GIFT OF MICHAEL REESE DIOLOG* LIBRARY G EXPEKIMENTAL MORPHOLOGY EXPERIMENTAL MORPHOLOGY BY CHARLES BENEDICT DAVENPORT, PH.D. INSTRUCTOR IN ZOOLOGY IN HARVARD UNIVERSITY ' PART FIRST ' EFFECT OF CHEMICAL AND ' PHYSICAL AGENTS UPON PROTOPLASM gork THE MACMILLAN COMPANY LONDON: MACMILLAN & CO., LTD. 1897 AH rights reserved £S5 COPYRIGHT, 1896, BY THE MACMILLAN COMPANY. J. S. Cushing & Co. — Berwick & Smith Norwood Mass. U.S.A. SDelricatet) TO THE MEMORY OF THE FIRST AXD MOST IMPORTANT OF MY TEACHERS IX NATURAL HISTORY MY MOTHEK Die morphologische Betrachtung setzt also eine genaue chemisch physikalische Kenntniss, 1. des betreffenden Korpers selbst, und 2. aller der bei seiner Entstehung auf ihn einwirkenden Stoffe und Korper voraus. — JAEGER, Zoologische Brief e, p. 9. La vie ne se co^oit que par le conflit des proprietes physico-chimiques du milieu exterieur et des proprietes vitales de 1'organisme reagissant les unes sur les autres. — BERNARD, Rapport sur lea progres de la physiologic generate en France, 1867, p. 5. There can be little doubt, indeed, that every science as it progresses will become gradually more and more quan- titative. Numerical precision is doubtless the very soul of science, as Herschel says. — JEVONS, Principles of Science, chap. xiii. PEEFACE THE problem which, since the time of Aristotle, has stood first in interest and importance among the great questions of Biology is that of the causes which direct the development of the individual — that marvellous process by which the germ is built up into the complex organism, by which the embryo clothes itself with the characters peculiar to its species, by which even minute individual traits of form and action are exactly reproduced in the offspring from its parents. The burden of clearing up this problem has fallen, naturally enough, upon the shoulders of students of morphology. For since morphologists deal with form, they are properly especially concerned with the interpretation of form — they may well be asked to account for it. Thus the problem of development is an acknowledged morphological problem. Several distinct steps can be recognized in the progress which has been made in the interpretation of form. The earlier studies were concerned chiefly with answering the ques- tion. What are the differences between the various adult forms ? The results of observations and reflections relating to this question constitute the sciences of descriptive and comparative anatomy. Xext, a more fundamental inquiry was entered upon : Sow are these forms produced or developed ? The results of observations and reflections upon this subject con- stitute the science of comparative embryology. Finally, in these later days a still more fundamental question has come to the front : Why does an organism develop as it does ? What is that which directs the path of its differentiation? This is the problem which the new school of " Entwicklungsmecha- nik " has set for itself — it is likewise the problem with which this book is concerned. viii PREFACE The causes which determine the course of an organism's development are numerous, but fall into two general categories ; namely, internal causes, which include the qualities of the devel- oping protoplasm ; and external causes, which include the chemical and physical properties of the environment in which the protoplasm is developing. The internal and external causes may be studied separately, and in order to disentangle their effects they must needs be studied separately. It is the pur- pose of the present work to consider the effects resulting from external causes. When we wish to isolate the separate effects in any complex of causes, we must resort to the well-known procedure of experimentation, — and we find, indeed, that these external causes lend themselves readily to this method of treatment. Accordingly we call in experiment to get an insight into the causes of organic form, and thus justify the name which we have applied to our study, — Experimental Morphology. The primary subdivision of the subject is based upon the morphogenic processes to be treated of; and of these, four principal classes may be recognized. The first includes those processes which are characteristic of all living protoplasm; the second, those connected with growth ; the third, those involved in cell-division; and the fourth, those producing differentiation. It is proposed to devote one part of the work to each of these four classes of processes. The secondary subdivision may be based upon the chemical and physical agents whose effects we wish to isolate. These may be grouped into eight categories, determined largely by convenience ; namely, 1, chemical substances ; 2, water ; 3, den- sity of the medium ; 4, molar agents ; 5, gravity ; 6, electri- city ; 7, light ; and 8, heat. It is proposed to devote one chapter to a consideration of the effects of each of these agents upon protoplasm, upon growth, upon cell-division, and upon differentiation. Two words should be said about the point of view from which this book has been written. In the first place, the developing organism is regarded as a living organism, and as such endowed with irritability and capacity of response ; con- sequently, at the outset, we must especially consider the phe- PREFACE ix nomenon of response to external stimuli. Again it is with living organisms that we have to deal, and, accordingly, no distinction should be made between animals and plants. I have, indeed, made no such distinction ; nevertheless, tastes and training have led me to lay especial stress upon animals. Even this is unfortunate, for the problem with which we are concerned is precisely the same problem in all living organisms. In the second place, much stress is laid upon the quantitative measurement of agents and effects. The lack of precision in many investigations can hardly be too strongly decried ; for it often results in confusion and useless disputes. On the other hand, there is good reason for believing that exact measure- ment is the key to many of the most puzzling of our problems, and important results are to be expected from its use. As for the aim of the book, it is twofold. I have hoped on the one hand that it might be readable to those who are inter- ested in the general matters of which it treats — matters of importance for philosophy, for psychology, and for pedagogy. For man is an organism, and the development of his qualities is modified by just those agents which guide the development of other organisms. My primary aim, however, has been a dif- ferent one. It is this aim to which other purposes have been made subservient, which justifies the historical treatment that has been often adopted, and justifies also the detailed descrip- tions of methods which the lay reader will, naturally, omit. This aim is so to exhibit our present knowledge in the field of experimental morphology as to indicate the directions for further research. A few words of explanation and acknowledgment are neces- sary : It was planned at the first to issue all four parts of the work at once ; but the task grew in the doing, while the need of its publication became more pressing. So it was decided to issue the work in parts as soon as each should be done. Even under this arrangement it has not been possible to include some of the papers of the last six months ; especially I regret the omission of important papers by VERWORX and LOEB upon Galvanotaxis. In writing a book of this sort, which draws upon several sciences, I have had recourse to the kind assistance of several of my colleagues in the physical and x PREFACE chemical departments of the University. I must especially thank for favors Professor W. C. SABINE, Dr. G. W. COGGESHALL, and Dr. H. E. SAWYER. Of my zoological associates, I am greatly indebted to Dr. G. H. PARKER, who has read nearly the entire manuscript and has offered valuable criticisms on it, and to Professor E. L. MARK, who has read parts of the manuscript and proof and has made important suggestions and emendations. I am also greatly indebted to Mr. CHARLES BULLARD for his kindness in making photo- graphs of figures from which most of the illustrations of the First Part were reproduced. Finally, I cannot forbear to men- tion the painstaking work of my wife, GERTRUDE GROTTY DAVENPORT, in preparing the manuscript for the press and revising the proofs. As I send out this work I do so with the hope that it may stimulate to even greater activity in the field of experimental morphology. The subject is new, its importance hardly yet generally recognized, its needs incompletely appreciated. In its scope it embraces much of physics and chemistry, for life and development are to be studied as the physicist studies light and heat, or as the chemist studies solutions and combustion. They are phenomena which must be analyzed by the use of instruments of precision to determine the quality and quantity of the acting agents, and to measure the change in the phe- nomena resulting from a change in these agents. No other field offers a better opportunity for the utilization of a broad scientific training. The times, too, are auspicious. Biology has never before attracted so many enthusiastic workers as it does to-day. As DRIESCH has said, uDie Lust an thatsach- licher exacter biologischer Forschung ist erwacht"; and the greatest problem of morphology is ever more and more the object of this biological experimentation. CHARLES BENEDICT DAVENPORT. CAMBRIDGE, MASS., Dec. 1, 1890. CONTENTS PAGE PREFACE • . vii CHAPTER I ACTION OF CHEMICAL AGENTS UPON PROTOPLASM § 1. Modification of Vital Actions 1. Oxygen .... 1 2 2. Hydrogen 5 3. Oxides of Carbon .... 6 4. Ammonia ..... 6 5. Catalytic Poisons .... 7 6. Poisons which form Salts 12 a. Acids 12 b. Soluble Mineral Bases . 13 c. Salts of Heavy Metals . 13 7. Substitution Poisons • . 15 8. Sodic Fluoride .... 21 9. Special Poisons .... 22 § 2. Acclimatization to Chemical Agents . 27 § 3. Chemotaxis 32 Summary of the Chapter .... 45 Appendix to Chapter I .... 52 Literature 54 CHAPTER II EFFECT OF VARYING MOISTURE UPON PROTOPLASM § 1. On the Amount of AVater in Organisms 58 § 2. On the Effect of Desiccation upon the Functions of Protoplasm . 59 1. Effect of Dryness on Metabolism 59 2. Effect of Dryness upon the Motion of Protoplasm . . 60 3. Desiccation-rigor and Death 60 § 3. On the Acclimatization of Organisms to Desiccation ... 65 § 4. The Determination of the Direction of Movement by Moisture — Hydrotaxis 66 Literature .... 67 XI xii CONTENTS CHAPTER III ACTION OF THE DENSITY OF THE MEDIUM UPON PROTOPLASM PAGE § 1. Introductory Remarks upon the Structure of Protoplasm and the Physical Action of Solutions ....... 70 § 2. Effect of Varying Density upon the Structure and General Functions of Protoplasm ....... 74 § 3. Acclimatization to Solutions of Greater or Less Density than the Normal . . . . . . . . . . .85 § 4. Control of the Direction of Locomotion by Density — Tonotaxis . 89 Literature 93 CHAPTER IV ACTION OF MOLAR AGENTS UPON PROTOPLASM § 1. Effect of Molar Agents upon Lifeless Matter .... 97 § 2. Effect of Molar Agents upon the Metabolism and Movement of Protoplasm 98 § 3. Effect of Molar Agents in Determining the Direction of Locomo- tion — Thigmotaxis (Stereotaxis) and Rheotaxis . . . 105 Literature 110 CHAPTER V EFFECT OF GRAVITY UPON PROTOPLASM § 1. Methods of Study 112 § 2. Effect of Gravity upon the Structure of Protoplasm . . .113 § 3. Control of the Direction of Locomotion by Gravity — Geotaxis . 114 Literature 124 CHAPTER VI EFFECT OF ELECTRICITY UPON PROTOPLASM § 1. Concerning Methods 126 § 2. The Effect of Electricity upon the Structure and General Func- tions of Protoplasm 129 § 3. Electrotaxis ........... 140 Summary of the Chapter 151 Literature 152 CONTEXTS xiii CHAPTER VH ACTION OF LIGHT UPON PROTOPLASM PAGE § 1. The Application and Measurement of Light .... 154 § 2. The Chemical Action of Light upon Xon-living Substances . 161 1. The Synthetic Effects of Light 162 2. Analytic Effect of Light .... . 163 3. Substitution Effects of Light 164 4. The Isomerismic and Polymerismic Changes produced by Light 164 § 3. The Effect of Light upon the General Functions of Organisms . 166 1. Effect of Light upon Metabolism 166 a. The Thermic Effect of Light on Metabolism . .166 b. The Chemical Effect of Light on Metabolism . . 170 2. Vital Limits of Light Action on Protoplasm . . . 171 3. Effect of Light upon the Movement of Protoplasm . . 175 a. Effect of Low Intensity of Light on Movement — Dark- rigor .......... 175 b. Effect of High Intensity of Light on Movement — Light-rigor 178 §4. Control of the Direction of Locomotion by Light — Phototaxis and Photopathy * . . .180 1. False and True Phototaxis . . . . . . .181 2. Distribution of Phototaxis and Photopathy . . . 182 a. Protista .182 b. Cells and Cell-organs 189 a. Chlorophyll Bodies 189 (3. The Rearrangement of Pigment in Animal Cells in Response to Light ..... 192 y. The Migration of Pigment Cells in the Metazoan Body .193 c. Metazoa 194 3. The General Laws of Phototaxis and Photopathy . . 196 a. The Sense of the Response . . . . . .196 b. The Effective Rays .201 c. Phototaxis vs. Photopathy ...... 203 (L The Mechanics of Response to Light .... 207 Summary of the Chapter . 210 Literature -1- xiv CONTENTS CHAPTER ACTION OF HEAT UPON PROTOPLASM PAGE § 1. Nature of Heat and the General Methods of its Application . 219 § 2. The Effect of Heat upon the General Functions of Organisms . 222 1. Effect of Heat upon Metabolism ...... 222 2. Effect of Heat upon the Movement of Protoplasm and its Irritability ......... 225 § 3. Temperature-Limits of Life ....... 231 1. Temporary Rigor and Death at the Higher Limit of Tem- perature, Maximum and Ultramaximum . . . .231 2. Temporary Rigor and Death at the Lower Limit of Tempera- ture, Minimum and Ultraminimum ..... 239 § 4. Acclimatization of Organisms to Extreme Temperatures . . 249 1. Acclimatization to Heat ....... 249 2. Acclimatization to Cold ....... 257 § 5. Determination of the Direction of Locomotion by Heat — Ther- motaxis . . . . ' . . . . . . . 258 Notes to Table XXI .......... 263 Literature ... . 267 CHAPTER IX GENERAL CONSIDERATIONS ON THE EFFECTS OF CHEMICAL AND PHYSICAL AGENTS UPON PROTOPLASM § 1. Conclusions on the Structure and Composition of Protoplasm . 274 § 2. The Limiting Conditions of Metabolism 275 § 3. The Dependence of Protoplasmic Movement upon Metabolism and upon External Stimuli 277 § 4. The Determination of the Direction of Locomotion 278 EXPERIMENTAL MORPHOLOGY CHAPTER I ACTION OF CHEMICAL AGENTS UPON PROTOPLASM IN this chapter it is proposed to consider (I) the effect of the various chemical agents upon the chemical constitution of protoplasm, as revealed by the results of their application, — death, modification of the metabolic processes, and of rate of movement ; (II) the phenomena of acclimatization to chemi- cal agents ; and (III) the effect of such agents in determining the direction of locomotion, — chemotaxis. § 1. MODIFICATION OF VITAL ACTIONS* The vital processes are chemical processes, taking place in a highly complex, very unstable, constantly changing substance, whose activities we call life. It is not easy to study this living substance chemically by the ordinary methods ; for these usually, first of all, kill the substance. That the living substance and the dead are quite different is illustrated, for example, in the action of diamid (N2H2) and hydroxylamine (XH2 — O — H), which show no action upon dead protoplasm, but are powerful poisons for all living plasm. The instability of protoplasm enables us, on the other hand, to make use of * In the preparation of this section, much use has been made of the admirable work of LOEW ('93). Not only is the adopted classification of poisons for the most part his, but also, in a few cases, passages from his book have been translated in toto here. Most of the determinations of killing strengths of the various re- agents for which no other authority is given have been taken from LoEw'sbook. B 1 2 CHEMICAL AGENTS AND PROTOPLASM [Cn. I certain indirect means for determining its constitution. Since death is due to chemical change, we ought to determine what substances are fatal poisons to protoplasm ; and since every activity of protoplasm is a chemical process, we ought to study the modifications of these processes by the action of various chemical reagents. In studying the behavior of protoplasm in the presence of various reagents, we shall make use especially of observa- tions upon Protista, sexual cells, and tissue cells. In cases where sufficient observations on isolated plant or animal cells are wanting, use will be made of observations upon Metazoa.. At the outset, attention should be called to the necessity of a more quantitative study of the subject. A quantitative study demands, especially, a careful noting of the conditions of the experiment ; for the various physical conditions under which the reagent is applied modify the result. Thus, it has been shown, for example, by RICHET ('89, p. 212) that with various poisons the toxic dose diminishes in amount with the elevation of the temperature of the body. 1. Oxygen. — It is almost certain that no protoplasm can long survive in the absence of oxygen. Apparent exceptions are found in the case of the anaerobic bacteria, some of which are killed in the presence of free oxygen, but multiply rapidly when the oxygen supply is cut off. It has been suggested that, in the case of these and some other parasitic organisms, oxygen is derived from the breaking down of O -containing compounds in the nutritive medium. (Cf. LOEW, '91, p. 760.) The effect of diminished oxygen upon protoplasm is described by CLARK ('89, pp. 370, 371) and by DEMOOR ('94, p. 191). CLARK determined the minimum oxygen pressure necessary for the vital movements of the plasmodia of Myxomycetes and the protoplasm of plant hairs and tissue cells. This he found to range from 1 mm. (plasmodia of Myxomycetes) to 3 mm. (leaf hairs of Urtica) of mercury. DEMOOR* subjected Tradescantia stamen hairs, in water, to a * In studying the effect of a vacuum, DEMOOR employed a piece of apparatus constructed essentially on the plan of an ENGELMANN'S chamber. This consists of a box whose top is a centrally perforated metallic diaphragm and whose bottom is a circular glass. The vertical walls consist of an outer cylinder, at § 1] MODIFICATIOX OF VITAL ACTIONS 3 pressure of 6 to 8 cm. of Hg, or 0.08 to 0.1 of an atmosphere (p. 71). In one hour, on the average, the protoplasmic move- ments were affected, and in most cells ceased in 2 to 3 hours, slight oscillations only of granules occurring. Thus, not death, but arrest of activity, occurred during this period as a result of reduction of atmospheric pressure — upon which the amount of oxygen held in the water depends. That death did not occur is shown by the fact that when air was readmitted at the normal pressure the protoplasm promptly regained its nor- mal activity. Cells immobilized during 24 hours regain their movements in less than 5 minutes, and these become normal in from 10 to 20 minutes. Pure oxygen acts in an opposite fashion from diminished oxy- gen tension, exaggerating the activity of protoplasm. Under its action the protoplasmic movements are much accelerated, but preserve, meantime, their normal character. (Tradescantia hairs, leucocytes; DEMOOK, '94, pp. 192, 218.) In Ciliata the rate of the contractile vesicle does not, however, seem to be altered. (RossBACH, '72, p. 40.) Ozone and hydrogen peroxide produce atomistic "active" oxy- gen by becoming split up in the plasma. Ozone (O3) is said to kill quickly bacteria in water, if the latter does not contain too much organic substance ; in the dry state, however, bacte- ria are injured only slowly by it. (OHLMULLEK, '92, p. 861.) Other substances which, with a greater or less degree of probability, may be said to act through oxidation of the pro- toplasm, may be treated of here. Hydrogen peroxide (H2O2). — PAXETH ('89) added one part of neutralized H2O2 to 10,000 (0.01%) of hay infusion, and found that all Ciliata were dead within 15 to 30 minutes. Stronger solutions act more rapidly ; and even in a 0.005% solution, only part of the animals survived. Algse survived only 10 to 12 hours in a completely neutral 0.1% solution. A 10% solution is fatal in a few minutes. (Cf. BOKORNY, '86, p. 355.) Salts of chromic, manganic, permanganic, and hypochlorous the periphery of the diaphragm, and an inner cylinder at the inner margin of the diaphragm. An inlet and an outlet tube communicate with each of the spaces, — the central space and that between the two cylinders. 4 CHEMICAL AGENTS AND PROTOPLASM [Cn. I acids act as intense poisons, apparently by directly yielding oxygen atoms to the plasma proteins. Sodic chr ornate (Na2CrO4). — Many anaerobic Schizophytes are killed even by a 0.05% solution of this salt. Splenic fever bacteria do not develop in a 0.05% solution in bouillon; in an agar-agar solution, they do not cease to develop until a con- centration of 0.5% is reached, although they no longer produce spores in a 0.05% solution. Sodic chromate also acts strongly on algae. (LoEW, '93, p. 16.) Potassic dichromate (K2Cr2O7). — A 0.1% solution kills algae (Spirogyra) in a few hours. Potassic permanganate (KMnO4) is an energetic poison for algae and Infusoria. A 0.2% solution kills Infusoria (Para- mecium) in one minute. Chlorine, bromine, and iodine, as well as hypochlorous acid salts, act, even in very considerable dilution, fatally upon all organisms, by splitting water, forming hydro-halogen com- pounds, and leaving the oxygen to unite with the living proto- plasm. The action of bromine upon glucose may be written — C6H1206 + Br2 + H20 = C6H1207 + 2 HBr. glucose. (LoEW, '93, p. 15.) BINZ has pointed out that (on Infusoria) the poisonous action of these three halogens, like their other chemical prop- erties, diminishes with increase of atomic weight, in the series Cl, Br, I. (Compare the osmotic effects of the halogens, p. 72.) Potassic chlorate (KC1O3) — also similar salts of I and Br — oxidizes in an essentially different fashion from the permanga- nates. For the latter oxidize even dead organic matter, but the former does not. This reagent may be considered a pas- sively oxidizing one. Concerning its visible effects, we find that bacteria in general are injured by a 2% solution; with weaker solutions in nutrient media the bacteria reduce it to KC1. The anaerobic forms are affected by a 0.5% solution; the aerobic withstand up to 3%. Algae (Spirogyra) die after a few days in a 0.01% solution of the salt. (LoEW, '93, p. 17.) Arsenious acid (H3AsO3) and to a less degree arsenic acid (H3AsO4) are poisons which BIKZ and SCHULZ ('79) believe § 1] MODIFICATION OF VITAL ACTIONS 5 to act by oxidizing the protoplasm. Thus H3AsO3 can take up free oxygen as it would be found in water, and it can part with it readily to the protoplasm, thus oxidizing and eventually wholly consuming it. Such is one theory of its action. A few words as to effects upon Protista: Infusoria survive in 0.1% potassic arsenite in spring water only a short time, but live for weeks in a 0.1% solution of the potassic arsenate. (LoEW, '83, p. 112.) Algse (Spirogyra) are killed by a 0.1% solution of potassic arsenite in six days, — the protoplasm contracts and shows formation of granules, the death of the chlorophyll bands preceding that of the cytoplasm. The same solution of potassic arsenate, meantime, shows no injurious action, (LoEW, '87, p. 445.) Still other arsenious acid salts tried upon other algae (Zygnema, Diatomacea), upon Infusoria, and upon tadpoles showed themselves, uniformly, more powerful agents than the corresponding arsenic salts. The lower fungi are only slightly affected by arsenious salts ; not at all by those of arsenic acid. 2. Hydrogen. — KUHNE ('64, p. 52) subjected Amoeba to H for 24 minutes. At the end of that time, some individuals had assumed a spherical shape, others appeared unchanged in form, but were motionless. Similar results were obtained with Actinophrys, the plasmodium stage of Myxomycetes, and with the stamen hairs of Tradescantia. DEMOOR ('94, p. 190) also experimented upon the latter object, and his results are worth giving in detail.* During the first moments of the passage of the gas, the protoplasmic movements are slightly accelerated. Soon the protoplasm be- comes very granular, and, after a variable time, 15 to 40 minutes, is quiet. The aspect of the protoplasm at this time varies with the character of the cell. If it is young, having a large nucleus and without a primordial utricle, the protoplasm ap- pears uniformly granular. If, on the contrary, the cell possesses a great reserve of water, with long, protoplasmic filaments, the protoplasmic granules become more refringent, increase in volume, and accumulate around the nucleus, — the peripheral * Method : The hydrogen gas may be generated in a KIPP'S apparatus, and should pass through a series of washing flasks containing, e.g., solutions of potash and acetate of lead. See VERWORX : Allgemeine Physiol., p. 285. DEMOOR kept his stamen hairs in sugared water in an EXGELMAXN'S chamber. 6 CHEMICAL AGENTS AND PROTOPLASM [Cn. I protoplasm appearing hyaline. The living substance is in repose. The hydrogen may be passed through the apparatus containing the stamen hairs for from 1 to 5 hours without any movement or other change appearing in the protoplasm. Air is now admitted. The protoplasmic movements rapidly return ; the granules at first oscillate in their places, then gradually extend the range of their movement. In 5 to 6 minutes the cell has regained all of its anatomical and physiological charac- ters. A similar immobility affects also leucocytes subjected to hydrogen. This occurs in about an hour; but there is great individual variation in this respect. Upon substituting air, the activity of the protoplasm is resumed in from 10 to 20 minutes. Protoplasm which has been subjected to the action of hydrogen thus appears not to be permanently modified, since normal movements recur rapidly upon readmitting air. It seems probable, therefore, that the temporary cessation in move- ments in the presence of hydrogen is due to the exclusion of oxygen from the protoplasm. 3. The two Oxides of Carbon, CO2 and CO, have very dif- ferent effects upon protoplasm. Thus DEMOOR ('94, pp. 191, 202, 219) found that whereas the former immobilizes quickly, but kills very slowly, perhaps chiefly by asphyxia, the latter seems in some cases actively to attack the protoplasm. In leucocytes, the ectosarc is separated from the endosarc in a number of completely hyaline fragments ; the endosarc becomes vacuolated, and death ensues in from 20 to 60 minutes. Many bacteria are only slightly affected by CO. 4. Ammonia (NH3). — A 10% solution provokes vacuoli- J zation, partial coagulation, and irregular movements in the I protoplasm of the Tradescantia hair. The cell finally enters into repose, all the granules accumulating around the nucleus. Washing the preparation with water restores the original char- acters of the protoplasm. Thus, ammonia at first energetically excites protoplasm, later producing anaesthesia. (DEMOOR, '94, p. 193.) Even with very weak aqueous solutions (0.005%), which do not kill the protoplasm, BOKORNY ('88) has observed the production in Spirogyra cells of granules, which process does not, however, seem to modify the normal activities of the cell. These granules, " proteosomes," are intensely blackened § 1] MODIFICATION OF VITAL ACTIONS 7 by alkaline (0.001%) silver solutions. Other basic substances, like potash and organic amine bases, and various alkaloids, produce the same effect. (Cf. LOEW and BOKORNY, '89.) Azoimid. — LOEW ('91) has studied the effect upon proto- NX plasm of this somewhat close ally of ammonia, || /NH. The N' poisonous action of this substance seems to depend upon its excessively unstable structure, for it easily disintegrates with violent explosion and production of ammonia. This latter then produces the characteristic granulations. Infusoria are killed in 2 to 2J hours by a 0.1% solution of N3Na, and the water-living Nematodes, Planaria, Ostracoda, Copepoda, and young Planorbis and Lymnsea are killed by a 0.05% solution in 30 to 40 minutes. Algae are more resistant. 5. Catalytic Poisons. — There is a large number of unstable C-compounds which are neither acid nor basic nor characterized by chemical energy, which are, nevertheless, intense poisons for all living cells. Here belong the anaesthetics — ethylether, chloroform, chloral, carbontetrachlorid, methylal, alcohols, car- bon disulphide, etc.* NAGELI believes these to act as poisons by virtue of an in- herent lively condition of molepular movement, which disturbs the normal condition of movement in the living plasma body, and, on that account, produces death. LOEW believes, more precisely, that the transmitted condition of violent movement leads to chemical transformations in the unstable albumen of the protoplasm. As examples of the effect of the mere presence of many un- stable carbohydrates upon chemical processes, it has been found that HC1 and prussic acid, which unite alone only at a high temperature, unite in the presence of various ethers at —15°. Again, the mere presence of some CH compounds transforms a substance into its isomeric condition. Thus, thiourea is trans- formed by an alcoholic solution of ainylnitrite into its isomer rhodanammonium. Such poisons, which change the protoplasm by transmission of molecular movements, may be called cata- * This paragraph and the two following are largely translated from LOEW ('93). 8 CHEMICAL AGENTS AXD PROTOPLASM [Cn. I lytic poisons ; the process which they inaugurate being known as catalysis (apparently produced by mere contact). First will be considered the laws of relation between molecular composition and strength of action. We may begin with the me than series. This series, which has CH3 — as its base, runs as follows : — CH4 C2H6 C3H8, etc. In the members of this series, the poisonous action increases up to a certain limit, with the number of O atoms ; above that limit the compounds are more stable and are more indifferent ; e.g. paraffine (C21H44 to C27H56). Beginning with methan, CH4, we find this substance — marsh gas — innocuous when mingled with air. As the If atoms become replaced by one or more chlorine atoms, the poisonous qualities increase, — CH3C1 is slightly anaesthetic, CHC13 = chloroform, CC14 is very dangerous, stupefying involuntary muscles. If the H atoms are replaced by any other halogen, — e.g. I, — anaesthesia is produced among some Vertebrates. Thus, 0.5 to 1 grain of CH2I2 kills a rabbit. In ethan (C2H6) also, when Cl replaces H, the substance be- comes a more active poison ; e.g. C2H3C13, methal chloroform, acts like chloroform. Also, among the sulphur hydrocarbons we observe the same fact of increase of poisonous action with increase in the number of Cl atoms to the molecule ; thus, — sulphur ethyl, . C2H5— S— C2H5 is a weak poison, monochlorsulphurethyl, C2H5— S— C2H4C1 is a stronger poison, dichlorsulphurethyl, C2H4C1 — S — C2H4C1 is a very powerful poison. In the more complex sulphonic hydrocarbons of the methan series, where the alkyls CH3— , C2H5 — , etc., are introduced, the rule holds that the more atoms in the alkyl the more- active the substance as a poison; thus, — § 1] MODIFICATION OF VITAL ACTIONS 9 /S02CH3 / C \ is not poisonous ; CH3/ \S02CH3 v /S02C2H5 sulphonal : / C \ is poisonous ; CH/ /S02C2H5 trional : / C \ is poisonous ; and C2H/ \S02C2H5 C2H5x /S02C2H5 tetronal : / C \ is more poisonous. C2H/ \S02C2H5 The same holds for the acetals ; thus, — H\ /O.CH3 Hv /O.C2H5 >C C is half as active as ) C C H/ \O.CH3 CH3/ \O.C2H5 We find the same thing in the ethyl group, — CH3\ C2H5\ CH3— C - OH is less active than CH3— C - OH. CH3/ CH3/ trimethalcarbinol. ditnethalethylcarbinol. And also in the alcohols, — methyl alcohol, CH3OH, weak action ; ethyl alcohol, C2H5OH, weak action ; isopropyl alcohol, C3H7OH, stupefymg. A few words now concerning the morphological changes ob-' served in protoplasm subjected to the action of poisons belonging to this group. Here belong especially the various anaesthetics. Chloroform and ether seem to affect all protoplasm anaes- thetically. that of the higher plants as well as that of the higher animals. (BERNARD, C., 78, and ELFIXG, F., '86.) KUHXE (T>4, p. 100) first studied the effect of chloroform vapor upon Tradescantia hairs, but DEMOOR ('94, p. 193) has since described the action of this reagent in much more detail. \ chloroform water at first (2 to 5 minutes) produces a very intense excitement in the movements of the protoplasm, a strong vacuolization occurs, and then the cytoplasm gradually 10 CHEMICAL AGENTS AND PROTOPLASM [Cn. I becomes immobile and dies in from 15 to 30 minutes. The nucleoplasm is less energetically acted upon than the cyto- plasm. Upon swarm-spores, which are highly responsive to light (p. 182), weak solutions of ether and chloroform have such an effect that without preventing locomotion they destroy the power' of responding to the stimulus of the external agent. So, too, the migrations of the chlorophyll in Metaphyta * under the influence of light (see p. 189) is prevented. Ciliata are slightly paralyzed, for the period of the contractile vacuole is diminished. The whole cell body becomes distended with water, and the trichocysts are exploded. (SCHURMAYER, '90, p. 453.) When chloroform water is slowly applied to leucocytes they acquire a spherical form ; when quickly applied they become immobile without change of form. The first effect is a very intense increase in movements, especially of the ectosarc. Ultimate washing in serum suffices to restore the leucocyte to its wonted activity ; so that it has not been killed, but only ansesthetized. (DEMOOR, '94, p. 217.) /OH Chloral hydrate, CC13 — C — OH, which is closely related to \H /Cl chloroform, Cl — C — Cl, acts similarly as a protoplasmic anses- \H the tic. A 0.1% solution kills Infusoria, Rotifera, and diatoms in 24 hours, but filamentous algse and Nematoda withstand it. Sulphonal in 0.1% solution is less injurious than the pre- ceding, since during 24 hours the above-mentioned organisms are uninjured. (Losw, '93, p. 25.) Upon the effect of alcohols on protoplasm, extended experi- ments have recently been made by TSUKAMOTO ('95). These reveal in much detail the peculiarities of action of the different kinds. I give three tables showing the time of resistance in hours of various organisms to the various alcohols, constructed from data furnished by his paper. * ELFING, F. ('86, pp. 47-51). The swarm-spores employed belonged to the species Chlamydomonas pulvisculus. The strengths of the solutions which inhibit their response without stopping locomotion are : of ether, 1% to 5% ; of chloroform, 12% to 25%. The migration of chlorophyll in Mesocarpus is in- hibited by a 1% to 2% ether solution. §1] MODIFICATION OF VITAL ACTIONS 11 TABLE I TIME (IN HOURS) OF RESISTANCE OP TADPOLES OP BUFO VULGARIS, LAUR. (HIND LEGS HAD JUST APPEARED) TO VARIOUS ALCOHOLS STRENGTHS. O.01% 0.1% 0.3% 0.5% 0.7% 1.0% 1.5% 2.0% 2.6% methylic* .... ethylic 4-5' 1.0 0.2-0.5 1.5-2.0 0.08 0.15-0.25 0.6 0.5 0.05-0.15 0.15-0.33 0.3 0.2-0.3 0.05-0.25 0.15 0.15 stupor 0.3-0.8 0.16 0.07-0.3 propylic, norm. . propylic, iso. . . . butylic, norm.. . butylic, iso. . . . butylic, tertiary, amylic, norm. . . allylic TABLE II TIME (IN HOURS) OF RESISTANCE OF INFUSORIA AND OSTRACODA TO VARIOUS ALCOHOLS STRENGTHS. O.005% 0.1% 0.5% 1.0% 3.0% methvlic 20 ethylic 4 propylic, norm *. . 72 1 propvlic iso 18 butylic norm 48 18 butylic iso 72 48t butylic tertiary . 48 + t amylic norm. . . 24 + 24 allylic 24 * The structural formulas of these alcohols are given here for reference : — methylic H - CH2OH ethylic CH3 - CH2OH propylic, norm CH3 . CH2 - CH2OH propylic, iso (CH3)2 - CHOH butylic, norm CH3 . CH2 . CH2 - CH2OH butylic, iso. CH3 CH3 > CH - CH2OH butylic, tertiary . ^3>COH-CH3 L/.H.3 amylic, norm CH3 . CH2 . CH2 . CH2 - CH2OH allylic CH2.CH-CH2OH t Ostracoda only ; the Infusoria died after 18 hours. OF THF UNIVERSITY 12 CHEMICAL AGENTS AND PROTOPLASM [Cn. I TABLE III TIME (IN HOURS) OF RESISTANCE PERIOD OF SPIROGTRA COMMUNIS TO VARIOUS ALCOHOLS STKENGTHS. 0.005% 0.01% 0.05% 0.1% 0.5% 1.0% 2.0% 3.0% 4.0% methylic ethylic 120 72 96 72 48 48 propylic, norm. . propylic, iso. . . . butylic, norm. . . butylic, iso. . . . butylic, tertiary, amylic, norm. . . allylic 66 72 24 24 72 96 24 72 48 48 48 48 i From these experiments it appears that allylic alcohol is more injurious than the others, so that TSUKAMOTO ('95, p. 281) believes it to attack the protoplasm directly rather than to act merely catalytically. We see also that the rule enunciated above about the greater activity of substances with more com- plex alkyls holds true in general. Of the butylic alcohols the normal is the most poisonous ; the tertiary, least. Carbonic disulphide (CS2) is one of the more powerful cata- lytic poisons. A saturated aqueous solution, which contains only a trace of CS2, nevertheless kills quickly algse, bacteria, and the lower water animals. (LoEW, '93, p. 29.) 6. Poisons which form Salts. — This is the third group recog- nized by LOEW. In this case we have to do with acids and bases which unite with the protein substances of the pro- toplasm-producing salts. Thus disturbances leading to death are produced. In addition this group comprises the poisonous metallic salts. So we may recognize three groups: a. acids; b. the soluble mineral bases ; c. salts of heavy metals. a. Acids. — The strong inorganic acids act, in general, more powerfully than the organic. Most bacteria, algae, and Infusoria are very sensitive to inorganic acids (see MIGULA, '90), but splenic fever bacteria resist 1% HC1 for 24 hours, and their spores 2% HC1 for 48 hours. Mold withstands 1% phos- phoric acid. Certain tissues have gained a high resistance capacity to inorganic acids. Thus, the gland cells of marine § 1] MODIFICATION OF VITAL ACTIONS 13 Gastropoda (Dolium, Cassis, Tritonium, Natica heros) secrete -2% to 3% H2SO4. (LoEW.) To organic acids many algae are little resistant. Thus Spiro- gyra and Sphseroplea die in 0.1% malic or tartaric acid after 30 minutes ; in 0.05% malic or tartaric acid after 24 hours ; in 0.01% of these same acids in a few days. Formic acid pre- vents development of bacteria even in small percents — 0.05% to 0.006%. On the other hand, some protoplasm has acquired a resistance to organic acids. The vinegar eel — Rhabditis aceti — lives in 4% acetic acid. The protoplasm of the Dro- sera tentacle resists 0.23% tartaric, citric, and other organic acids. b. Soluble mineral bases, including those of corrosive alkalies and the alkaline earths : Ca, Ba, and Sr. The corrosive alka- lies cause a swelling of the protoplasm, but the primary effect is rather a chemical one. (Cf. FROMAXX, "84, p. 90.) The lower water animals and plants are quickly killed by 0.1% potassic or sodic hydrate. Thus, the movements of Chara cease in 0.05% KOH in 35 minutes. Bacteria are more resistant ; the limit for the typhus bacilli being between 0.10% and 0.14%, and for the cholera bacillus, between 0.14% and 0.18%. Ascaris is still more resistant, living for 20 minutes in a 2% solution of XaOH. CaO is still more powerful. A 0.007% to 0.025% solution in bouillon kills bacilli. A 0.013% solution is fatal to alga3 like Spirogyra. K2CO3 kills bacteria in 0.8% to 1.0% solutions. Xa2CO3 kills Ascaris in a 5.8% solution after 5 to 6 hours. (LoEW, '93, pp. 33, 34.) FROMAXX has discussed the histo- logical changes in protoplasm after treatment in Na2CO3. It is difficult to say whether the action of some of these re- agents may not be an osmotic, rather than a chemical one. The action of Xa2CO3, for example, as described by FROMAXX, is very similar to that of NaCl, whose action is probably solely osmotic. c. Salts of Heavy Metals. — The method of action of these poisons has been accounted for upon the following grounds: When amido-acids (which are found as disintegration products of all animal tissues) are treated Avith salts of the lieavy metals, 14 CHEMICAL AGENTS AND PROTOPLASM [Cn. I the hydrogen in either the carboxyl-group or the amido- group can be replaced by the metal. Likewise the hydrogen of the amido-group in urea derivatives and many bases are replaceable by metals. In the still more complicated protein stuffs the H bound to the N or O can be replaced. Many metals, indeed, like silver or mercury replace preferably the H of the amido-groups, and on this account, perhaps, their salts are especially poisonous. (LoEW, '93, p. 34.) Salts of Hg, Ag, and Cu cause death to Spirogyra even in a dilution of 1 : 1,000,000 ; the chlorophyll bodies being first affected.