLACCID SHOOT. [CH. IV wood, A piece of yew-branch (2—3 inches) is fitted to a rubber tube of about 3 feet in length. The tube is now filled with water and closed by a ‘clip. If the wood is held vertically with tube hanging straight down, the upper surface of the wood, which must be cut smooth, is dry. If the closed end of the tube is raised until it is slightly above the top of the branch, the surface of the young wood is seen to blush or change colour, even before the water can be seen to actually ooze from it. (104) Recovery of a flaccid shoot. De Vries has shown? that when a shoot is cut in the air it frequently withers after it has been placed in water. This has usually been explained as being due to the air rushing in under negative pressure and filling the vessels. It is not quite clear that this is the cause, but whatever the explanation may be, it is interesting to note that a shoot which has been rendered flaccid, by being cut in the air and allowed to partially wither, can be rapidly restored to turgescence by forcing water into its vessels under pressure,—which takes the place of the suction of nega- tive pressure. The cut end of such a withered shoot is attached by a rubber tube to the short arm of a U tube containing water. The position of the end of the shoot, which droops flaccidly over, is noted on a vertical scale, and then mercury is poured into the long arm. Under the pressure of about 10 cm. mercury, the plant recovers and the end of the shoot can be traced with the naked eye rapidly travelling up the scale. 1 Sachs’ Arbeiten, 1. p. 287. CH. IV] EMULSION EXPERIMENT. 83 (105) Sachs’ emulsion experiment? To illustrate the fact that the pits of coniferous wood are closed and that the water-current must therefore filter through them, the following experiment is useful. Prepare an emulsion of vermillion by adding a few paint brushes full of good colour to a beaker of water, and filtering it through coarse filter paper. Take a piece of yew 3 or 4 inches in length and attach it by a rubber tube to the lower end of a glass tube 3 feet in length held vertically in a clamp. Fill the tube with emulsion and observe that colourless water drips from the lower end of the wood. After an hour or so remove the wood: note the red colour of the young wood due to injection of the cut tracheids with vermillion. Cut a shaving off the surface to show that the colour only extends to a small depth. An interesting modification of the experiment is to plunge the cut ends of transpiring branches into emulsion : in this way the distribution of the transpiration current in the cross section may be studied in various plants. Diluted skim milk stained black with osmic acid makes a good emulsion. (106) Injection of vessels’. ” Cut two similar branches of Portugal laurel, place one in water, the other in melted cocoa-butter, into which it must dip as deeply as the vessel allows. After an hour, during which the cocoa-butter is kept melted, take the 1 Arbeiten, 11. p. 299. 2 Elfving, Bot. Zeitung, 1882. 84 COMPRESSION. (cH. Iv branches out of the fluids, and let them lie on the table till the cocoa-fat is quite cold: cut fresh surfaces to both and place them in watery solution of eosin. After an hour or two the progress of the eosin may be compared by cutting off shavings of bark at various heights in the two branches. (107) Compression. To prove that the transpiration current travels in the vascular cavities, it may be shown that squeezing the tissues in a vice checks the upward stream of water’. Cut, under water, a leafy stem of Helianthus tuberosus, and fit it to Kohl’s apparatus. A plant with well formed wood must be chosen,—young stems are too brittle. Branches of bramble also serve, but are awkward to work with ; in winter, last summer’s shoots of ivy answer fairly well. Do not attempt to compress the whole stem but cut away half of it before applying the vice. The exposed surface may for greater security be rubbed with lard to prevent air leaking into the vessels exposed ; in any case the part selected for compression must be as far as convenient from the cut end, so as to avoid the chance of air being sucked back into the apparatus. The vice should be a light one so that it may support itself when it is screwed on to the stem. " When the rate of absorption is steady, compression may be applied: it will be found necessary to screw the vice with great force so that the compressed tissues are squeezed to a mere plate. If the compression has been 1 F, Darwin and R. Phillips, Cambridge Philosoph. Soc. 1886. CH. IV] INCISION. 85 continued for some time, and the vice is then unscrewed, it will be noted that the absorption is very rapid and that it soon slows down. This shows that negative pressure rises during compression and falls when water is allowed freely to enter the vessels. (108) Incisions. In some trees it is obvious that the amount of wood in the transverse section is far greater than is absolutely needed to carry the transpiration current. Fit a branch of yew! (Taxus) to the potometer, and take a few readings, then saw it half through and read again. The rate of absorption will be unaltered, and the branch may indeed be almost severed before the rate of absorption-is seriously depressed. The branch must be firmly supported in two places and the incision made between them, otherwise the weight of the branch will break the thin bridge of wood which is ultimately left. When a slowing of the current has been clearly pro- duced, cut the bridge through and compare its area with that of the rest of the splint wood of the branch. (109) Cross-cuts. Take a branch of Portugal laurel (or of ordinary laurel) which has stood some hours in water; fit it up in the potometer and, as in experiment 108, support the branch in two places, so that it may not break when incisions are made. Having taken a few readings, saw the branch " In the case of yew it is better to remove the bark (at any rate in the spring) because it is easily detached from the wood, and this makes it difficult to slip on the rubber tube. 86 EOSIN ABSORBED. [CH. IV half through at a spot between the two supported points, and 10—15 cm. from the cut end. To see clearly how far the saw-cut penetrates, and on which side of the branch it lies, it is advisable to push a square piece of cardboard (for instance a post-card) into the cut. This serves as a guide in making the next cut, which must be exactly opposite incision (i), and 2cm. above it. It must be slightly deeper than incision (i), so as to overlap it; it is easy to make sure of this if a second card is placed in the second incision: the edges of the cards should be parallel and should slightly overlap each other. The points to note are that cut (ii) depresses the rate of absorption very much more than cut (i), and that after the fall, a rise in absorption-rate comes on. (110) Course shown by eosin solution. Remove from the potometer the branch used in ex- periment 109, and place the cut end in strong watery solution of eosin, taking off the bark of the part in which the saw cuts were made, leaving, however, 2 or 3 inches at the base unpeeled. The course of the fluid as it passes up the stem is now traced by the eosin, the manner in which the colour spreads at the doubly-cut region being of course the chief point to be noticed. The reason for leaving the bark on the terminal two inches (which is not an essential precaution) is simply to ensure that any superficial rise of fluid shall take place on the bark instead of on the wood. (111) Atr-pump. Cut a branch of Portugal laurel of the same size as CH. IV] AIR-PUMP. 87 that used in experiment 109, and select one having a part of about 12 inches in length bare of side branches; leave it in water for some hours, then cut off the 12 inches and attach one end of it to a potometer, and the other to a water air-pump. When the air-pump is in action water will be sucked through the branch out of the poto- meter and readings can be taken with a stop-watch. Adjust the suction of the pump so that the readings of the poto- meter are roughly the same as those obtained in experi- ment 109. Now make the two overlapping saw-cuts as explained under experiment 109 and note the result. The point of interest is that here there is no recovery after .. the depression in rate of absorption, because there is nothing corresponding to the increased negative pressure due to continued transpiration in experiment 109. (112) Strasburger’s air-pump experiment}. The last experiment depends on a current of water being drawn through wood by diminished air pressure. In the following experiment the current moves in spite of negative pressure. Cut a sound branch of yew, peel the lower 4 or 5 inches and place it in water for about 12 hours; cut a clean surface and fix it tightly in a perforated rubber cork fitted into a bottle with a ground mouth. The cork is also perforated for a tube connected with an air-pump. The tube must only just project below the cork, while the yew branch must be thrust through far enough to dip 1 Leitungsbahnen, p. 795. A similar experiment is given by Janse, Pringsheim’s Jahrb. 