* Upon the bacteria of splenic fever the double cyanides of Ag, Hg, and Au are the most injurious, next those of Cu, Pb,' and Zn, and, finally, those, of Pt, Ir, and Os. Tad- poles and Tubifex are killed in 24 hours by solutions of CuSO4 weaker than 0.00005%. (LOCKE, '95, p. 327.) Among mer- curic salts, splenic fever bacteria do not develop in 0.0003% HgCl2 in nutritive bouillon, nor 0.0125% in blood. Lactic acid bacteria do not reproduce in 0.0007%. Mold spores are killed in * In this connection reference must be made to the posthumous paper of NAGELI ('93), " Ueber oligodynamische Erscheinungen in lebenden Zellen." This author found that water distilled in copper vessels or 1 litre of water in which 12 clean copper coins had stood for four days acted fatally upon Spiro- gyra. The water was found in one such case to contain 1 part Cu in 77,000,000 of water. It was believed to be in solution in the form of the hydroxyd (CuH2O2). Similarly produced solutions of other metals, Ag, Zn, Fe, Pb, Hg, had a simi- larly fatal effect upon Spirogyra. NAGELI believed that the effect of the metals was not a chemical one, but was due to a new force — " oligodynainic." Besides the fact of the action of very dilute solutions, the only evidence he adduced for the new force was based on the difference of action on the chlorophyl bands of solutions of 1 : 1000 or 1 : 10,000 and 1 : 10,000,000. In the weaker solutions (" oligodynamic " action) the bands alone were drawn away from the cell- wall, in the stronger solution (chemical action) the whole peripheral protoplasm was shrunken away. It does not seem necessary to invoke a new force to explain the action of weak solutions: first, because the two actions are not sharply separated, according to NAGELI'S own data; secondly, because the chlorophyll bands are in general more sensitive than the rest of the protoplasm (p. 5) ; and, thirdly, because the action of so weak a solution is not surprising in view of the fact that Spirogyra is one of the least resistant of organisms. Even in a comparatively resistant organism, like Stentor, a solution of 1 : 80,000,000 HgCl2 produces acclimatization to the poison and 1 : 10,000,000 has an injurious effect. Yet between the action of such solutions and those of 1 : 1000 there is a complete graduation in increasing effect. (Seep. 30.) Even in NAGELI'S experiments- solutions of less than 1 : 100,000,000 had little action. § 1] MODIFICATION OF VITAL ACTIONS 15 0.1%. NEAL and I have found that Stentor cceruleus is killed by a 0.001% solution HgCl2 in a few seconds. (Cf. p. 30.) Ascarids die in a 0.1% solution in an hour. (SCHRODER, '85.) Silver salts occasionally act upon bacteria more energetically that those of Hg. Cadmium and zinc salts are poisonous — the former more so than the latter. Thus, whereas 0.015% of cad- mium sulphate inhibits reproduction of lactic acid bacteria, 0.1% of zinc sulphate is not injurious. Many salts of thallium are likewise active. Thus LOEW ('93, p. 37) found that in 0.1% thallium sulphate Spirogyra died in 4 to 6 hours. 7. Substitution Poisons. — In this group LOEW places cer- tain nitrogenous substances which attack the ainido and alde- hyde groups of living protoplasm. These are extremely unstable substances and may therefore be transformed by agents which have no effect upon dead protoplasm. The supposed method of action of a poison upon an aldehyde may be illustrated in the case of the poison hydroxylamine (H2N — OH); which justifies at the same time the term "substitution poisons." ,0 ^N + H>0. - 2- - \H \H any aldehyde. hydroxylamine. an aldoxim. Hydroxylamine. — This is a general and powerful poison. Thus, among the lower organisms, a solution of neutral hy- droxylamine of — 0.001% kills diatoms within 24 hours. (LOEW, '85a, p. 523.) 0.005% kills in 36 hours Infusoria which withstand a similar con- centration of strychnine. (LOEW.) 0.01% kills diatoms in something less than 15 hours; Planaria and leeches in 12 to 16 hours. (LOEW.) 0.1% paralyzes the muscles of Rotifera in 10 to 15 minutes ; those of Xais in 20 to 30 minutes. (HOFER, '90, pp. 324, 325.) 0.2% kills Rotifers, Copepoda, and Isopods in 1 hour (LOEW) ; stupefies Yorticella in from 2 to 10 minutes. (HOFER, '90, p. 325.) 0.25% stupefies Stentor in 10 to 20 minutes. (HOFER.) Benzenylamidoxim and acetoxim, more complex derivatives of hydroxylamine, are somewhat less poisonous. 16 CHEMICAL AGENTS AND PROTOPLASM [Cn. I Diamid, or hydrazin (H2N — NH2) in the form of neutral solutions of the sulphate is a rapid poison. A solution of - 0.01% kills various alga species in 1 to 2 days. 0.02% is injurious to bacteria. 0.05% kills various water animals within 12 hours. (LOEW, '93.) Phenylhydrazin , H f solution of — N-NH, is more powerful. A 0.0067% kills Infusoria and algae within 18 hours. 0.05% prevents the development of bacteria and mucors. As free ammonia (NH3) is a far weaker poison than diamid (H2N — NH2), so anilin (C6H5NH2) is far weaker than phenyl- hydrazin (C6H5 . NH . NH2). Passing now to the more complex nitrogenous compounds, we find, first, that bodies which possess slight or no poisonous power, and contain tertiary N, can become strong poisons by addition of H and formation of imido-groups (i.e. groups which can be derived from ammonia by the substitution for two H atoms of bivalent acid radicals) ; thus, — /CH = CH\ /CH2 - CH2\ CH/ )K CH/ O ^CH - CH^ \CH2 - CH/ pyridin (weaker poison). piperidin (stronger poison) /HC = HC\ /H,C - H,C\ " >N CH/ ) C^ \CH9 - CH/ I I CH CH2 / \ I 3 CH3 CH2 coUidin (weak). CH3 coniin (violent). In the preceding and the two following cases it is seen that, in general, when there is a hydrogen atom of the amid radical (NH2) unreplaced by an alkyl, the substance is poisonous. § 1] MODIFICATION OF VITAL ACTIONS 17 ONLY SLIGHTLY POISONOUS. VIOLENT POISONS. )CH - C6H5 I /CH 6H5-CH = N/ C6H5-C = W - C6H5 hydrobenzamid. amarin. /CH = CH\ CH = CH\ CH/ I >H CH = CHX pyridin. pyrrol.* This increased poisonousness, correlated with the presence of H joined to N, may be accounted for by the union of this H with the O of the ketons or aldehydes of the living substance. Likewise when one or more H atoms of the amido-group are replaced by an acid radical (e.g. that of acetic acid, CO . CH3), the poisonous qualities of the substance are con- siderably diminished ; thus, — MORE POISONOUS. LESS POISONOUS. C6H5NH2 CeHsNH . CO - CH3 anilin. antifebrin.t . NH2 C6H5ira . NH . CO . CH3 phenylhydrazin. pyrodin. /NH« HN = C( HN = C( .C0.^xx2 guanidin. dicyandiamidin. In like manner when, in an imido-group, the H (of the NH radical) is replaced by alkyls (e.g. CH3), the substances become less poisonous ; thus, — * While a 0.07 % solution of pyrrol kills Isopods, Rotifers, Planaria, etc., in about 1 hour, these organisms withstand a solution of pyridin of the same strength. (LoE\v, '87, p. 444.) t SCHURMAYER ('90, p. 445) finds that upon Ciliata (Carchesium) a 0.1% solution slightly accelerates at first the action of the cilia, diminishes the rate of succession of the phases of the contractile vacuole, and leaves the protoplasm permanently more or less paralyzed, c 18 CHEMICAL AGENTS AXD PROTOPLASM [Cn. ] NH-CH N.CH3-CH I II I II CO C - KH CO C - N . CH3 I I ^>CO I I \CO NH-C=NX NH C = N ' xanthin. theobromine. N.CH3-CH I II CO C-N.CH, I I ^CO N . CH3 - C = N ' coffein. are successively less poisonous. (LoEW, '93, p. 46.) H I HV /Cv /a \c/ \c/ While benzol \ \ is rather inactive, 8 grammes /C\ /Gv H/ \C/ ^H I H per day being withstood by the human organism, with the replacement of the H atoms by OH the substance becomes more poisonous in direct proportion to the number of H atoms thus replaced. (LoEW, '87, p. 440.) Thus there fol- low in order of poisonousness : — H H H I I I HC/ \C-OH HC/ \0-OH HO-C/ \C-OH II II II HC \ /CH HC >. I I I H OH OH phenol (monoxybenzole). resorcin (dioxybenzole). phloroglucin (trioxybenzole). Phenol (or carbolic acid) and its derivatives attack unstable substances, especially aldehydes, forming insoluble products. Phenol itself produces in the higher animals a paralysis of the nerve centres. Algae die in a 1% solution after 20 to 30 § 1] MODIFICATION OF VITAL ACTIONS 19 minutes; in a 0.1% solution after 3 days. Infusoria die quickly in a 1% solution. Ascaris lives only 3 hours in a 0.5% solution. Resorcin (0.5 g.) is hypnotic to man; 0.085 g. per kg. is fatal to dogs. Its isomer — pyrocatechin — is more active. 0.1% of it in spring water kills diatoms and Infusoria after a few minutes, and filamentous algae in a few hours; while, with resorcin, Infusoria, diatoms, and green algae live several hours — even as long as 18 hours. By replacing one of the H atoms of phenol by CO OH (or carboxyl), thus producing salicylic acid, the poisonous qualities are reduced. Hydrocyanic Acid, CNH. — The action of this substance is peculiar in that, acting on the central nervous system, it is in small quantities a more violent poison for Vertebrates than for Invertebrates. Hydrocyanic acid acts upon aldehydes — in dilute solutions upon the most unstable compounds only ; in stronger concentration upon all aldehydes. Its peculiar work- ing may be hypothetically explained by assuming the aldehydes of the ganglion cells to be more unstable than those of other cells, so that traces of CNH which do not injure other cells destroy quickly the nerve cells. (LoEW.) The action may be shown by the equation, — E\ K\ )C = 0 + CNH = )C— OH. H/ H/ aldehyde. The degree and variety of its action may be inferred from the following data, taken from LOEW ('93): Infusoria die quickly in a 0.1% solution, but Ascaris resists a 3% solution for 75 minutes. The resistance of the hedgehog to CNH is remarkable ; five times the dose which killed, in 4 minutes, a cat weighing 2 kg. produced in the hedgehog only a slight sickness. A myriapod (Fontaria) excretes CNH when irri- tated. Certain salts of CNH act as poisons ; e.g. (CN)2Hg, Na2Fe(CN)5NO. Hydric sulphide acts as a poison either by deoxidizing the plasma, H2S + 0 = H20 4- S, 20 CHEMICAL AGENTS AND PROTOPLASM [Cn. I or by acting on the aldehydes, E\ Bv )C = O + H2S = )C = S + H20. H/ H/ It acts rather energetically upon algse and Infusoria. In Ver- tebrates, the central nervous system is attacked and the oxy- hsemoglobin of the blood is altered. Sulphurous oxide (SO2) attacks members of the aldehyde c = o + SOSKH = ;c— OH H/ H/ \H aldehyde. 0.1% kills lower fungi in a few minutes, 0.01% in a few hours. Selenous oxide (SeO2), which acts chemically much like SO2 and has a much greater molecular weight (64 : 111), acts less energetically as a poison. A 0.1% solution kills Spirogyra and Zygnema in 3 hours, while 0.01% is scarcely injurious. Tellurous oxide (TeO2, mol. wt. = 157) is non-poisonous, although chemically closely allied to the two preceding. (BOKORNY, '93.) Aldehydes. — The poisonous action of these substances de- rived from oxidation of alcohol is dependent upon their insta- bility. So we find that an aldehyde, which, like grape sugar, is fairly stable, is likewise non-poisonous ; while formaldehyde, which is very unstable and active, is correspondingly poisonous. Aldehydes attack especially the unstable amides, affording ni- trogenous compounds ; e.g. — C6H5 • NH2 + CH20 = C6H5NCH2 + H2O. Now, even in passive albumens, part of the N is in the form of amido-groups ; for, in treating with nitric acid, much nitro- gen is set free, which would not occur were all of the N second- arily or tertiarily bound up. (LoEW, '93, p. 58.) Hence the poisonousness of aldehydes for living albumens. Formaldehyde. — This substance (H — CH : O) acts upon propeptones and upon albumen, affording compounds which are not readily soluble. An aqueous solution of — § 1] MODIFICATION OF VITAL ACTIONS 21 0 0.01 % is fatal to bacteria. .05% kills worms, molluscs, and isopods in 2 hours. (LOEW, '88, p. 40.) 1.00% kills Spirogyra very quickly. (CoHN, '94, p. 5.) A weak solution seems to act anaesthetically upon Noctiluca. (MASSART, '93, p. 65.) When various radicals are substituted in H — CH : O, the substance acts more like a catalytic poison. Thus, ethyl- aldehyde (CH3 — CHO) is anaesthetic ; and paraldehyde (CH3-CHO)3 kills algae in a solution of 0.002% in 24 hours and causes protoplasm to become immobile either (leuco- cytes) after momentary stimulation (DEMOOR, '94, p. 218) or (Xoctiluca) at once (M ASSART, '93, p. 66). Several derivatives of ethylaldehyde are poisonous. LOEW ('93, p. 60) has shown that in a 0.1% solution of the neutral sulphate of NH2 — CH2 — CH : (OC2H5)2, amidoacetal, Infu- soria, and diatoms die within 15 hours, and, somewhat later, filamentous algae. Nitrous acid, as is well known, produces, even in great dilu- tion, OH-cornpounds from amido-compounds (R — NH2) ; or else, under certain conditions, especially with aromatic amido- compounds, diazo-compounds result ; e.g. — C2H5 . NH2 + HO . NO = C2H5 . OH + NT, + H2O, amine. alcohol. and C6H5 . XH2 . HN03 4- HO . NO — C6H5 . N2ON02 + 2 H2O. aniline nitrate. diazobenzene nitrate. Thus a solution of 0.001% of free nitrous acid is poisonous to algae, and more so than nitric acid. The lower fungi are also very sensitive to nitrous acid. Its action as an acid is weak, so that its salts are set free in the presence of even the weaker organic acids. On this account, even neutral nitrates kill such plants (some algae, e.g. Spirogyra) as have an acid cell-sap. 8. Sodic Fluoride. — The poisonous action of the fluorides of the light metals, and especially sodium, has not hitherto been explained. A 0.2% solution of NaF kills various algae (Oscillaria, Cladophora, CEdogonium, diatoms) within 24 hours, 22 CHEMICAL AGENTS AND PROTOPLASM [Cn. I producing a change in size of the nucleus. (LoEW, '92.) A 1% solution kills the nerves of a frog in 2 hours. 9. Special Poisons. — Toxic Protein Compounds. Little need be said here concerning the recent discoveries of poisonous albuminoids excreted by disease-producing bacteria, or of those secreted by the parasitized body (alexines) . Similar compounds, highly poisonous to Vertebrates, have been extracted from the seeds of some Phanerogams, e.g. ricin, from the seeds of Rici- nus communis (castor-oil bean); abrin, from the seeds of the leguminous Abrus precatorius, L. ; and phallin, from the toad- stool Agaricus phalloides, Fr. Finally, in this group may be placed a large number of protein substances derived from animals, which are more or less poisonous to a greater or smaller number of kinds of protoplasm. The poison of the rattlesnake (Crotalus) and of the cobra (Naja) is fatal to Vertebrates in small, hypodermically injected, doses. Hydra, Turbellaria, Rotifera, and Crustacea are also affected by it ; but Infusoria and Flagellata are apparently unaffected. (HEi- DENSCHILD, '86, p. 330.) It is important that, according to the experiments of several investigators, among the earlier of whom may be mentioned DAREMBERG ('91) and BTJCHNER ('92), the various species of Vertebrates possess protein substances in their blood serum which are to a certain extent injurious to other species, since the blood serum of any one species will destroy the red and white blood corpuscles of another. The poisonous action of these animal protein substances seems to be due to their un- stable character, whereby they easily form unions with the unstable groups of the protoplasm, frequently producing thereby violent poisons which work as substitution poisons. (LoEW, '93, pp. 81-84.) Alkaloids. — These basic, nitrogenous compounds have, for the most part, very complex molecules, so that their structure has, in many cases, not been determined. Consequently the nature of their chemical action upon protoplasm is, in general, unknown. LOEW suggests ('93, p. 85) the following theory of action of alkaloids. The bases unite with the active protein substances of the cell, and thereby introduce a disturbance of equilibrium § 1] MODIFICATION OF VITAL ACTIONS 23 in the plasma body — a disturbance which is especially mani- fest in their action upon the protoplasm of ganglion cells. The capacity of this union is influenced by various factors : by the configuration and degree of dilution of the poison ; by the degree of instability of the kind of protoplasm acted upon ; by the configuration of the molecule of the active protein substance in the cells ; and by the specific (micellar) structure of the plasma body. That organic bases can unite with active albumen is known from observations upon plant cells contain- ing stored-up active protein substances. If the configuration of the albumen molecule and the general texture of the pro- toplasm favor the attack by the base, a disturbance of the equilibrium of the protoplasm will result, even in considerable dilution of the poison. The alkaloids affect chiefly the nervous tissue in the higher animals, producing, in some cases, paralysis ; in others, increased activity. Thus, curarin is paralytic in its action, while the closely allied strychnin is (in dilute solutions) stimulating upon nerve cells. Since action is almost confined to nerve tissue, additional evidence is afforded of the extraordinary instability of the nervous protoplasm. The dissimilar effect of an alkaloid upon the different sub- stances constituting nerve protoplasm gives an idea of the complexity of the latter. Thus, an alkaloid may stimulate nerves with certain functions to increased activity, and may reduce nerves in the same body, having other functions, to depression and paralysis; e.g. nicotin excites sensory nerves, and depresses the activity of the cardio-motor nerves. It is important that many of these alkaloids act also upon Protozoa and the lowest plants, in which nervous substance is still undifferentiated. Other of these alkaloids, however, do not act upon Protista. We will now proceed to an examination of the action of the principal vegetable alkaloids, arranged according to the sys- tematic position of the plants from which they are obtained. Nicotin. — The effect produced by nicotin is directly pro- portional to the differentiation of nervous substance ; thus, it is almost inoperative on Protozoa and Actinia. Hydra is not very sensitive, 0.5% being a fatal dose. A solution of 0.05% 24 CHEMICAL AGENTS AND PROTOPLASM [Cn. I causes Medusae to become quiet in 30 minutes, and is fatal to the earthworm in a few hours ; Echinoderms are paralyzed by a 0.05% solution in 30 minutes ; Palsemon (after temporary stimulation) is paralyzed by a 0.01% solution in 30 minutes ; Sepiola is killed by 0.005% in less than a minute. (GREEN- WOOD, '90.) Veratrin, Atropin, and Cocaine act upon Vertebrates so as to excite the central nervous system at first, and then to paralyze it. All act, however, as poisons upon undifferenti- ated protoplasm (Protozoa). Thus, ROSSBACH ('72) found that when Ciliata were subjected to veratrin chloride and to atropin sulphate, a peculiar rotary movement took place about one end as a fixed axis. Then imbibition of water with great vacuolation of the protoplasm occurred. Later, the contractile vacuole fails to contract, and protoplasmic movements cease a few seconds after. (Cf. KUHNE, '64, pp. 47, 65, 100.) Cocaine is apparently a benzol derivative, closely related, chemically, to atropin. Its formula is thus given: — CH H2C CH2 CH CO.C6H5. C - COO . CH Its action upon Protista has been studied by CHAKPENTIER ('85), ADDUCO ('90), SCHURMAYER ('90, pp. 438-448), AL- BERTONI ('91, p. 318), DANILEWSKI ('92), and MASSART ('93, p. 66); upon sexual cells, by O. and R. HERTWIG ('87, p. 159) and ALBERTONI ('91, p. 309); and upon tissue cells by ALBERTONI. The result has been to show that cocaine first stimulates for a very short time to excessive activity, and then stupefies and paralyzes. With the paralysis, a strong vacuolation of the protoplasm occurs, since the excretory func- tion of the contractile vacuole is inhibited (SCHURMAYER, '90, p. 439). Cocaine acts similarly upon the nerve centres and muscles of the more differentiated animals. 11 j i :; § 1] MODIFICATION OF VITAL ACTIONS 25 Morphin acts less violently upon the nervous tissue of Ver- tebrates. It has a very weak action upon Protista. Strychnin, chemically considered, is an alkaloid with the formula: C21H22X2O2 ; specific gravity, 1.359 at 18°; soluble to about 0.025% in water at 14.5°; has a very bitter taste. The nitrate is generally employed. The action of strychnin upon Protista is known through the studies of MAX SCHULTZE ('63, p. 32), BINZ ('67, pp. 384-389), ROSSBACH (72, pp. 52-54), and SCHURMAYER ('90, pp.. 423-433). KRUKEXBERG ('80) has studied its effects upon higher Invertebrates. Its action upon sexual cells has been studied by the brothers HEBTWIG ('87, pp. 153-156, 164). Although not fatal to bacteria and only in strong solutions fatal to the large fungi, strychnin is a nearly universal proto- plasmic poison. It kills the protoplasm of the Drosera ten- tacles and hinders the development of peas, corn, and lupines. The amount of strychnin that Protozoa can withstand has been variously stated, while all authors admit considerable individ- ual variation in this respect. Probably Protozoa cannot ordi- narily resist a saturated solution for one minute. ROSSBACH (72, p. 52) found that no infusorian of his cultures survived a "0.1% solution" long enough to be placed under the mi- croscope. A 0.02% or 0.01% solution can be withstood for a few minutes (0.01% solution was withstood for 5 minutes, SCHURMAYER). As for the weakest solution that will kill, SCHURMAYER found that a Paramecium resisted for only 15 to 20 minutes so weak a solution as 0.0005%, while ROSSBACH found Stylonychia little affected by a 0.0055% solution. The spermatozoa of Echinoids, according to the HERTWIGS ('87, p. 164), so resist a 0.01% solution that after 180 minutes the movement is only somewhat retarded. Echinoid eggs are in- jured in a few minutes by 0.005%. The injurious action of strychnin on Protozoa varies in the different groups, the resistance capacity increasing, in general, with the height of systematic position of the group. The first effect in Ciliata is an increased activity of the cilia; if irri are present, these strike more powerfully; locomotion is abnormally rapid ; but the movements lack coordination, and a rotation takes place about the axis of progression. Next, 26 CHEMICAL AGENTS AND PROTOPLASM [Cn. I the movements become so disorganized that locomotion is im- possible, despite accelerated cilia-motion. Finally, the move- ments suddenly cease, death intervening. (SCHURMAYER, '90, pp. 423-426.) The process of excretion seems to be especially affected. Immediately after the addition of a 0.02% solution, the contractile vesicle increases to from 4 to 10 times its normal diameter, and loses its spheroidal form. In a slightly greater dilution, 0.014%, the contractile vesicle momentarily constricts, but in diastole gains twice its normal diameter, and the time between phases of contraction is greatly increased. Thus, the normal rate of contraction for Euplotes at 15° C. is between 30 and 35 seconds ; but in a 0.014% solution of strychnin this is diminished to 1 in 500 seconds. Frequently, several vacuoles are formed, and, eventually, the whole body becomes greatly vacuolated, and death intervenes. (RossBACH, '72, pp. 52, 53.) Many higher organisms are not very sensitive to strychnin. Thus, Ascaridse have a considerable resistance, which SCHRODER ('85, p. 307) ascribes to their not opening their mouths in the solution, the poison being thus obliged to pass through the skin. So, too, snails were found by KRTJKENBERG ('80, p. 100) to be very resistant to strychnin. Curare, or urare, is an alkaloid, derived from Strichnos species. The commercial substance is very variable in com- position. NIKOLSKI and DOGIEL ('90) have studied the effects of this drug upon various organisms. Upon adding a few drops of a 0.8% solution of curare to water containing an amoeba, the first effect is a shrinking towards a spherical form and a cessation of all movements. Subsequent washing in water ultimately restores the normal movements. As is well known, it paralyzes also the protoplasm of the nerve endings. Quinine, or chinin (C20H24N2O2). — The "sulphate," which is first produced in the process of extraction, is commonly employed. This is, moreover, more soluble than the pure alkaloid, 1 part dissolving at 9.5° in 788 parts of water. In- vestigations on the action of this poison upon protoplasm have been made especially by BINZ in a series of papers beginning with '67; by ROSSBACH ('72) on Protozoa; by TEN BOSCH ('80) on leucocytes; by O. and R. HERTWIG ('87) on sexual cells; and by KRUKENBERG ('80) on higher Invertebrata. TJN- § 2] ACCLIMATIZATION TO CHEMICAL AGENTS Amoeba, Actinophrys, and various Infusoria are killed by a 0.1% solution in a few minutes, and leucocytes and eggs of Echinoicls are paralyzed even by a 0.005% solution. Its action is thus more powerful than that of strychnin. The proto- plasm at first contracts, then gradually dissolves and streams away. Upon the higher animals, quinine so acts as to paralyze the central nervous tissue (in Mollusca, KRUKENBERG, '80, p. 10), and it affects the cerebrum and heart ganglia of mammals. Antipyrin, or phenyldimethylpyrazolon, is an alkaloid de- rived from and belonging clearly to the benzol type,, in which one atom of H is replaced by a complex atom-group, as may be seen from the formula — .pTT TTP r* r'Ti /\jjn.\. ±1 \J \j VyXlg HCX ^ULC | | I I OC\ /N-CHS HCv ,G ^NX The effect of this agent upon Protozoa has been studied by SCHTTRMAYER ('90, pp. 434-437) and M ASSART ('93, p. 64). A solution of 0.1% acting for 80 minutes, caused Oxytricha at first to move more rapidly, but eventually to become transformed into a shapeless mass, whose protoplasm disinte- grates. Acting upon Noctiluca, a 0.25% solution causes a bright glimmer immediately after applying, followed by dark- ness again. Thus there is here a momentary hyperesthesia. § 2. ACCLIMATIZATION TO CHEMICAL AGENTS It is clear that the protoplasm of different organisms is dis- similar. We see this in the different reactions to the same chemical agent. Not only is the reaction of the various spe- cies unlike, but individuals of the same species from different localities differ widely. (Cf. LOEW, '85.) We are, naturally, most familiar with this phenomenon in the case of man. Thus, the common North American poison ivy (Rhus toxicodendron) produces, in some persons, extensive inflammation in parts which have come even indirectly in con- 28 CHEMICAL AGENTS AND PROTOPLASM [Cn. I tact with it ; while, by other persons, it may be taken into the mouth with impunity. The phenomenon shown by man is found in other animals also. Thus, among Invertebrates, although few bacteria can resist 1% .Na2CO3, and even the extremely resistant Ascaris lives only 5 to 6 hours in a 5.8% solution of this salt, LOE\V ('77, p. 137) has found in Owen's Lake, California (an alkaline water containing among other things 2.5% Na2CO3), numerous living Infusoria, Copepoda, larvae of Ephydra, and molds. Again, the vinegar eel, Rhabditis aceti, lives in a 4% solution of acetic acid, although this strength is usually fatal ; e.g. a 0.23% solution of acetic acid kills the tentacles of Drosera. (DARWIN, '75, p. 191.) What is true of the whole organism is true also of its parts. The gland cells of some marine Gasteropoda (Dolium, Cassis, Tritonium, Natica heros) secrete H2SO4 of a strength (2% to 3%) which is fatal to most protoplasm; the myriapod Foiitaria excretes, when irritated, the extremely poisonous CHN; and, according to LOEW ('87, p. 438), the plant Oxalis produces potassic oxalate, which is a violent poison to most protoplasm. One general law of high resistance is worthy of notice: an organism which produces an albuminoid poison is strongly resistant to that poison. Thus, FAYRER ('74) has shown that venomous serpents are not destroyed by the secretion of their poison glands when it is injected into them ; and BOURNE ('87) has shown that scorpions are not injured by their own venom. An explanation of the facts of varied resistance capacity is first gained through experiment. We all know that, among men, a high resistance capacity to a poison may be acquired by taking a small quantity of it at frequent intervals. Thus, users of tobacco, alcohol, and various alkaloids become, in time, capable of taking, without apparent injury, quantities which would at first have proved fatal. Arsenic eaters may eventu- ally swallow without injury four times the ordinarily lethal dose, i.e. as much as 0.4 gramme. (BiNZ and SCHULZ, '79.) Results similar to those observed in man have been obtained by experiment upon other animals. Thus, SEWALL ('87) inoculated a pigeon hypodermically with sub-lethal doses of ACCLIMATIZATION TO CHEMICAL AGENTS 29 rattlesnake poison (Crotalophorus tergeminus). While no unacclimatized pigeon could resist 1 drop of a 6.8% solution of venom in glycerine, pigeons inoculated with at first weak, then gradually increasing solutions, came at last (after 178 days) to resist 4 drops of the glycerine venom mixture. Likewise KAN- THACK ('92) succeeded in acclimatizing two rabbits and a hen to serpent's venom. Very similar are the experiments of EHRLTCH ('91). This investigator fed white mice (which are killed by ^j- of their weight of a 0.0005% solution of ricin, hypodermically in- jected) upon food cakes soaked in a weak solution of the poison. After feeding them for a varying length of time upon constantly increasing solutions, he determined the maximum solution which, hypodermically injected, they would withstand. If we call the maximum solution which the unacclimatized organism will withstand our unit of immunity, we can express the degree of immunity of the acclimatized organisms by the strength of solution (expressed in terms of that unit) which they can resist. The following table, taken from EHKLICH'S paper, shows the gradual increase of immunity as a result of feeding on the poison : — TABLE IV No. OF EXPERIMENT DAY. STRENGTH OF LAST DOSE GIVEN. LN MG. NUMBER OF INDI- VIDUALS EXPERI- MENTED ON. MAXIMUM IN- JECTED SOLUTION BORNE, %'S. DEGREE OF IMMUNITY. IV 4 8 j Die in 1 V 5 16 1 0.0005 0.0007 1.3 VI 6 23 0.0066 13.3 YII 7 5 0.005 10 VIII . . . 8 18 0.01 20 x 12 9 0.02 40 XII 20 3 0.033 66.6 XV . . 50 1 0.05 100 XVIII. . . 80 4 0.1 200 XXI 80 1 0.2 400 Thus after the first 4 or 5 days the immunity rapidly in- creased; so that, while the solution of oWoo~o ^^s norma^ 30 CHEMICAL AGENTS AND PROTOPLASM [Cn. I mice, those acclimatized during 21 days resist yoVo to occasionally giro, corresponding to a grade of immunity of 200 to 800. By fundamentally similar procedures CALMETTE ('94) has rendered rabbits immune to the action of strong doses of the venom of Naja tripudians (cobra) and of Pelias berus. Still more recently MARMIER ('95) has isolated a toxin produced by anthrax bacteria reared in a peptone-glycerine solution. Inoculated into an animal sensitive to anthrax, this toxin produces, in certain doses, death by cachexy. By employing suitably graduated doses, however, one can obtain immunity of the organism to anthrax, as one does to the venom of serpents. Some attempts to produce acclimatization in lower organisms have been made by Dr. H. V. NEAL and myself. Stentor was employed as the object of experimentation. We reared two lots of Stentors under similar conditions except that Lot 1 was cultivated in water and Lot 2 in 0.00005% HgCl2. After the lapse of two days both were put into a killing solution of 0.001% HgCl2, and the second lot was found to survive longer than the first. The mean resistance period to the killing solu- tion of the lot reared in water was 83 seconds; of that reared in 0.00005% corrosive sublimate, 304 seconds. Similar results were obtained in other experiments. In Fig. 1 a curve is given showing the relation between strength of culture solu- tion and period of resistance. From this curve, based upon 132 determinations, it appears that the resistance period varies directly with the strength of the solution in which the protoplasm has been cultivated. This law holds good, however, only within certain limits. If the culture solution is too strong, above 0.0001%, the organism will be weakened by it so that it cannot resist the killing solution so long as those reared in water can. A similar effect of heightened resistance to quinine is ob- tained by cultivating organisms in quinine. Experiment shows that a slight increase of the resistance period follows subjection to the culture for one hour only; and that the degree of acclimatization varies directly as the time of subjection. „ ACCLIMATIZATION TO CHEMICAL AGENTS 31 The facts obtained by us clearly indicated, then, that, without selection, — for no deaths occurred in our culture solutions, — the protoplasm may become modified merely by subjection to the poison, so as to gain an increased resistance to it. Hence the acclimatizations that we find in nature need not have been brought about by natural selection — must have occurred, indeed, even without selection, if the organisms had been gradually subjected to their environment. \ 90 SECS. 70 1 .50 40 SECS. STRENGTH OF CULTURE SOLUTIONS FIG. 1. — Curve of resistance periods to a 0.00125% solution of HgCl2 of Stentors reared in various solutions of HgCl2 during 20 to 96 hours. We did not determine for how long a time the acclimatized Protozoa retained their heightened resistance capacity. The only data we have upon the subject of persistence of acclima- tization is derived from studies on Vertebrates. Thus it is the familiar experience of arsenic eaters that, after they have broken off their habit, the body does not quickly return to a normal condition. Even after a considerable period of self-denial the taking of large doses may be recommenced — must be recommenced, indeed, or illness sets in. EHBLICH ('91) has studied experimentally the phenomenon 32 CHEMICAL AGENTS AND PROTOPLASM [Cn. I of acclimatization to ricin. Mice which had gained an immu- nity of over 200, and were then kept for 6.5 months on normal food, had still a resistance, although not precisely determined, certainly far above 50. It is an important question : Is an organism acclimated to one poison thereby rendered more resistant to poisons in gen- eral, or only to the specific poison to which it has been accli- mated? EHRLICH found that mice acclimated to ricin were just as sensitive to abrin as the normal animals, and the same is true, mutatis mutandis, for mice which resist abrin. Concerning the changes in the protoplasm brought about by acclimatization little is known. EHRLICH ('91) and CALMETTE ('94) have shown that in the blood of the immunized animal a substance, antitoxic to the specific substance employed, is produced, and this apparently prevents the action of the strong poison by transforming its molecules. The antitoxic substance is of such a nature that when blood containing it (from an acclimatized animal) is injected into an unacclimatized one, the latter becomes immune to the poison. For Protista another hypothesis is admissible ; namely, that the weak solution of the poison, which is used in acclimatiza- tion, gradually destroys those compounds upon which the strong solution would have acted suddenly and, therefore, fatally. The gradual destruction is not fatal because of its slowness. At the same time it prevents the violent action of the strong poison, since it leaves it nothing to be acted upon. The parallelism between the results of experiments upon acclimatization to poisons and those upon immunization through vaccination, leads to the suspicion that, at bottom, the two processes are closely akin. § 3. CHEMOTAXIS ENGELMANN ('81) seems to have been the first to show that the direction of locomotion of simple protoplasmic masses is de- terminable by chemical agents in the environment. He found that Bacterium termo is thus acted upon by oxygen which is not uni'formly distributed. Like many Infusoria, these bacteria § 3] CHEMOTAXIS * 33 gather at the margin of the cover-glass, where oxygen is more abundant than elsewhere. If oxidized blood is introduced under the cover-glass, they move toward it, but not toward blood containing much CO2 in place of oxygen. If green algie are introduced, the bacteria move towards them so long as they, under the influence of sunlight, are producing oxygen. In the dark the algae have no effect. During the decade and a half which have elapsed since Ex- GELMAXN'S first paper appeared, chemotactic phenomena have been observed among nearly all kinds of motile organisms and with reference to the most diverse kinds of chemical substances. EXGELMAXX ('82) has studied the chemotactic movements of diatoms ; STAHL ('84), of Myxomycetes ; PFEFFER ('84, '88), of plant spermatozoids, zoospores, Flagellata, Infusoria, and bacteria ; ADERHOLD ('88), of Euglena viridis ; VERWORN ('89, p. 107), of Cryptomonas ; STAXGE ('90), of zoospores and Myxomycetes ; and MASSART ('91), of Spirillum, Heteromita, and Ciliata. Within the last five years a voluminous literature has grown up on the medical side relating to chemotaxis in leucocytes and pathogenic bacteria. Into this literature we cannot penetrate deeply, but refer to some of the principal papers : LEBER, '88 ; BUCHXEE, '91 ; ROEMER, '92 ; METSCHNIKOFF, '92. It thus appears that chemotactic phenomena show themselves among all the groups of lower motile organisms : Rhizopoda (Myxomycetes), Flagellata, Ciliata, bacteria, diatoms, zo- ospores, and sperinatozooids. It can hardly be questioned that the phenomena shown by these organisms are of the same order as those seen in Metazoa — in those ants which LUBBOCK ('84, p. 283) has shown to move from chemical agents (essence of cloves, lavender water, and other scented stuffs), saturating a camel's-hair brush placed about ^ inch above their path ; and in the larvae of flies with which LOEB ('90, p. 79) has experi- •nted. LOEB found that these crept towards a piece of flesh brought nearer to them than the distance of 1.5 cm. Even just hatched larvae (which had therefore never been stimulated by meat) reacted in this way. Not meat only, but a trace of meat juice on glass attracted the larvae strongly. A\7hile decaying flesh ; and cheese allure, neither fat, asafoetida, nor ammonia do so. !> 34 CHEMICAL AGENTS AND PROTOPLASM [Cn. 1 Returning now to the simple organisms, let us consider the kinds of chemical substances which incite to a response. Oxygen is for almost all organisms a means of attraction. Various methods of demonstrating this have been used. Thus STANGE ('90, p. 139) filled capillary tubes with pure oxygen, under an air-pump, and brought them to the water containing zoospores, which then penetrated into them. The aggregation of zoospores and bacteria to the edges of the cover-glass, to the open end of a capillary tube (ADEKHOLD, Air bubble /# , Zone of Spirillum **' ;V Zone 0/ Anophrys . . *'« *• V^35^ A Gt,. VV.-:V:^C.T.::;V^ FIG. 2. — a. Corner of the glass slip covering a drop of liquid containing Spirillum and Anophrys, showing their aggregation with reference to the aerated bound- ing film of the drop. b. An air-bubble in the drop, showing aggregation of the organisms about it. (From MASSART, '91.) '88, p. 314), or to an enclosed air-bubble, are well-known phe- nomena. (Cf . MASSART, '91, p. 159 ; VEEWOEN, '89, p. 107 ; and see Fig. 2.) ENGELMANN ('94) has employed a still more refined method of studying attraction towards oxygen. A drop of foul water is put on a glass slide with an alga cell in the centre, and is covered by a cover-glass whose edges are hermetically sealed by vaseline. The bacteria are uniformly distributed in the water, moving in a lively manner, since they gain oxygen everywhere. If the slide thus prepared is kept in the dark, the oxygen is gradually consumed and the bacteria become quiescent, showing no distribution with reference to the cen- tral chlorophyllaceous body (Fig. 3). If the slide is now exposed to the light, oxygen is produced by the alga and a regular distribution of the bacteria in two distinct regions — in a mass around the central alga, and in a i § 3] CHEMOTAXIS 35 peripheral zone — is apparent (Fig. 4). The peripheral zone contains bacteria which are beyond the tactic action of the oxygen. The central mass of bacteria have emigrated from what is now a clear ring between the centre and the peripheral zone. If the light be temporarily cut off, the central bacteria V*^ •;."'*-' ".'^1? ;0v',.^' -:5ft *•":'.'•/ r . ".*7»7 . . » .y'«V/ ^»- • *.. * . •-• «•.*;. « »* . ..»\s , . •. ••• ^fv?:* ''V; r»*"f/:V"C 4 ?^:f 'W.Hk-li:.' .V*3 i^^l'wr ^ :^.%fv--i»- ^ •{^KV o'^^^' -^- FIGS. 3-5 a. — Bacteria surrounding an algal cell. Fig. 2 shows the uniform distribu- tion of the bacteria \vhen the drop of water is kept in the dark. Fig. 3 shows the aggregation of the bacteria towards the algal cell when this has emitted oxygen rapidly in the strong light for two minutes. Fig. 4 shows the same preparation shortly after the light has been cut off. Fig. 5 shows the same preparation when a fainter light is now permitted to fall upon the green cell. Magnified about 170 diameters. (From ENGELMANX, '94.) begin to disperse (Fig. o). If, a minute after, a dim light be let through, the radius of its activity will be relatively small, so that a central aggregation will be found and also an inner peripheral zone, comprising those dispersing central bacteria which are not affected by the small oxygen tension resulting from the dim light (Fig. 5 a). 36 CHEMICAL AGENTS AND PROTOPLASM [Cn. I Inorganic Salts. — PFEFFER* ('88, p. 601) tried various salts of potassium; viz. chloride, phosphates, nitrate, sulphate, car- bonates, chlorate, ferrocyanide, and tartrate, and found that all attracted various bacteria (B< termo, Spirillum undula), and the flagellate Bodo saltans with greater or less strength, when the solution in the capillary tube contained 0.1% K. So, likewise, various salts of sodium, rubidium, caesium, lithium, ammonium, calcium, strontium, barium, magnesium, especially the chlorides, were employed. All of these solutions at a concentration of 0.5% exhibited a marked attractive influ- ence upon Bacterium termo ; a weaker one, upon the two other species. STANGE ('90) experimented with the action of various phos- phates upon zoospores of a Saprolegnia belonging to the ferax group of DE BARY.| Sodic, ammonic, lithic phosphate, calcic phosphate held in solution by CO2, as well as phosphoric acid were employed and found to act attractively. Other salts, KNO3, K2SO4, KC1, HKCO3, BaClO3, SrCO3, MgSO4, had either a negative or indifferent action upon the zoospores. The attractive action of the phosphates is correlated with the fact that phosphates are abundant in the muscles of insects. The following table shows the effect of the different strengths of solutions of four phosphates upon zoospores of Saprolegnia. In this table, constructed from STANGE, the symbol r indicates repulsion ; 0, no action ; a, attraction ; a^ indicates a slight attraction ; #2, a strong attraction ; a2rv an attraction which is partly balanced by a repulsion due to density, so that the * The method employed by PFEFFER in his experiments was as follows : Glass capillary tubes with a lumen of from 0.03 to 0.14 mm. diameter and a length of 7 to 12 mm., and sealed by fusion at one end were employed. To fill the capillary tube, it was placed in a watch-glass containing the experiment solu- tion, and the whole was placed in a vessel from which air was pumped. Under the diminished atmospheric pressure, 2 to 4 mm. of fluid entered the capillary tube ; the rest of the tube contained air, which kept the fluid oxidized. After rinsing, the free end of the tube was plunged into the drop culture, whence the solution diffused out. t The species were cultivated upon carcasses of flies thrown into glasses filled with bog water. After good colonies were, obtained, the carcasses were washed, to rid of Infusoria. Such colonies may be employed to infect sterilized flies' legs placed in sterilized bog water, or they may be transferred directly to wounds in flies' legs. ,. CHEMOTAXIS 37 organisms pass only into the first part of the tube ; «3r3, such a balancing of the opposing forces that the organisms stand before the mouth of the capillary tube. STP.EXGTH OF SOLUTION, C" 'c SODIC DIPHOS- PHATE POTASSIC MONO- PHOSPHATE AMMONIUM PHOS- PHATE PHOSPHORIC ACID 70 °- (HXa2P04). (H2KP04). (H2NH4P04?). (HSP04). 0.8 to 0.4 .... «3r2 a3r2 asr3 r 0.4 to 0.08 . . . °2rl Ofl 0.08 to 0.04 . . . «2 a, °2 air2 0.01 to 0.02 . . . 0 ai °1 fllrl 0.02 to 0.008 . . . 0 0 «2 0.008 to 0.004 . . . ai 0.004 to 0.002 . . . 0 It will be noticed that the various substances produce dif- ferent effects in the same strength of solution ; and it is interesting to observe (a point to which further reference will be made) that the strength of solution required to produce a given response is roughly proportional to the molecular weight of the substance employed. Inorganic acids and hydrides seem, in general, to act repul- sively, but phosphoric acid is an important exception to this rule. DEWITZ ('85, pp. 222, 223) states that mammalian spermatozoa are attracted by KHO. Organic Compounds. — Alcohol, in grades between 10% and 1%, acts repulsively towards bacteria. G-lycerine is neutral to the same organisms and to zoospores of Saprolegnia. (STANGE, '90.) The sugars, etc., dextrose, milk sugar, dextrin, act attractively upon Bacterium termo in 10% or weaker solutions. Many or- ganic acids are among the most attractive reagents. It was with malic acid that PFEFFEK ('84) tried his earlier fundamental experiments upon the spermatozoids of ferns. The attraction exerted is very great, so that a capillary tube of 0.1 to 0.14 mm. calibre, containing a 0.05% solution of malic acid, attracts from a drop of water full of spermatozoids at the rate of 100 individuals in one hour. Even a 0.001% solution acts chemo- tactically. Now, malic acid is of very wide distribution among plants, and it occurs in the fern prothalli upon which the sexual 38 CHEMICAL AGENTS AND PROTOPLASM [Cn. I organs arise, so that it seems probable that it occurs in the mouth of the archigonium, and that by its presence sperma- tozoids are attracted towards the egg cell. STANGE ('90, p. 155) has experimented much more fully with the action of organic acids upon zoospores of Saprolegnia and upon myxamcebse. To the former, acetic acid (0.01%) and tartaric acid (0.0125%) act attractively. Upon the latter, still other acids were tried; butyric, lactic, and valeric acids cause response in concentrations between 0.2% and 4% ; malic acid, between 0.5% and 4%. Other attracting acids are : propionic, citric, tartaric, and tannic. Acetic acid repels the amoebae of JEthalium, its repellent action being about equal to the attrac- tive action of an equal amount of butyric acid. Nitrogenous Compounds. — Urea, asparagin, kreatin, taurin, hypozanthin, carnin, and peptone have been found by PFEFFER ('88) to exert an attractive influence, especially in the case of the reagents italicized. Benzol Derivatives. — PFEFFER found that sodium salicylicate and (commercial) sulphate of morphine are clearly attractive to Bacterium termo in 1% solutions. From the foregoing list of organic compounds whose effect upon Protista has been tested by PFEFFER and STANGE, it appears that except alcohol and sometimes acetic acid, none acts repulsively, and that glycerine alone is neutral to all proto- plasm. It is further true that we do not find here any strict relation between the chemotactic action of a substance and its advantage to the organism. Substances which have a nutri- tive value for the organism, such as glycerine has for bacteria, may be wholly neutral, while solutions which act fatally, like 1% sodic salicylicate and 1% morphine, attract. In the same way, many of the organic salts which act attractively cannot be considered as of importance to the organism. On the other hand, as already pointed out, the attraction of most Protista to oxygen, of Saprolegnia zoospores to phosphates, as well as the cases of attraction of bacteria (PFEFFER, '88, p. 605) and of fly larvae (LoEB, '90, p. 79) to meat extract, and of Myxomycetes to bark extract (STAHL, '84 ; STANGE, '90), is advantageous. Chemotaxis is, therefore, in some cases, a response to the stimulus afforded by substances which can be employed by „ CHEMOTAXIS 39 the organism as food ; under which circumstances it can be called " Trophotaxis." * In other cases it is a response to chemical substances which have no significance as food, and have no other importance for the organism. It is clear that we cannot assume that response to injurious substances is an adaptation which has been brought about by natural selection. If a response occurs in one case independent of the action of selection, we should hesitate to ascribe to this cause the origin of other, even favorable, responses. General Remarks on the Relation between Molecular Composi- tion and Response. — PFEFFEB, ('88, pp. 608-612) has pointed out that the capacity of any substance to stimulate cannot be inferred from its chemical constitution and relationships. Thus, the minimum congentration of milk sugar which will produce a response is 1%, while in the case of the closely allied grape sugar it is 10 % ; but in the very different kreatin it is also 1 % • Also, the action of any chemical compound is determined not by the elements, such as C, H, O, which it contains, but by the entire molecule ; in other words, the atomic composition is less important than the structure of the molecule or the arrange- ment of its atoms. Thus, malic acid and its compounds with neutral ammonium, sodium, barium, and calcium containing 0.001% parts of the acid, have an equal action upon the sperma- tozoids of ferns, which do not react to the diethylester of malic acid, even in strong solutions. (PFEFFER, '88, p. 655.) Again, nitrogenous organic compounds are, in general, more active than the non-nitrogenous ones ; but this cannot be held to be due alone to the presence of N ; for dextrin (C6H10O5) is nearly as active as the nitrogenous peptone, and, on the other hand, the nitrates of metals are not more active than their chlorides, while the ammonia salts are relatively weak. Relation between the Strength of the Stimulus and that of the Response. — When we say that malic acid attracts sperma- tozoids we mean that under certain physical conditions of the water which we may call normal it does so. And under normal conditions of the water, it is only within certain limits * STAHL ('84, p. 164) called the attraction of plasmodium of myxomycetes to bark extract " Trophotropism." 40 CHEMICAL AGENTS AND PROTOPLASM [Cn. I that malic acid attracts. The strengths of solutions which attract under such conditions lie between 0.001% and 10%. The weaker solution may be designated the minimum ; the stronger, the maximum concentration which provokes a re- sponse. The minimum solution provoking response is also often called by the Germans the " Reizschwelle," or "stimula- tion threshold " ; * the optimum, the " Reizhohe," or " stimu- lation acme"; the range, the "Reizumfang." The character of the responses observable at the two limits is very different ; at the minimum, attraction is very feeble ; thus, while a capillary tube containing 0.01% neutral sodic malate, plunged into water at 14°-20° C., swarming with spermatozoids, attracts 400 of them in 10 minutes, a 0.001% solution attracts only 10-25 individuals during the same time, and a 0.0008% exerts little attractive effect, the spermatozoids remaining undirected in their movements. At the maximum, on the contrary, repulsion is observed. The spermatozoids move from the mouth of the capillary tube. Between the two extremes lies the concentration of greatest attraction — the acme. As we pass from the acme towards the minimum, the attraction becomes less and less. As we pass towards the maximum, the attraction remains the same, or increases ; but repelling influences are now at work, which eventually entirely counteract the attractive influences. A satisfactory method of expressing quantitatively the facts just mentioned has not been invented. PFEFFER ('88, p. 599) has employed the nomenclature which we have used above (p. 36) — a± to as being combined with r1 to rs to indicate the coworking in varying proportions of attraction and repul- sion. Using this nomenclature, we may illustrate the state- ments made in the last paragraph with examples taken from PFEFFER'S work: — * The following substances at the solutions named produce the threshold attraction («i) in Bodo saltans: KC1, 0.02%; K3PO4, 0.002%; KH2PO4, 0.0035%; KN03, 0.26%; K2S04, 0.22%; KC103, 0.3%; K4(CN)6Fe, 0.235%; K2.C4H406, 0.02%; BbCl, 0.14%; LiCl, 0.6%; LiN03, 3%; NH4C1, 0.3%; neutral ammonium phosphate, 0.08%; SrCl2, 0.2%; Sr(N03)2, 0.4%; BaCl2, 0.17%; dextrin, 0.1%; urea, 1%; asparagin, 0.1%; taurin, 1%; sarkin, 0.33%; pepton, 0.01%; meat extract, 0.01%. CHEMOTAXIS 41 GRADE OF SOLUTION. EESPOXSE OF BACTERIUM TERMO. SPIRILLUM. 9.53 % KC1 = 5%K a3 a3r 1.906 %KC1 = 1%K «3 «3ri 0.191 % KC1 = 0.1% K °3 an 0.019 % KC1 = 0.01%K «2 ai 0.0019 % KC1 = 0.001 % K «1 0 BODO SALTASS. 3.48 % KH2PO4 = 1.0%K a3r3 0.348 %KH2PO4 - 0.1 %K <*3r2 • 0.035 %KH.,PO4 = 0.01%K a2 0.0035 %KH2PO4 = 0.001 % K ai 0.00067 % KH2PO4 = 0.0002 %K 0 Compare also the table on p. 37. For all reagents which exert an attractive influence there exists the maximum (repelling) and minimum (indifferent) limits referred to. In the case of reagents, which, like alco- hol, repel bacteria at between 1% and 10%, there is doubtless an indifferent limit, but it is not necessary that there should be a degree of concentration at which attraction takes place. In the one case, then, the phenomena of indifference, attraction, repulsion, follow each other with increasing concentration ; in the other case, only indifference and repulsion. The difference in action of the two cases is due, in part at least, to the fact that all solutions, independently of their chemical constitution, become repellent when they become concentrated enough. The repulsion, then, of high grades of chemical solutions is purely an osmotic phenomenon, and, as such, will come under discussion in the third chapter. It follows, also, from what has been said, that, in the case of those reagents which exert no attraction at any concentration, the acme and maximum coin- cide and lie at the saturation point of the solution. Finally, we may discuss the third case in which the reagent acts indifferently, as glycerine does upon bacteria between 17% and 0.86%. It is clear, that if the density of the solution can 42 CHEMICAL AGENTS AND PROTOPLASM [Cn. 1 be rendered great enough, a repulsion due to osmosis must occur. If the substance is, however, only soluble slightly or miscible, it may be that repulsion will never occur. Whether or not attraction will occur before the repulsion point is reached will have to be determined experimentally for each reagent. Thus the action of an untried substance upon any organism may be any one of three kinds : (1) It may be indifferent at all grades; (2) it may be indifferent at lower and repellent at higher grades ; (3) it may be indifferent, attractive, and repellent at successive grades. Of two substances belonging to the second or third class, one may act upon an organism at a certain concentration with indifference, the other at the same concentration with repulsion. Likewise the same solution of a substance may attract one kind of protoplasm and repel another, under otherwise similar conditions. We have seen above that the same reagent acts upon the same kind of protoplasm similarly only when the other con- ditions of the experiment are also the same. Among the varying conditions which have been especially investigated is that of the chemical constitution of the medium. The experi- ment has generally been made as follows : A particular species, let us say Bacterium termo, is to be subjected to the action of a particular reagent, e.g. meat extract. The bacteria are reared in cultures containing a varying quantity of the meat extract, and the concentration of the capillary fluid producing the threshold stimulation is measured in each case. We may compare not only the threshold stimulations but also the concentrations necessary to produce the response indicated by av «2, etc. The following table, from PFEFFER ('88, p. 634), gives some of such determinations: — CULTURE FLUID — MEAT EXTRACT. CAPILLARY FLUID — MEAT EXTRACT. 3 x cult. cone. 5 x cult. cone. 8 x cult. cone. 10 x cult. cone. 0.01 % 0.1% 1% 0.03 o/o (?) 0.8% (?) 3%(?) 0.05 %(at) 0.5%(0l) 5%(«i) 0.08% (a,) 0-8% (a,) 8%(«2) 0.1% (aj !%(«*) io%(«2) ,. ] CHEMOTAXIS 43 From this table it appears that the strength of solution necessary to provoke a certain response depends upon the strength of solution to which the protoplasm has been pre- viously subjected and increases proportionately with it. It is clear that the capillary solution of 0.1%, which produces a marked chemotaxis in bacteria reared in a culture solution of 0.01%, would awaken no response in bacteria reared in 0.1%. We are now in a position to appreciate the importance of still another addition to our terminology of stimuli — the dif- ferential threshold stimulation (Reizunterscheidsschwelle) — which may be denned as the minimum increase of a preexisting stimulus which is capable of calling forth a just noticeable reaction. In the case just cited, the differential threshold stimulation lies just above 3 times the preexisting (culture) stimulus, and this is true whatever the degree of the preexist- ing stimulus ; and it is shown by experiment that, in general, as the preexisting stimulus increases, the differential threshold stimulation increases in the same proportion. This observa- tion is in perfect accord with the law formulated long ago bj WEBER with especial reference to sight. This law runs : The smallest change in the magnitude of a stimulus which will call forth a response (differential threshold stimulation) always bears the same proportion to the whole stimulus. We may express this law mathematically, as FECHNER has done, by the following considerations : Let us take the case of a protoplas- mic body, as, for example, that of a spermatozoid, living in a stimulating medium (s) of a certain concentration and experi- encing a certain reaction rr ; then s corresponds to r'. In order just to get a chemotactic response (threshold stimulation), a solution of say 30 times the concentration must be brought to the solution affording the stimulation s. This will give a reaction which is greater than r' by a quantity which we may designate r, so that the quantity of the whole reaction may be designated as r' + r. If the organisms are now placed in this stronger solution (31 s), the solution in the capillary tube must be 30 times stronger (30 x 31 s) in order to give the differential threshold stimulation. The reaction following this stimulation may be designated, according to FECHNER 's conception, as r' + r + r. The relation of the successive stimuli and the reac- 44 CHEMICAL AGENTS AND PROTOPLASM [Cn. I tions may be shown by the following table, following one given by PFEFFER ('84, p. 401) : — s corresponds to r'. s-f- 30s = 31s corresponds to r'+r. 31s-f 30x31s= 31 x 31 s corresponds to r'+r +r. 31 x 31 s+30 x 31 x 31 s = 31 x 31 x 31 s corresponds to r'+r+r+r. That is to say, while the stimulation increases geometrically, the reaction increases arithmetically. In the preceding table the second term of the left-hand member of the equation is always the differential threshold stimulation. The most important objection that can be urged against this formula of FECHNEB is that there is not sufficient reason for believing that the various reactions (r) to the differential threshold stimulations of various strengths are equal, nor that the stronger reaction to the strong stimulus is composed of many weak reactions. If these assumptions were true, it would follow that when the successively higher stimuli increase as a series of numbers the reactions increase as the logarithms of these numbers. If now we adopt as a unit in this phenomenon the quantity of the threshold stimulation (estimated in units of concentration of solution, of mass, light intensity, heat inten- sity, etc.), which we may call s, the strength of any stimulus ($) may be estimated in those units, and the strength of the corresponding reaction (.72) will be indicated by the equation R = c • log S, in which c is a constant to be determined empiri- cally, and S the strength of the stimulus expressed in units of the threshold stimulation.* While we are not yet in a position to understand the signifi- cance of WEBER'S law, we cannot fail to be struck with the resemblance of the phenomena with which it concerns itself to those of acclimatization referred to in the second section of this chapter. We there showed that organisms subjected for a while to a chemical agent no longer reacted as at first to that reagent. We have here shown that organisms subjected for * Since the German word for stimulus is Heiz (initial .R), and since the re- action is usually indicated by the initial letter in Empfindung, in German text- books this formula usually runs E = c • log 7?, which differs from the above equation only in the symbols employed. SUMMARY OF THE CHAPTER 45 while to the action of a certain stimulating agent respond no longer to a concentration which would at first have .provoked a reaction. In both cases it is the action of the chemical agent which modifies the subsequent action of the protoplasm, without doubt by changing the chemical constitution of the protoplasm. Mechanics of Response. — Having considered the general relation between strength of stimulus and of reaction, it now becomes necessary to examine more in detail into the way in which the reaction takes place. A variety of kinds of locomotion exists among chemotactic organisms — that of the Myxomycetes is amoeboid, that of Infusoria is by flagella or cilia. In all cases the first and perhaps the only effect of the acting reagent is to determine the position of the axis of the body, in the case of bodies with fixed form ; or to determine the pole of outflow in the case of amoeboid organisms. The axis lies in the line of flow of the diffusing solution or perpendicular to the isotonic lines, or lines of equal concentration. Whatever movement now occurs must be either towards or from the source of stimulus. I have said above that the axis orientation is perhaps the only effect of the acting reagent. PFEFFER ('84, p. 463 ; '88, p. 631), indeed, maintains that the stimulus does not directly cause a markedly more rapid locomotion in the case of bacteria and Flagellata ; but in the case of plasmodia it seems possible that such a hastening of movements occurs. (SxAHL, '84.) However, it is necessary that measurements should be made in this matter. Further observations on the mechanics of taxis must be deferred to the general treatment of the subject in Chapter IX. SUMMARY OF THE CHAPTER We attempted in the first section to bring together observa- tions relating to the action of various chemical substances upon protoplasm with the aims of discovering the general laws of poison-action on protoplasm and of gaining an insight into the chemical structure of protoplasm and the chemical operations involved in the elementary vital processes. We ought now, therefore, to attempt to draw such conclusions as the imperfect and often confusing data we have collected will permit. 46 CHEMICAL AGENTS AND PROTOPLASM [Cn. I All the reagents with which we have dealt have been sub- stances capable of absorption by, mixture with, or solution in, water; and the reason for this is that almost all proto- plasm is itself enveloped by water and largely composed of water. For the most part we have dealt with mixtures or solutions. Now the action of these is a double one. They exhibit, first, an osmotic action, and, secondly, they may attack the molecules of the protoplasm, transforming them. The osmotic action we will consider in the third chapter ; the transforming one alone concerns us now. It is not easy, without experiment, to say to which of these two categories of action the change wrought by any substance is due. To determine between the two possible causes it would be desirable in each case to treat the protoplasm to a control solution having the same osmotic action as the first, but no transforming effect. If such a solution produces no modification of the protoplasm, then the effect wrought by the first reagent is due purely to molecular trans- formations. It is not easy to find a reagent of which we may be certain that it acts only osmotically. NaCl is probably more generally useful in this way than any other substance. In the experiments which have hitherto been made, this double action of solutions has not been sufficiently considered. Hence, a doubt concerning the immediate cause hangs about many of the phenomena described in the first section. The first principle which the data collected establish is that the protoplasm of different organisms is dissimilar. This is shown by the diversity in their chemical reactions ; by the fact that whereas, in one case, a 'certain percent solution causes so extensive a molecular transformation as to result in death, in another, no injurious effect is produced. Thus, according to BOER ('90, p. 479), it takes of gold chloride to kill — TABLE V Anthrax bacillus 0.0125% Cholera spirillum 0.1% Diphtheria bacillus 0.1% Typhoid bacillus 0.2% Glanders bacillus 0.25% SUMMARY OF THE CHAPTER 47 Thus the weak solution, 0.0125%, of AuCl3, which is fatal to anthrax, does not injure the glanders bacillus, which requires a solution 20 times as strong ; and we conclude that the chemi- cal constitution of the glanders bacillus must be different from that of anthrax. The dissimilarity of the different protoplasms may be either a qualitative or a quantitative one. That is to say, the kinds of molecules, or the proportions of the different molecules, may differ in the two cases. If we assume that gold chloride acts upon protoplasm by the Au replacing some of the H in an amido-acid, then the diversity in action of AuCl3 upon anthrax and typhoid may be accounted for by assuming that the amido- acids are dissimilar in anthrax and typhoid or that the propor- tion of the kinds especially affected is different in the two cases. To which of these two causes the diverse reactions to AuCl3 are due cannot yet, in any given case, be determined. A second principle which we may draw from our data is this : Some kinds of protoplasm have a general high resistance to all chemical agents, while other kinds have a high or low resistance to particular agents only (specific high or low resistance). Thus, in the case of pathogenic bacteria, the experiments of BOER ('90) show that, in general, the anthrax bacillus has a low resistance, and glanders a high one. His experiments were made with 10 reagents upon five kinds of bacteria. Table VII gives in modified form the results obtained by BOER. His results are given in the form of Table V; the present table is constructed from the original by making the mean of the five observations in each column unity and reducing the sepa- rate observations proportionately. Thus Table V becomes — TABLE VI Anthrax bacillus 0.09 Cholera spirillum 0.75 Diphtheria bacillus 0.75 Typhoid bacillus 1.51 Glanders bacillus 1.88 All the other determinations have been treated in like manner. Throughout the table the numbers in each column stand for 48 CHEMICAL AGENTS AND PROTOPLASM [CH. I relative resistance capacity. The reagents are placed with the weakest-acting first. TABLE VII CAUSTIC CARBOLIC MURIATIC SULPHURIC METHYL SUBSTANCES. SODA ACID ACID ACID VIOLET. (NaHO). (C6H60). (HC1). (H2S04). MOLECULAR WEIGHTS. 40 94 37 98 Anthrax bacillus .... 0.46 0.95 0.40 0.37 0.07 Cholera spirillum . . . 1.38 0.71 0.32 0.37 0.33 Diphtheria bacillus . . 0.69 0.95 0.63 . 0.94 0.17 Typhoid bacillus .... 1.09 1.43 1.46 0.94 2.22 Glanders bacillus .... 1.38 0.95 2.19 2.36 2.22 SILVER GOLD MALACHITE OXTCYANIDE SUBSTANCES. NITRATE CHLORIDE GREEN. OP Hg. AVERAGE. (AgNOg). (AuCl,). MOLECULAR "WEIGHTS. 170 304 Anthrax bacillus .... 0.20 0.09 0.02 0.93 0.388 Cholera spirillum . . . 1.04 0.75 0.17 0.62 0.632 Diphtheria bacillus . . 1.67 0.75 0.11 0.93 0.760 Typhoid bacillus .... 1.04 1.51 1.75 1.25 1.415 Glanders bacillus .... 1.04 1.88 2.92 1.25 1.802 From this table we see that the bacillus of glanders is more resistant than that of anthrax (except in one instance, in which the resistance is equal in the two .cases) whatsoever be the poison employed. The bacillus of glanders affords, thus, a good illustration of an organism with a general high resistance capacity. The diversity in general resistance capacity which is found among bacteria exists also among other organisms. Thus, the parasitic Ascaris has shown itself highly resistant in all cases in which the action of a poison on it has been compared with that on another species; for instance (p. 10) 0.1% chloral hydrate kills Infusoria, Rotifera, and diatoms in 24 hours, but •Ascaris withstands this solution. Again, while 0.1% HCN kills Infusoria quickly, Ascaris resists 3% for 75 minutes. The general higher resistance may be due to one of three causes : - SUMMARY OF THE CHAPTER 49 ther to the fact that the protoplasm is protected from attack, as is the case with the encysted forms of Protozoa, which are very resistant ; or to the fact that the protoplasm is not so readily acted upon by reagents brought actually in contact with it, clue to diminished amount of water or other structural modifications ; or, finally, that the protoplasm has a different composition, certain unstable molecules found in other kinds of protoplasm being absent. We will now consider the phenomena of diversity in specific resistance of protoplasm. A case of specific low resistance is found in the nervous tissue. Thus many of the alkaloids, e.g. nicotine and cocaine, are almost indifferent to the protoplasm of Protista, but act towards the nervous system as powerful poisons. Hence we are led to conclude that nervous protoplasm contains especially unstable compounds, upon which its action depends. When they are subjected to the action of very weak — towards most substances, indifferent — reagents, extensive and fatal transformations occur. Cases of specific high resistance are apparently found in some glands which secrete intense poisons, or in some organisms which live in solutions of some usually poisonous agent. Ex- amples of this class are the HCl-secreting glands of the Vertebrate alimentary tract, the poison-glands of venomous serpents, and the H2SO4-secreting glands of Gasteropoda; also the vinegar eel, which lives in 4% acetic acid. It ought to be said that it is largely an inference based upon experi- ments on acclimatization, that these glands or organisms will not show a general high resistance. Experiments are needed to determine this point. As to the cause of specific high resist- ance, I believe that much light is gained from the facts of acclimatization, and that any sufficient theory of the latter would serve also to explain the former (see p. 30). Under the general poisons we have distinguished four main groups : a. oxidizing poisons ; b. salt-forming poisons ; c. sub- stitution poisons ; d. catalytic poisons. I will comment briefly upon the action of the poisons of each of these groups. a. Oxidizing Poisons. — The ordinary oxidation processes in [living protoplasm involve the consumption not of the proto- 50 CHEMICAL AGENTS AND PROTOPLASM [Cn. I plasm itself, but of the thermogenic substances stored therein (sugar, yolk). After these have been consumed in starvation, or when the organism is subjected to the action of oxidizing poisons, the molecules of the protoplasm become oxidized. All protoplasm which is readily accessible must be injured by the direct attacks of " active " oxygen. b. Salt-forming Poisons. — The facility with which an acid or a base forms salts with the protein substances of the proto- plasm must depend, in large part, upon the quality of the protein molecules. It is well known that certain protein sub- stances, such as keratin, chitin, and fibrin, are not readily acted on by acids or bases, and it seems necessary to suppose that some such resistant proteids are the essential parts of glands which secrete these reagents. Into this group fall the salts of heavy metals characterized by their extraordinary fatalness. c. Substitution Poisons. — This group comprises, besides a few sulphur compounds, almost exclusively nitrogenous sub- stances, and among these a large proportion of compounds with closed chains. As many of these are indifferent to dead albumen, but violent poisons to living protoplasm, it is clear that the latter must contain certain extremely unstable groups (amido-, aldehyde-, and keton-groups, LOEW, '80). Among these poisons the relation between molecular structure and poisonous action is very marked, especially in the nitro-com- pounds. Thus, bodies containing H united with N are poison- ous in direct proportion to the number of H atoms so combined. It seems probable that H so combined is very easily given up to the molecules of the living substance, destroying them. H in the hydroxyl radical seems also more easily parted with than H joined to C. d. Catalytic Poisons. — Chiefly organic compounds of the fat series, which have little chemical energy and produce, for the most part, anaesthesia. The poisonous action seems here proportional to the complexity and instability of the compound. Thus, in many groups, when the alkyls CH3 — , C2H5 — , etc., are successively introduced, the substance grows more poison- ous as the number of atoms in the alkyl increases. In the methan series and among sulphureted compounds the substi- tution of Cl for H increases the poisonous action. TJ SUMMARY OF THE CHAPTER (The study of the action of poisons upon protoplasm gives us insight into the extreme complexity of the living substance its composition out of numerous kinds of compounds, many which are extremely unstable. Not all protoplasm contains the same compounds, hence it must be a very dissimilar thing in different organisms. Not all of the compounds in any pro- toplasmic body are essential to life, for we may act upon a protoplasmic body by a weak reagent, and gradually change its composition so that it will no longer be killed by the strong solution, and all of this without perceptible injury — at least, this is the conclusion to which the study of acclimatization of Protista leads us. The altered chemical constitution will be transmitted in the division of the individual, and thus the composition of the protoplasm of a race will have been deter- mined by the medium in which it and its ancestors have been living. Finally, we may consider what light the action of reagents throws upon the processes involved in the elementary vital functions. The normal movement of protoplasm is profoundly modified by interfering with the oxygen supply. Thus, when the oxygen pressure is diminished, movements are retarded ; in the presence of pure oxygen they are accelerated. Some anaes- thetic or paralyzing agents — e.g. chloroform and some alka- loids, veratrin, atropin, cocaine, strychnin, and antipyrin — give rise first to acceleration, then to disappearance of movements in the protoplasm. Protoplasmic movement is, consequently, closely associated with oxidation, and it does not occur in the absence of irritability. Normal locomotion is interfered with by strychnin and co- caine. Their stimulating action produces accelerated move- ments, and these are accompanied by loss of coordination. Since many catalytic poisons (anaesthetics) destroy irrita- bility, one may conclude from the action of these chemical agents that (p. 7) stability of molecular movement is essential to the performance of this function. Disturbance of the excretory function results from the action of CO, NH3, chloroform, cocaine, strychnin ; at least, an ex- cessive vacuolation of the protoplasmic body occurs under the action of these agents. 