1887. 88 LOSS OF WEIGHT. [CH. IV well below the eosin solution which should not more than half fill the bottle. When the air-pump is set in action, it is obvious that its tendency is to suck out the contents of the tracheids at the cut end, and as a fact air bubbles are seen to issue at that point. Nevertheless, in spite of this the eosin rises in the branch. Leave the pump running for 6 or 7 hours, when the branch should be sawn off above the cork, without stopping the pump, so as to avoid injection of the wood with eosin. Readings of the baro- meter should be taken in the course of the experiment and compared with the readings of the pump-manometer'’. Section B. Loss of Water by Transpiration. (113) Loss of weight. To get a general idea of the amount of loss due to transpiration it is well to take a series of weighings of a plant growing in a flower pot. Select a plant? with a large leaf surface, in a small flower pot so that it may not be too heavy for the balance*, In order to confine the loss by evaporation to the plant, the sur- face of the earth must be covered with a divided disc of sheet-cork painted over with wax mixture. The pot is wrapped in sheet india- Fie. 15. Exp. 113. 1 We have only used a negative pressure of 50 cm., but Strasburger used 72 cm. 2 Jerusalem artichoke or Chrysanthemum. 3 We use a French druggist’s balance capable of carrying 4 or 5 kilograms, and of turning with 0-5 gram when loaded with 1000 grams in each pan; it is a useful form of balance for the purpose in question. The beam, etc. being below in the box, and the pans there- fore free to take a tall plant. : CH. IV] LOSS OF WEIGHT. 89 rubber which may be held in its place as shown in fig. 15; the glass vessel c grips the rubber sheet, and also serves to prevent evaporation from the bottom of the pot. Since it may be necessary to water the plant during the course of the experiment, a corked tube must be fitted into a hole in the cork plate. (114) Transpiration compared with evaporation of a surface of water. To estimate the transpiration from a given leaf surface it will be necessary to take a plant small enough to be placed on more delicate balance. Detmer recom- mends a Phaseolus grown in a glass vessel having a ground edge so that it can be covered by a divided glass plate. We have found it a simple plan to make use of Lambert and Butler’s 4 1b. tobacco tins. A small plant such as a Pelargonium can be knocked out of its pot and trans- planted to one of these tins. Owing to the stopper-like arrangement by which the tins are closed, it is easy to replace the tin-lid by a split cork through which the stem, and a watering-tube pass. Ascertain the loss by transpiration in say 12 hours, and at the same time ascertain the loss of weight from a shallow dish of water of known area. Now calculate the transpiring area of the plant and compare its loss of weight per unit of area with that of the water. If a planimeter is not available the area may be calculated by tracing the form of a leaf on stout paper, cutting it out and comparing its weight with that of a 90 LOSS COMPARED {CH IV piece of known area. If the plant has leaves of various sizes, the leaves are classified into two or three sizes, and the area of one of each heap is taken. If the amount of stem or branches is considerable an estimate should be made of the area of these, and the amount added to the area of the leaves. (115) Loss of weight compared with absorption. To demonstrate that the loss of weight is roughly equal to the amount of water absorbed by a cut branch, Fic, 16. Exp. 115. ° select a branch of Portugal laurel which has no young CH. Iv] WITH ABSORPTION. 91 growing shoots, or remove any that may be present. Let it remain in water for 24 hours, cut a fresh surface and fit it in a bottle arranged as in the fig. 16. The branch B fits a tube which pierces the cork and should dip well into the water in the bottle. Through another hole in the cork a tube 7 graduated into j,cc. and holding about 20c.c. is passed: this serves to record the amount of water absorbed by B: the opening of 7 must be closed with a plug of cotton wool to allow air to enter, and yet to hinder evaporation from 7. In one experiment we found that a branch of laurel with 30 leaves weighed, with the bottle of water, 287 grams: by using one of Becker’s balances without a glass case, and having a beam sup- ported 40 cm. above the table of the balance, it was possible to place the bottle on the pan and arrange the leaves and branches so as to be clear of the scale-pan knife-edges. Weighings and readings should be taken at hourly intervals, The burette ought to be read to 0°01lc.c., if the weight is recorded to 0°01 gram. It is instructive to repeat the experiment with a freshly cut branch in which the negative pressure is not satisfied. It will be seen that the absorption is much greater than the loss by weight, it may, for instance, be three times as great. After taking a few hourly readings the apparatus should be left to itself for 12 hours when equality between gain and loss should be fairly established. (116) Spring Balance. We have found the following arrangement, fig. 17, useful 92 SPRING BALANCE. - [CH. IV for demonstrating the loss of weight due to transpiration, and it is probable that it may prove to be useful for Fic. 17. Exp. 116. research purposes under certain conditions. A cut branch in a test-tube of water 7 is suspended by a wire to a spiral spring S. At P the wire passes through a small hole in a metal plate: at 7 a fine spun glass filament is fastened horizontally to the wire. As T loses weight the index rises and its movement is recorded by means of a horizontal microscope. With the weakest power of our microscope one degree of the ocular micrometer equals 0:044:mm.: the following readings (expressed in gradua- tions of the micrometer) were obtained by adding a CH, IV] STOMATA. _ 98 decigram at a time to a scale-pan suspended to the spring, PS 03 5 Pe VS TOs 18g CEs Poe TO. When the whole weight was put on at once the index moved through 66° of the micrometer, giving 7:3° as the average value of 0'1 gram’. When a transpiring plant is suspended the loss of weight may be read every 5 minutes with less disturbance to the plant and with less labour to the observer than with a balance. SEcrION C. Stomata. Bloom. Lenticels. (117) Stomatal transpiration. Cut a pair of similar well-grown leaves of Ficus elastica and when the bleeding of latex from the cut ends has practically ceased, slip about an inch of tightly fitting rubber tubing over the leaf stalk, leaving 4 inch of tube projecting; then fold the free end down and wire it tightly to the tube-covered stalk. In this way evaporation from the cut end of the stalk is prevented: the wire ties will also serve to hang up the leaves, and by twisting the free end of the wire into a loop for one leaf and into a hook for the other, obvious and permanent marks are provided for the distinction of the leaves. Having weighed them, hang them up close together in a dry room for 2 or 3 hours when they must be again weighed. These weighings give the ratio between the normal transpiration 1 The springs we use are made by Salter of Birmingham: they are about 5em. in length, when unstretched. 94 STIPA HYGROMETER. [cH. Iv of the two leaves. Now smear the lower surface of A and the upper surface of B with vaseline, which should be carefully rubbed on with a finger. Weigh the specimens and leave them for 24 hours. It will be found that B loses in weight something like 10 times as much as A. (118) Stomatal transpiration (observed by another method). For demonstration purposes the well-known experi- ment of Garreau? can be repeated in a very simple manner, with a rough sort of hygrometer represented in the sectional dia- gram, fig. 18. It consists of a small 3 9 glass cylinder g across the mouth of \(—+—I which a glass tube is fixed: from the centre of the tube a piece of Stipa-awn § projects at right angles and bears at its end an index 7 which may conveniently be made of thin iron wire. The awn is sensitive to hygrometric change, in damp air it untwists, in dry air it twists up again. If the vessel is therefore placed mouth downwards on damp blotting paper or on a transpiring leaf, the index 7 will rotate and its movement can be read off on a graduated ring of paper fastened to the bottom of the vessel. If two hygrometers are made, one may be placed on each surface of a leaf and the difference in the movement of their indices compared. Certain precautions are necessary : in the first place, it is difficult to get two pieces of awn’, which behave similarly, 1 Ann. Sc. Nat. 1850. 2 The awn should be thoroughly ripe, brown in colour, not yellow, and stiff not weedy in texture. Fie. 18. Exp. 118. CH. Iv] STIPA HYGROMETER. 95 so that it is necessary to graduate the two hygrometers to make their readings comparable. Take a filter paper and damp it carefully, making sure that it is not wetter in one part than the other, place it on a flat glass plate and having marked the position of the index with pencil on the paper rings in both hygrometers, place them side by side on the wet paper. After from 4 to 8 minutes mark the position of the index again. If, for instance, the movement of hygrometer A is only 3 of that of B, it is clear that the paper ring on A must be marked out in divisions each of which is 2 of the unit used for hygrometer B. Our hygrometer scales are usually divided into 60—100 divisions. To fit the hygrometers on to the leaf (we use laurel leaves?) two plates of cork are wanted, each having a circular opening slightly smaller than the hygrometer: one plate has a groove running across the middle which receives the midrib of the leaf, and allows it to lie flat between two plates. One hygrometer is placed mouth upwards on the table, then the leaf between the plates, then the other hygrometer mouth downwards: the whole being kept steady by a weight of 2 or 3 oz. placed on the top. For laurel leaves 7 or 8 minutes is generally long enough to wait before reading the hygrometers. [The general behaviour of stomata may be conveniently studied here.] (119) Stomata: connection with intercellular spaces. For this experiment the choice of a suitable leaf 1 Ivy leaves are equally good, and if the apical part of the leaf is used, the cork plates may be dispensed with. 96 INTERCELLULAR SPACES. (CH. IV is important. The following answer well: Arwm macu- latum, Ranunculus ficaria, Eranthis hiemalis, Caltha palustris, Primula sinensis, Iimnanthemum sp. The leaf stalk is fixed air-tight in a rubber cork which fits a bottle filled with water. The leaf can be fixed by piercing the cork with a hole too small for the stalk, and dividing the cork longitudinally down one side till the hole is laid open. Or an undivided hole may be used if made air tight with wax mixture. Through a second hole in the cork passes a glass tube (it must not dip into the water) connected with the air-pump. When the pump is set in action a stream of bubbles emerges from the cut end of the stalk which is below the surface of the water. The reverse experiment may also be made by placing the lamina in the water and the cut end in the air. (120) Injection with water. Many leaves e.g. Ficaria, Limnan- themum, Hydrocharis can be injected by sucking the stalk with the mouth while the lamina is in water. Or the air pump may be applied. It is easy to ascertain the pressure necessary for injection by the following arrange- ment. The leaf is attached to one arm of a T tube: by means of the second arm suction is applied, and the third ends in a bent tube dipping into mercury. If the junction between the stalk and Fie. 19. Exp. 120. CH. IV] FROST EFFECTS. 97 the T tube is not quite air-tight it is of no consequence, since the leak affects the leaf and the manometer equally. A good air-tight junction may however be made by Devaux’s method’ of melting the leaf stalk into a funnel with gelatine G as shown in fig. 19. (121) Frost effects. The injection of the intercellular spaces with water can be observed on the frozen leaves of certain evergreens. In a hard frost the leaves of the ivy have a semi-trans- parent, dark green appearance like, but not so dark as, the colour of a water-logged leaf. If the leaf is pinched between the finger and thumb the normal light green colour returns to the under surface: the same effect may be produced by dipping a corner of the leaf in lukewarm water. If however the whole leaf including the cut stalk is thawed under water, it does not become light green, but assumes the very dark tint of an injected leaf. In the first case thawing produces injection with air, in the second with water. The explanation given by Moll? is that the cells of the mesophyll, in freezing, give up water to the intercellular spaces, and that when they are thawed the cells absorb the melted ice in the intercellular spaces, which then fill up with air or water as the case may be. (122) Blocking of stomata by water’. One arm of a bent glass tube is gently pushed into the cavity of an onion leaf and is there firmly secured by a ligature of soft cotton or worsted. The other end of 1 Ann. Sc. Nat. 1889. 2 Archives Néerlandaises, Vol. xv. 3 Sachs’ Physiologie Végétale, p. 280. e ( D, A. 98 MOVEMENTS OF STOMATA. [cH. Iv the tube is held in the mouth, and the leaf is immersed in water: by blowing gently, bubbles are forced out of the stomata on the external surface. Now clean the bloom from a zone of the leaf, which may be done by gently rubbing it with a plug of cotton wool dipped in warm water. On again immersing the leaf and blowing, it will be seen that the air does not come out of the cleaned zone, -which is now thoroughly wetted owing to the removal of the bloom. When onions are not available, the flower stalk of Narcissus answers well. A rubber tube can be slipped over the cut end, and the stalk plunged upside down flower and all, into a jar of water. The stalk should not be cut too near the ground but where it begins to be hollow. (128) Opening and closing of stomata. The majority of stomata close when surface-sections of the leaf are placed in water. Some leaves however behave in the reverse way: of these the most easily accessible are those which form the floating rosettes of Callitriche. If the tissue of the lower surface of the leaf is gently scraped away with a needle, and the leaf is mounted in water with the upper surface upmost the Stomata are visible; they can be made to close by irriga- tion with 2-5 NaCl solution and again to open by replacing the salt solution with water. The stomata of Trianea bogotensts are also useful for similar experiments. CH. Iv] LENTICELS. 