52 CHEMICAL AGENTS AND PROTOPLASM [Cn. I Experiments on chemotaxis show that many substances brought near to protoplasmic bodies control their locomotion. The effect upon locomotion depends both upon the kind of protoplasm and the strength of the reagent. In many cases, a certain strength of reagent attracts an organism, while a stronger solution repels, and a weaker solution is indifferent. In such a case we may speak of the protoplasm as being attuned to the attracting strength of the reagent. We find great diver- sity in the strength of solution of a reagent to which different protoplasms are attuned. This difference of attunement to chemotactic reagents is parallel to the difference in strength of the killing solution of various protoplasms. As the latter is probably due to the past action of chemical agents upon the protoplasm, so is also the former. APPENDIX TO CHAPTER I Cytotaxis (= Cytotropisvi) Roux ('94) has given the latter name to a phenomenon which is probably only a special case of chemotaxis, but which may be better considered apart. He isolated, in an indifferent medium, two or three cells from the egg of a frog (Raiia fusca) at the morula or blastula stage of development. These he placed near each other upon a glass slide, and found that they moved slowly, and that the direction of the movement of any one cell was, under certain conditions, determined by the position of the other cell or cells. In order to perform the experiment a proper medium in which to study the movement of the cells must be prepared as follows: a small quantity — 5 to 10 ccm. — of fresh egg albumen (not cut up, but with the albumen threads intact) is filtered through clean wadding, and the completely clear filtrate is used. In other cases a more or less strong salt solution is employed. The cleavage cells are isolated in the filtrated albumen, on a glass plate, by means of needles. To diminish evaporation the glass plate is put into a shallow glass vessel containing several drops of water. When two cells were placed near each other (about one-fourth of their diameter apart), the distance between them diminished. The approach took place along a line joining the two cells ; APPENDIX,— CYTOTAXIS 53 and when several pairs of cells were in the field, this movement took place in various directions, indicating that their move- ment was not determined by conditions outside the approaching cells. To get further light on the migration of the cells, their distance apart was meas- ured at short intervals of time. The results of two series of such meas- urements are represented graphically in Figs. 6 and 7. In both of these dia- grams the heavy lines indicate the successive positions assumed by four points ; namely, the points of the two cells which are nearest each other and those which are most distant. In the first case the cells traverse the distance of their diameters (58 //,) in about 10.5 minutes. The rate of migration is, however, extremely variable. In some cases the cells seem even to move apart (negative cytotaxis ?) . Certain special cases are worthy of considera- tion. When a third cell lies near an approaching pair, the path of migra- tion of the pair may become convex towards the third cell. Two cell-complexes, each composed of three or four cells, may approach and connect. But masses composed of a larger number of cells form " closed complexes " which show no cytotactic activity. The isolated cells of differ- FIGS. 6, 7. — Two sets of curves, showing the course of "cytotactic" movements of the cleavage cells of the frog. In each figure the dotted line represents a diameter of the cell. The full line represents the successive positions of the extremities of the diameters as the cells approach. The distances between horizontal lines = 4/n ; between vertical lines, 75 seconds. (From Roux, '94.) 54 CHEMICAL AGENTS AND PROTOPLASM [Cn. I ent eggs of the same species behave like cells from the same egg* By these important experiments it is established that, inside of the body, parts may act upon parts, determining the direc- tion of motion. The importance of this fact will be discussed in a later Part of this book. LITERATURE ADDUCO, V. '90. Sur 1'existence et sur la nature du centre respiratoire bulbaire. Arch. Ital. de Biol. XIII, 89-123. 21 March, 1890. ADERHOLD, R. '88. Beitrag zur Kenntnis richtender Krafte bei der Bewe- gung niederer Organismen. Jena. Zeitschr. XXII, 310-342. ALBERTONI, P. '91. Wirkung des Cocains auf die Contractilitat des Proto- plasma. Arch. f. d. ges. Physiol. XL VIII, 307-319. 28 Jan. 1891. BERNARD, C. '78. Le9ons sur les phenomenes de la vie communs aux ani- maux et aux vegetaux. Tome I, 404 pp. Paris. BOER, O. '90. Ueber die Leistungsfahigkeit mehrerer chemischer Desin- fectionsmittel bei einiger fiir den Menschen pathogenen Bacterien. Zeitschr. f. Hygiene. IX, 479-491. BOSCH, C. TEN '80. De physiologische werking van chinamine. Onder- zoek. Physiol. Lab. Utrecht. V, 248-292. BINZ, C. '67. Ueber die Einwirkung des Chinin auf Protoplasma-Bewe- gungen. Arch. f. mik. Anat. Ill, 383-389. BINZ, C. and SCHULZ, H. '79. Die Arsengiftwirkungen vom chemischen Standpunkt betrachtet. Arch. f. exper. Path. u. Pharm. XI, 200- 230. BOKORNY, T. '86. Das Wasserstoffsuperoxyd und die Silberabscheidung durch actives Albumin. Jahrb. f. wiss. Bot. XVII, 347-358. '88. Ueber die Einwirkung basischer Stoffe auf das lebende Protoplasma. Jahrb. f. wiss. Bot. XIX, 206-220. '93. Ueber die physiologische Wirkung der tellurigen Saure. [Abstr. in] Bot. Centralbl. LVII, 16. BOURNE, A. G. '87. The 'Reputed Suicide of Scorpions. Proc. Roy. Soc. London. XLH, 17-22. BUCHNER, H. '91. Die chemische Reizbarkeit der Leukocyten und deren Beziehung zur Entziindung und Eiterung. Sb. Ges. Morph. u. Physiol. Miinchen. VI, 148-152. '92. Die keimtodtende, die globulicide und die antitoxische Wirkung des Blutserums. Miinchener Med. Wochenschr. XXXIX, 119-12:'.. CALMETTE, A. '94. L'immunisation artificielle des animaux contre le venom . des serpentes et la therapeutique experimentale des morsures veni- meuses. C. R. Soc. de Biol. (10) I, 120-124. LITERATURE 55 CHARPENTIER, A. '85. Action de la cocaine et d'autres alcalo'ides sur cer- tains infusoires a chlorophylle. C. R. Soc. de Biol. XXXVII, 183, 184. CLARK, J. '89. Protoplasmic Movements and their Relation to Oxygen Pressure. Proc. Roy. Soc. Lond. XL VI, 370, 371. June 20, 1889. COHN, F. '94. Formaldehyd und seine Wirkung auf Bacterien. Bot. Centralbl. LVII, 3-6. DANILEWSKI, B. '92. Ueber die physiologische Wirkung des Cocains auf wirbellose Thiere. Arch. f. d. ges. Physiol. LI, 446-454. DAREMBERG, G. '91. Sur le pouvoir destructeur du serum sanguin pour les globules rouges. C. R. Soc. Biol. XLIII, 719-721. DARWIN, C. '75. Insectivorous Plants. 462 pp. New York : Appleton & Co. DAVENPORT, C. B. and NEAL, H. V., '96. On the Acclimatization of Organisms to Poisonous Chemical Substances. Arch, f . Entwick. d. Organismen. II, 564-583. 28 Jan. 1896. DEMOOR, J. '94. Contribution a 1'etude de la physiologic de la cellule (in- dependance functionnelle du protoplasma et du noyau). Arch, de Biol. Xin, 163-244, Pis. IX, X. 28 Feb. 1894. DEWITZ, J. '85. Ueber die Vereinigung der Spermatozoen mit dem Ei. Arch. f. d. ges. Physiol. XXXVII, 219-223. 29 Oct. 1885. EHRLICH, P. '91. Experimentelle Untersuchungen iiber Immunitat. I Ueber Ricin. II Ueber Abrin. Deutsche med. Wochenschr. 976-979 ; 1218, 1219. ELFVING, F. '86. Ueber die Einwirkung von Ather und Chloroform auf die Pflanzen. Ofversigt af Finska Vetensk. Soc. Forh. XXVIII, 36-53. ENGELMANN. T. W. '81. Neue Methode zur Untersuchung der Sauer- stoffauscheidung pflanzlicher und thierischer Organismen. Arch. f. d. ges. Physiol. XXV, 285-292. 20 June, 1881. '82. Ueber Licht- und Farbenperceptiou niederster Organismen. Arch. f. d. ges. Physiol. XXIX, 387-400. 3 Nov. 1882. '94. L'emission d'oxygene sous I'mfluence de la lumiere, par les cellules a chromophylle, demontree au moyen de la methode bacterienne. Arch. Neerland. XXVHI, 358-371. FAYRER, J. '74. The Thanatophidia. 2d ed., 178 pp., 31 pis. London : Churchill. FROMANN, C. '84. Untersuchungen iiber Struktur, Lebenserscheinungen und Reaktionen thierischer und pflanzlicher Zellen. Jena. Zeitschr. XVII, 1-349. Taf. I-TII. 19 Jan. 1884. GREENWOOD, M. '90. On the Action of Xicotin upon Certain Invertebrates. Jour, of Physiol. XI, 573-605. Dec. 1890. HEIDENSCHILD, W. '86. Untersuchungen iiber die Wirkung des Giftes der Brillen- und der Klapperschlange. Jahresber. d. Thier-Chem. XVII, 330. [From Inaug. Diss. Dorpat. Lookmann, 1886.] HERTWIG, O. and R. '87. Ueber den Befruchtungs- und Teilungsvorgang des tierischen Eies unter dem Einfluss ausserer Agentien. Jena. Zeitschr. XX, 120-241. 8 Jan. 1887. 56 CHEMICAL AGENTS AND PROTOPLASM [Cn. I HOFER, B. '90. Ueber die lahmende Wirkung des Hydroxylamins auf die contractilen Elements. Zeitschr. f. wiss. Mikr. VII, 318-326. 18 Dec. 1890. KANTHACK, A. A. '92. The Nature of Cobra Poison. Jour, of Physiol. XIII, 272-299. May, 1892. KRUKENBERG, C. F. W. '80. Vergleichend-physiologische Studien. 1 Reihe, 1 Abth., 77-155. KUHNE, W. '64. Untersuchungen liber das Protoplasma und die Contrac- tilitat. 158 pp., 8 Taf . Leipzig : Engelmann. LEBER, T. '88. Ueber die Entstehung der Entziindung und die Wirkung der entziindungserregenden Schadlichkeiten. Fortschritte d. Medicin. VI, 460-464. LOCKE, F. S. '95. On a Supposed Action of Distilled Water as such on Cer- tain Animal Organisms. Jour, of Physiol. XVIII, 319-331. 5 Sept. 1895. LOEB, J. '90. Der Heliotropismus der Thiere und seine Uebereinstimmung mit dem Heliotropismus der Pflanzen. 118 pp. Wiirzburg : Hertz. LOEW, O. '77. Lieutenant Wheeler's Expedition durch das siidliche Cali- fornien im Jahre 1875. Petermann's Geogr. Mitth. XXIII, 134-140. '83. Sind Arsenverbindungen Gift f iir pflanzliches Protoplasma ? Arch. f. d. ges. Physiol. XXXII, 111-113. 12 Sept. 1883. '85. Ueber den verschiedenen Resistenzgrad im Protoplasma. Arch. f. d. ges. Physiol. XXXV, 509-516. 30 Jan. 1885. 85a. Ueber die Giftwirkung des Hydroxylamins verglichen mit der von anderen Substanzen. Arch. f. d. ges. Physiol. XXXV, 516-527. 30 Jan. 1885. '87. Ueber Giftwirkung. Arch. f. d. ges. Physiol. XL, 437-447. 18 May, 1887. '88. Physiologische Notizen iiber Formaldehyd. Sb. Ges. f. Morpol. u. Physiol. Miinchen. IV, 39-41. '91. Die chemischen Verhaltnisse des Bakterienlebens. Centralbl. f. Bakteriol. u. Parasitenk. IX, 659-663 ; 690-697 ; 722-726 ; 757-760 ; 789-790. May-June, 1891. '92. Ueber die Giftwirkung des Fluornatriums auf Pflanzenzellen. Miinchen er Med. Wochenschr. XXXIX, 587. '93. Ein naturliches System der Gif t-Wirkungen . 136 pp. Miinchen, Wolff u. Liineburg, 1893. LOEW, O. and BOKORNY, T. '89. Ueber das Verhalten von Pflanzenzellen zu stark verdiinnter alkalischer Silberlosung. Bot. Centralbl. XXXIX, 369-373 ; XL, 161-164, 193-197. LUBBOCK, J. '84. Ants, Bees, and Wasps. Internat. Sci. Ser. XLII, 448 pp. 5 pis. New York : Appleton. MARMIER, L. '95. Sur la toxine charbonneuse. Ann. de 1'Inst. Pasteur. IX, 533-574. MASSART, J. '91. La sensibilite a la concentration chez les etres unicellu- laires marins. Bull. 1'acad. roy. Belg. (3) XXII, 148-167. LITERATURE 57 CHAPTER II EFFECT OF VARYING MOISTURE UPON PROTOPLASM IN this chapter it is proposed to speak (I) of the amount of water in organisms ; (II) of the effect of desiccation upon the functions of protoplasm ; (III) of the acclimatization of organ- isms to desiccation, and (IV) of the control of the direction of locomotion by moisture — hydrotaxis. § 1. ON THE AMOUNT OF WATER IN ORGANISMS Any theory of the structure of protoplasm must recognize that water forms the greater part of the whole mass ; between 60% and 90%. In the case of dry seeds and grains, however, it may fall below 15%. Many determinations have been made of the proportion of water in the body of entire organisms and in their organs. I give in tabular form some of these determina- tions, which were made by BEZOLD ('57), designated by (B) ; KRUKENBERG ('80), designated by (K); and LIEBERMANN ('88), designated by (L). TABLE VIII SPECIES. CONDITIONS OF WEIGHING. % WATJSU. Various sponges (K) Medusa : Rhizostoma Cuvieri (K) . Various Actinia (K) Alcyonium palmatum (K) .... Asteracanthion glacialis (K). . . Lumbricus complanatus (K) . . . In most cases, kept a short time in fresh sea water ; dried on surface and weighed. Whole animal, directly from water Piece of disc. A few minutes after removal from sea. A little water lost from central cavity Weighed when fresh, 73.5 g. Weighed 850 g. 2 large specimens 58 84.0 to 74.5 95.4 95.0 87.7 to 83.2 84.3 82.3 87.8 >] DESICCATION AND PROTOPLASMIC FUNCTIONS 59 SPECIES. CONDITIONS OP "WEIGHING. % WATEE. Oniscus murarius (B) 200 young individuals 68.1 1 individual 81.9 3 individuals weighing from 16.6 to Doris tuberculata (K) 27.4 g. 71.1 88.4 3 individuals 86.5 6 individuals weighing from 4.3 to Lininx maximus (B) 27.1 g. 4 individuals weighing from 0.1 to 86.8 Botryllus (K) 17.1 g. 4 individuals weighing from 111.2 82.1 Various Vertebrates (B) to 35.2 g. 93.6 58.4 to Chick (L) 80.1 7 days old Embryo only, yolk removed 92.8 21 days old Embryo only, ready to hatch 80.4 Turnip (root) From Goodale's Physiolog. Bot., p. 236 91.0 These determinations suffice to show that water immensely predominates over any other substance in active organisms, and indicate that it plays an important role. The role played by water is, in fact, extremely varied. It serves to maintain that unstable, foam-like structure of the protoplasm upon which its capacity for movement depends ; it acts as a solvent for matter taken into the protoplasmic body; and it serves to transport dissolved substances from place to place in the organism. In a word, it is essential to movement and to those chemical processes which constitute metabolism. § 2. Ox THE EFFECT OF DESICCATION UPON THE FUNC- TIONS OF PROTOPLASM We may consider this topic under the following heads : (1) effect upon metabolism ; (2) effect upon the motion of protoplasm; and (3) the production of desiccation-rigor and death. 1. Effect of Dryness on Metabolism. — Since water is so essential to metabolism, we should expect that a diminution of metabolism would accompany dryness. And this is clearly the case. Thus dry seeds, in which the water is reduced to only 10 60 MOISTURE AND PROTOPLASM [Cn. II to 15%, when placed under conditions of temperature favor- able to metabolism, show almost no change in the course of days. This has been indicated also by an experiment of KOCHS' ('90, p. 685), who placed seeds, which had been dried in a vacuum, in a receptacle connected with a GEISSLER'S tube, such as is used in the spectroscopic study of gases. The air was completely pumped out of both vessels, and after some months a spectroscopic study of the gases in the GEISSLER'S tube showed no trace of nitrogen or carbon, yet the seeds later germinated. This experiment can hardly be considered to demonstrate KOCHS' point, however, since the seeds were deprived of oxygen, as well as moisture. The act of drying may, on the contrary, induce the manu- facture and elimination of certain secretions. This occurs apparently in many Protista, which form cysts in the drying pools. This phenomenon is seen again in some of the higher animals, — such as our garden slugs, — which secrete slime in large amount when kept for a short time in a dry place. In both cases the result is of immense importance for the con- tinued life of the organism, — in both cases it is to be consid- ered a response to the stimulus afforded by evaporation of water. 2. Effect of Dryness upon the Motion of Protoplasm. — We have seen that water plays an important role in the movement of protoplasm. When by any means the water is partly with- drawn, the protoplasmic currents will be slowed. When, on the contrary, protoplasm, which is lying in an " indifferent " medium, such as blood serum, is placed in distilled water, unusually active movements occur. This has been shown by ENGELMANN ('68, p. 446) in the case of the spermatozoa of the frog, and the ciliated epithelium of the frog's oesophagus just removed from its body. Similarly, DEHNECKE ('81) found that protoplasm of the tissue cells of the higher plants exhibited abnormally rapid movements upon adding water. These obser- vations indicate that water may act as a stimulus to the move- ment of protoplasm. 3. Desiccation-rigor and Death. — It is a familiar fact which has been established by over a hundred and fifty years of experimentation, that some organisms, when gradually dried, may cease from movements. This immotile condition has §2] DESICCATION AND PROTOPLASMIC FUNCTIONS 61 sometimes been regarded as death. By PREYER ('91) it has been called " anabiosis." I shall call it desiccation-rigor, and correlate it with phenomena, produced by various other agents, which cause the cessation of a movement that is restored again when the action of the untoward agent is withdrawn. And these are just the conditions we meet with here, — ces- sation of activity without loss of power of revival. This was very evident from the work of SPALLAXZAXI (1787, Tom. II, pp. 212, 213). This author showed clearly that one and the same adult rotifer can be observed in the evaporating drop, until all the water is gone, and it has lost all movement and its normal form. If, after an hour, the slide is moistened again, the rotifer reassumes, by degrees, its natural form and activities. SPALLAXZAXI noticed, what has been the nearly unanimous testimony of subsequent observers, that a rotifer dried for hours on clean glass does not revive ; revivification occurs only when the rotifers have crawled into sand. As for the length of time during which desiccation-rigor may persist in rotifers without death occurring, we know only that it may be considerable, extending through months, and even years. Similar phenomena to those observed in rotifers have been described for tardigrades and certain nematodes, although these organisms have not been studied in so much detail. Among the tardigrades only those species which live in moss, and are thus especially liable to desiccation, withstand drying. (LAXCE, '94.) Among nematodes, Tylenchus devastatrix, KUHX, which lives in grains of wheat, is a classic object of study. Strongylus rufescens is, according to RAILLIET ('92), capable of resisting dry ness for 68 days or more. We may thus conclude that adult organisms of certain species may be subjected to desiccating influences, and that those same indi- viduals may resist them so as to reexhibit activities after the return of favorable conditions.* While the results of these drying experiments are scarcely doubted, much difference of opinion has arisen concerning the interpretation of the results. The first moot point is the degree of desiccation which the protoplasm of the organisms * See in connection with this the valuable report of BROCA ('61). 62 MOISTURE AND PROTOPLASM [Cn. II experiences. Many writers have assumed that this has been rendered in their experiments very great or nearly perfect. Thus DOYERE ('42, p. 28) says: "What is the condition of the animalcules in the dried sand of the gutters? I have never seen them, at such times, in any other condition than reduced to spangles as fragile and more deformed than when dried free on the glass. I have never discovered a single one which manifested any traces whatever of life, or which did not present all the appearances of a complete desiccation. Never- theless, I do not pretend by this to invalidate all contrary asser- tions ; the principal fact, that of the return to life after an absolute desiccation, is not affected thereby." The physicist GAVARRET ('59, p. 317) subjected, for 34 days, moss contain- ing rotifers to a vacuum having a pressure of only 4 mm. of mercury, and other experimenters have likewise employed a similar "chemically drying" device, which they believed capa- ble of extracting all water from the protoplasm. The evidence that all water is withdrawn from the body of the organism is often very slight. The fact that the seeds or plant tissues in which nematodes or tardigrades are living, have been dried until they lose no appreciable weight, is not sufficient evidence that their inhabitants are completely dried. On the other hand, there is positive evidence that one, at least, of the organisms which has been considered as having been absolutely dried, can protect itself from this condition. It is especially DAVIS ('73) who has shown this. This author has experimented with the rotifer Philodina roseata. When dried on a glass plate with sand, it assumes a spherical form. At the same time, however, it secretes a gelatinous envelope. Thus encapsuled it may rest for days until upon the addition of water it reassumes its active, adult form. That a layer of such gelatinous substance is sufficient to resist the drying action of a vacuum-chamber with sulphuric acid, was illus- trated by putting grapes varnished with gelatine in such a dry chamber for one week. They emerged in a fresh, juicy con- dition. - One of the encapsuled rotifers was crushed after "desiccation" and yielded under the cover-glass a drop of fluid. In this case then the rotifer was not fully dried. DAVIS accounts for the fact that isolated rotifers dried on a clean § 2] DESICCATION AND PROTOPLASMIC FUNCTIONS 63 slide will not survive desiccation, on the ground that the sand forms a necessary retreat in which the organism can quietly encapsule itself. Of the fact of such encapsuling there can be no doubt ; it is abundantly substantiated by the testimony of HUDSON ('73 and '86). There is a doubt, however, whether this encapsuling is a phenomenon common to all organisms which can resist desiccating influences, and, therefore, whether DA vis's explanation is generally applicable. To sum up : I believe there is no sufficient evidence that an adult organism or active protoplasm of any sort can rapidly lose all of its " free water" without such a destruction of its finer structure as would make it incapable of exhibiting vital activities upon moistening again. A much greater capacity undoubtedly inheres in spores and seeds. Thus KOCHS ('90) subjected perforated seeds of Zea mais, Phaseolus, and Triticum vulgare to an almost perfect vacuum (made by a mercury pump) for 8 days, and they nearly all germinated. Even the small radish seed, with part of the cuticula removed, subjected to a vacuum for three weeks ger- minated perfectly. Probably there is no limit to the amount of desiccation which seeds and other masses of protoplasm especially adapted to resist desiccation can withstand. The second moot point is this : Is the protoplasm, rendered immotile by drying, living or not ? SPALLANZANI prejudiced the question by the title of his chapter on this matter, — " Observations and experiments on some marvellous animals which the observer can at his will make pass from death to life " ; and he and many of his successors argue that death has truly occurred. PREYER ('91), however, prefers to reserve the term " dead " for protoplasm which is at the same time lifeless and incapable of life ; while to protoplasm which is lifeless but capable of revivification he applies the term " anabiotic." The question then is this, is life truly suspended during the immo- tile state ? If we think of life as the sum of the chemical changes occurring in the protoplasm, we shall realize that all degrees of vitality, even to complete cessation of activity, may occur without our being able anywhere to say at this point life becomes extinct. We can hardly hope ever to deny that mini- mum vital changes are occurring ; since the minimum changes 64 MOISTURE AND PROTOPLASM [Cn. II must be beyond our ken. That these vital changes are some- times exceedingly slight, is sufficiently indicated by the experi- ments upon seeds performed by KOCHS ('90), and referred to on p. 60. That, however, slight changes are occurring even in seeds is indicated by the fact that dessication-rigor cannot continue indefinitely without loss of the power of revivifi- cation. In the case of the rotifers, tardigrades, and nematodes, months, and even years, may elapse without complete loss of capacity for revivification. It is generally admitted, however, that in cases of long-continued drought, the chances of revitalizing upon moistening are much diminished.* In the case of seeds it has been maintained that under certain conditions, such as are realized in the mummy-graves of Egypt, life may persist for more than a thousand years. However, the experiments of MUNTER ('47), and more especially of KOCHS ('90, p. 683), throw doubt upon this assertion, since they found that the ancient, charred seeds fell to pieces in water like lime. As for seeds preserved above ground in the ordinary way, KOCHS was assured by seedmen that they could not remain capable of germination over 10 years. These facts go to show that gradual changes occur in the dry protoplasm which are prob- ably metabolic changes, i.e. vital changes ; and that therefore life is hardly extinct in the very dryest protoplasm. The whole matter of desiccation-rigor is, after all, one with which we are familiar in nature's larger laboratory. Many Protista, when the ponds in which they live dry out, encyst themselves and enter into a motionless condition in which they resist the hot and dry summer winds. Thus, they may lie for weeks, and, as experimentation has shown, they may be dried for several years (see BUTSCHLI, '89, p. 1663, for references) without loss of capacity for revivification. The same device * Thus RAILLIET ('02) says of Strongylus rufescens : "I have seen them regain their activity after 42 and even 68 days of desiccation. However, this activity is much slower in manifesting itself. After the course of a month a contact of 8 to 10 minutes is sufficient to bring them back to movement. . . . After 68 days at least 50 minutes are required, and certain individuals have shown activity only after 1 hour and 20 minutes. Moreover, the movements were limited, and only a small number of cases contorted themselves like ordi- nary Anguillulidse." J] ACCLIMATIZATIOX TO DESICCATION 65 >r resisting desiccation is seen in the gemmules of sponges, id Bryozoa, the eggs of many animals, and the spores of many mts. Thus some protoplasm normally responds to the :imulus of drought by going into desiccation-rigor. While, as we have seen, some protoplasmic bodies may be dried as far as possible by the ordinary methods used in chem- istry without death ensuing, other bodies, especially the adult forms of higher organisms, whose cellular respiration is de- pendent upon a circulating fluid, are killed by desiccation. For loss of this fluid or desiccation-rigor in the pumping muscles will produce asphyxia. But these conditions do not militate against the belief that there is no necessary lower limit to the amount of water which must occur in quiescent protoplasm in order that it may retain vitality during a lim- ited period. § 3. ON THE ACCLIMATIZATION OF ORGANISMS TO DESICCATION We have seen in the last section that certain organisms are more capable of resisting desiccation without fatal effect than others; e.g. rotifers, tardigrades, and Tylenchus. Now it is clear that these organisms are especially apt to become dried, so that it is possible that their high capacity for resistance has been produced by acclimatization without selection. I shall here add certain other cases of resistance to dryness which I believe, but cannot prove, to have been thus produced. LANCE ('94) has mentioned, as already stated, that only those tardi- grades which live in the moss of gutters (where they are alternately wet and dried), and not those living in water, show the phenomenon of revivification. CERTES ('92) has found that, although marine Ciliata cannot, in general, withstand desiccation, those from the chotts and saline lakes of Algeria may be dried like those from fresh-water ponds and swamps. The difference in resistance between the forms dwelling in the sea and in inland salt-water ponds is doubtless due to the fact that the former are not regularly desiccated, while the latter are ; consequently the latter alone have had a chance to become acclimated to desiccation. 66 MOISTURE AND PROTOPLASM [Cn. II § 4. THE DETERMINATION OF THE DIRECTION OF MOVE- MENT BY MOISTURE, — HYDROTAXIS This phenomenon has been described by STAHL ('84) in ^Ethalium. When this Myxomycete is placed in the dark upon a glass plate covered with several layers of moistened filter paper, it expands uniformly over the homogeneously moistened substratum. If, now, the plate be placed in a drying chamber, the paper dries slowly, and one can see that the mass of the plasmodium draws towards those places which remain longest damp. If a dilute gelatine jelly is smeared upon a glass slide supported in a horizontal position about 2 mm. above the plasmodium, still in the dark chamber, the plasmodium sends up branches, some of which may touch the gelatine and spread out over it. If the water dries still further, the entire Myxomy- cete may become transferred to the slide above. If, now, the paper be moistened again, the plasmodium sends branches down to it. STAHL'S explanation is that of the old mechanical school. He says, the peripheral protoplasmic layer lying next the dryer region is poorer in water; while that next the damper part of the substratum contains much water. If it be assumed that the internal streaming tends to occur uniformly towards all points of the periphery, it is clear that the dryer, more consistent part will offer greater resistance than the more fluid part, and in this part, therefore, branches will tend to arise. In correspondence with the interpretations which we have hitherto placed upon similar phenomena I prefer to call this a case of response to the stimulus of excessive moisture — in any case it may be designated positive hydrotaxis. When, however, the plasmodium of jEthalium is in the fruit- ing stage, it retreats from the moister part of the substratum, and other Myxomycetes in the fruiting stage show the same negatively hydrotactic tendency. Thus the same agent, water, stimulates the same organism, at different stages, to reverse movements. I will now summarize the conclusions concerning the effect of water upon protoplasm. Water constitutes by far the larger part of protoplasm and of all active organisms. Metabolism is LITERATURE 67 lireetly dependent upon it, and certain excretory processes are imulated by it. The motion of protoplasm is likewise de- jndent upon water, which determines the unstable condition that substance. Desiccation, therefore, produces a rigor, ind this may continue, in the absence of water, for months, and even years, the organism being, meanwhile, ready to awake to activity upon the return of moisture. The degree of desic- cation which organisms can resist varies. In the case of the higher organisms, it is slight ; in the case of bodies especially adapted to resist dryness (spores, seeds, statoblasts), there is, perhaps, no practicably attainable limit to the dryness which their protoplasm may undergo without loss of power of revivifi- cation. The condition of desiccation-rigor is not known to be one of death which is replaced by life upon return of moisture. It is probably rather a condition of minimum metabolism. The great resistance capacity exhibited by certain organisms is correlated with their liability to desiccation in their natural surroundings. Finally, Myxomycetes (and probably other or- ganisms) respond to inequalities in the amount of moisture in their environment, moving either towards or from greater moisture. We recognize thus that the activities of protoplasm are to a large extent dependent upon the existence of water in it ; and that protoplasm reveals itself as sensitive to differences in the amount of moisture, responding by secretions, by the assumption of a quiescent condition, and by locomotion with reference to water. ' LITERATURE BLAINVILLE, H. DE '26. Sur quelques petits Animaux qui, apres avoir perdu le mouvement par la dessication, le reprennent comme aupara- vant quand on vient a les mettre dans 1'eau. Ann. des Sci. Nat. IX, 104-110. BEZOLD, A. VON '57. Untersuchungen liber die Vertheilung von Wasser. organischer Materie und anorganischen Verbindungen im Thierreiche, Zeitsch. f. wiss. Zool. VIII, 487-524. 26 Feb. 1857. Bos, R. '88. Untersuchung iiber Tylenchus devastatrix, KUHN. Biol. Cen- tralb. VII, 646-659. 1 Jan. 1888. BROCA, P. '61. Rapport sur la question soumise a la Societe de Biologie par MM. POUCHET, PENNETIER, TIXEL et DOYERE au sujet de la revi- viscence des animaux desseches, lu par M. PAUL BROCA au nom d'une 68 MOISTURE AND PROTOPLASM [Cn. II commission comp. de MM. BALBIANI, BERTHELOT, BROWN-SEQUARD, DARESTE, GUILLEMIN, CH. ROBIN et BROCA. Mem. Soc. de Biol. (3), 1-139. BUTSCHLI, O. '89. Protozoa (part). BRONN'S Klass. u. Ord. d. Thier-reichs. I Bd. 1585-2035. 1889. CERTES, A. '92. Sur la vitalite des germes des organismes microscopique des eaux douces et salees. Bull. Soc. Zool. France. XVII, 59-62. DAVIS, H. '73. A New Callidina : with the Result of Experiments on the Desiccation of Rotifers. Monthly Micr. Jour. IX, 201-209. 1 May, 1873. DEHNECKE, C. '81. Einige Beobachtungen iiber den Einfluss der Prapa- rationsmethode auf die Bewegungen des Protoplasma der Pflanzenellen. Flora. LXIV, 8-14, 24-30. 1, 11 Jan. 1881. DOYERE, M. P. L. N. '42. Memoire sur les Tardigrades. Ann. des. Sci. Nat. (2) XVIII, 5-35. ENGELMANN, T. W. '68. Ueber die Flimmerbewegung. Jena. Zeitschr. IV, 321-479. FAGGIOLI, F. '92. De la pretendu reviviscence des Rotiferes. Arch. Ital. de Biol. XVI, 360-374. 31 Jan. 1892. FROMENTEL, E. DE '77. Recherches sur la revivification des rotiferes, des anguillules et des tardigrades. C. R. Assoc. fran9- 1'avanc. des sci. VI (Le Havre), 641-657. GAVARRET, J. '59. Quelques experiences sur les rotiferes, les tardigrades et les anguillules des mousses des toits. Ann. Sci. Nat. (Zool.). (4), XI, 315-330. HUDSON, C. T. '73. Remarks on Mr. Henry DAVIS' Paper "On the Desic- cation of Rotifers." Monthly Micr. Jour. IX, 274-276. 1 June, 1873. '86. [Desiccation of Rotifers.] Jour. Roy. Micr. Soc. (2) VI, 79. KOCHS, W. '90. Kann die Kontinuitat der Lebensvorgange zeitweilig vol- lig unterbrochen werden? Biol. Centralbl. X, 673-686. 15 Dec. 1890. KRUKENBERG, C. F. W. '80. Ueber die Vertheilung des Wassers, der organ- ischen und anorganischen Verbindungen im Kb'rper wirbelloser Thiere. Vergl.-Physiol. Stud. I, 2 Abth. 78-106. LANCE, D. '94. Sur la reviviscence des Tardigrades. Comp. Rend. CXVIII, 817, 818. 9 Apr. 1894. LIEBERMANX, L. '88. Embryochemische Untersuchungen. Arch. f. d. ges. Physiol. XLIII, 71-157. 7 Apr. 1888. MUNTER, J. '47. Flora. XXX, 478. POUCHET, F. A. '59. Recherches et experiences sur les animaux ressusci- tants. Paris : J. B. Bailie re et fils. 92 pp. 1859. PREYER, W. '91. Ueber die Anabiose. Biol. Centralbl. XI, 1-5. 1 Feb. 1891. RAILLIET, A. '92. Observations sur la resistance vitale des embryons de quelques Nematodes. C. R. Soc. de Biol. XLIV, 703, 704. LITERATURE 69 RYWOSCH, D. '89. Einige Beobachtungen an Tardigraden. Sb. Naturf. Ges. Dorpat. IX, 89-92. SPALLANZAXI, L. 1787. Oeuvres : Opuscules de physique, animale et vege- tale, etc. Trans. Jean SENEBIER. 3 tomes. Pavia and Paris. STAHL, E. '84. (See Chapter I, Literature.) ZACHARIAS, O. '86. Kommen die Rotatorien und Tardigraden nach vollstan- diger Austrocknung wieder aufleben oder nicht? Biol. Centralb. VI, 230-235. 15 June, 1886. CHAPTER III ACTION OF THE DENSITY OF THE MEDIUM UPON PROTOPLASM IN this chapter we shall consider (I) the structure of proto- plasm and the physiological action of solutions ; (II) the effect of density upon the structure and general functions of proto- plasm ; (III) acclimatization to solutions of greater or less density than the normal ; and (IV) control of the direction of locomotion by density — tonotaxis. § 1. INTRODUCTORY REMARKS UPON THE STRUCTURE OF PROTOPLASM AND THE PHYSICAL ACTION OF SOLUTIONS It is now generally recognized that protoplasm consists of two substances closely interwoven : the living plasma and a watery chylema. The relation of the plasma and the chylema is still a debated matter. Since the only theory of the struct- ure of protoplasm which has been experimentally tested is that of BUTSCHLI, his theory is especially worthy of recognition. According to this theory, the relation of plasma and chylema is that of water and air in a foam- work. The whole protoplasmic mass is bounded and penetrated through and through by plasma films which envelop watery globules. It is with membranes constructed of such protoplasm that the physical phenomena of osmosis are exhibited.* Osmosis occurs when two aqueous solutions of different density are separated by an animal membrane, f Such a mem- * Excellent treatises on the physical and chemical nature of solutions, in- cluding a discussion of osmosis, are : OSTWALD, '91, and WHETHAM, '95. t Osmosis occurs likewise when such solutions are separated by inorganic walls containing pores of extreme fineness ; e.g. a wall of porous clay in which copper ferrocyanide has been precipitated. 70 1] PHYSICAL ACTION OF SOLUTIONS 71 *ane permits the free passage of water, but not of the dissolved ibstance, or rather, of the dissolved substance but slowly. rnder these conditions, the water flows more rapidly towards solution containing the greater number of molecules (per cc.). 'he theory of this movement is that upon the side containing ie greater number of molecules of salt fewer water molecules -ill in a given time strike the membrane than upon the other side ; and since the number passing through is proportional to the number striking, relatively fewer molecules of water will consequently pass out, and so there will be a resultant flow of water to that side ; and if the mass of water is confined, it will exert great pressure. This phenomenon of osmosis plays an important part in organic life. Thus, under certain conditions, cells take in the surrounding water, so that their walls are put under tension (turgescence). The tension thus gained may be considerable, amounting to 6 or 7 atmospheres. Under other conditions the cells give up their water to the surrounding medium, thus losing their turgescence. This occurs when they are put into certain solutions of KNO3 or NaCl. The relation between the density of the internal and external fluids thus determines the internal pressure experienced by the cell. A quantitative method of determining this pressure in the presence of various solutions has been employed by PFEFFER ('77). Solutions of different dry salts in different proportions, enveloped by a semi-permeable membrane, were placed in pure water, and the pressure upon a column of mercury determined. It was found, for example, that with a 1% solution of cane sugar a pressure of 47.1 cm. of mercury* was produced ; with a 1% solution of K2SO4, a pressure of 193 cm. of Hg. He concluded, as a result of his various experiments, (1) that the pressure is proportional to the concentration of the solution, and (2) that as the temperature rises the pressure increases. DE VKIES ('84) made a noteworthy advance, using plant cells as objects of experimentation and subjecting them to various solutions of substances freed of water. He determined the degree of concentration which a solution of KC1 must have * The pressure of 76 cm. of mercury equals that of 1 atmosphere. 72 SOLUTIONS AND PROTOPLASM [Cn. Ill in order that no endosmosis or exosmosis should occur through the cell wall.