99 (124) Electric effect. Strips from the under surface of the leaf of Ranunculus ficaria are mounted dry under a cover-glass, on a slide bearing a pair of microscopic electrodes. On passing the induced current the stomata close. A current, slightly stronger than that bearable on the tongue, is necessary. If Callitriche is used it must be mounted in water: a stronger current is needed. (125) Lenticels. The fact that lenticels communicate with intercellular spaces may be conveniently studied in connection with the parallel results obtained with stomata. Fit a woody dicotyledonous branch (dog-wood, Cornus sanguinea, does well) to the short arm of a U tube by means of firmly wired india-rubber tube. The vessels and intercellular spaces at the upper (free) end of the stick are to be secured by an india-rubber tube wired on and closed by being folded down parallel to the branch and again wired. The U tube is placed in a jar of water so that the stick is immersed, and mercury is poured into the long arm: after a varying time air is seen to issue in fine streams from the lenticels—that is if they are open. Fifteen or 20 cm. of mercury is sufficient pressure. (126) Bloom. The character of the leaf-surface has an effect on transpiration, as may be shown in the following way’. Bring into the laboratory a pot of Klemia or Cotyledon, 1 See Garreau, Ann. Sc. Nat. S. 3, T. xiii., p. 339. 7—2 100 BLOOM. [cH. IV or some other succulent plant with a good bloom. It is best to bring the pot into the laboratory because if the leaves are cut before they are wanted they are likely to get rubbed. Cut off two similar leaves (or two similar twigs) and pierce each specimen with a piece of thin copper wire to serve as a hook by which to hang it up. Paint the cut surfaces with lard: weigh the specimens, hang them in a dry room for 12 or 24 hours, and weigh them again. This will give the normal transpiration of the two specimens. Now remove the bloom from one specimen by delicate sponging with water at 35°C. When it is dry weigh both again, and once more expose them to the dry air of the laboratory for 12 or 24 hours. The cleaned specimen should now transpire relatively more than the other. CHAPTER V. PHYSICAL AND MECHANICAL PROPERTIES. Section A. Imbibition, Hygroscopic_ movement, Polariscope, Osmosis. Srction B. Turgor. Section C. Tensions of tissues. Section A. (127) Laminaria, microscopic observation. The thallus of Laminaria is useful for the demonstra- tion of some of the phenomena of imbibition, especially the great increase in size which takes place when the dry tissue is placed in water. Cut transverse sections of the dry stalk of Laminaria, mount them in methylated spirit, and irrigate them, while under the microscope, with water by placing a drop of water on one side of the cover-glass and a strip of filter paper on the other. Observe that the cell-walls increase enormously in thickness. 102 LAMINARIA. [cH. v (128) Increase of size not uniform in direction. Cut a rectangular piece out of the thallus of Laminaria, choosing a part free from wrinkles; let it be slightly oblong so that the longitudinal axis of the thallus may be distinguishable. Measure the length and breadth with a millimeter scale and mark, by means of a pin hole in the corner, the two edges along which the measurements were taken. Place it in water and measure it again in a quarter of an hour, and again in an hour. It will be found to have increased far more in the transverse than in the longitudinal direction. (129) Effect of temperature. Weigh, to 0°1 gram, about 30 grams of air-dried peas: place them in water at about 26° C., and let them remain at that temperature for 2 or 3 hours. Dry them first with a soft cloth, then with filter paper, and weigh them again. Place at the same time a similar weight of peas in water at 10°—14° C. and compare the gain in weight in the two cases. The peas, which have been in warm water, will have absorbed much more water than the second lot. (130) Salt solution. Weigh about 30 grams of peas, taking care to use the same material as that employed in experiment 129; place them in 10 per cent. NaCl solution, which must be kept at the same temperature as the cool water in experiment 129. After 2 or 3 hours (as the case may be) dry and weigh them. The peas will be found to have increased in weight, but much less than the control material in experiment 129. CH. V] STIPA. 103 (181) Stipa pennata. The awn of Stipa pennata is, as previously explained, extremely hygroscopic, untwisting when wetted, and twisting again when dried. To observe its movements it must be fitted up as shown in fig. 20. The awn S is P Fic. 20, Exp. 131. lashed with fine wire to a strong straight wire above, and below to a hooked wire W: the latter, W, is fixed into the cork C, which is attached underneath the stout paper or cardboard P: in the middle of P is a hole through which the straight wire passes, bearing at right angles the index J, On the upper surface of P a circle is marked, and is divided into degrees which may be 2 or 3 mm. in width. When the Stipa-awn is dipped in water the index moves round the clock-face in one direction, which 104 STIPA. [cH. Vv we may call the “wet” direction; when it is removed it will, after a time, reverse itself and move in the “ dry” direction. (132) Stipa: effects of temperature. As in the case of experiment 129, so here, it may be shown that warmth increases the action of water very greatly. Prepare 2 beakers of water, one at 14°—15°C.,, the other at 40°-—45° C.; place the awn in the cold water, and when the index has clearly begun its slow movement, plunge it in the warm water, when the untwisting is at once accelerated. (133) Stipa: effects of temperature. Place the Stipa in water at about 15°C. and allow it to come to rest: then transfer it to water at about 40° C., there will be a sudden deflection in the “wet” direction and a return to a position slightly on the “dry” side of the original position of rest. A similar result may be obtained with a dry awn, by holding it high above a spirit lamp or a small gas flame, taking care not to scorch it; the first sudden move will be in the “wet” direction, the heat will then dry the awn and a steady “dry” movement will follow. These effects of temperature are not understood’. (134) Stipa: salt-solution. A Stipa which has come to rest in water can be made to twist in the dry direction by transferring it to 10 per cent. NaCl solution. 1 Francis Darwin, Transactions of Linnean Society, 1876. CH. V] NOBBE'S EXPERIMENT. 105 (1385) Stipa: mechanism of the movement. The twisting power of the awn depends on the hygro- scopic torsion of its individual cells. To show this it is necessary to isolate some of the elements. Prepare Schulze’s macerating fluid by dissolving in 50 cc. of nitric acid, 1 grm. of potassium chlorate ; add to this half its volume of water, and boil a ripe awn cut into two or three pieces in a test tube half full of the diluted liquid. It is best not to boil it too much; as soon as the awn is clearly beginning to disintegrate it must be removed, thoroughly washed in water!, and teased out with needles. A small portion is now dried on a glass slip over a flame and examined under the microscope. Cells will be found which are obviously twisted on their axes, and which at once untwist when water is added. (1386) Nobbe’s experiment? Take two stoppered bottles of about 400 cc. capacity: fit each with a rubber cork through which passes a narrow graduated tube. Half fill bottle A with whole peas, and place the same quantity of split peas in B. Fill both bottles with water which has acquired the temperature of the room, and take care to get rid of any air adhering to the peas; force in the corks firmly, and note the height of the water column in each bottle. If the peas increase in volume by the amount of the water absorbed, the level of 1 Because the fumes of Schulze’s finid are bad for the lenses of microscopes. 2 Handbuch der Samenkunde, p. 126. 