* He next determined the same thing for some other substances, e.g. KI, and found that the degree of con- centration which produces no osmosis is, for two different solutions, proportional to the molecular weights of the salts dissolved in them. Solutions which produce the same osmotic effect DE VRIES called isotonic. A solution of 0.746% KC1 is isotonic with a solution of 1.661% of KI, for the molecular weight of KC1 is 74.6, and that of KI is 166.1. Thus the first result which DE VRIES gained was that the osmotic effect of solutions of salts of similar structure depends upon the number of their molecules in the solution. The second conclusion of DE VRIES was that salts of dis- similar structure have different osmotic properties, even when the number of molecules in the two solutions is the same. Thus, he found that with an equal number of molecules to the solution (molecular-weight solutions f ) : — (1) All salts of alkalis with one atom of metal to the molecule are isotonic (formula, E'A9 [composed of a monad metallic radicle, R, and a monad acidic radicle, A]) ; (2) All organic compounds with no metal radicle, have two-thirds the osmotic action of the first group ; e.g. cane sugar, C^H^Ou- $ * As is well known, when a fully developed plant cell is put into a strong saline solution the living plasma sac separates from the cell wall and contracts, eventually, into a ball, — the result of the chylema flowing out of the protoplasm (plasm olysis) . The weaker the concentration, the less marked the plasmolytic phenomena. Finally, a concentration is reached so weak that the separation of the plasma sac hardly occurs or is limited to a single corner. This concentra- tion may be regarded as equal to that of the cell-sap — as that at which no osmosis occurs. (See Fig. 8.) t I shall use the phrase " molecular- weight solution " to indicate solutions in the making up of which the molecular weight of the substance in grammes, dis- solved in 100 g. of water, is used as the unit of concentration. It will often be convenient to abbreviate it as MW% sol. Chemists frequently use as a unit solution, called "normal" solution, the molecular weight in grammes dissolved in 1000 g. of water. Our M W % sol. is therefore equal to one-tenth of a " normal ' ' solution. J The fact that glycerine can be absorbed by some plants has introduced a complexity into the determination of its isotonic coefficient. This determina- tion has been made the subject of a special investigation by DE VRIES ('88), who, by the use of slowly absorbing plants, has found the isotonic coefficient to be 1.78, which agrees approximately with the number given above for organic compounds. § 1] PHYSICAL ACTION OF SOLUTIONS 73 (3) All salts of alkalis with two atoms of metal to the molecule have four-thirds the osmotic action of (1) (formula, R2'A")', e.g. K2S04. (4) Salts of alkalis with three atoms of the metal to the molecule have five-thirds the osmotic action of (1) (formula, RB'A'"); e.g. K3(C6H507). In other words, the osmotic action of groups (2), (1), (3), and (4) are in the proportions of 2, 3, 4, 5. These last num- bers are the Isotonic Coefficients of DE VRIES. In addition to these substances, DE VRIES determined that the isotonic coeffi- cient is, in the case of — salts of earthy alkalis with 1 acid radicle ; e.g. MgS04 . . 2 salts of earthy alkalis with 2 acid radicles ; e.g. CaCL, ... 4 In the third place DE VRIES established the law that each acid group and each metal has, in all compounds, the same par- tial isotonic coefficients ; the coefficient of any salt is the sum of these partial coefficients of the constituent components. These partial coefficients are : — for each atom-group of an acid 2 for each atom of an alkaline metal (Li, Na, K, Kb, Cs) . . 1 for each atom of an earthy metal (Ca, Sr, Ba, Mg) .... 0 while of the compounds the isotonic coefficients are — KC1 = 1 + 2 = 3, MgSO4 =0 + 2 = 2, K2S04 = 2x1 + 2 = 4, MgCl2 = 0 + 2x2 = 4, K,(C6HA) = 3x1+2 = 5, etc. The determination of isotonic coefficients has subsequently been extended by several authors, especially by HAMBURGER ('86 and '87) and by MASSART ('89). The work of HAMBURGER was done upon blood corpuscles. The method employed by him was as follows : In certain weak solutions the haemoglobin passes out of the red blood corpuscles of ox blood. The concentration at which it just began to ex- trude was determined for various salts, and it was found that these concentrations were usually proportional to the molecular weights of the substances divided by certain whole numbers, which are the same as the isotonic coefficients of DE VRIES. 74 SOLUTIONS AND PROTOPLASM [Cn. Ill The work of MASSART was done chiefly upon bacteria. He made use of the fact demonstrated by PFEFFER (see p. 41) that substances, which at a low concentration attract bacteria chemotactically, at a higher concentration repel them. He found that, in general, the repulsions exercised by the various dissolved substances are proportional to their isotonic coef- ficients, when the solutions are made up as MW solutions. Thus, when a 10 MW % concentration of a substance with iso- tonic coefficient 2 just begins to repel bacteria, a substance which just begins to repel in a 5 MW % concentration has an isotonic coefficient of 4.* § 2. EFFECT OF VARYING DENSITY UPON THE STRUCTURE AND GENERAL FUNCTIONS OF PROTOPLASM Under this head we may consider, (a) the effect upon the general structure of protoplasm ; (5) the modification of gen- eral functions, and (n p. 82. 84 SOLUTIONS AND PROTOPLASM [Cn. Ill The curve shown in Fig. 10 is constructed from the second and third lines of this table. The table shows that, within the limits of 2.8% and 0.9% concentration, the curve is a logarith- mic one, i.e. as the ordinates increase the abscissae increase as the logarithms of the ordinates. In line 4 are given the (BRIGGS') loga- rithms of the num- bers in line 3, and in line 5 these loga- rithms are each mul- tiplied by a constant, 1.7, which gives a series of numbers closely similar to that of line 2. The relation between density and resist- ance period can thus be expressed by the equation D = k. log. R, in which D stands for density ; R, for re- sistance period; and k is a constant whose value depends upon the system of loga- rithms employed. This formula may be 2 ", in which e is the Since the 40 30 20 10 0 < FK | / / / \f\ i / i / / OA / 7 1 I 1 on / / / 1 J / / / r / — — • . — jn " J .9 1.2 1.9 2.5 2.8 3.7 }. 10. — Curve showing relation between the per- centage of salt in mixtures of fresh and salt water (abscissae) and the mean resistance periods in hours of various organisms plunged therein (ordinates). Constructed from the table. (After data of Go- GORZA, '91.) transformed into the equivalent: R = base of the NAPERIAN system of logarithms. osmotic pressure is proportional to the concentration (p. 71), Q it follows also that R = ek> where 0 stands for the osmotic pressure and k' for a new constant. The same relation holds when we compare the reciprocals of the relative resistance periods — or the relative rapidity of killing — and the abso- lute diminution of concentration. ACCLIMATIZATION 85 3. ACCLIMATIZATION TO SOLUTIONS OF GREATER OR LESS DENSITY THAN THE NORMAL In the preceding section we saw that different organisms had diverse resistance period to the same density of solution. In rt, this may be accounted for, as we have seen, on the ground a difference in the rapidity of osmotic action — thick-skinned inimals resisting longer than thin-skinned ones. All diversity the effect of solutions, cannot, however, be accounted for on is ground. Thus, the molluscs of the sea and those of fresh ater appear to have an equally pervious epidermis, yet the rruer will, of course, withstand a much stronger solution of lt than the latter. This difference in resistance capacity ems closely correlated with the conditions of the medium in hich the organism has been reared. Thus, BEUDANT ('16) und that littoral species (living, therefore, in a part of the sea here the water is much diluted by rivers), e.g. Ostrea, Mytilus, atella vulgata, resist fresh water better than deep-sea species; d this discovery has been abundantly confirmed by DE AKIGNY ('88).* That the conditions of density of the culture medium deter- ine the resistance capacity is proven by experiment, for, by rying the density of the culture solution, we may vary the istance period of the individuals experimented on. BEUDANT 16) was the first to show this. He used Lymnea, Physa, lanorbis, Ancylus, Paludina, and some other fresh-water Mol- sca. He began in April by putting these organisms into a XaCl solution, and, continuing to add salt slowly, by Sep- mber many of these withstood a 4% solution — a solution kills animals suddenly subjected to it. He performed ewise the reverse experiment upon marine Mollusca (Patella, extremes of density in which organisms are capable of living are often nsiderable. On the one hand, the individuals of some species, especially fish, able to migrate from fresh to salt water and back, with impunity. On the er hand, many species of a family, the other members of which are marine, ve become accustomed to fresh water. Examples of this last case are the droid Cordylophora lacustris, the mollusc Dreissena, and the endoproctan ozoan Urnatella. Likewise, some marine species have come to live in exces- ively salt water. Such, for example, is the case with Artemia salina which ives in Salt Lake, Utah, containing over 22% of salts. (LEIDY, '72, p. 165.) 86 SOLUTIONS AND PROTOPLASM [Cn. Ill Turbo, Area, Cardium edule, Mytilus edulus, etc.) bringing them to live in fresh water by gradually diluting the medium. PLATEAU ('71) gradually accustomed the fresh-water Asel- lus aquaticus to pure sea water, so that even in mixtures con- taining between 20% and 80% of sea water they laid eggs and produced a second generation. The second generation lived 108 hours in pure sea water, while Asellus freshly taken and plunged into sea water live only about 5 hours. Not only the larger organisms, but also tissues and Protozoa may become acclimated. ROTH observed in '66 (p. 190) that cilia become "accommodated" to gradually increasing densi- ties ; ENGELMANN ('68, p. 343), however, denied, though with- out critical experiments, the validity of this conclusion for the case of the ciliated epithelium of the frog's throat. Later, CZERNY ('69, p. 161) succeeded in acclimating Amoeba to a 4% solution of NaCl, although Amoeba rarely resists 1 % when sud- denly subjected to it. These early experiments have since been greatly extended, observations having been made upon nearly all groups of organ- isms — upon algae, by RICHTER ('92) ; upon Myxomycetes, by STAHL ('84) ; upon Actinospherium, by VERWORN ('89, p. 10) ; upon bacteria, Flagellata, Ciliata, and Hydra, by MASSART ('89); upon Ciliata, by FABRE-DOMERGUE ('88); upon Crus- tacea, by PLATEAU ('71), SCHMANKEWITSCH ('75 and '77), and BERT ('83); upon the tadpoles of frogs, by YUNG ('85, p. 520) ; and upon representatives of almost all of the principal groups, by BE VARIGNY ('88) and GOGORZA ('91). The aims and methods of these experimenters have been very diverse. Some have sought merely to illustrate how marine organisms may have come to live in fresh water, or the reverse. Such have usually made mixtures of fresh and salt water, the proportions of the one gradually increasing (DE VARIGNY, SCHMANKEWITSCH, GOGORZA), or they have added sea salt, dry or in solution, to the normal fresh-water medium of the organism (YUNG). MASSART, on the other hand, having in mind the more fundamental problem of the action of density upon protoplasm, has employed solutions of a single salt at a time — solutions, moreover, based usually upon the osmotic index of the salt as a unit of concentration. §3] ACCLIMATIZATION 87 Athough there has been a gradual improvement in methods, the conditions other than that of concentration have too often been omitted from consideration. The omission of the tempera- ture of the experiment solutions is especially unfortunate, for according to GoGORZA ('91, p. 270), acclimatization is more easily effected at a low temperature than at a high one. Of the papers mentioned above, that of MASS ART is especially worthy of extended notice from its quantitative nature. He subjected cysts of Ciliata to various concentrations of KNO3 and noted the effect upon the protoplasm. In the following tables, the first line of numbers names the solution in parts of the molecular weight expressed in grammes. The symbols in the columns headed by these numbers have the following signifi- cations: 0, no effect; v, the cysts possess a large vacuole whose pulsations are infrequent; v p, the vacuole is still prominent but plasmolysis is occurring ; p, the plasmolysis is more marked and the vacuole is gone; P, the plasmolysis is so marked that the form of the infusorian is lost. The results given in the third and fourth lines were obtained from individuals acclimated for 22 hours to a 1.8 MW % and to a 3 MW % solution of KXO3 respectively. The observations were made immediately after immersion of the cysts. No mention is made of the temperature. TABLE X — VORTICELLA HUXDREDTHS OF MOLECULAR WEIGHT. O.5 1.0 1.5 2.0 2.5 3.O 3.5 4.O 5.O Unacclimated y V V D V V UTSCHLI, O. '92. Untersuchungen iiber mikroskopische Schaume und das Protoplasma. Leipzig. 232 pp. 1892. 94 SOLUTIONS AND PROTOPLASM [On. Ill CERTES, A. '84. Note relative a 1'action des hautes pressions sur la vitalite des micro-organismes d'eau douce et d'eau de mer. C. R. Soc. de Biol. XXXVI, 220-222. '84a. De 1'action des hautes pressions sur les phenomenes de la putri- faction et sur la vitalite des micro-organismes d'eau douce et d'eau de mer. Comp. Rend. XCIX, 385-388. 25 Aug. 1884. COUTANCE, H. A. '83. Action biologique des sels de 1'eau de mer au point de vue de 1'entretien des animaux marins. Bull, de la Soc. d'Acclimat. (3) X, 98-106. Feb. 1883. CZERNY, V. '69. Einige Beobachtungen iiber Ambben. Arch, f . mik. Anat. V, 158-163. EMERY, H. '69. Notes physiologiques. Ann. des Sci. Nat. (Zool.). (5) XII, 305-325. ENGELMANN, T. W. '68. (See Chapter II, Literature.) FABRE-DOMERGUE '88. Recherches anatomique et physiologiques sur les infusoires cilies. Ann. des Sci. Nat. (7) V, 1-140. FREDERICQ, L. '85. Influence du milieu ambient sur la composition du sang des animaux aquatiques. Arch, de Zool. (2) III, xxxiv- xxxviii. GOGORZA Y GONZALEZ, D. J. '91. Influencia del aqua dulce en los Animales Marinos. Annales de la Soc. Esp. Hist. Nat. XX, 220-271. 1891. GRUBER, A. '89. Biologische Studien an Protozoen. Biol. Centralbl. IX, 14-23. 1 March, 1889. HAMBURGER, -H. J. '86. Ueber den Einfluss chemischer Yerbindungen auf Blutkbrperchen im Zusammenhang mit ihren Molecular-Gewichten. Arch. f. Anat. u. Physiol., Physiol. Abth. Jahrg. 1886. 476-487. '87. Ueber die durch Salz- und Rohrzucker-Lb'sungen bewirkten Ver- anderungen der Blutkorperchen. Arch. f. Anat. u. Physiol., Physiol. Abth. Jahrg. 1887. 31-50. JANSE, J. M. '87. Plasmolytische Versuche an Algen. Bot. Centralbl. XXXII, 21-26. KOFOID, C. A. '95. On the Early Development of Limax. Bull. Mus. Comp. Zool. XXVII, 35-118. KUHNE, W. '64. (See Chapter I, Literature.) LEIDY, J. '72. On Artemia from Salt Lake, Utah. Proc. Acad. Nat. Sci. Philad. 1872. 164-166. MASSART, J. '89. Sensibilite et adaption des organismes a la concentra- tion des solutions salines. Arch, de Biol. IX, 515-570. '91. (See Chapter I, Literature.) OSTWALD, W. '91. Solutions. Translated by MUIR. 316 pp. London i Macmillan. 1891. PLATEAU, F. '71. Recherches physico-chlmiques sur les articules aquatiques. Mem. cour. 1'Acad. Roy. Belgique. XXXVI, 68 pp. PFEFFER, W. '77. Osmotische Untersuchungen. Leipzig. 1877. '84. (See Chapter I, Literature.) '88. (See Chapter I, Literature.) LITERATURE 95 REGXARD, P. '84. Recherches expefimentales sur 1'influence des tres hautes pressions sur les organismes vivants. Comp. Rend. XCVIII, 745- 7-47. 21 March, 1884. '84a. Note sur les conditions de la vie dans les profondeurs de la mer. C. R. Soc. de Biol. XXXVI, 164-168. '84b. Note relative a Faction des hautes pressions sur quelques pheno- menes vitaux (mouvement des cils vibratiles, fermentation). C. R. Soc. de Biol. XXXVI, 187-188. '84C. Sur la cause de la rigidite des muscles soumis aux tres hautes pressions. C. R. Soc. de Biol. XXXVI, 310-311. '84d. Effe\t des hautes pressions sur les auimaux marins. C. R. Soc. de Biol. XXXVI, 394-395. '86. Action des hautes pressions sur les tissues animaux. Comp. Rend. CII, 173-176. RICHTER, A. '92. Ueber die Anpassung der Siisswasseralgen an Kochsalz- losungen. Flora. L, 4-56. RIXGER, S. and BUXTOX, D. W. '85. Concerning the Action of Small Quantities of Calcium, Sodium, and Potassium Salts upon the Vitality and Function of Contractile Tissue and the Cuticular Cells of Fishes. Jour, of Physiol. VI, 154-161. July, 1885. ROGER, H. '95. Action des hautes pressions sur quelques bacteries. Arch. de Physiol. (5) VII, 12-17. Jan. 1895. ROSSBACH, M. J. '72. (See Chapter I, Literature.) ROTH, M. '66. Ueber einige Beziehungeii des Flimmerepithels zum con- tractilen Protoplasma. Arch. f. path. Anat. u. Physiol. XXXVII, 184-194. Oct. 1866. SCIIMANKEWITSCH, V. '75. Ueber des Verhaltniss der Artemia salina Miln. Edw. zur Artemia Miihlhausenii Miln. Edw. und dem Genus Bran- chipus Schaeff. Zeitschr. f. wiss. Zool. XXV, Suppl., 103-116. '77. Zur Kenntniss des Einflusses der ausseren Lebensbedingungen auf die Organisation der Thiere. Zeitsch. f. wiss. Zool. XXIX, 429-494. 6 Sept. 1877. '79. [Abstr. in Nature. XXIX, 274. 1884.] STAHL, E. '84. (See Chapter I, Literature.) VARIGXY, H. DE '88. Beitrag zum Studium des Einflusses des siissen Wassers auf die Seethiere. Centralbl. f. Physiol. I, 566-568. 21 Jan. 1888. VERWORX, M. '89. (See Chapter I, Literature.) VRIES, H. DE '84. Eine Methode zur Analyse der Turgorkraft. Jahrb. f . wiss. Bot. XIV, 427-601. '88. Le Coefficient Isotonique de la Glycerine. Arch. Neerland. XXII, 384-391. '89. Ueber die Permeabilitat der Protoplaste fur Harnstoff. Bot. Ztg. XL VII, 309. WHETHAM, "W. C. D. '95. Solutions and Electrolysis. . Cambridge Nat. Sci. Man. Cambridge, Eng. 296 pp. 1895. 96 SOLUTIONS AND PROTOPLASM [Cn. Ill YUXG, E. '85. De 1'influence des variations du milieu physico-chemique sur le developpement des animaux. Arch. Sci. phys. et nat. (3) XIV, 502-522. 15 Dec. 1885. ZACHARIAS, O. '84. Ueber die amoeboiden Bewegungen der Spermatozoen von Polyphemus pediculus de Geer. Zeitschr. f. wiss. Zool. XLI, 252-258. '88. Ueber Pseudopodien und Geisseln. Biol. Centralbl. VIII, 548, 549. 15 Nov. 1888. CHAPTER IV ACTION OF MOLAR AGENTS UPON PROTOPLASM THIS subject is so ill-defined that it is impossible to draw any line of distinction between contact on the one hand and a crushing pressure, or wounding, on the other. The molar agents may be solid or fluid. The methods of application may vary from a blunt contact or a sharp cut or puncture to the impact of flowing liquid. All these agents have this in common, however, that they act in a gross, mechanical way. The sub- ject will be discussed under the following heads : (I) The effect of molar agents upon lifeless matter; (II) effect upon the metabolism and movement of protoplasm ; and (III) effect in determining the direction of locomotion, — thigmotaxis (stereotaxis) and rheotaxis. § 1. EFFECT OF MOLAR AGENTS UPON LIFELESS MATTER Mechanical disturbance can induce in certain lifeless com- pounds violent chemical changes. Compounds which are so affected are preeminently unstable. This instability, however, varies greatly in degree. In some cases, the blow of a hammer is required to upset the molecules ; the result being often a violent explosion. In other cases (#•#• chloride or iodide of nitrogen), the slightest touch of a feather suffices to produce an explosion. Now, most of the substances which explode upon impact, and which are used in the arts, are organic com- pounds, — fulminate, nitro-glycerine, gun-cotton, and picric- acid derivatives, — and therefore it is not surprising that we find the notoriously unstable protoplasm violently affected by contact. Especially important for biology is the fact that undulatory motions and other periodic disturbances produce very important H 97 98 MOLAR AGENTS AND PROTOPLASM [Cn. IV molecular changes in chemical compounds. Certain substances have a specific rate of vibration, so that when this is reproduced by a vibrating cord or plate, explosion of the substance may occur. Iodide of nitrogen is one of these substances which is exploded by a high note. (CHAMPION and PELLET, 72, p. 212.) Upon this property of explosive compounds depends, apparently, the efficacy of "detonators," the explosion of a small quantity of which is capable of producing the explosion of -a great mass of a second compound. Living protoplasm is, likewise, espe- cially affected by periodic disturbances, and it is doubtless due to the peculiarities of its chemical structure that the auditory epithelium is so affected by sound waves in all their modifica- tions of pitch, volume, and timbre. § 2. EFFECT OF MOLAR AGENTS UPON THE METABOLISM AND MOVEMENT OF PROTOPLASM We shall first consider the effect on metabolism, and then on movement. The principal metabolic effects that will be con- sidered are phosphorescence and secretion. The phosphorescence of organisms is usually regarded as a slow combustion (oxidation) of organic substances. This chemical process is apparently accelerated by mechanical irritation, as every one must have noticed who has rowed a boat on a quiet summer's evening upon the sea. At every stroke of the oar, a gleam is sent along its length. An analytical study of this phenomenon has been made by MASS ART ('93, p. 62). When a drop of water containing Noctiluca is put on filter paper, and the liquid is absorbed, there comes a moment when the surface film of the water flattens the spherical body of Noctiluca. At that moment of pressure light is emitted. If, however, the water is put into a slight vibration by a needle attached to a tuning-fork, and if the agitation is insufficient to deform the body, no light will be given forth. Deformation of the body, but not slight agitation, is, consequently, accompanied by those metabolic processes which result in the production of light. Secondly, contact may induce the production and discharge of secretions. VERWORN ('89, p. 81) has called attention to this phenomenon in the cases of Actinosphserium and Thalassi- § 2] EFFECT ON METABOLISM AXD MOVEMENT 99 cola. When Actinosphserium is subjected to a slight stimula- tion, such as would be produced by other Protozoa wandering among its pseudopodia, it shows no response. But when an infusorian or a rotifer swims against the pseudopodia with force, they discharge a sticky substance which holds the dis- turbing organism fast. The same result follows the irritation of one of the pseudopodia by touching it with a fibre of cloth or filter paper. Like effects follow the irritation of Thalas- sicola. Thus, some Protista respond to particular kinds of contact by the excretion of a sticky substance. In the higher animals, also, contact may call forth secretions ; thus, the stolons of many hydroids secrete a cement from the surface applied to the substratum. Among the higher plants, also, contact has sometimes a similar effect. Examples appear in DARWIN'S ('75, p. 393) work on the gland cells of insectivorous plants. In many species, to be sure, e.g. Drosera, Dionsea, Drosophylluin, mere contact of inorganic bodies has no effect upon the secretions of the glands of the leaves. In the case of Pinguicula lusitanica, however, fragments of glass, as well as seeds and albumen, caused the glands with which they came in contact to secrete more freely than before. This response to contact by secretion is, for the most part, an advantageous one. It enables the Protista and the insec- tivorous plants to hold their prey or their enemy, as the case may be ; and it enables the stolon to hold fast to the sub- stratum. The change in metabolism may be so profound as to lead to death. HOKVAKTH ('78) and MELTZER ('94) have shown that when bacteria are violently shaken, not only is growth interfered with, as we shall see in the second part of this book, but death may ensue, so that cultures of bacteria may be sterilized. We now turn to consider the modification of movement by molar agents. The general phenomena are familiar. An amoeba, any other rhizopod, or a white blood corpuscle con- tracts when the cover-glass over it is disturbed. The stream- ing in the plasmodia of Myxomycetes is retarded or inhibited 100 MOLAR AGENTS AND PROTOPLASM [Cn. IV x • upon shaking. When alga cells, such as those of Chara or Vallisneria, are freshly transferred to the slide, the disturbance causes cessation of movements (HOFMEISTER, '67, p. .50). When the stamen hairs of Tradescantia are crushed, the stream- ing of the plasma ceases. When Chara is cut across or punct- ured, rotation stops for a longer or shorter time (DUTROCHET, '37, p. 780). Even when a stem of Chara is pricked at the node by a needle, without penetrating into the cavity, move- ment ceases for a minute or two. Thus, mechanical disturbance profoundly af- fects protoplasm. Let us now consider more in detail the changes which take place in the protoplasm. VERWOKN ('92, p. 24) has given us data 011 this matter. Orbitolites is a rhizopod having extremely deli- cate, filamentous pseudo- podia. If one of these pseudopodia be cut across as at #, Fig. 13, a, the following changes occur : the protoplasm lying next the cut directly collects into small spherical or fusiform masses which be- gin to migrate centripe- tally (Fig. 13, 5). This movement meets with the normal centrifugally migrating plasm and turns the latter towards the centre again (Fig. 13, c). Gradually the thickenings elongate until, before they have reached the central body, they are no longer visible (Fig. 13, d). In about 2 minutes normal move- ments are completely restored (Fig. 13, e). Slightly different results are gained from Cyphoderia (Fig. 14). When the large pseudopodium of this organism is touched with a needle near its distal end, it thickens (as in the case of Obitolites) and FIG. 13. — Pseudopodium of Orbitolites, re- tracting as a result of local stimulation. The arrows give the direction of the streaming of protoplasm. At the left is shown the beginning of the excitation; at the right, its end. (From VERWORN, '92). §2] EFFECT OX METABOLISM AXD MOVEMENT 101 the thick region, together with all the proximal lying proto- plasm, begins to flow towards the centre. The whole plasma thread retracts. Again, if an individual of Difflugia (Fig. 15) be slightly shaken, the pseudopodium contracts into the shell ; if it be FIG. 14. — Cyphoderia margaritacea, showing the retraction of its pseudopodium as a result of irritation at the point indicated by the arrow. (From VERWORN, '92.) FIG. 15. — Dimugia urceolata : at a, stimulated by a weak local irritation ; at 6, by a somewhat stronger one. (From VERWORX, '89.) violently shaken, the following changes occur : drops of a less highly refractive substance seem to gather on the surface of the filamentous pseudopodium and unite to form a sheath sur- rounding a more highly refractive axis. At the same time, axis and sheath retreat into the central mass. In this case, then, we have a segregation of dissimilar protoplasmic sub- stances, and a tendency to collect about centres along the 102 MOLAR AGENTS AND PROTOPLASM [Cii. IV FIG. 16. — A series showing seven phases in the contraction of a pseudopodium of Difflugia lobostoma, following total stimulation. The series passes from left to right. (From VERWORN, '92.) pseudopodium and in the whole mass. The same thing is seen in the widely dissimilar Actinospherium (Fig. 17). Here is Fia. 17. — Actinosphaermm Eichhornii, unirritated. Natural size about 0.5 mm. (From VEBWORN, '89.) especially noticeable (Figs. 18, 19) the tendency to produce fusiform or spherical aggregations, and to retract the pseudo- podia. So, too, in the irritated stamen hairs of Tradescantia ; 2] EFFECT ON METABOLISM AND MOVEMENT 103 FIG. 18. — Actinosphaerium Eichhornii, at the beginning of irritation. The proto- plasm is accumulated along the pseudopodia in drops and spindles. (From VERWORN, '89.) says HOFMEISTER ('67, p. 50), "The threads become knotty, tear apart, draw together into short clubs or balls, and fuse FIG. 19. — Three pseudopodia of the same individual, much enlarged, a, normal condition; the axial thread is seen, surrounded by protoplasm. 6, the pseudo- podia at the beginning of stimulation, c, d, the stimulation is continuing, and the axial thread is shortening, e, the three pseudopodia are almost completely retracted. (From VERWORN, '89.) partly with the collection of protoplasm lying about the cell- nucleus and partly with the peripheral protoplasmic layer." These similar phenomena from various organisms are funda- mental ; how are they to be interpreted? It is well known 104 MOLAR AGENTS AND PROTOPLASM [Cii. IV that non-vital semi-fluid substances tend to assume a spherical form by virtue of the property of surface tensions. That pro- toplasm does not always assume this form is due to special causes. When a Protist or one of its pseudopodia is irritated by contact, it tends to assume a spherical form or a thread tends to aggregate into spherical drops. It seems probable, we can- not say more than that, that this aggregation is due to a dimi- nution in the activity of those causes which oppose the action of surface tension; and so the latter reasserts itself. It is likewise possible that new attractive centres arise. That a thread should break up into drops indicates, moreover, a loss in cohesion. Loss of cohesion, formation of new centres of attraction, arid diminution of the form-maintaining forces, — these seem to be the effects of contact. They must be due to the chemical changes wrought by contact. The changes just referred to constitute the essence of con- traction, a phenomenon of widespread occurrence not only among Protista, but among the higher plants and animals ; for ex- ample, in the sensitive plant and in Vertebrate muscle. Into these contraction phenomena which follow contact in the higher organisms we cannot go ; their study belongs to the field of plant and animal physiology. At bottom, however, we must believe many of these phenomena in the higher organisms to be due to the same causes as contraction in Protista. A few words concerning rhythmically repeated disturbances. A single disturbance gives rise, as we have seen, to a series of phenomena producing contraction; but in a few seconds the effects of the disturbances are past and the protoplasm returns to its uncontracted form. If, however, the shock is repeated before relaxation has fully occurred a new contraction is super- imposed on the first, and the resulting contraction is more violent than a single one. If now shock follow shock in quick succession, a violently contracted condition, known as tetanus, results. Under the condition of tetanus the amoeba becomes a spherical mass, Actinosphserium retracts all of its pseudopodia, a branching Carchesium stock forms a little ball, and muscle fibres are greatly shortened. In a word, rhythmically repeated shocks are accompanied by an exaggeration of those changes which result from a single shock. § 3] THIGMOTAXIS 105 § 3. EFFECT OF MOLAR AGENTS IN DETERMINING THE DIRECTION OF LOCOMOTION — THIGMOTAXIS (STEREOTAXIS) AND RHEOTAXIS * We have already seen that when a pseudopodium of an amoeba is touched by a solid body it retracts. In this retrac- tion the centre of mass is transferred to a new point. If the stimulation is often repeated upon the same side, contraction continues on that side, until eventually the amoeba will have migrated a considerable distance. In this case the determina- tion of the direction of locomotion is closely allied to the phe- nomena of contraction as a result of stimulation, considered in section 2. The retraction of the protoplasm which follows its irritation is the cause of the migration of the amoeba in a defi- nite direction. This direction is away from the touching body. The response may consequently be called negative thigmo taxis. The phenomenon of negative thigmotaxis is widespread. There are almost no free-moving organisms which do not move away from contact or molar disturbance of an unusual or vio- lent sort. Thus you may very definitely control the direction of movement of a planarian or a slug by touching the body upon the side opposite the direction in which you wish it to move. In such cases, also, there is first a contraction of the body upon the irritated side. The opposite phenomenon of movement towards, or clinging to, the irritating body — positive thigmotaxis ' — is less common and therefore more striking. It has long been known, I imag- ine, — it certainly is an observation easily made, — that an amoeba which has come in contact with a solid body clings close to it and moves over its surface. LE DANTEC ('95, p. 211) has described the action in much detail. An amoeba descending in the drop touches the glass slide first by a single protruding pseudopodium. Xext, the pseudopod elongates hori- zontally, and at the same time affixation takes place, so that the organism does not roll about when the water is agitated. The * Thigmotaxis, under the different form " thigmotropism " (from "contact") was first applied to these phenomena by VEKWORN ('89, p. 90); stereotaxis, under the form " stereotropism " (from o-repeos, "solid"), was intro- duced by LOEB ('90, p. 28), and is practically synonymous with thigmotaxis. 106 MOLAR AGENTS AND PROTOPLASM [Cn. IV pseudopod gradually extends itself, and new ones are formed, until at last the whole substance of the amosba is spread out parallel to the glass, over whose surface it moves. That there is a considerable adherence is shown by the fact that the amoeba is not disturbed by an appreciable current. If, how- ever, it is made to contract, it looses its hold at once. Very similar phenomena occur, according to VERWORN ('95, p. 429), in Orbitolites also. Such an organism lying in a watch glass begins to send out pseudopodia which, so long as they move free in the water, are simple straight threads ; but when they touch the glass they adhere to it, stream out along it, and send out branches. In these Rhizopoda, consequently, the presence of a solid body is a stimulus to the spreading out of the pseudopodia and to those changes by which close adhesion is effected. We now pass to the other simple organisms. Among Infu- soria, PFEFFER ('88, pp. 618-621) has found that Glaucoma scintillans and, to a less degree, Colpidium colpoda, Parame- cium aurelia, and Stylonychia mytilus aggregate about solid bodies in the water, such as fragments of soaked filter paper or particles of barium sulphate. Since these cannot supply oxygen or soluble substances, the effect produced is doubtless due to contact. The aggregated organisms tend, in moving, to keep upon the surface of the solid. Thus PFEFFER ('88, p. 619) found that Urostyla weissii, coming in contact with glass threads, moved along them on their ventral surfaces ; and MASSART ('91) observed some Chlamydomonades remain hanging to objects with which they came in contact. VERWORN ('95, p. 431), likewise, finds that Oxytricha travels over the surface of Ano- donta eggs or particles of detritus which it happens upon in the water. In one instance, the organism ran for some time over the surface of an egg of Anodonta without being able to leave it. After four hours, it was able, by the aid of a piece of slime which came in contact with the egg, to free itself from that body. Phenomena similar to the above-described for bacteria and Infusoria are found in spermatozoa also. DEWITZ ('85 and '86) first noticed this in the case of the cockroach, Periplaneta §3] THIGMOTAXIS 10T orientalis. When an 0.8% or 0.9% NaCl solution contain- ing spermatozoa was put under a cover-glass, the spermatozoa arranged themselves in two layers, one in contact with the cover-glass, the other in contact with the slide. By isolating some of the spermatozoa at the upper surface and putting them under a cover-glass, he found that they likewise distributed themselves at both upper and lower surfaces. Hence the segre- gation into two layers was not due to a difference in kind between the spermatozoa occupying the two positions, but to the fact that there were here two surfaces of contact, separated FIG. 20. — A, Oxytricha seen from below; B, from the side; C, crawling over the egg of Anodonta. (From VERWORN, '95.) by a water-film. If a spherical grain be placed in the drop of water, aggregation takes place about that also. A similar experiment, with similar results, was made by MASSART ('88) with frog spermatozoa. Here, too, the active spermatozoa kept in contact with the upper and lower glass surfaces, whilst the weak forms lay midway between. The fact that only active spermatozoa show this tendency to keep in contact with solids, indicates that we are here dealing with irritability to contact. The quality of the surface influences its capacity for stimu- lating to positive thigmotaxis. Thus, while mere roughness has no effect, if the surface of glass be smeared with a slimy mass, so thick that the spermatozoa can hardly penetrate it, 108 MOLAR AGENTS AND PROTOPLASM [Cn. IV they may no longer cling to the glass, but wander, undirected, through the water. Again, while the surface film of water often acts thigmotactically, if the surface tension is reduced by a thin covering of oil, it no longer holds the organisms. It would seem that a certain minimum difference in rigidity, between any surface and the medium, is necessary in order that the surface should act thigmotactically. Once in contact with a sufficiently attracting surface, the organism may move to and fro over it, but it can hardly leave it. It is, as DEWITZ ('86, p. 366) says, as though the sperma- tozoa were attracted by a magnet. This close adhesion of the organism to the irritating surface is a remarkable phenomenon. LE DANTEC ('95) suggests that the amoeba adheres to the glass by molecular attraction. On the other hand, it may be doubted whether the close adhesion signifies anything else than the absence of a sufficient stimulus to leave the surface of contact. When an organism has been stimulated by contact for some time, it at last becomes changed so that it no longer responds as it did at first. Thus Dr. W. E. CASTLE has informed me that he has seen a colony of Stentors, in an aquarium, being constantly struck by Tubifex waving back and forth, yet the Stentors did not contract as they usually do when struck. PFEFFER, ('88, p. 619) has observed that Urostyla retreats, after a time, from the surface with which it was in contact. These facts indicate that protoplasm can become acclimatized to contact so as to be no longer stimulated by it. We now turn to the consideration of Rheotaxis, which may be regarded provisionally as a form of thigmotaxis, although the possibility of its being rather a case of chemotaxis is not excluded. ROSANOFF ('68) was the first to notice the rheotaxis of the large plasmodium of ^Ethalium septicum, but he ascribed it to geotaxis. The correct interpretation was first given by STRASBURGER ('78, p. 62), and has been confirmed by JONX- SON ('83), and STAHL ('84). When ^Ethalium is placed on a strip of saturated filter paper, the upper end of which is dipped in a beaker of water, it is subjected to a current of water in the substratum. At the same time it moves § 3] RHEOTAXIS 109 against the current. The current controls the direction of locomotion. The evidence that it is indeed the current is partly gained by exclusion. It cannot be geotaxis, for if the current is flowing upwards on any arm of the strip, the plasmodium flows down. It can hardly be hydrotaxis, for the strip is uni- formly saturated throughout. The action of light may be excluded by shutting the whole apparatus in the dark, when the same response occurs. When the direction of the current in the strip is reversed, the movement of the plasmodium is reversed also. Thus no other cause will explain the result but that of the moving water. Satisfactory evidence that it is the current as such which acts will not be forthcoming until it has been shown that other fluids than water, e.g. oil, provoke a similar response. Until such an explanation has been tried, it must remain uncertain whether the phenomenon is not perhaps due to a difference in the quality of the afferent and the efferent water. Finally, it must be mentioned that higher organisms, espe- cially fish, are rheotactic. Whoever has seen fish ascending streams from the sea in the spring has had this vividly impressed upon him. Before some dam thousands of fish will be seen, all facing the torrent of water against which they can hardly hold their own. It is the current which determines their position. They are responding to the direction of flow of the waters. To recapitulate : In many non-living substances, especially organic compounds, violent chemical changes (explosions) are brought about by contact and especially by repeated vibra- tions. So, too, in protoplasm, chemical change, exhibiting itself in modified metabolism, frequently follows contact. The . explanation adapted to the non-living series of phenomena is adapted to the living series also, — the molecules of the sub- stance are complex, loosely associated, very unstable, so that even a slight mechanical disturbance will serve to dissociate their atoms. Protoplasm is a mixture of so many substances that the whole mass does not become changed at once ; but continued stimulation may eventually produce such wide- spread changes as to lead to death. One of the most evident 110 MOLAR AGENTS AND PROTOPLASM [Cn. IV results of contact upon protoplasm is modification of move- ment,— momentary quiet, followed by contraction. Rapidly repeated shocks lead to a summation of responses called teta- nus. Slowly repeated shocks may lead to acclimatization to contact. Finally, the direction of locomotion is in some cases controlled by contact ; many organisms move from the touch- ing body — negative thigmotaxis ; others may face the impact of flowing water or keep close, as though attached, to the rigid surface — positive thigmotaxis. If the changed chemical con- dition following contact be called the "response," then all changes wrought by contact on protoplasm may be considered as responses. LITERATURE CHAMPION, P. and PELLET, H. 72. Sur la theorie de 1'explosion des com- poses detonants. Compt. Rend. LXXV, 210-214. DANTEC, F. LE '95. Sur 1'adherence des amibes aux corps solides. Compt. Rend. CXX, 210-213. 28 Jan. 1895. DARWIN, C. 75. (See Chapter I, Literature.) DEWITZ, J. '85. Ueber die Vereinigung der Spermatozoen mit dem Ei. Arch. f. d. ges. Physiol. XXXVII, 219-223. 29 Oct. 1885. '86. Ueber Gesetzmassigkeit in der Ortsveranderung der Spermatozoen und in der Vereinigung derselben mit dem Ei. Arch, f . d. ges. Physiol. XXXVIII, 358-385. 31 March, 1886. DUTROCHET '37. (See Chapter VIII, Literature.) HOFMEISTER, W. '67. Die Lehre von der Pflanzenzelle. Leipzig: Engel- mann. 664 pp. 1867. HORVATH, A. 78. Ueber den Einfluss der Ruhe und der Bewegung auf das Leben. Arch. f. d. ges. Physiol. XVII, 125-134. 21 May, 1878. JONSSON, B. '83. Der richtende Einfluss strb'menden Wassers auf wachsende Pflanzen und Pflanzentheile (Rheotropismus). Ber. D. bot. Ges. I, 512-521. LOEB, J. '90. (See Chapter VII, Literature.) MASSART, J. '88. Sur 1'irritabilite des spermatozoides de la grenouille. Bull. 1'Acad. roy. Belg. (3) XV, 750-754. '91. La sensibilite tactile chez les organismes inferieurs. Jour, de Medecine de Bruxelles. 5 Jan. 1891. [Abstract only seen in Centralb. f. Bacteriol. XI, 566.] '93. (See Chapter I, Literature.) MELTZER, S. J. '94. Ueber die fundamentale Bedeutung der Erschiitterung fur die lebende Materie. Ztschr. f. Biol. XXX, 464-509. PFEFFER, W. '88. (See Chapter I, Literature.) LITERATURE 111 ROSAXOFF, S. '68. De I'influence de 1'attraction terrestre sur la direction des plasmodia des myxomycetes. Mem. Soc. Sci. nat. Cherbourg, XIV, 149-172, Tab. I. STAHL, E. '84. (See Chapter I, Literature.) STRASBURGER, E. '78. (See Chapter VII, Literature.) VERWORX, M. '89. (See Chapter I, Literature.) '92. Die Bewegung der lebendigen Substanz. 103 pp. Jena : Fischer. 1892. '95. Allgemeine Physiologic. 584 pp. Jena : Fischer. 1895. CHAPTER V EFFECT OF GRAVITY UPON PROTOPLASM WE shall consider this subject under three heads : (I) Methods of Study ; (II) Effect of Gravity upon the Structure of Proto- plasm; (III) Control of Locomotion by Gravity — Geotaxis. § 1. METHODS OF STUDY Under normal circumstances gravity acts upon organisms continuously, uniformly, and in one direction only at a time. In this respect it is widely different from most of the agents FIG. 21. — Diagram of the essential part of a klinostat. A rotating block or drum, to which tubes containing the geotactic organisms may be attached in the position indicated. which we have to consider. Since its action is uniform it can be varied only in an indirect way ; i.e. by turning the organism or by replacing gravity in part by a force working in another direction. One of the simplest ways of turning the organism so as to eliminate gravity is by means of the klinostat (Fig. 21). This is made in various forms, and consists essentially of a horizontal rod supported near the ends and made to revolve about its long axis by clockwork. Towards the middle of the rod, or at one end, is rigidly affixed a block to which may be 112 §2] EFFECT OX STRUCTURE 113 fastened, radially, the vessels containing the objects of experi- mentation. When the rod revolves, all sides of the object are brought successively and equally under the influence of gravity's pull. By this means the directive action of gravity is eliminated. ) In the case of organisms living in water, the effect of gravity may be overcome by the buoyancy of the medium. It is clear that an organism floating in a medium of its own weight can- not be affected by gravity. This condition can be brought about by increasing the specific gravity of water by adding soluble substances such as gelatine and gum arabic. Since the specific gravity of the organism tends gradually to change with that of the medium, this method does for rapid experi- ments only. In dealing with larger organisms, which, like slugs, can keep affixed to glass or other smooth surfaces, the inclination of the surface may be varied from a vertical position to a horizontal one, thus varying the active component of gravity. Finally, gravity may be replaced by centrifugal force by rapidly rotating either about a horizontal or a vertical axis. By varying the rate of rotation the centrifugal force will vary, in accordance O 2 with the formula, / = ~ r, in which r is the rotating radius L ' (in meters) and t2 the square of the time of a rotation (in seconds). This varying centrifugal force will act exactly in the same way as gravity, only from the centre of rotation. § 2. EFFECT OF GRAVITY UPON THE STRUCTURE OF PROTOPLASM Very few observations have been made upon this subject, and yet indications are not wanting that the field would well repay working. Thus, where the cell contains specifically heavier and lighter substances the two will be separated by the action of gravity. This occurs in plant cells in which, according to DEHNECKE ('80), various contained bodies, e.g. chlorophyll granules and starch grains, tend to sink to the lower side of the cell. This result is produced in from a few minutes to several hours. This effect is likewise seen in many 114 GRAVITY AND PROTOPLASM [Cn.V ova in which the yolk sinks to the lower pole and the proto- plasm floats on top, in whatever position the egg may be held. This fact undoubtedly has an important effect upon develop- ment, as we shall see later. Of the specifically heavier bodies above referred to, the nucleolus is a striking example, as HERRICK ('95) has recently shown. Thus, when the — • ovary of a lobster is killed, the nucleoli of all the nuclei are found in contact with that part of the nuclear membrane which was the lowest at FIG. 22. — Section through the ovary of a lobster hardened with its dorsal surface (D) upper- most. The nucleoli lie against the ventral surface of the nucleus. Magnified 50 diame- ters. (From HERRICK, '95.) FIG. 23. — Section through the nucleus of a young ovum (£ mm. in diameter) showing the nucleolus, which has, ap- parently, caused a distention of the nuclear membrane by the pressure of its own weight. Arrow shows the direction of the earth's centre. Magnified 248 diameters. (From HER- RICK, '95.) the moment of killing (Fig. 22). The weight of the nucleolus is relatively so great as sometimes to cause a depression in the part of the nuclear membrane upon which it rests (Fig. 23). § 3. CONTROL OF THE DIRECTION OF LOCOMOTION BY GRAVITY — GEOTAXIS * The control of the movements of Protista has been investi- gated chiefly by four naturalists : SCHWARZ ('84), who studied Euglena and Chlamidomonas ; ADERHOLD ('88), who studied So called by SCHWARZ ('84, p. 71). § 3] GEOTAXIS 115 Euglena and desmids ; MASSART ('91), who worked upon bacteria, and ciliate and flagellate Infusoria ; and JENSEN ('93), who experimented with Euglena, Chlamydomonas, and eight species of Ciliata. The observation that led SCHWARZ to his study was that Euglena and Chlamidomonas, shaken up with sand and covered by it, constantly, even in the dark, rose to the surface. The experiments now made by SCHWAKZ to determine the true cause of the pheriornenon were a model of experimental investi- gation. In the first place only fresh and actively moving individuals were used, and light was carefully excluded, either by enveloping the culture vessel in black paper, or by working in a dark chamber. I shall now give in detail the experiments and their results. When the Flagellata were placed in water they responded like those1 in sand — they soon came to the upper surface. But may not this upward movement be purely passive due to the small specific gravity of the algse or to currents in the water ? To get an answer to this question SCHWARZ heated the sand to 70° C — a fatal temperature — and no aggregation occurred. Again, the algae were subjected to vapor of chloroform ; no aggregation. Again, to a low temperature (5° to 6°) ; no aggregation. An aggregation occurred, however, when the temperature of the same culture was raised to 22°. Finally, Lycopodium spores and Euglena in the resting stage do not move upwards ; hence no currents are passing in this direction. On the contrary, these experiments show that the upward movements of the algae are the results of its own active loco- motion. Nor can it be that anything else than gravity determines the direction of the locomotion. That the greater amount of oxy- gen at the upper level is not the controlling agent was shown by smearing the sides of a glass cylinder with a thin layer of sand containing the algae. In this thin layer, permeated by oxygen, they still accumulated at the upper margin. That the locomotion was not directed by currents in the water (Rheo- taxis, p. 108) was indicated by the fact that whether the free end of the tube, at which evaporation is occurring, be up or down, migration is always upwards. Thus, since the stimulus 116 GRAVITY AND PROTOPLASM [Cii. V of chemical agents and currents was eliminated, gravity seemed to remain as the only directing force. It only remained to show that the attractive force of the earth can be replaced by centrifugal force, and this SCHWARZ was able to do by means of the klinostat. By varying the rate of rotation of this machine he varied the centrifugal force and was able to determine the limits within which the Infusoria move against an opposing force. The acceleration of the rota- tion-force may be expressed in terms of the attraction of gravity as a unit by the formula c = -, when c equals the acceleration y of centrifugal force in the required units ; /", the centrifugal force found, as on p. 113 ; and g the acceleration due to gravity. , It appeared from the experiments that, in both living Euglena and Chlamidomonas migration took place towards the central end, thus against the centrifugal force, when the latter was over 0.5 g., and under 8.5 g. Under the lower limit no migra- tion occurred ; near the upper limit aggregation occurred at both ends ; above the upper limit aggregation took place at the peripheral end — that is to say, with the centrifugal force. Clearly, then, geotaxis is in these cases a movement against an opposing force, provided that force is considerable (over 0.5 g.) but not too great (over 8.5 g.). The workers in this field who followed SCHWARZ advanced our knowledge of geotaxis in two principal ways : first, by increasing the number of organisms known to be geotactic, and, secondly, by revealing the fact that closely allied species may have geotaxis of opposite sense. MASSART ('91, pp. 161—167) employed a simple but satisfac- tory method. He placed Protista in a capillary tube which was open, hence equally oxygenated at the two ends. By invert- ing the tube the ends were brought into different relative posi- tions with respect to the earth, causing the geotactic organisms to migrate throughout its length. As a result of his experi- ments it appeared that Spirillum; the flagellata, Polytoma, Chlamydomonas, and Chromulina ; and the ciliata, Anophrys and Euplotes, are geotactic^ The sense of geotaxis may be different between individuals of the same genus ; thus, under similar conditions Spirillum separated into a lot lying at the 3] GEOTAXIS 117 upper part of the tube and a lot at the lower part ; and the individuals of both the upper and lower lot were active. The sense of response depends upon temperature also. Thus Chro- mtilina woroniniana is negatively geotactic at 15° to 20° C., and positively geotactic at 5° to 7° C. The other species men- tioned above are negatively geotactic — i.e. move in the direc- tion opposite to that in which the force tends to carry them. JEXSEX* ('93) finally has greatly extended our knowledge of the spe- cies responsive to gravity, has shown the necessity for regarding carefully the other agents acting during the experiment, and has entered more carefully into the cause of the phe- nomenon than previous authors. \The new forms which JEXSEX worked with were these Ciliata : Paramecium, Urostyla, Spirostomum, Colpoda, Col- ^ ^.-GlaJtubes, about 0.5 pidium, Ophryoglena, and Coleps ; also the more commonly used species, Euglena and Chlamydomonas. The other agents whose action may mod- ify that of gravity are chemical stuffs, density, warmth, light, etc. Light may be easily excluded. yOn warm days the typical geotactic phenomena are often absent, the Paramecia sink- ing to the deeper, cooler layers. The Infusoria aggregate around bacteria in the water, — chemotaxis (Fig. 24, &), — and they shun the uppermost layer, apparently because, owing to evaporation, this layer is denser — tonotaxis (Fig. 24, 0; at any angle! As explained on p. 113, the intensity of gravity's action will diminish as the sine of the angle of inclination of the plate is diminished from 90° to 0°. We determined the deviation of the slug from a vertical position upon plates at various inclinations, and after the lapse of a constant Oo time (45 seconds^ . The 0 So0 experiments were per- J * formed in a dark box. £ The number of tests 3 _0 made at each inclina- 5 tion was sixty. The 1 time required to re- 1 25° spond fully to gravity Q did not vary appreci- ably with the angle of inclination. ^The re- sults obtained indicated that the deviation of the slug from vertical- ity diminished with the cosine of the angle made by the plate/\ The relation between the angular deviation from vertically and the sine of inclination of the glass plate is graphically represented in Fig. 25. The con- clusion from the ex- periments is that the lower limit of the sen- sitiveness of Limax to gravity is extremely small, below 0.13 g., and that as the angle of inclination of the plate diminishes the deviation from 45° towards verticality dimin- ishes in accordance with the relation : S = a- sin 0, in which 90° 80° 70° 60° 50° 40" 30° 20° 10° 0° ANGLE OF INCLINATION OF SURFACE TO HORIZONTAL FIG. 25. — Curves showing relation between the sine of the angle of inclination of a glass plate and the angular position of a slug which was first placed on it in a horizontal position and then left for 40 seconds in the dark. Curve A is constructed by drawing ordinates from the heavy horizontal line, 0°-45°, corresponding to each angle of inclination of the surface (laid off as abscissas) . The lengths of the ordinates are determined by the number of degrees of de- viation of the axis of the slug from 45° towards 90°. 45° is taken as a base, since it would be the mean angular deviation from the initial position of a slug crawling undirected upon a horizontal plate. Curve B is constructed by drawing down from the base ordinates propor- tional to the natural sines of the different an- gles of inclination of the glass plate. 120 GRAVITY AND PROTOPLASM [Cn.V S is the angular deviation of the slug from 45° towards 90°, expressed in degrees ; 6 is the angle of inclination of the plate to the horizontal, and a is a constant. \ In inquiring into the cause of geotaxis in animals it seems best to consider chiefly the phenomenon as exhibited in Protista, for in the higher animals this capacity seems bound up with the possession of special organs of orientation^ { In this group the first and apparently most important part played by gravity is the determination of the axis of the individual, which comes to lie vertical and with the head end up or down according to the conditions of the protoplasm. ' After the positions of the axis and poles are determined, or- dinary locomotion produces the geo- tactic phenomena. 'That gravity may determine a vertical position without locomotion occurring is shown in the ciliate infusorian Spirostomum (Fig. 26), which at times occurs in large numbers in ordinary aquaria, sus- pended almost motionless in mid- water, having a distinctly vertical position and with the head end directed upward.^ They cannot be said to be strictly motionless, since by carefully attending to them one FIG. 26. — Spirostomum ambi- guum, side view, az, ado- ral zone of cilia ; o, mouth ; 08, gullet; n, nucleus; ck, contractile canal; cv, con- tractile vacuole; a, anus. Magnified about 120 diam- eters. (From BUTSCHLI [BRONN'S Thier-reicb : Pro- tozoa], after STEIN.) can see them slowly rising or falling or alternately, perhaps, rising and falling in their almost im- perceptible movements. Miss JULIA B. PLATT, who has studied carefully the movements of Spirostomum, found that of 78 individuals observed all but 7 had the anterior extrem- ity directed upwards and the 7 exceptional individuals were all moving downwards. ( It therefore seems quite certain that Spirostomum tends in water to orient itself with reference to gravity, although without aggregating at the upper surface^ § 3] GEOTAXIS 121 To explain the phenomenon of axis-orientation, two principal theories have been advanced. The first may be called the mechanical theory; the second the response-to-stimulus theory A The first theory is that once suggested by VERWORX ('89, p. 122). It appeared to him that it was self-evident from purely physical grounds that, in complete quiescence of the flagellum, the hinder end of the protist should be directed downwards, and not the anterior flagellum-bearing end. If one conceives such an individual to move its flagellum, which precedes in locomotion, it must move towards the surface of the water; thus against gravity. \VERWORX finds the stimulation theory inconceivable, since gravity cannot even be compared with stimuli. In falling, the body of the protist might rub against the water particles, which would offer a stimulus, but this would be more allied to rheotaxis~7\ It might seem an easy thing to determine whether geotactic Protista artificially rendered quiescent (e.g. killed or stupefied) would stand with their anterior ends uppermost; but the killing is apt to distort the form, and the organisms being heavier than water * fall to the bottom. Something might be gained from an observation of how they fall, but there is very great discordance among authors upon this point, probably in part due to difficulties of observation. Thus SCHWARZ ('84, p. 68) says that both Euglena and Chlamidomonas assume all positions in falling; MASSART ('91, p. 164) finds that Chlamido- monas falls with flagellum directed upwards and JENSEN ('93, p. 451) declares that Euglena viridis killed by iodine falls * Few determinations seem to have been made of the specific gravity of living Protista. JENSEN ('93a) attempted to do this for Paramecia, but his method was bad and his results bad likewise. He made solutions of potassium carbonate, of varying specific gravity, and found that Paramecium just floats in a solution whose sp. gr. is 1.25. The difficulty of the method is that solutions of salt having a relatively small molecular weight act so powerfully in withdrawing water from the organism as to cause it to shrink and increase in relative weight. Miss PLATT has used solutions of gum arable whose osmotic action is so slight that organisms live in it for hours. In such solutions, paralyzed but living Spirostoma and Paramecia neither sank nor rose when the specific gravity was between 1.016 and 1.019 ; so that it seems probable that the specific gravity of Infusoria lies near 1.017. Tadpoles recently hatched and having a length of 9.5 mm. had a sp. gr. of 1.044, while those 12 mm. long had a sp. gr. of 1.017. 122 GRAVITY AND PROTOPLASM [Cn. V almost without exception with the broader flagellate pole down- wards, f Both' from the fact that it can be easily demonstrated that when a body heavier than water falls in that medium its larger end will precede, and from the fact that JENSEN was especially careful that the killed organism should not be deformed, his results must be considered the best established.^ VNow, since the dead Euglena tends to sink with .flagellum downwards whereas the active Euglena stands flagellum upwards, we must conclude that the orientation of Euglena and probably other Protista is not passive but due to their activity and must be regarded as a response more or less directly due to gravity.^ (But just how does gravity act as a stimulus to determine the direction of orientation of the body? We have two principal theories to examine. (First, that of JENSEN, that gravity acts indirectly on the organism by directly causing a difference in pressure in the water at different levels. \ This difference in water pressure, at various levels, affects directly the two poles of the organisms, which stand at different levels, and the organism responds to this difference in pressure. (Th& second theory, which I adopt, is that the organism, owing to its specific gravity being greater than the medium, experiences greater h resistance (friction -f weight) in going upwards even to the slightest extent than in going downwards (friction — weight). (Another stimulus, which is probably associated with this, de- pends upon the fact that an unsymmetrical body, heavier than water, tends to fall with its larger end downf^ Those nega- tively geotactic- organisms, which stand with their larger end up, will be consequently in a condition of unstable equilibrium; those organisms which stand with their larger end down will be in stable equilibrium. In the first case a deviation from verticality would be accompanied by relatively diminished resistance on one side ; in the second by relatively increased resistance on one side. In either case, the distribution of the mass of the animal may give the organism the means of deter- mining, but not in a mechanical way, the position of its axis. (The evidence for the first theory JENSEN finds especially in a fact which he believes opposes the second. Negatively geotactic organisms, placed in an inclined tube, move towards GEOTAXIS 123 upper side and then travel obliquely, not vertically, along toward the upper part of the tube, thus into strata of con- stantly diminishing pressure.^) If weight controlled in any way their movements, they should move vertically as from 1 to 2 (Fig. 27) until they meet the side of the glass. Then they should move off, as to 3, then vertically to 5, and so on. Since they do not so move, gravity, JENSEN thinks, cannot be said to act directly. In criticism of this conclusion it may be urged that it is without proper foundation, for if an organism whose irritability (in- stincts) would lead it to move verti- cally is mechanically unable to do so exactly, it will do so as far as practi- cable. This observation cannot, there- fore, be said to militate against the second theory. Finally, there is this positive objection to JENSEN'S theory that it is applicable only to geotaxis in water animals, and can therefore be only a special explanation of geotaxis. f On the other hand, there is evidence which is opposed to the first theory and favors directly the second^ And JEN- SEX has himself contributed some of this evidence. He put Urostyla into a glass tube containing a 0.5% aque- ous gelatine solution. They showed no tendency to go upwards. At the expiration of 20 hours many deaths had occurred, but some normally ac- tive individuals were still at the lower end of the tube. Why this loss of geotaxis ? JENSEN believes it due to the fact that the difference in pressure of the successive layers did not increase proportionally to the increase in resistance of the solution.^ I would suggest that it may be due to the fact that the weight of the body of the Protista is now relatively less than that of the solution, so that the organism, tending to move against resistance, comes to lie at the bottom of the buoyant fluid, hence appears positively geotactic. FIG. 27. — Hypothetical line of migration of Parame- cium in an inclined tube, upon the assumption that gravity acts directly to determine direction of locomotion, according to the conception of JENSEN. The arrow at p indicates the direction of the pull of gravity; 1, 2, 3, 4, 5, successive positions occu- pied by the Paramecium. (From JENSEN, '93.) 124 GRAVITY AND PROTOPLASM [Cn. V ( Geotaxis in the higher organisms, especially Vertebrates, cannot here be discussed at length. It is sufficient to state that as LOEB ('91, p. 189) concludes, it is probably dependent upon the internal ear. Miss PLATT has, at my suggestion, sub- jected young negatively geotactic tadpoles to solutions of gum arabic of the same specific gravity as themselves, and has found that they still migrate upwards. This result makes it probable that here also orientation is effected by the internal ear, and hence is independent of the action of gravity upon the entire body?) ( Finally must be mentioned, the phenomenon of acclimatiza- tion to a central pressure, ^his has been observed by JENSEN ('93, p. 470), who says, when Paramecium or Urostyla has been strongly " centrifugated " towards the peripheral end of the tube, where it is subjected to a high pressure, it shows, when the tube is then placed vertically, a much livelier geotaxis than it would have done without " centrifugating." Clearly the temporary action of the high pressure has increased the irritability to gravity?^' 1 To recapitulate : Gravity affects the structure of protoplasm by separating the lighter and heavier substances. It may determine the direction of locomotion by determining the ver- ticality of the axis of the body. Varying the intensity of gravity's attraction diminishes the precision with which this determination takes place. The determination of the vertical position is, in the lower organisms, probably due to difference in ease of movement when going up and going downX LITERATURE ADERHOLD, R. '88. (See Chapter I, Literature.) DEHNECKE, C. '80. Ueber nicht assimilirende Chlorophyllkorper. Inaug. Diss. Koln. Abstr. in Bot. Ztg. XXXVIII, 795-798. Also in Bot. Centralbl. I, 1537. HERRICK, F. H. '95. Movements of the Nucleolus through the Action o-f Gravity. Anat. Anz. X, 337-340. 8 Jan. 1895. JENSEN, P. '93. Ueber den Geotropismus niederer Organismen. Arch, f . d. ges. Physiol. LIU, 428-480. 5 Jan. 1893. '93a. Die absolute Kraft einer Flimmerzelle. Arch. f. d. ges. Physiol. LIV, 537-551. 24 June, 1893. LITERATURE 125 LOEB, J. '88. Die Orientirung der Thiere gegen die Schwerkraft der Erde. (Thierischer Geotropismus.) Sb. Wiirzb. Phys.-med. Ges. '90. (See Chapter VII, Literature.) '91. Ueber Geotropismus bei Thieren. Arch. f. d. ges. Physiol. XLIX, 175-189. 1891. MASS ART, J. '91. Recherches sur les organismes inferieurs. III. La sensibilite a la gravitation. Bull. 1'Acad. roy. Belg. (3) XXII, 158-167. 1891. SCHWARZ, F. '84. Der Einfluss der Schwerkraft auf die Bewegungsrichtung von Chlamidomonas und Euglena. Ber. bot. Ges. II, 51-72. VERWORN, M. '89. (See Chapter I, Literature.) CHAPTER VI EFFECT OF ELECTRICITY UPON PROTOPLASM IN this chapter we shall consider (I) some methods em- ployed in the investigation of this subject ; (II) the effect of electricity upon the structure and general functions of pro- toplasm ; and (III) the effect of electricity in determining direction of locomotion — electrotaxis. A § 1. CONCERNING METHODS While the phenomena of magnetism and electricity are closely allied, their effects upon protoplasm seem to be widely dis- similar. Thus no certain action of magnetism has hitherto been observed, but electricity, however produced, causes nearly uniformly an effect?) ^ Any experimental work with the electric current involves apparatus for its production, application, and measurement ; namely, batteries or other sources of electricity ; electrodes for applying the current to the organism ; troughs to contain the free swimming animals used for experimentation ; a galvanom- eter for measuring the current ; a rheochord for varying the intensity of the current ; a reversing key ; and, for interrupted currents, an induction machine with interrupter, and an elec- trometer for measuring such currents. A description of the principal forms of these instruments and the methods of con- structing some of them will be found in VERWORN, '95, Chapter V, and in OSTWALD, '94, Chapter XV. Since the works just named are easily accessible, it will be unnecessary here to describe these instruments in detail. A few additional suggestions, the result of my experience, may, however, be found helpful. Concerning latteries, first; accumu- lators are without doubt to be preferred, where practicable, on 126 • 1] METHODS 127 account of the strength and continuance of their currents. In other cases, CLAKK or DANEELL elements, if enough of them are united in series, will meet the requirements. The character of the electrodes, next, will depend upon the nature of the in- vestigation. Xonpolarizable ones of hair (camel's-hair brush), clay, or paper (plug of filter paper in glass tubing drawn out to a cone) are usually employed, but all of these offer consider- able resistance. The troughs will vary in form and size with the organisms to be contained in them ; some of them will be described in connection with the experiments in which they have been employed. (They are all rectangular enclosures having clay ends when it is desirable that these should be nonpolariz- able^j /For large troughs, sheet-zinc electrodes are used, cover- ing the smaller sides of the trough.) Although some of the reflecting galvanometers are more sensitive, a " millammeter " such as that made by the WESTON Electrical Works is a much more convenient instrument and sensitive enough for most work of this sort. The rheochord is practically a low-resistance box, capable of indefinitely fine gradations. This is introduced into the short branch of a divided circuit, so that by varying its resistance a varying share of the current shall be forced into the longer circuit. A very simple and excellent device for altering the strength of current is the " Compression-rheostat n of BLASIUS and SCHWEIZER ('93). This consists of a piece of rubber tubing filled with zinc sulphate, stopped at the ends and introduced into the circuit. By means of a thumbscrew the walls of the middle of the tube may be pressed together, the lumen correspondingly reduced, and the resistance increased. The induction apparatus usually employed is one invented by DU BoiS-REYMOND. In this the secondary coil may be with- drawn from the primary coil to any desired distance, thereby diminishing the intensity of the induced current. Through the action of such an instrument the current is alternately made and broken, and each electrode becomes in quick suc- cession anode and kathode. Since alternating currents cannot be measured by an ordinary galvanometer, an electrometer must be employed. So much concerning apparatus. ' A word should be said about the method of stating the cur- rent employed?) Very many authors have been satisfied with 128 ELECTRICITY AND PROTOPLASM [Cn. VI saying that the current was strong or weak, others have given the kind and number of elements employed. Such statements are wholly inadequate to give an accurate idea of the strength of current to which the organisms under experimentation were subjected. (Even merely to state the galvanometer reading in milliamperes is insufficient^ We must know as nearly as pos- sible what strength of current is passing through the organism, and this involves knowing the density of the current passing through the water in the trough. Now it is obvious that a current passing through a mass of water of small cross-section is stronger per square millimeter than an equal current dis- tributed over a large cross-section. It is necessary, conse- quently, to know the cross-section of the mass of water through which the stimulating current is passing, in order to determine the " density " or strength at any point. For technical pur- poses the unit of current-density is taken at 1 ampere to the square millimeter. HERMANN and MATTHIAS ('94, p. 394) propose for physiological purposes a unit one-millionth as great, to be designated as S. & then indicates a current of yoVo milli- ampere per square millimeter of cross-section. It is very de- sirable that, when practicable, currents should hereafter be expressed in 8's. More than one useless discussion has been precipitated by not giving a sufficiently accurate quantitative expression to the current employed. (See, for illustration, below, p. 149.) Finally, the strength of current necessary to produce a certain result depends upon the relative conductivity of the organism and the surrounding water. If, through the presence of substances in solution, the conductivity of the water is abnormally great, one must use a greater current (as read off from the -galvanometer) than otherwise to produce a certain effect. (WALLER, '95, p. 97.) It would probably be best, when possible, to use in the trough the water in which the organism has been living, since the quantity of salts in the organism has been shown to vary with that of its medium. (See p. 88.)* * See KAISER, Wien Akad. CIV, p. 17, 1895, for a new trough adapted to the stage of the microscope. ] EFFECT ON STRUCTURE AND FUNCTIONS 129 § 2. THE EFFECT OF ELECTRICITY UPON THE STRUCTURE AND GENERAL FUNCTIONS OF PROTOPLASM The fundamental phenomenon of the action of an electric current upon protoplasm may be seen while watching a helio- zoaii (Actinosphaeriuni), lying in a drop of water, through which a weak, constant current is "made." We find that the filamentous pseudopodia begin quickly to retract at the two poles lying in the axis of the current ; and as the current continues, this contraction continues likewise. The primary effect of a weak constant current is thus a centripetal flowing of the protoplasm. The current stimulates to contraction. If, now, the current be increased, or be longer continued, further changes occur. The pseudopodia lying in the current become varicose, and break up into a chain of drops; the vacuoles on the periphery begin to burst, emptying out their fluids; and in these regions the protoplasm collapses. Thus, the stronger current produces continued contraction, accom- panied by collapse of the protoplasmic foam-work. Finally, the plasma itself begins, upon the anode side, to dis- integrate, and the loose particles to move towards the positive electrode. As the plasma of this side is gradually eaten away, the outline of the Actinosphserium passes through phases like those of the waning moon, until, finally, the last thin crescent fades away. The particles of the mass have wholly lost their cohesion (Fig. 28). The facts just given concerning the behavior of Actino- sphaerium to the constant current are gathered from the observa- tions of KUHNE ('64, p. 59) and VERWORN ('89a, pp. 8, 9). Fundamentally similar observations have been made by KUHNE ('64, p. 79) and VERWORN ('89b, p. 274) on Myxomycetes, and by VERWORN ('89% pp. 13, 17) on the rhizopods, Poly- stomella and Pelomyxa. So these data may be considered as of general worth for naked protoplasm. Also upon ciliated epithelium, the constant current acts as a very strong excitant, producing an active movement in cilia which had previously nearly ceased to beat. This excita- tion occurs, especially about the two poles, immediately upon •• making " the current. (KRAFT, '90, pp. 234, 235.) 