106 SWELLING OF SEEDS. [cH. v the water will not change. This is what happens’ in B, which contains the split peas, but in A the level rapidly rises and then falls. This curious phenomenon is said to be due to the expansion of the testas of the peas producing a temporary increase in size. (137) Variability in the swelling of seeds. When seeds of certain plants are placed in water there is great variability in the time which elapses before they become imbibed. This is especially the case with legu- minous seeds. Nobbe? describes the phenomenon in clover seeds; we use those of a Lupin with a rough sur- face to the testa. Take 100 Lupin seeds and place them in a flat vessel (so that they may be easily examined) and add about a liter of tap-water. After 24 hours the majority of the seeds will be swollen, the minority which have not yet swollen can be easily distinguished by their smaller size. The swollen seeds should be removed and the water renewed to minimise decomposition, and this should be done at intervals of 12 hours until all the seeds are swollen, noting at each examination the number of freshly imbibed seeds. The cause of the individuality in imbibition seems to depend, not on the cotyledons, but on the seed coats, This may be demonstrated on a similar number of seeds, 1 Reinke shows that the increase in volume is slightly less than the water absorbed. 2 Handbuch der Samenkunde. CH. V] SWELLING OF WOOD. 107 after the first interval of 24 hours has elapsed. Pick out all the seeds which have not swollen, and in half of them pierce the testa with a needle, leaving the other half intact. It will be found that all the punctured seeds swell within 12 hours, whereas only a percentage of the intact seeds are swollen’. (188) Rise of temperature accompanying imbibition. Prepare enough dry powdered starch? to make a layer about an inch thick at the bottom of a beaker, and place a similar quantity of water in a second beaker. When starch and water are at the same temperature, pour the water into the first beaker, stir with a thermometer bulb, and note the rise of a few degrees which takes place. (139) Work done during imbibition. Saw out a square inch from a deal board of about 8 of an inch in thickness. Put it in a flat photographic dish and let it serve as a support for a 28 lb. weight. On adding water the wood swells and raises the weight, a movement which may be recorded in various ways, e.g. by a horizontal microscope, or by the micrometer-screw described below, experiment 155. The weight will com- press the dry wood slightly, so that it is necessary to wait until the index comes to rest before the water is added. 1 See Detmer, Praktikum, p. 131. 2 The starch should be dried at 100°C. and may be allowed to cool, without special precautions, to the room-temperature, when it will still be sufficiently dry for our purpose. 108 POLARISCOPE. [CH. v (140) Observations with the polariscope. The apparatus consists of two parts, the polariser and the analyser. In the Zeiss pattern of instrument, the former of these is to be fixed axially on the substage of the microscope above the mirror; the analyser separates into two pieces, one, a disc—which should be graduated— fastens on to the upper end of the tube like a collar; into this an ordinary ocular is slipped, and the other piece is fitted on to it like a cap. The essential part of each is a-so-called Nicol’s prism —pieces of doubly refractive Iceland spar so cut and disposed that the light transmitted is all polarised in one particular axial plane. The central upper part of the analyser rotates on the collar-like disk and bears a pointer which records the amount of the rotation. If, with the parts placed in position and the eye at the ocular, the analyser be rotated until its plane of polarisation becomes identical with that of the polariser, the light will be transmitted through it undiminished and the field of the microscope appear bright. On now slowly turning the analyser either way through a right angle, the light ‘will gradually fade until the field is completely dark. In this latter position the polarising planes of the two prisms are at right angles and the analyser intercepts all the light transmitted by the polariser. On continuing the rotation of the analyser through a further 90°, maximum brightness from coincidence of the planes will be again obtained. With the dark field, place on the stage of the microscope a slide on which is mounted some anisotropic, CH. V] POLARISCOPE. 109 1é. doubly refractive, object such as almost any vegetable tissue, and it will be seen that parts of each of its elements will appear black and parts white. The object thus seen is said to be viewed “with crossed Nicols” and the appearance is due to the arrangement of the planes of polarisation in the object so altering the polari- sation of the light that part of it and part only is capable of passing through the analyser in its present position. Hence the alternating light and dark markings. On shift- ing the analyser through a right angle these markings are now seen reversed in their optical properties. The arrangement of the markings is constant with given objects. Starch grains exhibit on a black field a large black and white Maltese cross with its centre at the hilum of the grain. To see this well starch grains of potato should be mounted in Canada balsam so as to be quite transparent and when examined in ordinary light quite invisible. Advantage may be taken of this method for detecting anisotropic bodies otherwise small and difficult to perceive. Thus the very minute crystals of calcium oxalate occurring in many leaves are often difficult to make out but if the leaf be decolorised and rendered quite transparent by soaking it in strong chloral-hydrate solution the distribution of these crystals may be easily observed by their light crossed markings on a black field. This is a valuable method for tracing the seat and causes of the formation of calcium oxalate in the plant}. 1 Schimper, Bot. Zeitung, 1888, p. 81. 110 POLARISCOPE, [cH. v (141) Tension. All crystalline bodies are anisotropic and it is one view of the significance of the anisotropism of organised bodies, cell-walls, starch grains, etc, to attribute this to a crystalline structure of the ultimate particles—micellae of Nageli—of which these bodies are held to be built up. Another view denies this crystalline structure and attributes the anisotropism to the tensions produced between the strata of the substance of the cell-wall or the starch grain as a result of their particular structure. In order to realise that tension may produce double refraction ina substance that is not in itself anisotropic, for example glass, the experiment should be performed of stretching a fine glass filament while it is under observation in polarised light in the field of the microscope. To make the effect more apparent use should be made of the selenite discs generally supplied with the polarising apparatus. On placing one of these on the stage of the microscope the field will appear of a certain colour which changes on rotation of the analyser. The various colours which bodies exhibit when viewed in these coloured fields give a measure of their respective anisotropism, but the theory of this cannot be entered into here. To perform the stretching experiment a piece of glass rod, drawn out at the blowpipe to a fine filament in its middle part, should be so clamped at one end that the fine part lies across the field of the microscope and can be focussed with a low power. The selenite disc No. L, which gives a red purple field, should be suitably placed below the glass thread, CH, V.] TRAUBE’'S CELL, 111 which then appears as a double black contour with red- purple between. The free end of the glass rod should rest on some guiding support which will keep it in focus but allow it to be stretched. If the observer looks down the microscope while the rod is steadily pulled, the colour of the centre of the thread will be seen to change distinctly, the nature of the change depending on the amount of traction exerted upon the filament: on releasing it the purple colour reappears. Compression should give a different colour-change but this cannot be easily exhibited. Other transparent bodies such as gelatine films show similar effects. We have thus seen that both crystalline structure and internal tensions may be accountable for the anisotropism of organised bodies. (142) . Traube’s artificial cell. Traube’s method is of great interest as a graphic way of demonstrating the possibility of pressure arising osmotically inside a cell. The method is moreover capable of giving results of great value, especially as modified by Pfeffer. The following experiment is merely meant to serve as a demonstration. Fill a beaker with a solution (2 or 3 per cent.) of potassium ferrocyanide and drop into it a crystal of copper sulphide. The sulphide is instantly coated with a pre- cipitated membrane of copper ferrocyanide. . In the artificial cell so produced osmotic pressure 1 Traube in Archiv fiir Anatomie und Physiologie (Reichert and Du Bois-Reymond), 1867, 2 Osmotische Untersuchungen, 1877. 112 SLOW DIFFUSION. [CH. V arises by which the brittle cell-wall is broken, but is instantly mended by a fresh precipitate forming: as soon as the wall is mended the pressure inside again increases, and again ruptures the cell-wall, and thus by a series of breaks, healed as soon as made, an apparently continuous growth of the cell takes place. (148) Slowness of diffusion’. Fill a tall narrow jar with water and with the help of a long funnel run in very slowly and carefully a stratum of concentrated solution of potassium bichromate, which accumulates at the bottom of the jar. It will be seen that the colour spreads to the upper stratum with extraordinary slowness. The chief physio- logical interest of the result is that it serves to suggest the value, to the living cell, of protoplasmic circulation. (144) Relation of membrane to diffusing fluid®. A dialyser made of vegetable parchment is filled with a 1 per cent. solution of di-sodic phosphate coloured with methylene blue, and is placed in distilled water; after some hours the blue colour is visible in the water. If, however, a precipitation membrane of calcium phosphate is pro- duced in the wall of the dialyser, the methylene blue is unable to pass. The precipitate is produced by immersing, in 1 per cent. calcium nitrate, a dialyser filled as before with 1 per cent, di-sodic phosphate coloured with methylene blue. The importance of the experiment is to show that 1 See de Vries, Bot. Zeitung, 1885, p. 1. 2 Taken from Detmer’s Praktikum, p. 96. CH, V] TURGOR. 113 by the formation of a precipitation membrane the osmotic quality of the parchment is changed. (145) Absorption of methylene blue. It is interesting to note in connection with the last experiment that methylene blue, as Pfeffer’ has shown, can pass a living protoplasmic membrane. Two or three sprigs of Elodea are placed in about a liter of tap-water containing 00008 per cent. of methylene blue, after from 24 to 36 hours the living cells will be found to contain blue cell sep. Section B. Turgor. (146) Plasmolysis, microscopic observation. In order to realise the existence of turgor the well- known microscopic observation of the effect of salt solution on turgescent tissues should be repeated. Plas- molysis is easily seen in Spirogyra, or any tissue with coloured cell sap may be used; it is only necessary to irrigate a preparation with 5°/, NaCl solution. It is instructive to compare the result of plasmolysis with the change produced by death. In the first case the cell sap remains within the protoplasmic sac, in the killed cell it escapes and moreover stains the dead protoplasm. (147) Recovery after plasmolysis. It is important to realise that plasmolysed parts are in no way injured, and that they recover their normal \ Untersuchungen aus dem Bot. Institut zu Tiibingen, ii. p. 223. D. A. 8 114 ISOTONIC COEFFICIENT. [cH. v condition when the plasmolysing fluid is replaced by water. A few simple observations on roots of V. faba serve for this purpose. A bean root about an inch in length is placed in 5°/, NaCl solution, where it almost immediately becomes soft and flaccid. When replaced in water it quickly becomes turgid again’. The observations here suggested are meant as illustra- tions of the very simplest aspect of turgor, chiefly to show that turgor is an osmotic phenomenon, since the condition of the cell is clearly regulated by the relation between the cell sap and the environing fluid. (148) Osmotic strength of cell sap in terms of KNO,, The method of de Vries’ depends on the fact explained in experiment 163 (Section C) that when a turgescent shoot is bisected longitudinally each half curves outwards, ue. with the epidermis on the concave side. If the curved portions are put in water the curvature increases greatly: if they are placed in strong NaCl solution (5°/,) they uncurl, ¢.e. become straight again, or they may even become convex on the epidermic side. Therefore an ‘intermediate strength of salt solution must be discoverable which equals the cellsap im osmotic force, and which neither produces increase nor decrease in curvature. ; In “summer we use the scape of the dandelion, 1 We have observed the root of the bean, if placed alternately in salt solution and water several times, becomes translucent, being in fact injected with water. It would seem that the collapse and re-turgescence of the cells act like a pump and fill the intercellular spaces. 2 Pringshein’s Jahrbiicher, xiv. CH. V] ISOTONIC COEFFICIENT. 115 dandelion is split longitudinally into four strips which, on being dipped for a moment into water, curl up into spirals and can then be cut up into some 7 or 8 rings, b, fig. 21: these are delicate tests of changes in turgescence since a small increase or decrease in the curvature of the turgescent tissue is at once perceptible. Thus s is in too (JO $ w b Fic. 21, Exp. 148. strong a solution, w is in too weak a solution, while 6 is in one that almost exactly balances the osmotic power of the cell sap. The process with Ricinus is a little more troublesome ; the hypocotyl is split in 4 or more longitudinal portions, and the form of each is traced with a paint-brush (which answers better than a pencil) on paper. We now have a number of curved bits of tissue (whose form is known) each one of which must be placed in a solution of a different strength. These solutions are made according to equivalents, and in the case of KNO; (which forms the standard) may contain 0°05, 0°10, 0°11, 0°12, 0:18, 0:14 gram-molecules per liter; stronger solutions may however be needed. After a quarter of an hour the result may be noted: if the material consist of dandelion rings the result is obvious on inspection; with Ricinus the seg- ments must be compared with the sketches. 8—2 116 ISOTONIC COEFFICIENT. [CH. Vv Fig. 22 gives tracings of pieces of split Ricinus hypocotyl before and after immersion. The upper row of tracings gives the form of the pieces before being placed in the solutions, the lower row shows the change of form produced by the immersion. The numbers 0°10, 0°12, ete. give the strength of the KNO, solution in which each was placed. OC ( Fie. 22, Exp. 148. 0.10 It will be seen that the first two have increased in curvature, while the last two have uncurled and the middle piece (ae. that in the 0°13 solution) remains unchanged. Therefore 0°13 expresses the osmotic quality of the cell sap in terms of KNO,,. (149) Isotonic coefficient. The same experiment must be made with cane sugar, using solutions 0°16, 0°18, 0:20, 0°22, 0°24. From the results CH. V] ISOTONIC COEFFICIENT. 