130 ELECTRICITY AND PROTOPLASM [Cn. VI FIG. 28. — Actinosphserium eichhornii in four successive stages of polar excitation by means of the constant electric current. Disintegration begins at the anode (+) pole. (From VERWORN, '95.) Allied, apparently, to the foregoing phenomena are the pro- toplasmic changes which follow the sudden breaking of the current. Unless the current has been very feeble, the pseudo- podia of Actinosphserium begin, at the moment of breaking, to contract and become varicose upon the kathode side, while the formerly irritated anode side is quiet. Thus, the breaking of the current also acts as a stimulus, but this is, in general, weaker than that caused by making. If, now, a current which endures for only an instant — if a single induction shock — is sent through, the making and break- ing stimuli are practically coincident, and a violent response may be called forth. Thus, ENGELMANN ('69, p. 317) found that Amoeba, subjected to a strong shock, retracted its pseudo- podia, and assumed a spherical form within two seconds ; and GOLUBEW ('68, p. 557) has described a similar response in leucocytes. Under similar circumstances, the flagellum of the flagellate Peranema (Fig. 29) made an energetic stroke. (VER- WORN, '95, p. 414.) I have spoken above as though there were both a making and a breaking stimulus ; but this is not known to be the case. It is generally recognized from experiments I EFFECT ON STRUCTURE AND FUNCTIONS 131 on muscle, that it is the " making " only of a single induction shock which produces the response ; but VEKWORN ('89% pp. 19-22) has found that in the rhizopod Pelomyxa it is, on the contrary, the breaking excitation which causes the response. The subject deserves further study. Finally, the effect of an alternating current must be con- sidered. This current is characterized by the fact that it is composed of a series of rapidly repeated instantaneous shocks spK&Vs* S^o^S^'^ ?. ?. : FIG. 29. — Peranema. a, quietly swimming; b, irritated by an induction stroke. (From VERWORX, '95.) FIG. 30. — Actinosphaerium eichhornii, STEIN. Showing effect of the alternating current. At both poles the pseudopodia are undergoing a disintegration, which proceeds equally at the two poles. (From VERWOBN, '89.) which alternately reverse their direction. Thus, each pole of the organism subjected to such a current receives alternately the making (or breaking) effects at anode and kathode. The maximum action is thus obtained. When an Actinosphserium is stimulated by such a current, the pseudopodia at both poles contract and become varicose ; and, finally, the protoplasmic substance begins to disintegrate and to flow out from the cell towards the two electrodes, until the body acquires a biconcave form. (VERWOKN, '89% p. 11.) In this case the disintegra- OF THF " "\ UNIVERSITY ) 132 ELECTRICITY AND PROTOPLASM [Cn. VI tion takes place at both poles, since both are, alternately, anodes (Fig. 30). Similar effects have been observed in other cases. Thus, when an amoeba is subjected to an alternating current, it be- comes spherical ; the protoplasmic streaming of the plasmodia of a myxomycete ceases, and, with stronger currents, the whole mass contracts, water being forced out.^ Finally, an attempt at a similar re- sult is seen in the stamen- hair cells of Tradescantia, in which, under stimulation, the protoplasmic threads segre- gate into irregular or sphe- roidal clumps. (KtiHNE, '64, pp. 30, 31, 75, 99.) In all these cases we see that the action of a violent current, like repeated contact, leads (as ENGELMANN, '69, p. 321, has suggested) to results which can be accounted for on the ground of reduced cohesion, — first, tendency to spherical aggregation, and, finally, disintegration (Fig. 31). After having studied the effect of the electric current upon Protista and simple cells, it remains to consider, very briefly, its effect upon muscle and upon nerve. Since CALDANI discovered, in 1756, that frogs, shortly after death, could be stimulated to movement by frictional electricity, and GALVANI and VOLTA, towards the end of the last century, discovered, by the same response, the phenomenon of galvan- ism-, these tissues have frequently been made the subject of careful experimentation. It has been shown, not merely that FIG. 31. — One of the cells of a stamen hair of Tradescantia virginica. A, unstimu- lated; B, stimulated by an induction current. At a, b, c, d, the protoplasm has aggregated into drops and clumps. (From VERWORN, '89, after KUHNE, '64.) §2] EFFECT ON STRUCTURE AND FUNCTIONS 138 the nerve can be stimulated to its functions, but that muscle from which the activity of the nerve has been excluded by the use of curare (which inhibits the action of the nerve, but not of the muscle), will contract upon the passage of a current.) Upon the character of the current, however, depends that of the response ; thus, although, as we have seen, a closed constant current continues to stimulate Protista, it has been said not to stimulate nerve or muscle. A contraction follows, it has been maintained, only upon considerable variations in the electrical condition, such as result from making or breaking the current. It is probable, however, that there is not so great a difference in responsiveness of muscle and Protista as would seem to be implied, for BIEDERMAXN ('83) has shown that the constant current produces a whole series of slight contractions in muscle which cannot be regarded merely as a secondary result of the making shock; and FICK ('63) has observed contraction due to the constant current in muscles of Lamel- libranchia. So that even in muscles, there is an actual, though weak, response to a steady, constant current, j There are two phenomena following momentary shocks ap- plied to muscles which deserve notice in passing. First, when a single induction shock is passed directly through a muscle, we notice that the contraction is not simultaneous with the shock, but follows only after the lapse of a certain ''latent period." This latent period represents, it is believed, time spent in transformations going on in the plasma preparatory to contraction. Secondly, when we pass (especially in a muscle- nerve preparation) a series of induction shocks, closely following one another, as in the alternating current, a very violent con- traction is produced, since the new shock comes to the muscle before it has had time fully to relax, and causes a contraction of the already contracted tissue. Thus stimulus is superimposed upon stimulus, and a summated response (tetanus) takes place. » We must now consider more carefully a subject to which we have hitherto merely alluded, namely, the relation to the electrodes of the point of the organism at which the response first appears. Thus, when the amoeboid Pelomyxa is subjected to the constant current, a contraction appears, at the time of 134 ELECTRICITY AND PROTOPLASM [Cn. VI making the current, at that pole only which is turned towards the anode. When the current is broken, on the contrary, a contraction occurs at the kathode, the pseudopodia next the anode becoming quiet. (VERWORN, '89a, p. 19.) This relation may be expressed in tabular form as follows : — AT ANODE. AT KATHODE. Upon making excitation rest Upon breaking rest excitation All Protista do not, however, according to VERWORN, respond in the same way as Pelomyxa ; thus, with the constant current of a certain intensity, he got in both Polystomella crispa, and Actinosphserium, the following reaction : — AT ANODE. AT KATHODE. Upon making excitation rest Upon breaking rest rest It must be said, however, that the reactions obtained in any case are dependent upon the strength of current employed ; thus, with a stronger current, the following result was obtained with Actinosphyerium : — AT ANODE. AT KATHODE. Upon making . excitation excitation Upon breaking rest excitation A comparison of the last two tables seems to indicate that, very probably, with a current intermediate between the weak and the strong current employed, we should get a result like that obtained with Pelomyxa. At any rate, we may say that all these cases tend to group themselves about the Pelomyxa for- mula ; — making : anode, excitation ; kathode, rest ; breaking : anode, rest ; kathode, excitation. A brief designation of this type is desirable. Since the condition at the anode upon § 2] EFFECT ON STRUCTURE AND FUNCTIONS 135 making is distinctive, we may, call this the anode-excitation tyr>e, or, briefer still, anex type. C Turning, now, to nerve and muscle tissue, we meet with a type of response, on making and breaking the current, alto- gether irreconcilable with this. As is well known, when a constant current is made or broken, all the tissue lying between the electrodes is not stimulated at one time, but the excitation makes its appearance at the anode or kathode, and thence is transmitted to the other pole. One can demonstrate this on slow-moving (e.g. extremely tired or dying) muscle at the extremities of which the electrodes are placed. The contrac- tion begins at one electrode, and travels towards the other. By using more refined methods, this relation, which holds for nerves, striated and smooth muscle (cf . ENGELMANN, '70, p. 302) has been formulated as follows : — AT ANODE. AT KATHODE. Upon making T6St 6xcit3/tion Upon brGctkin0" sxcitcition l*6St This is seen to be the very opposite of the response given by Pelomyxa. It may be called the kathode-excitation type, or, in brief, the katex type. Having now seen that two fundamentally different types exist in the response of the two extreme groups of the animal king- dom, the question arises, what is the distribution of these types amongst the intermediate forms — the Invertebrate Metazoa? j Fortunately, through the investigations of NAGEL ('92 and '92a), we have data upon this subject. In NAGEL'S experiments, the whole animal was employed, the two electrodes were placed at the opposite ends of its long axis, the metallic circuit was then made or broken as required, and the pole (anode or kathode) at which contraction first occurred was noted. Thus, NAGEL found that when the current was made through the sea-hare, Aplysia, there was strong excitation and momentary retraction of the parts next to the ariode, while next to the kathode the body showed a considerably weaker contraction. Upon break- ing the current, there was some excitation of the parts of the 136 ELECTRICITY AND PROTOPLASM [Cii. VI body next to the kathode, but none at the anode end. The result that one obtains depends, ho\vever, to a certain extent, upon the strength of the current that one employs. But NAGEL did not, apparently, measure his currents, so there is no cer- tainty that his results can be at once duplicated. Taking the results for various Invertebrates as they are given, however they are instructive. TABLE XIII UPON MAKING. UPON BREAKING. AT ANODE. AT KATHODE. AT ANODE. AT KATHODE. LinmsBus . . excitation rest rest excitation Planorbis . excitation rest rest excitation Aplysia punctata . . excitation > excitation rest excitation Schseurgus (octopod) excitation > excitation rest excitation Helix hortensis . . . excitation > excitation rest slight excitation Ciona intestinalis . . excitation > excitation rest rest Janus cristatus . . . excitation = excitation rest slight excitation Pleurobranchia . . . excitation = excitation * rest slight excitation ISTassa reticulata . . . weak excit. rest rest rest f All the species in this table (all of which, except Ciona, are Mollusca) show in their response a more or less close approach to the type of Pelomyxa (excitation, rest ; rest, excitation) ; and we may believe that with appropriate stimulus they would re- spond in precisely that way. In a second class of cases the response of the whole animal belongs to the katex type; thus two species examined by NAGEL showed the following responses : — TABLE XIV UPON MAKING. UPON BREAKING. AT ANODE. AT KATHODE. AT ANODE. AT KATHODK. Pagurus striatus . . Triton cristatus . . . rest excitation < excitation excitation excitation rest * Inconstant in occurrence. §2] EFFECT ON STRUCTURE AND FUNCTIONS 137 These are representatives of the groups Crustacea and Ver- tebrata. Among all the species studied by NAGEL there was only one which gave results not easily assignable to either of the two types. This organism is the larva of a dragon-fly, JEschurea. Its formula is excitation = excitation ; rest, rest. NAGEL says, however, that these results were uncertain and variable. In some other groups studied — Ccelenterata and Echinoder- mata — the current used provoked no response at either pole ; while with Amphioxus the current employed produced excita- tion at both poles on both making and breaking the circuit. Such variations as these, are, however, easily accounted for on the ground that different species require currents of different strengths to call forth what may be termed the typical response. Not merely between different groups do we find a difference in the type of response, but even inside the group of Protozoa dissimilarity has been shown to occur. Thus VERWOKX ('896, p. 301) has given reasons for believing that three flagellate species, the ciliate Opalina, and some bacteria belong to the katex type, although as just stated (p. 133) other Protozoa exhibit the anex type of response. Finally it appears that individuals of one and the same species subjected to different intensities of current may give rise to responses belonging to the opposite types. Thus when a medium current is " made " through Triton cristatus the exci- tation is greater at the kathode than at the anode ; but when the weakest current is employed a making response occurs at the anode only, and when a slightly greater intensity is used the continuance of the current provokes a continuance of the exci- tation at the anode. (XAGEL, '92% p. 341.) Likewise the reaction of Vertebrate muscle varies with its internal condition. Thus degenerated or over-stimulated muscle shows predominat- ing anode stimulation on making the current ; and transverse stimulation of the muscle fibre gives the same result.* (To sum up, two principal types of response to the electric current may be distinguished : the first or anex type character- izing most Protozoa, Mollusca, Vertebrates (slightly stimulated), * For a discussion of these cases see VERWOEN ('89a, p. 24). 138 ELECTRICITY AND PROTOPLASM [Cn. VI and weak muscle fibres ; the second or katex type found espe- cially among some Flagellata, Arthropoda, and Vertebrata. A third possible type (katanex type) is certainly of very limited distribution. Between the two types we notice this connecting link, that in some Vertebrates a weak current produces one type of response ; a strong current the other. The reason for the existence of these two distinct types — one of which char- acterizes animals with less differentiated, the other those with more differentiated, muscular and nervous systems — is still greatly in need of investigation. The nature of the protoplasmic change wrought by the current is an important matter. We have already accounted for the effect that we see in the Protista, on the ground of re- duced cohesion. It is probable also that the current gives rise in the cytoplasm to chemical changes which are different at the two poles. It is well known that when a current is passed through a neutral solution of a salt there is produced an acid at the anode, and an alkali at the kathode. Since in the higher animals, at least, the body contains such solutions, it seems probable that acid and alkaline substances are here likewise produced by the passing current. This probability is supported by an observation of KUHNE ('64, p. 100), who found that the violet coloring matter of the stamen hairs of Tradescantia v become changed by the action of a very strong induction shock. He says that a change in the violet fluid, like that which occurs at the anode, can be brought about by dilute hydrochloric acid ; while a change like that appearing at the kathode can be pro- duced by potassic hydrate. An observation of NAGEL ('92a, p. 346) suggests also that the current acts by producing a chemi- cal change. He finds that that part of the body of the snail and the leech which shows most markedly the anode-making excitation is coincident with that which is most sensitive to chemical substances. So that the reaction to a galvanic, still more to a faradic, stimulation resembles that to a strong, dis- agreeable taste (quinine). In this case the response may result either from the chemical substance acting directly on the muscles or attacking first the sense organs. In the latter case the response would occur through the mediation of the nervous system? „ EFFECT OX STRUCTURE AXD FUXCTIOXS 139 So far then we have distinguished two chief effects of the current on protoplasm — a dissociation effect and a chemical effect. It may now be worth while to mention that there is good reason for believing that in the more highly differentiated animals, like Vertebrates, not all protoplasm is affected to the same degree nor in the same way. Thus a nervous and a muscular effect can be clearly distinguished in frogs, for ex- ample. The principal effect is exerted upon the central nervous system, for HERMANN ('86, p. 415) found that the tail of tadpoles is responsive only so long as it contains a piece of spinal nerve ; and upon frogs subjected to curare, which inhib- its the action of the nerve alone, the current produces a much- diminished effect, giving rise merely to muscular twitchingSo (BLASIUS and SCHWEIZER, '93, p. 528.) Very little progress has been made, however, upon the determination of the action of different intensities upon the different tissues of which the Vertebrate body is composed. We have seen that the electric current provokes a response, and we have seen also that organisms vary in their responsive- ness so that a current strong enough to call forth a response in one species is not sufficient to excite another species. We may say that the one species is attuned to a different strength of current from the other. This difference in responsiveness indi- cates, of course, a corresponding difference in composition of the protoplasm. Such a difference may, moreover, be produced in a single individual by artificial means. These means are the subjection for a considerable period to the electric current. Suppose we subject an organism to a current of a strength only slightly greater than that just necessary to provoke a response. After the current has acted for some time we find that it no longer excites. This phenomenon of acclimatization to the galvanic current was first observed among the Protista, so far as I know, by KUHXE ('64, pp. 76, 78), who found that in Mvxomycetes, after a few induction shocks had been sent through the plasmodium, additional shocks of the same intensity were without effect, and stronger shocks had to be sent through to cause contraction. Similar results were obtained by VER- X ('89% p. 10; '89b, p. 272) in subjecting Actinosphserium 140 ELECTRICITY AND PROTOPLASM [CH. VI and Amoeba to a weak constant current. At first the pseudo- podia on the anode side plainly retracted, but later ceased to do so, and, finally, the current still passing, the retracted pseudopodia began to extend again. The action of the current so modified the protoplasm as to change the attunernent of the organism. § 3. ELECTROTAXIS In studying the subject of aggregation with reference to the electric current, we shall consider first the simplest case of this phenomenon as it is exhibited in Amoeba ; then pass to the more complex forms of Protista, especially the Ciliata, and after FIG. 32. — Galvanotaxis of Amoeba diffluens. A, Amoeba creeping, unstimulated ; B, after closing of the constant current. The arrow indicates the direction of locomotion. (From VEBWOBN, '95.) that to the Metazoa. After dealing with the phenomena we must attempt to explain them. When such an amoeba as that shown in Fig. 32, A, is sub- jected in a drop of water to the action of a weak constant current, as already indicated (p. 129), it contracts, especially upon the face turned towards the anode. If the current is not strong enough to produce disintegration at that pole, but only repeated contraction, and if meanwhile the kathode pole retains its power of throwing out pseudopodia, the amoeba must grad- ually move from the anode (Fig. 32, jB), and if several Amoebae are under the cover-glass, they will eventually aggregate about the kathode. Here we have, then, in its simplest form, a case of electrotaxis, and, since the organism moves toward the negative electrode, we may call it negative electrotaxis. If now, instead of an amoeba, we watch a free swimming flagellate Infusorian, — Trachelomonas hispida (Fig. 33), — we § 3] ELECTROTAXIS 141 see the long- flagellum which precedes in locomotion coming to lie in the current and directed towards the kathode, so that the animal migrates in that direction. The following explanation of the observed fact that the flagellum becomes directed towards the kathode has been offered by VERWORN ('89b, p. 298). ^The flagellum and the pole from which it arises constitute the most sensitive end of the body. When the flagellum is stimulated it beats violently, and since it is stimulated most when turned towards the anode, it beats most violently when in this attitude. A position 180° from this is one of comparative rest. In interme- diate positions the degree of stimulation is intermediate. After a f CAV strokes the body will " naturally " come to assume and to retain that position in which the flagellum is least stimulated. More detailed still is our knowledge of electrotaxis among FIG. 33. — Trachelomonas hispida, swimming towards the kathode (— ) upon closure of the current. The arrow shows the direction of locomotion. (From VER- WORX, '89.) the ciliate Infusoria. The authors who have worked upon this group are chiefly VERWORX ('89* and '89b) and LUDLOFF ('95). The work of the former shows that the phenomenon of electrotaxis is exhibited by many species, especially Para- mecium aurelia and P. bursaria, Stentor coerulens and S. polymorpha, Pleuronema chrysalis, Opalina ranarum, Bursaria truncatella, Halteria grandinella and Stylonichia mytilus. LUDLOFF employed only Paramecium, but studied it much more completely, especially using various currents of known relative intensity.* He found that the precision with which * LUDLOFF employed a trough with wax walls, clay ends, and glass bottom, and used brush electrodes. The intensities of current given by him are the readings of the galvanometer. The cross-section of the water mass in which the Paramecia were, and over which the current spread itself, is not exactly given, but was probably about 20 sq. mm. If we employ the unit of strength recommended by HERMANN and MATTHIAS (p. 128), namely, 1 one-millionth of 142 ELECTRICITY AND PROTOPLASM [Cn. VI the Paramecia aggregated at one pole was determined by the strength of the current, as follows : A current of 3 8 caused in general a movement towards the kathode, although many individuals appeared not to be affected by it. In 20 seconds the anode end of the fluid was almost free from Infusoria. With currents of 6 $ and 15 8 the aggregation at one pole be- came more complete and took place in a short time. Indeed, there was a relation found between the time required for f— ^ o^uicon^oiOi-^i RELATIVE RATES OF MIGRATION 1 \ / \ / \ / f \ 1 v \ \ \ ^* ^ 5 ^v. 10(5 20(5 30d 40(5 50(5 STRENGTHS OF CURRENTS 60(5 70(5 FIG. 34. — Curve showing relation between strength of currents and relative time elapsing before Paramecia have aggregated at the kathode. The ordinates are measured by the reciprocals of the number of seconds elapsing ; the abscissae, by the strength of current in 5's. aggregation at the kathode and the strength of current employed, which is instructive, and is given above in graphic form (Fig. 34). This curve shows that as the current increased from 3 8 to 21 8 the rapidity of aggregation increased, but as the current increased still further this rate diminished until locomotion nearly ceased at above 60 8. The intensity, therefore, of 21 8 produced the most rapid movements. opening the current, the Infusoria in all cases swim, \ Upon 1 ampere per sq. mm. (designated 5), then we must divide LUDLOFF'S galva- nometer readings (given in milliamperes) by Tf£o» or (which is the same thing) multiply them by 50. That will give us the current in 5's per sq. mm. All of the numerical data given in the text have undergone this operation. B] ELECTROTAXIS 143 rapidly for a moment, towards the opposite pole (anode), but then quickly begin to redistribute themselves throughout the water. This redistribution has occurred in about 20 seconds FIG. 35. — Apparatus for studying electrotaxis of Paramecium. A rectangular trough, whose ends are of clay and sides of wax, is built upon a glass plate. The current is applied by means of brush electrodes. The direction of the migration of the paramecia is indicated by the arrow. They move towards the kathode. (From VERWORX, '95.) after the current is broken, and the time is independent of the strength of the preexisting current. The movement of the Infusorian from one pole to the other takes place along the lines of flow of the current. If the ter- minals are two parallel plates, these lines are about parallel (Fig. 35); if they are two points near the opposite sides of A B FIG. 36. — Curves made by Paramecia in its galvanotactic response when pointed electrodes are used in the drop of water. A, beginning of migration • B, com- plete aggregation. (From VERWORN, '95.) a water drop, the lines have the direction of the lines made by iron filings scattered on a plate over the two poles of a magnet (Fig. 36). Besides this path of general migration, the form of the path followed by individuals varies with the current. Normally Paramecium moves in a long spiral. As the current is in- 144 ELECTRICITY AND PROTOPLASM [Cn. VI creased, however, this spiral becomes shorter, i.e. has more turns per centimeter of progression from one pole to the other \ FIG. 37. — Form of the path of Paramecium under different conditions, a, when not subjected to the constant current ; &, when subjected to a slight current; c, when subjected to a still stronger one. (From LUDLOFF, '95.) (Fig. 37), until at 6.0 S, in making one turn of the spiral, the organism progresses hardly more than its own length. Finally, the effect of the current upon the movement of the cilia must be considered.* In the resting Para- mecium the cilia rise perpendicularly from the surface of the body (Fig. 38). If an individual stands with its ante- rior (blunt) end towards the anode, and a current of 8 8 passes through, the cilia at the posterior (kathode) end begin to vibrate. If the individ- ual lies transverse to the current and the current is closed, the cilia on the kathode side vibrate, those on the a cur_ FIG. 38. -Paramecium, show- anode gide being. ing position of cilia when . . unstimuiated. The blunt rent of 16 o one can see that the kath- end is anterior. (From ode stimulation increases the forward (anteriad) phase of the cilium move- ment (the "recovery"). With an intensity of 24 8, vibration of cilia occurs at both kathode and anode. It is, however, more * LUDLOFF was enabled to make a careful study of the effect of the current on the cilia by making use of gelatine solutions such as have been recommended by JENSEN ('93, p. 556). . 3] ELECTROTAXIS 145 intense at the kathode and is also in opposite directions at the two poles. This law is important, and may be thus formulated: The current intensifies at the anode the backward movements and at the kathode thefonvard movements of the cilia, and the latter are more intensified than the former ; or, in other words, the anode stimulation increases the effectiveness of the normal stroke, the kathode stimulation diminishes the effectiveness of the normal stroke, and the diminishing effect is the greater of the two. On the basis of these observed facts, LTJDLOFF has proposed a theory which accounts for several of the electrotactic phe- nomena in the Ciliata, especially the fact that with strong currents there is a diminution in the rate of locomotion, and that at a lower intensity the axis of the organism is placed in the axis of the current, with its anterior end towards the kathode. This theory may be stated as follows: In every com- plete swing of a cilium two phases may be distinguished — the backward "stroke" and the forward "recovery." Nor- mally the stroke is the more effective, otherwise forward loco- motion would not occur."N> The excess in effectiveness of the stroke may be designated by the quantity x. Let us assume a Paramecium lying in the axis of the current with its anterior end towards the kathode. Then the stimulus received at the anode or hinder end increases the effectiveness of the stroke by a quantity which we may designate m. Thus the excess energy of the stroke over recovery is for these hinder cilia x + m. The stimulus received at the kathode or anterior end diminishes the effectiveness of the stroke by a quantity which we may call n, which is larger than m. Here the excess energy of stroke over recovery is x — n. If at any intensity of cur- rent n exceeds x, the anterior cilia will work to oppose the forward motion of the individual, and when n — x = x + m loco- motion will not occur. Such a strength of current probably occurred in the experiment given on p. 142, where locomotion ceased at above 60 B. To account for orientation of the axis and its anterior end, we have merely to apply the general law given above. Let us suppose that we are observing a Paramecium lying in the axis of a current of medium intensity, with its anterior 146 ELECTRICITY AND PROTOPLASM [CH. VI end towards the anode. Since n is here supposed to be less than x, the resultant effect is to move the animal forward. In moving forward in its spiral course, one side becomes presented to the anode. On this side the excess of energy of the stroke is x + m, while on the kathode side it is x — n. The resultant effect of the cilia on the two sides forms a couple which revolves the organism about one of its short axes until it comes again into the axis of the current, but with its anterior end towards the kathode. The beating of its cilia must now carry it towards the kathode (Fig. 39). FIG. 39. — Diagram showing the successive attitudes (a, 6, c, d, and e) assumed by Paramecium when its head is turned towards the anode at the beginning (a). It rotates till its head is next the kathode (e) . (From LUDLOFF, '95.) Before leaving the Protozoa, we ought to look over the whole field. We have hitherto considered only cases of migration towards the kathode — negative galvanotaxis. VERWORN ('89b), however, has found that some Protista are positively electrotactic ; namely, the flagellata, Polytoma uvella, Crypto- monas ovata, and Chilomonas paramecium; the ciliate, Opalina; and some bacteria. Finally, VERWORN ('95, p. 446) describes one of the elongated Ciliata — Spirostomum ambiguum — which places its long axis across that of the current and migrates towards neither pole, a condition which may be called (after VERWORN) transverse electrotaxis. Passing now to the Metazoa, we find investigations concern- ing electrotaxis among Invertebrates by NAGEL ('92 and '92a ~,q ELECTROTAXIS 147 v t $ and '95), and BLASIUS and SCHWEIZER ('93) ; and among Vertebrates by these authors and also HERMANN ('85 and '86), AVALD ('94, '94% and '94b), HERMANN and MATTHIAS ('94), and ALLER ('95). The number of species investigated has been considerable. I give below a table of the Invertebrate genera studied and the sense (-f t>r — ) of their response at the given intensity of currents. Barely rough quantitative expression of strength of current can be deduced from NAGEL'S paper. Where no data are given the currents are supposed to be of intermediate strength. N. stands for NAGEL, B.S. for BLA- SIUS and SCHWEIZER. The numbers which follow give the year of publication and the page. TABLE XV SPECIFIC NAME. STRENGTH OF CURRENT. SENSE OF RESPONSE. AUTHORITY. Mollusca : Limn86a stagnalis weak N. '95 Var. other Gastropoda (probably) . Annelida : Lunibricus — N. '92»; '95 N '95 6°6 Tubifex rivuloriim . N '95 631 Hirudo medicinalis 0.88 B.S. '92, 516 Branchiobdella parasitica B.S. '92, 516 Crustacea : Cyclops strong -}. N. '92, 629 Asejlus aouaticus • strong 4- N. '95 633 Astacus fluviatilus 048 _i_ B S. '92 518 In sect a : -f ? N. '95, 636 -f N. '95, 636 Dytiscus marginalis 1.98 + B.S. '92, 519 Hydrophilus piceus 198 B.S. '92, 519 From this table it appears that Mollusca and Annelida are usually negatively electrotactic to a current of medium inten- sity, while Arthropoda are mostly positively electrotactic to such a current. It is noteworthy, however, that two quite 148 ELECTRICITY AND PROTOPLASM [Cn. YI closely allied beetles like Dytiscus and Hydrophilus should by the same observers be found to react in different ways. In addition to the species named, some have been studied which have given no results. Thus NAGEL ('95, p. 639) ob- tained no response from the larva of Libellula depressa, even with a wide range of current-intensities. Passing now to the Vertebrata, we enter a region in which, as a result of more numerous studies, the data are more volu- minous, but at the same time less in accord. Since many mat- ters are here still in dispute, we may best consider historically, i.e. in chronological sequence, what has hitherto been done in this field. vThe first person to describe the phenomenon of electrotaxis in Vertebrates — as, indeed, in any organism — was HERMANN ('85, '86). j He used frog larvae 14 days old, held in a shallow rectangular porcelain trough, along the two small sides of which thick zinc wires were placed, connected with a chain of 20 small zinc-carbon elements. No mention of the strength of the cur- rent except such as can be gained from these facts was made. This omission of quantitative details is to be regretted, since had the strength of current to which the organisms were sub- jected been given, much subsequent confusion might have been avoided. With this current, then, of unknown intensity, flow- ing through the water containing the larvae, all of the latter were seen to place themselves in the axis of the current with their heads directed toward the anode. This orientation was the consequence of the fact that a current passing cephalad through the larva acted as a violent stimulus ; but while passing caudad it brought stupefaction or even (temporary) paralysis. The results obtained by HERMANN were now confirmed and extended by BLASIUS and SCHWEIZER ('93). They employed a wooden trough with sheet zinc electrodes of nearly the cross- section of the smaller ends of the trough, and experimented upon fishes, Salamandra, and the frog. The weakest current employed was 0.35 B to 0.47 8, which merely affected the char- acter of the swimming of the fish subjected to it, without determining its direction. The next stronger current men- tioned was 1.58S. With this current a marked orientation of I 3] ELECTROTAXTS 149 the fish with their heads to the anode was noticed. With Salamandra larvae currents of 2.38 to 4.78 were chiefly em- loyed. In the experiments of BLASIUS and SCHWEIZER the organisms sometimes migrated toward the anode, if the cur- rent was not so strong as to stupefy, but they lay stress upon the point that the migration is a secondary phenomenon — that the orientation is the primary effect of the current. Xext came the observations of EWALD ('94), who used very young tadpoles (5 days) and non-polarizable points as elec- trodes. Thus he was not able to give the strength of current to which the individuals were subjected. His results seemed directly to oppose those of the two preceding authors, for with his (unknown) current, the tadpoles were stimulated when the current passed caudad and stupefied by one passing cephalad ; also they placed themselves in the axis of the cur- rent with their heads towards the kathode. The larvae did not seem to find this position as a direct response to stimulus, but whenever an individual, in its turnings to the right and left, fell into this — electrotactic — position it was no longer stimulated, but stupefied, and so came to rest. These discordant results of EWALD led HERMANN, with his student MATTHIAS, again into the discussion. By careful measurements of the strength of current, they found that between 0.38 and 1.5 & frog tadpoles of from 1 to 3 weeks old did face the kathode, as EWALD fojund, and did move towards it. But HERMANN and MATTHIAS ('94) believed this result to be due to the fact that only cephalad-flowing currents of this intensity excite, and thus only such currents are able to pro- duce locomotion. EWALD ('94b), however, cannot accept their idea that a caudad-passing current of small intensity produces no excita- tion, for, he says, he has seen small fish, lying with face to the anode, made to move towards the kathode by the action of the weak current. Since HERMANN and others have shown that very strong currents cause paralysis even when flowing cepha- lad, EWALD concludes that we must recognize the existence of three different effects at three intensities; weakest, medium, and strongest. The medium current (which has the broadest range) is that by which the organisms are irritated as it flows 150 ELECTRICITY AND PROTOPLASM [Cn. VI cephalad, so that they come to lie with head towards anode ; the weakest current is that by which (following EWALD) the organisms are irritated as it flows caudad, so that they come to lie facing the kathode ; finally, the strongest cur- rent is that at which a violent stimulation leading to paraly- sis is produced by the cephalad-flowing current (as well as the caudad?). • In seeking for an explanation of electrotaxis in Metazoa, it is necessary, first of all, to notice that there is a close relation between response to the make-shock (as described on pp. 136, 137) and the direction of orientation of the body in electro- taxis. Thus all gastropods studied are, upon making, excited chiefly at the anode, and, correspondingly, all gastropods hitherto studied when subjected to the current face the kathode ; so on the other hand, such Crustacea as have been studied are stimu- lated at the kathode, and they accordingly come to face the anode. In regard to Vertebrates, we have apparently a double electrotactic orientation varying with the current, and corre- spondingly we have, as NAGEL has shown (p. 137), a double irritability depending on the current. A medium current pro- duces a kathode excitation and an anode orientation ; while the weakest current produces an anode excitation and a kathode orientation. So we may lay it down as a general law : Positively electrotactic organisms exhibit the katex type of irritability; and negatively * electrotactic organisms exhibit the anex type or, in general, the electrotactic organism turns tail to the exciting pole. EWALD ('94, pp. 611-615) accounts for this difference of response of Vertebrates to weak and strong currents, by the aid of certain observations that he made upon the excitation of the nerve cord. We have already seen that the making of the medium constant current stimulates at the kathode, so that an animal turns tail to the kathode. EWALD found that the two parts of the dorsal nerve were differently stimulated by the current ; the brain was stimulated chiefly by a caudad- passing current ; the spinal cord chiefly by a cephalad-passing current. This conclusion was established by two experiments. First, the two electrodes, placed a few millimeters apart, are brought into contact with different parts of the body of a fish. SUMMARY OF THE CHAPTER 151 Let the current be passing through the fish from the anterior to the posterior electrode. At the tail end we get no excitation ; and, as we pass forward, the body remains quiet until, pass- 'ing the region of the medulla oblongata, an excitation appears. If the operation is repeated with a reverse current, we get excitation behind the head and quiet on the head. Secondly, if a frog larva be cut in two transversely at the root of the tail, the head end is irritated only by a caudad-flowing cur- rent ; the tail end only by a cephalad-flowing current ; while in both cases the opposite current quiets. (Cf. also EWALD, '94b, p. 162.) These observations were now made use of to explain the opposite orientation of the tadpole in the presence of weak and strong currents. NAGEL assumed that the weak- est currents can affect the brain only. Now if that current runs caudad, it will strongly stimulate the body so that it turns tail to the anode. The medium currents, however, stimulate the whole dorsal nerve, but the spinal cord to a preponderating degree, so that a cephalad-passing current irritates more than a caudad current, and the animal will turn tail to the kathode. Thus weaker or stronger current will determine — or 4- electrotaxis. SUMMARY OF THE CHAPTER Electricity affects protoplasm in two principal ways : first,, by causing contraction ; second, by determining orientation. We can distinguish two principal types of contraction phe- nomena and, corresponding to and dependent upon these, two types of orientation phenomena. The first type of contraction is that which is produced, upon making the constant current, chiefly at the anode ; the second is produced chiefly at the kathode. The corresponding orientation or migration types are, facing the kathode and facing the anode. Since the orien- tation phenomena are dependent upon the contraction phe- nomena, the most important causes to be investigated are, first, that of contraction, and second, that of the difference in type of contraction exhibited by different organisms. The funda- mental teaching of this chapter is that the electric current acts as a stimulus upon protoplasm, and may determine the charac- ter of its activities. 