117 obtained (combined with experiment 148) it is possible to calculate the isotonie coefficient (I. C.) of cane sugar, i.e. the attraction for water of a molecule of cane sugar expressed in terms of the attraction of a molecule of KNO, for water. For the sake of convenience the value of this last quantity is taken as 3 instead of 1. We have then the following calculation. Assuming that we have found that the cell sap=0'13 KNO, and also = 0-20 cane sugar, IC. ofsugar 13... ag ge 8, I. C. of sugar = 1:95 or in round numbers = 2. In this way, using the plant as an index, it is possible to ascertain the osmotic intensity of solutions of a number of substances in relation to a living protoplasmic mem- brane. (150) Microscopic method. The principle of “de Vries’ second method is simple: small portions of tissue are put in a graduated series of salt solutions and the equivalence between one of them and the cell sap is estimated by the degree of plasmolysis observed microscopically. The tissue must contain coloured cell sap so that plasmolysis may be readily observed. De Vries recommends as material the epi- dermis of parts of the leaf of Tradescantia discolor, Begonia manicata, or Curcuma rubricaulis. Of these Tradescantia discolor is the most universally available and is the only one of which we have any experience. In Tradescantia the part of the leaf used is the 118 HYDROSTATIC PRESSURE. [CH. V epidermis of the under-surface: to get good results it is necessary to use closely adjacent parts of the epidermis taken from the midrib. De Vries makes parallel incisions 14 or 2 mm. apart in the epidermis of the midrib: the areas so marked out can-then be freed by a surface-cut with a razor. The fragments of the epidermis must remain in the solutions for at least an hour before being examined. The condition of each is noted as P. completely plasmolysed, H. P. half plasmolysed, or N. P. not plasmolysed. The solution which produces the H. P. effect is taken as osmotically equivalent to the cell sap. (151) Estimation of the hydrostatic pressure in turgescent tissue’. Take an actively growing flower stalk such as that of the cowslip (which must be in the budding condition): mark off 100 mm. near the upper end and place the stalk in 5°, NaCl solution. As soon as it is thoroughly flaccid it should be measured again, when it will be found to be shorter, owing to the elastic contraction of the cell-walls, which were previously stretched by the turgescence of the cells. If it can now be ascertained what force is needed to stretch the shrunken stalk to its original length, we shall know what was the force exerted by the turgidity “of the tissues. The bud of the cowslip is fixed in a screw-clamp lined with cork-plates and the clamp is slipped behind a couple 1 De Vries, Untersuchungen iiber die mechanischen Ursachen der Zellstreckung (1877), p. 118. CH. VJ GYPSUM METHOD, 119 of upright nails fixed in a horizontal board, or secured in some way so that when the other end is pulled the stalk will be stretched. The basal end of the stalk may be simply knotted to a piece of cord, which passes over a pulley let into the board, and supports a scale pan. A millimeter scale having been arranged so that the distange- between the marks on the stalk can be easily read off, weights are added to the scale pan until the marks are once more 100 mm. apart. The diameter of the stalk must be roughly measured, and the area calculated, so that the force which is equivalent to the hydrostatic pressure in the tissues, may be expressed in grams per square millimeter. It should finally be expressed in terms of atmospheric pressure-——which equals about 10 grams per sq. mm. Something between 3 and 6 atmospheres may be expected as the result. (152) Pfeffer’s gypsum method’. Pfeffer has devised a method of estimating the pressure exerted by growing plants of which we have no practical experience: the following description is taken from his paper. The principle will be understood from fig. 28. The cotyledons and the basal part of the radicle are contained in the pot x and kept damp by means of sawdust. The extremity of the root is contained in the two blocks of gypsum a and b, so that as the root grows a and b are separated. Since a is fixed against the pot n, the block 6 moves, and in doing so compresses the oval spring /f. 1 Druck- und Arbeitsleistung &c. Abhandl, d.k. Séchs. Ges. Bd. xx. 1893. 120 GYPSUM METHOD. [CH: ¥ The degree of compression, and therefore the force exerted, is estimated by reading, with a horizontal microscope, the distance between the needle points fitted to the inside of the spring. The following is the method of fitting the plant into the apparatus. Fic. 23, Exp. 152, The seedling bean is placed in the flower-pot n filled with damp sawdust so that 15—30 mm. of the root project through the hole. A lid is placed on the pot, which CH. V] TENSIONS OF TISSUES. 121 is turned upside down and the root (which projects vertically upwards) is covered with soft gypsum. A piece of waxed paper in which a hole has been made (with a hot needle) is slipped over the tip of the root and pressed down with a bored glass plate. In this way the block of gypsum a is formed ; when it is sufficiently set, the waxed paper is removed, and for it is substituted a piece of wet silk-paper on which the block b is added. The form of the blocks a and 6 is regulated by cylinders of paper acting as moulds. When block 6 is set hard it may be removed from the root and trimmed with a knife: at the same time the silk-paper may be removed. Before the flower-pot is placed in the supporting ring m the block of gypsum b must be secured in its place by tying it with a thread which will be cut when the arrangement is complete. The block 6 is fixed by fluid gypsum to the glass plate c which rests on the spring f. The plate J, forming part of the spring, is fixed by the small screws k, & to the solid plate g, which can be raised and lowered by means of the screws h,h,h. In this way the desired amount of pressure can be applied at the beginning of the experiment. The distance between the needle points is regulated by the screw 7 which moves the lower needle. SECTION C. ‘Tensions of tissues. (153) Longitudinal tensions. The fundamental experiment illustrating the condition 122 LONGITUDINAL TENSION. (cH. Vv of strain or tension! which exists in turgescent tissues may be made in summer or spring on any rapidly growing juicy shoot, e.g. elder, or with certain leaf stalks, e.g. that of the rhubarb. In winter it is sometimes difficult to find suitable material: if a green-house is available the leaf stalks of Richardia will answer well. It is best to get fairly long shoots, ¢. e. not less than 20 cm., so that measure- ments to 1 mm. may give perceptible results. The material must be as fresh as possible, and if it has to be brought from any considerable distance must be wrapped in a wet cloth and placed in a vasculum: in this case too, it is worth while to take care that the vasculum is held verti- cally, lest the shoots should také a geotropic curvattte, as they may do if kept horizontal for an hour. Place the shoot on the table, cut the ends as square as possible and measure its length with a millimeter scale placed lengthwise on it. Remove a strip of cortical tissue along the side measured; it will be shorter than the original shoot. Now remove the whole of the cortical tissue, and measure the length of the cylinder of pith remaining, which will be found to be longer than the intact shoot. This experiment shows that the internal tissues are in a state of compression, while the cortex is extended. It is important to note that the amount of extension of the freed pith need not by any means be the same as the contraction of the cortex®. If the experiment is repeated 1 See Sachs’ Text-Book, Sect. 14, 15. The whole discussion should be studied. 2 Sachs’ Text-Book, p. 797. CH. V] TURGESCENT PITH. 123 with a scape of Fritillaria imperialis which has ceased to grow, it will be found that the pith lengthens considerably while the contraction of the cortex is imperceptible. (154) Eatension of pith in water. When pith is placed in water it increases greatly in length in consequence of the increased turgescence of its cells. To show this, place the pith from experiment 153 in water, and measure it again after an hour. (155) Change in the transverse dimensions of pith}. ~ Suntlowér, Elder, and of Impatiens sultani, also from a Rhubarb leaf stalk, parallel-sided pieces about 10—15 mm, in length and 5 mm. in width, taking especial care that they are free from all cortical tissue. Place a piece on its side (i.e. with the 5 mm. dimension vertical) in a small flat-bottomed glass vessel, and lay on the pith an ebonite vessel measuring 4—5 mm. in diameter by 2—3 in depth, and containing oil. By means of the following arrange- ment the oil is made to serve as a delicate index of any shrinking or swelling of the pith. A vertical micrometer screw graduated to 0°01 mm. carries at its lower end a vertical-needle, which can be lowered until it dimples the polished surface of the oil; the moment of contact is sharply defined, and in this way changes of 0°01 mm. in the diameter of the pith are easily read. After taking a few readings, which usually indicate a slight shrinking, 1 See Miss Anna Bateson, Annals of Botany, Vol. iv. p, 117. A drawing of the micrometer screw is given in Chapter VI. fig. 25. 124 SHORTENING OF ROOTS. [cH. v water should be added. The results of the increased turgescence so produced vary with the material employed ; in the case of Elder and Sunflower the pith begins to shrink, 7.¢. diminish in transverse diameter ; Rhubarb-pith increases and afterwards diminishes; while Impatiens increases but does not diminish. (156) Change in tangential dimension. Cut with a dry razor sections (such as would be con- sidered very thick for microscopic purposes) of a fresh dandelion-stalk or (in winter) of the hollow hypocotyl of Ricinus. Place the rings, so prepared, on a glass plate, and with a scalpel divide each at one point. The divided rings are now placed in water, when their curvature is found to increase, the curling inwards being due to the shrinking in tangential direction of the turgescent tissue forming the inner part of the ring. (157) De Vries’ experiment on the shortening of roots. In the roots of certain plants a phenomenon has been observed by de Vries’ which seems to be of the same character as those described under experiments 155—56, The roots shorten along their longitudinal axes when tur- gescence is increased, and lengthen when turgescence is diminished, e.g. by immersion in 5 per cent. NaCl. De Vries describes the phenomenon in Carum, Dip- sacus and other plants. Full directions are given by Detmer? for observation on the roots of young (2—3 months) plants of Carum 1 Landw. Jahrb. ix. 1880. 2 Praktikum, p. 248. CH. V] ELASTICITY. 125 carvt. If suitable material is wanting for following out Detmer’s instructions, it is generally possible to find roots in which shortening has already occurred, and which are remarkable for their wrinkled exterior. The roots of hyacinths grown in water show the phenomenon well. (158) Imperfect elasticity of plant-tissues. The fact that the tissues of a growing shoot or leaf are extensible, but not perfectly elastic, can be demonstrated on a variety of material, e.g. a flower scape of Polyanthus, or the leaf of a Narcissus: the form of the last named makes it convenient for the purpose. For this and similar experiments a strong sheet of cork mounted on a board is convenient: one end of the leaf is clamped between the mounted cork and a free block of cork, in such a position that the other end of the leaf projects beyond the board. Two marks about 100 mm. apart are painted on the leaf, one being close to the clamped end. The distance between the marks having been read on a mm. scale, also clamped to the cork-board, the projecting end of the leaf is pulled with the hand; the distance between the marks is now to be read off without releasing the traction, and again when the leaf is left to itself. The leaf will be found to be permanently extended; the tem- porary and permanent extensions should be recorded in percentages of the original length. (159) Cyclometer’. Take a straight turgescent shoot, e.g. a young cabbage shoot, bend it forcibly, and then release it: it will be found 1 Sachs’ Text-Book, p. 784. 126 HOFMEISTER’S EXPERIMENT. [CH. v to have taken on a permanent curve. This is only another way of demonstrating what is shown in experiment 158: the cortical tissues on the convex side of the shoot are forcibly elongated by the ‘bending, and being imperfectly elastic do not return to their original length, thus pro- ducing a distortion of the shoot. To get an accurate notion of what occurs in this experiment it is desirable to measure the radius of (1) the curvature forcibly produced, (2) of the permanent curvature remaining. This may be done with Sachs’ cyclometer, which consists of a number of concentric circles drawn on a board. By applying the shoot to the board, the circle which corresponds to it most nearly in curvature can be ascertained and noted. In our Laboratory we have two boards, one bearing circles whose radii range from 1 to 20 cm. in length: while the circles on the other range from 21 to 45 cm. (160) Hofmeister’s experiment?. This is in principle the same as experiment 159; it has, however, a certain classic interest which makes it worth repeating. What is needed is a vertical turgescent shoot fixed firmly at its lower end: it. may be either a plant growing in a pot, or a shoot fixed into a clamp by its basal end. In either case the base of the shoot is smartly struck with a light stick so as to produce violent curvature of the free end of the shoot towards the side which is struck. The consequence is the same as that in experiment 159, ’ 1 Berichte d. k. Sachs. Gesell. d. Wiss. 1859. CH. V] LOSS OF RIGIDITY. 127 namely, that a permanent curvature is produced in consequence of the overstretching of the convex side of the shoot. (161) Loss of rigidity. The rigidity of a turgescent shoot is dependent on (among other factors) the resistance of the cortical tissues ; if, by overstretching, these are permanently lengthened, the rigidity of the system is lessened. Fic. 24, Exp. 161. A straight turgescent shoot is fixed firmly by means of a bored and split cork in a test-tube of water, 7, figure 24, and at a point which should be marked by a streak of Indian ink, it is further supported on a prism of wood, F, resting on a support S. At the free end, the shoot bears a needle acting as an index J, and a loop of wire JL, to 128 SPLIT STEMS, [cH. Vv which weights may be hung. Having noted the position of J on the scale, hang a small weight W (a coil of lead wire of 8—10 grams) on the loop, and read off the position of the index. Remove the weight, and bend the shoot two or three times backwards and forwards in the vertical plane. When the weight is once more attached, the index will move. through a greater distance than that at first recorded. (162) Increased length. Since the pith is in a state of compression, any in- creased length of the cortical tissue must result in an increase in the length of the whole shoot. Therefore bending a turgescent shoot backwards and forwards as in experiment 161, must lengthen it. The length must be accurately measured, say to 0'l mm., to make sure of a result. (163) Splitting turgescent tissues. The relation between the compressed pith and the stretched cortex can be demonstrated by dividing a shoot longitudinally. It is best to prepare the shoot by cutting it flat on two opposite sides. We now have a column of pith bounded on two sides by strips of cortical tissue: this is placed on a glass plate and bisected with a knife, when each half curves so that the pith is on the convex, the cortex on the concave side. The curvature can be greatly increased by putting the half-shoots in water. This increase is strikingly seen if a dandelion stalk is split into 4 or 5 longitudinal strips, which curl up in water into helices of many turns’. 1 This fact has already been utilised in experiment 148. CH. V] SPLIT ROOT. 129 (164) Splitting a root. In some turgescent organs the erectile (compressed) tissue is external, while the resisting or stretched tissue is internal. In such cases the result of splitting longi- tudinally must obviously be the opposite of that just described, the parts will curve inwards, towards the longitudinal axis, not away from it. Pull up a seedling bean (Faba) with a root about 14 inches long, split the apical half-inch with a scalpel, and put it in lukewarm (25°—30°C.) water. The halves will certainly not curve outwards, and will after a little time show a slight inward bend. The aerial roots of Aroids show the same tensions. (165) Splitting a pulvinus. Take a large pulvinus of Phaseolus and cut from it an axile slab as described under experiment 163. Split the slab down the central strand, and put the halves in water, when they will curve inwards, ¢.¢e. with the vascular tissue on the concave side. CHAPTER VI. GROWTH. Section A. Conditions of growth (experiments without special apparatus). Section B. Distribution of growth. Section C.