152 ELECTRICITY AND PROTOPLASM [Cn. VI LITERATURE BIEDERMANN, W. '83. Ueber rythmische Contraction quergestreifter Mus- keln unter dem Einflusse des constanten Stromes. Sitzb. Wien. Akad., Math.-Nat. Cl. LXXXVII, Abth. 3, pp. 115-136. Taf. I-II, 1883. BLASIUS, E. and SCHWEIZER, F. '93. Electrotropismus und verwandte Erscheinungen. Arch. f. d. ges. Physiol. LIII, 493-543. 10 Feb. 1893. ENGELMANN, T. W. '69. Beitrage zur Physiologic des Protoplasma. Arch. f. d. ges. Physiol. II, 307-322. 70. Beitrage zur allgemeinen Muskel- und Nervenphysiologie. Arch, f . d. ges. Physiol. Ill, 247-326. EWALD, J. R. '94. Ueber die Wirkung des galvanischen Stroms bei der Langsdurchstromung ganzer Wirbelthiere. Arch. f. d. ges. Physiol. LV, 606-621. 10 Feb. 1894. '94a. Berichtigung. Arch. f. d. ges. Physiol. LVI, 354. 11 Apr. 1894. '94b. Ueber die Wirkung des galvanischen Stroms bei der Langsdurch- stromung ganzer Wirbelthiere. II Mitth. Arch. f. d. ges. Physiol. LIX, 153-164. 30 Nov. 1894. •FiCK, A. '63. Beitrage zur vergleichenden Physiologic der irritablen Sub- stanzen. Braunschweig. 1863. (Not seen.) GOLUBEW, A. '68. Ueber die Erscheinungen, welche elektrische Schlage an den sogenannten farblosen Formbestandtheilen des Blutes hervor- bringen. Sitzb. Wien. Akad., Math.-Nat. Cl. LVII, Abth. 2, 555-572. HERMANN, L. '85. Eine Wirkung galvanischer Strome auf Organismen. Arch. f. d. ges. Physiol. XXXVII, 457-460. 2 Dec. 1885. '86. Weitere Untersuchungen iiber das Verhalten der Frochlarven im galvanischen Strome. Arch. f. d. ges. Physiol. XXXIX, 414-419. 21 Oct. 1886. HERMANN, L. and MATTHIAS, F. '94. Der Galvanotropismus der Larven von Rana temporaria und der Fische. Arch. f. d. ges. Physiol. LVII, 391-405. 20 July, 1894. JENSEN, P. '93. Methode der Beobachtung und Vivisektion von Infusorien in Gelatinelosung. Biol. Centralbl. XII, 556-560. 1 Oct. 1892. KRAFT, H. '90. Zur Physiologie des Flimmerepithels bei Wirbelthieren. Arch. f. d. ges. Physiol. XL VII, 196-235. 9 May, 1890. KUHNE, W. '64. Untersuchungen iiber das Protoplasma und die Con- tractilitat. Leipzig: Engelmann. 1864. LUDLOFF, K. '95. Untersuchungen iiber den Galvanotropismus. Arch. f. d. ges. Physiol. LIX, 525-554. 5 Feb. 1895. NAGEL, W. A. '92. Beobachtungen iiber das Verhalten einiger wirbelloser Thiere gegen galvanische und faradische Reizung. Arch. f. d. ges. Physiol. LI, 624-631. 26 March, 1892. '92a. Fortgesetzte Beobachtungen iiber polare galvanische Reizung bei Wasserthieren. Arch. f. d. ges. Physiol. LIII, 332-347. 24 Nov. 1892. LITERATURE 153 XAGEL, W. A. '95. Ueber Galvanotaxis. Arch, f . d. ges. Physiol. LIX, 603- 642. 5 Feb. 1895. OSTWALD, W. '94. Manual of Physico-chemical Measurements. Translated by J. Walker. 255 pp. London : Macmillan. 1894. VERWORX, M. '89*. Die polare Erregung der Protisten durch den galva- nischen Strom. Arch. f. d. ges. Physiol. XLV, pp. 1-36. 23 March, 1889. '89b. The same (continued). Arch. f. d. ges. Physiol. XL VI, pp. 267- 303. 18 Xov. 1889. '95. (See Chapter IV.) WALLER, A. D. '95. Galvanotropism of Tadpoles. Science Progress. IV, 96-103. Oct. 1895. CHAPTER VII ACTION OF LIGHT UPON PROTOPLASM IN this chapter it is proposed to discuss (I) the application and measurement of light ; (II) its chemical action ; (III) the effect of light upon the general functions of organisms; and' (IV) the control of locomotion by light — phototaxis and photopathy. § 1. THE APPLICATION AND MEASUREMENT OF LIGHT Light, which as a form of radiant energy is closely related to radiant heat, is always accompanied by a certain quantity of heat, whose action (in at least one control experiment in every set) should be eliminated. To cut out heat without great loss of light, we must employ transparent adiathermal media. Of these, a plate of ice is the most effective, but alum, on account of its higher melting point, is more convenient. A parallel- sided vessel full of distilled water, or, still better, a saturated aqueous solution of alum, forms an inexpensive, highly adi- athermal screen. The quality of the light used in any experiment should be carefully determined. If any other light than that of the sun or incandescent solids is employed, it should be subjected to spectroscopic analysis. For biological purposes a direct-vision hand spectroscope, such as BROWNING'S, is convenient and adequate. Often monochromatic light or a definite range of the spectrum is desired. This may be obtained in various ways. The purest monochromatic light can be got by making a long spec- trum and using the desired part of it. To make such a spec- trum one may employ, in a dark room, a lamp, followed in succession by a slit and a lens to form an image of the slit 154 § 1] APPLICATION AND MEASUREMENT OF LIGHT 155 on a prism of bisulphide of carbon,* which gives very great dispersion of the rays. In defining the regions of the solar spectrum which are employed in any study, it is usual to make reference to the dark absorption bands (FRAUEXHOFER'S lines) which cross the solar spectrum. The largest of these are lettered, begin- ning with A in the visible red and ending with H in the visible violet. At other times it may be more convenient to define any part of the spectrum by means of the extreme wave lengths between which it lies. Lithographs showing the spectral colors and the wave lengths corresponding thereto are given in encyclopaedias and most of the text-books on physics. A crude attempt is made to show the relation be- tween color and wave length in Fig. 40. The wave lengths at Red Orange Yellow Green Blue Indigo Violet 1 ! 1 \ V 1701 65 CO 1 1 5 5 i ! 5 0 \ i 5 4 1 | ! j i ! J i ! ! aBC Eb F G h FIG. 40. — Diagram of the solar spectrum showing the main absorption bands and the range of the various spectral colors. The numbers are wave lengths in hundred- thousandths of a millimeter. (From REIXKE, '84.) the different absorption bands are given more exactly (in thou- sandths of a millimeter, = /i) • in the following table, and also the number of waves per second in 1012ths. TABLE XVI ABSORPTION BAKD. WAVE LENGTH, A. VIBRATION'S PEK SECOND n x 1012. ABSORPTION BAND. WAVE LENGTH, A. VIBRATIONS PER SECOND n x 10". A 0.760 /A 392 E 0.527 p 566 B 0.687 fj. 433 F 0.486 fj. 613 C 0.656 p. 454 G 0.431 IJL 692 D 0.589 fj. 506 H. . . . 0.397 /x 751 * The bisulphide prism may be made as follows : Upon a thick glass plate three rectangular pieces of glass of equal size are placed perpendicularly, so as 156 LIGHT AND PROTOPLASM [On. VII Extreme ultra-violet X = 0.295/*; 1010 x 1012 vibrations per second. To obtain monochromatic light from the spectrum, REINKE'S ('84) spec- trophor will be found useful (Fig. 41). In this instrument a beam of sun- light cast by a heliostat through a slit at H is converged by means of an interpolated lens, 0, upon a prism, P, set at the angle of minimum devia- tion. Passing through this prism the rays are dispersed and a spectrum is formed upon a diaphragm D, Dl composed of halves bounding a second slit whose position and width may be varied at will so as to include any desired part of the spectrum. A large lens C known as the collector brings the rays which have passed through the second slit to a focus at E. Just in FIG. 41.— Diagram showing the construction of REINKE'S spectrophor and the path of the rays in it. H, slit next to heliostat ; 0, pro- jecting lens; P, prism; S, Si, scale marked with wave lengths ; D, Dlf diaphragm, including a variable slit ; c, GI, collecting lens ; E, posi- tion of object subjected to the rays. The spectrum ranges from A = 0.75 ^ to A = 40 n. (From REINKE, '84.) front of the diaphragm is placed a scale S, Sl of wave lengths fitted to the spectrum obtained. Such a scale may be constructed with reference to the position of FRAUENHOFER'S lines by interpolation from Fig. 40.* To in- clude rays whose wave lengths differ by exactly 0.05 /x the slit must be wider when at the blue end than when at the red end of the spectrum (Fig. 40). to form a hollow, triangular (60°) prism. The plates are fixed to each other and to the glass base by a pasty cement made by mixing plaster of paris and liquid glue. This cement soon hardens, and is not attacked by the carbon disulphide. The hollow prism is now filled with fluid, and a triangular glass plate is cemented on as a cover. * If artificial light is used, two points on the scale can be obtained as follows : Volatilize in a Bunsen flame, temporarily replacing the lamp, a salt of barium and one of calcium, and note the position of the extreme blue band on the former (line F) and the yellow band of the latter (line D). After determining these two points the remaining lines can be plotted upon the scale. 1-1] APPLICATION AND MEASUREMENT OF LIGHT 157 A second method of getting monochromatic light is by the use of flames tinged with various volatilized metals. Of these, lithium salts give reds at X = 0.67/* and X = 0.61/z,, sodium salts give a pure yellow light of X = 0.59/>t, thallium salts (poisonous vapor) a green at about X = 0.54/z, and indium salts a blue and a violet, both beyond A = 0.46//,. The number of metallic vapors which give even nearly monochromatic light is, however, not large. A third method for producing monochromatic light is found in the use of solutions of pigments. Such solutions may be held in deep glass vessels whose back and front glass surfaces are parallel and near together. In the following table are given in the first column the names which may be applied to differ- ent parts of the spectrum, following HELMHOLTZ (Handbuch, p. 251); in the second column the pigments which while dry give corresponding spectral colors in diffuse daylight and which may also be used in making solutions ; and in the third column certain pigments which in solution YUNG ('78, p. 251) found to transmit almost exclusively the part of the spectrum named in the first column. TABLE XYII From outer limit to line C, red . . . ( orange . Cinnibar, HgS (vermilion) Minium Alcoholic solution f uchsin * C to D,\ ( golden yellow . . . { yellow . . Litharge, PbO Chrome yellow Concen. Sol. potassic chromate D to E,\ y ;: 1 yellow-green .... E to 6, green PbO,CrO2 cupric arsenite (a little red and green) Nickel nitrate, NiO2(NO2), 6 to F, transition from blue-green to blue F to F$G, cyaniteblue .... F$ G to G, indigo blue SCHEEL'S green Berlin blue Ultramarine Bleu de Lyon (a little V) t G to H, violet Violet de Parme Solutions made up from these pigments should, however, be examined spectroscopically before using to make sure of the purity of the color. * Also a solution of iodine in carbon disulphide. (PRINGSHEIM, '80, p. 409.) t F to H is given by ammoniated copper sulphate, CuSo44 NH3 + H20 (PRIXGSHEIM). 158 LIGHT AND PROTOPLASM [Cn. VII Finally, a fourth and decidedly practical way of obtaining pure colors is by the use of transparent plates of colored glass or other transparent solids. It is very difficult to get monochro- matic glasses of certain colors in the market. A pure red is easily obtainable; the blue is apt to contain some red also ; and the green, both blue and yellow. Lord RAYLEIGH ('81, p. 64) has used " films of gelatine or of collodion, spread upon glass and impregnated with various dyes, gelatine being chosen when the dye is soluble in water and collodion when the dye is soluble in alcohol." This method seems to me to be of wide applica- bility in our light experiments. For solid media are, after all, far less troublesome than fluids, vapors, or spectra ; and con- venience is one of the most valuable qualities of a method. A brief statement must be made concerning the physical properties of the different light waves. An inspection of any prismatic solar spectrum shows that certain parts are brighter to our eyes than others, and a thermometer placed in different parts of the spectrum indicates a higher temperature towards the red end. Curves are given in Fig. 42 which show the relative warmth of different parts of the visible spectrum both when the spectrum is a normal one (i.e. such as is given by a diffraction grating, where all rays differing in wave length by O.I//, are equally distant) and when it is prismatic (in which there is a crowding of rays at the red end). Curves of relative brightness and of relative chemical (actinic) activity, so far as can be judged from the union of chlorine and hydrogen, are also given, for the prismatic spectrum. Being laid off on the " normal " scale the curves last mentioned are somewhat dis- torted. From these curves it appears that the brightest part of the spectrum lies between lines D and E, at X= 0.59 /,<,;* the warmest part is, in the normal spectrum, near X= 0.60/>c, but in the prismatic spectrum, beyond the visible red, at about X = 1.00/A. Finally, the chemical activity of the rays increases towards the blue end of the spectrum, but the relative activity is different for the different substances acted upon. Measured by their ability to unite chlorine and hydrogen, the rays having * MENGARINI ('89, p. 135) finds the point of maximum brightness to lie at about 0.57 M. §1] APPLICATION AND MEASUREMENT OF LIGHT 159 1 1 ( Jurve of Relative Warmth in Normal Spectrum. Curve of Relative "Warmth in Prismatic Spectrum. Curve of Relative Actinic Effect in Prismatic S jectrum. 3 L^- V_ —— •— *. •T!^, -^^ -^ ~~~ ^ •^ I ^ x i \ ' s> / \ / i 1 _^ \ / i I VN/ \, / I \ / / | / i ^ ^ ( ^^ •^ \ / ' i ^ \ / 1 i / ^ / ', . X _^ / /' 1 .' / / j X / \ 2 t j? / \ i v / ' jX / y (' t-'\l T \ ' >•• g / • - ^> / \ ,•' i / V i ^ \ / t ,- / \ \ A \ , ' \ t • * i ^* \ \ ^ ,' • « x / ,,- / \_ \ • _.. ._. _- -- — -' ~ ~- — __ .4 U/ ,~ t/ .6 )./ »/ .. H C i F E D c R r- FIG. 42. — Scale of normal solar spectrum, above which is drawn the normal curve of relative warmth ; also the curves of relative warmth, brightness, and actinism of the prismatic solar spectrum. The curves of warmth are taken from LAXGLEY ('84, p. 233) ; the curve of brightness is constructed from the data of VIKRORDT (73, p. 17) ; that of actinism is taken from BUXSEX and ROSCOE ('59, p. 268) and indicates the relative efficiency of the different rays of the midday sun in caus- ing the union of chlorine and hydrogen. The absolute value of the ordinates is entirely arbitrary. a longer wave length than 0.51//, have feeble chemical action ; at about X = 0.42//, this action reaches a maximum. Not only the quality but also the intensity of the light with which we experiment must be known. It is, fortunately, quite 160 LIGHT AND PROTOPLASM [Cn. VII easy to determine the intensity of white light in terms of a recognized unit ; namely, a paraffine candle burning at the rate of 7.78 grammes per hour. A paraffine candle burning at this rate has one candle power (C. P.) ; burning at twice this rate, 2 candle power, and so on. A comparison of any other light with this standard may be made by means of any of the well- kiiown photometers, of which text-books of physics give a description. The determination of the intensity of a colored light requires an additional piece of apparatus ; namely, a spectrophotometer. The two principal types of spectrophotometer are that of VIERORDT (73) and that of GLAN ('77), both of which have undergone important improvements. The principle in both types is the same. A spectrum of both the unmodified (standard) light and that which has passed through the colored screen are made side by side, so that their corresponding colors can be compared. Since the source of light is the same, every part of the spectrum of the unmodified light will be brighter than the corresponding part of the spectrum of the colored light. To bring the corresponding colors in the two spectra to the same intensity, the unmodified light must be made less intense to a measurable extent. In VIERORDT'S spectrophotometer this result is brought about by narrowing that half of the slit through which the unmodified light passes to get to the prism. In GLAN'S apparatus the diminution in intensity is gained by the polarization of both lights and the obscuring of the brighter by the rotation of its analyzing NICHOL prism, until equality of brightness is obtained. A modified form of VIERORDT'S convenient instrument is made by H. KRUSS of Hamburg, Germany. A modified form of GLAN'S photometer is de- scribed by VOGEL ('77). VOGEL'S apparatus (Fig. 43) consists essentially of a collimator contain- ing (1) a slit of changeable width, separated by a band q into an upper and a lower part to receive respectively the modified and the normal light ; (2) a lens to render the rays parallel before they impinge upon (3) a doubly refract- ing quartz prism, by which both upper and lower rays are broken into two polarized rays. Of these four rays the uppermost and the lowest are cut off by a diaphragm near F, so that only the middle two, which lie near together, pass eventually to the eye. These two rays are oppositely polar- ized and come, one from the upper, the other from the lower slit. The two CHEMICAL ACTION OF LIGHT 161 rays now pass through (4) a XICHOL prism (capable of being rotated along- side a graduated arc) set at 45°, in which position both rays pass through j-^l without changed relative intensity. The rays emerging from J) |L the collecting telescope are now dispersed by P_j j ^& passing through the vertically placed prism, and the adjacent parallel spectra are observed through a telescope. By a rotation of the XICHOL prism through an observed number of degrees the stronger light may be brought to the intensity of the weaker. The relative intensity of two lights with reference to a third (con- stant) is as the squares of the tangent of the angle through which the NICHOL prism has been rotated. Other modifications of GLAN'S photometer are those of Lord RAYLEIGH ('81) and of LEA ('85), upon which the spectrophotometer of the Cambridge (Eng.) Scientific Instrument Co. is based. FIG. 43. — Diagrams showing con- struction of VOGEL'S spectropho- tometer. 1. Horizontal section through the optical axis. M, mirror to reflect standard light, by aid of a totally reflecting prism p, into the optical axis. S, shutter with slit divided into an upper and a lower half by means of a band q ; C, colli- mating lens. D, doubly refract- ing quartz prism; m, m, the holder of the NICHOL prism X, which can be rotated through an arc that can be read off from a graduation on m, m; P, a flint glass prism ; B, F, 0, observing telescope, in which, at the focal point, near F, is a diaphragm cutting out the two outermost of the four spectra coming through B. 2. Front view of the shutter. (From VOGEL, '77.) § 2. THE CHEMICAL ACTION OF LIGHT UPON NON-LIVING SUB- STANCES The process of photography has made us familiar with the fact that daylight acts upon the halo- gen salts of silver, gold, platinum, and other metals, although the nature of the chemical change wrought by the light is uncer- tain. It is not, perhaps, gener- ally appreciated, but it is well known to chemists, that light can produce or further very many chemical changes, particu- larly among organic compounds. The effects are mainly due to the blue and violet rays, hence are 162 LIGHT AND PROTOPLASM [Cn. VII not the results of the heat of sunlight. Most of these chemi- cal effects may be grouped under four heads. 1. Synthetic ; 2. Analytic ; 3. Substitutional ; and 4. Isomerismic and Poly- mer ismic. A few others may be classed (5) as fermentative. Let us now consider each of these five classes.* 1. The Synthetic Effects of Light will be considered chiefly with reference to organic compounds. All the cases I have gathered fall into three groups : addition to the organic com- pound either (a) of oxygen, (/3) of chlorine or bromine, or (7) of another organic compound. Among the compounds which take up oxygen is bilirubin, C32H36N4O6, a solution of which, in sunlight, even when air is excluded, oxidizes to biliverdin, C32H36N4O8. In the absence of sunlight this change requires air (B. Ill, 418). DUCLAUX ('87, p. 353) finds that vegetable oils, such as olive or palm oils, are rapidly oxidized if exposed to light. CHASTAIGN ('77, p. 198) believes this oxidizing action of light upon organic com- pounds to be of very wide-spread occurrence ; the blue-violet part of the spectrum being, in this respect, the most active. The direct combination by means of light of a halogen and another substance is also not rare. Thus, in daylight, hydro- gen unites with chlorine explosively. It unites with bromine also, although with difficulty. Similarly, equal volumes of chlorine and carbon monoxide unite quickly in the sunlight or magnesium light to form carbon monoxid chloride, COC12 (B. I, 546). Again, when chlorine is passed through alcohol under the influence of strong sunlight or magnesium light the two substances unite and produce chloral hydrate (STREET and FRANZ, '70). Likewise, when chlorine is passed, in sunlight, through a solution of C3H2C12O2 in CS2, there is formed C3H2C14O2, two atoms of Cl having been added. Finally, C2C16 may be made by uniting C2C14 and C12 in sunlight (B. I, 158) ; and the compound C2H4 • FeBr2 • 2H2O may be made by passing, in sunlight, C2H4 through a concentrated aqueous solution of FeBr2 (B. I, 113). * Most of these cases were obtained by searching through BEILSTEIX ('86-'93). References to this book will be made throughout this section by the letter B, followed by the number of the volume and page upon which the statement may be found. § 2] CHEMICAL ACTIOX OF LIGHT Important cases of the direct synthesis of organic compounds are given by KLIXGER and STANDKE ('91). These authors have shown that in sunlight (and not in the dark) phenanthren- chinon unites directly with benzaldehyd to form a third com- pound phenanthrenhydrochinonmonobenzoat, in accordance with the formula : C14H8O2 + C6H5CHO = C14H8 (OH) (O • CO C6H5). Again, chinon (benzochinon) and benzaldehyd may unite in the sunlight to form benzohydrochirion, according to the formula : C6H4O2 + C6H5CHO = C6H5CO C6H3(OH)2. Finally, benzochinon and isovaleraldehyd may similarly unite to form isovalerochinhydron, thus : C6H4O2 4- C4H9CHO = C4H9CO C6H3(HO)2. These cases, then, are examples of organic compounds which are wholly indifferent in the dark, but which, subjected to strong sunlight, lose their identity by uniting directly; they may suffice to illustrate the important synthetic effect of sunlight on non-living, organic compounds. 2. Analytic Effect of Light. — Cases of this effect are nu- merous, varied, and striking. I will cite a few. The organic dibasic acids CnH2n_2O4 break up in the sunlight and in the presence of a small quantity of uranium oxide, into CO2 and an acid CnH2nO2 (B. I, 63). For example, oxalic acid, C2H2O4, breaks up thus into formic acid, CH2O2 and CO2. Also an aqueous solution of butyric acid, C4H8O2, in the presence of uranyl nitrate, breaks up, in the sunlight, into CO2 and C3H8 (B. I, 422). We have seen that chlorine will unite directly with organic compounds under the influence of light ; on the other hand, compounds containing chlorine may lose it in the sunlight. Thus under these conditions the ketone (C8H17XO • HCl)2PtCl4, an ammoniacal derivative of acetone, becomes (C8H1TNO • HCl)2PtCl2; and (C9H17XO - HCl)2PtCl4 becomes (C9H17XO - HCl)2PtCl2 (B. I, 982, 983). Again, chlo- rine acetate, Cl • O • C2H3O, undergoes slow decomposition, in the light (B. I, 462) ; C5H6C12, a derivative of pentine, C5H8, does the same ; and ethylester, CIO • C2H5, explodes in sunlight. Similarly explosive in sunlight is the greenish oil distilled when absolute alcohol is poured over dry calcium chloride (B. I, 223). Finally, sugar (DUCLAUX, '86, p. 881) and oxalic acid (DcrwxES and BLUXT, '79, p. 209) are oxidized and break up into water, carbon dioxide, and other compounds. These cases may serve 164 LIGHT AXD PROTOPLASM [Cn.VIl to show the important chemical effects of sunlight in the dis- integration of organic compounds. 3. Substitution Effects of Light. — The principal substitu- tion effect of light is the replacement of hydrogen in an organic compound by either chlorine or bromine. This occurs so fre- quently that examples are superfluous. The substitution takes place most rapidly and completely in direct sunlight, and it has been shown that the rays at the blue end of the spectrum are the most active in this process. The compounds affected belong to the most varied groups of both the fatty and aro- matic series — carbohydrates, acids, aldehydes, ketones, and sulphides. 4. The Isomerismic and Polymerismic Changes produced by Light are among the most interesting. I will cite some exam- ples. In the first place, it may be said that the changes in the elements phosphorus and sulphur by which they assume their red form have been ascribed to sunlight. Elseomargin acid, C17H30O2, is a compound found in connection with glycerine in the oil of the seeds of Eleeococca (Aleurites) vernica — Chinese oil tree — one of the Euphorbiacese. This acid crys- tallizes in rhombic plates which melt at 48°. When an alco- holic solution of this acid is placed in a bright light, leaf -like crystals of its isomere, elseostearin acid, which melt at 71°, are produced (B. I, 535). Again, thymochinon forms yellow crystals, which are soluble in alcohol. Subjected to a strong light, opaque, whitish-yellow crystals are produced, which are insoluble in alcohol. This substance, which does not arise in the dark, and is hence not merely the result of oxidation, is called polythymochinon (B. Ill, 180). Again, among the derivatives of ethylene, C2H4, is chlorethylene, C2H3C1, a gas. When placed in the sunlight this passes into a polymere, which forms a viscous, amorphous, insoluble mass (B. I, 158). In like fashion, bromethylene, C2H3Br, a fluid, is rapidly trans- formed in the sunlight into a polymere, which is solid, amor- phous, and insoluble in water, alcohol, or ether (B. I. 181), and bromacetylen, C2HBr, a gas, is gradually transformed, in the light, into a solid polymere. Finally, very many substances undergo a gradual change of color in the sunlight, but the nature of the accompanying molecular change is unknown. §2] CHEMICAL ACTIOX OF LIGHT 165 5. Changes resembling those brought about by fermentation are produced by light. Thus NIEPCE DE SAINT VICTOR and CORVISART ('59) have found that a 0.1% solution of starch, exposed during 6 to 18 hours to the summer sun, becomes trans- formed into sugar, while in the dark no such change occurs. The change is favored by a small quantity of uranium nitrate. In a similar fashion glycogen is transformed into sugar more rapidly in the light than in the dark. On the other hand, GHEEX ('94) finds that the ferment which normally transforms starch into sugar is destroyed by subjection to a strong light, the ^iolet rays being especially active in this process. Like- wise, ptyalin, the ferment of saliva, is destroyed by light. To sum up, light affects organic compounds in very varied and important ways. We are, accordingly, prepared to find that light exerts a very important influence on the activities of protoplasm. Nor is the influence necessarily confined to the surface, for most protoplasmic bodies are more or less translucent. Thus SACHS ('60) found by looking through a tube with one end fitted to the eye and the other directed towards the sunlight, that considerable layers of plant tissue, for example over 32 mm. of the tissue of the potato tuber, did not cut out all the light, and that red had the greatest pene- trating power, violet the least. Even the epidermis of man permits light to pass, and ONIMUS ('95) asserts that light can pass through the hand to such an extent as to affect during 26 to 30 minutes an orthochromatic plate kept in a tight wooden box perforated only by the opening which is covered by the hand. Whether the " RONTGEN rays," which have so striking a power of penetrating organic matters, are more of the nature of light than of other physical agents, is still a subject of debate. Whether they produce any important chemical changes in protoplasm has not yet been fully determined.* * During the six months which have elapsed since the above was written, accounts of marked physiological effects of the ROXTGEN rays have been pub- lished. Thus, exposure of the skin to them for an hour frequently causes loss of hair and finger nails, and produces symptoms resembling those of sunburn. AXENFELD (Centralbl. f. Physiol. X, 147) finds that many insects and a crusta- cean, Porcellius, kept in a box only one-half of which is penetrated by the rays, aggregated in this part. Several experiments upon the tropic influences of the rays have resulted negatively. 166 LIGHT AND PROTOPLASM [Cn. VII § 3. THE EFFECT OF LIGHT UPON THE GENERAL FUNCTIONS OF ORGANISMS In this section we shall consider in succession (1) the effect of light upon metabolism ; (2) the vital limits of light action on protoplasm ; and (3) the effect of light upon the movement of protoplasm. 1. Effect of Light upon Metabolism (including Assimilation). — Metabolism is a complex of chemical processes. Since, as we have already seen, light has important chemical effects, we are not surprised to find that it plays an important ^ole in metabolism. The effects of light are, however, of two distinct kinds. One is a thermic effect, due to the heat rays of white light ; the other is a chemical effect due to the " actinic rays V of the spectrum. a. The Thermic Effect of Light on Metabolism is shown chiefly in the assimilative processes of chlorophyllaceous plants. The facts of this assimilation are chiefly these : various simple com- pounds, water, carbon dioxide, salts of ammonia and nitrates, are used as food by plants. For every volume of the gas — carbon dioxide — taken in, one volume (nearly) of oxygen is excreted. Starch (C6H10O5) is the first visible product of the water and carbon dioxide taken in. Chlorophyll is essential to the absorption of carbon dioxide, to the giving forth of oxygen, and to the formation of starch. Finally, chlorophyll can as- similate only in the presence of sunlight and at a proper tem- perature. Now, not all the rays of sunlight with their varied wave lengths are essential to this process. Just what rays are the essential ones has been a point of some dispute. The earlier studies on the subject, made chiefly by DRAPER ('44), SACHS ('64), and PFEFFER ('71), were unanimous in declaring that the most active rays in assimilation were those occupying the yellow part of the spectrum at about line D — the region of maximum brightness to our eyes (Fig. 42). But these ob- servers were at fault in that, while they carefully determined the quality of light and the corresponding quantity of assimila- tion, none of them gave, in the experiments with color screens, any adequate data upon the intensity of the diversely colored EFFECT UPON GENERAL FUNCTIONS 167 lights employed ; and this is a fundamental matter, for it has been shown, for instance by REIXKE ('83 and '84), that, within certain limits, the rate of assimilation increases with the inten- sity of the light (Fig. 44). Even in experiments with the colors of prismatic spectra one must remember that the rays are crowded together at the red end, so that a given length of the spectrum contains more rays at that end than at the other (of. Fig. 40). Later investigations with improved methods have shown quite conclusively that it is especially the rays with X = 0.68/4, or those very close to the absorption band B, which are most FIG. 44. — Curve showing the relation between intensity of light (abscissae) and quan- tity of oxygen set free by Elodea canadensis. i indicates the unit intensity of the light from the heliostat. (From REINKE, '84.) active in assimilation. The methods employed have been most diverse, but they have yielded the same result. TIMIRIAZEFF ('77) studied the assimilative power of the different parts of the solar prismatic spectrum, determining by gasometric methods the quantity of gases decomposed in a given time. REINKE ('84) also used the spectrum, but by means of his spectrophor was able to get more strictly monochromatic light, to use more nearly comparable extents of the spectrum, and, especially, to get a more exactly comparable (in this case, optimum) assimilative intensity for each part of the spectrum than his predecessors. (See p. 156.) As the measure of assimilation, REIXKE used the number of gas bubbles set free 168 LIGHT AND PROTOPLASM [Cii. VII per minute by the submerged, illuminated plant. As is shown in Fig. 45, the maximum of gas production occurred at about absorption line B — and this is the more marked of the absorption bands of chloro- phyll. A similar result was reached by ENGELMANN and set forth in a long series of papers ('81, '82, '82a, '83, '83a, '84, '86, and '87). He found that certain bacteria are extremely sensi- tive to oxygen, moving in the direction of small increments of the oxygen density. Now, by putting a thread of alga in the same water with bacteria and subjecting the thread to a "microspectrum," that part in which assimilation is pro- ceeding most rapidly, and in which, therefore, oxygen is being most rapidly excreted, will be indicated by the great- est aggregation of bacteria. The microspectrum was pro- duced by means of an appa- ratus especially designed by ENGELMANN for his work and now manufactured by the ZEISS firm in Jena. The appearances seen under the microscope when the spec- trum falls upon the alga in the bacterium - water are shown in Fig. 46. The max- imum aggregation (hence, maximum assimilative activ- ity) at the red end occurs I j 1 j i ! |i so ]T X)[ '( 50 6001 .5, >o 5 >0| 4 >0 it a B C D E b 1 C ifc FIG. 45. — Curve whose ordinates are pro- portional to the number of gas bubbles eliminated per minute by leaves illumi- nated by the various rays whose wave lengths are given at the bottom of the diagram, and whose number of vibra- tions per second are given at the top. The background of the figure is com- posed of the absorption spectra of the chlorophyll in living leaves. 1, 2, etc., at the left, indicate the number of leaves of Impatiens parviflora, which, when superimposed, give the corresponding spectrum at the right of these numbers. The absorption at 0 is from a fern pro- thallus, that at Ale is derived from an alcoholic solution of chlorophyll. I to IV indicate absorption bands. Beyond F there is very general absorption of the highly refractive rays. (After KEINKE, '84.) §3] EFFECT UPON GENERAL FUNCTIONS 169 close to the absorption band of chlorophyll. Observations upon the chlorophylls of brown, blue-green, and red cells, which, as EXGELMAXX'S microspectro-photometer indicated, have a maximum absorption at other points, showed a maxi- mum of assimilative activity at these other absorption points. In bacterio-purpurin also, in which some of the most active assimilative rays are those of the invisible red at about X = 0.85/1, most oxygen is produced at this point. (ExGEL- MAXX, ?83, p. 709.) Finally, by an ingeniously devised experiment, TIMIRIAZEFF ('90) has settled this matter in the most direct and indubitable FIG. 46. — Piece of Cladophora with swarming bacteria in the microspectrum (gas- light) . The chlorophyll grains which fill the cells very uniformly are omitted ; and, instead, the absorption band between B and C, and the tolerably pro- nounced band at the violet end between E and F, are indicated by shading. (From EXGELMAXX, '82.) fashion. He kept a plant for two or three days in the dark, until the starch in its leaves had gone; then, in a dark room, a prismatic spectrum was thrown upon the leaf and the position of FRAUEXHOFER'S lines indicated on the leaf. After from three to six hours, starch had formed, under the influence of the light, only in the region of the absorption bands of chloro- phyll lying between B and D. This was determined by plung- ing the leaf into boiling alcohol, thus decolorizing it, and then staining in tincture of iodine, which combines especially with the starch. The deeply dyed places, where starch had been formed, reproduced the absorption spectra of chlorophyll. The concurrent testimony of these and other observers work- ing upon so diverse material and with such excellent methods 170 LIGHT AND PROTOPLASM [Cn. VII justifies the conclusion that it is the rays absorbed by the plant pigments which enable them to do their work in the decompo- sition of carbon dioxide. The effective absorbed rays are, moreover, chiefly those towards the red end of the spectrum, those having over 525 x 1012 vibrations per second (i.e. below the D line).* In conclusion it may be said that the greater proportion of the radiant energy entering the plant tissue is absorbed. Thus MAYER ('93) has shown that of dark radiant heat at 100° about 80% is absorbed by a leaf through which it passes, and this proportion is about the same whether the leaf is thick or thin. Of this absorbed heat perhaps less than 10% is absorbed by the chlorophyll. The rest must be used up in the vital processes other than assimilation. b. The Chemical Effect of Light on Metabolism must now be considered; and of this we must notice at the outset two degrees. The greater effect, which is a fatal one and the better known, will be treated of further on. The lesser effect is less striking, yet it must be included in the greater. It shows itself in a disturbance of metabolism. This disturbance of metabolism is evinced in some green plants by heightened production of carbon dioxide and the formation of chlorophyll ; and it is noteworthy that a similar result occurs among Infusoria, according to the observations of FATIGATI ('79), who finds the violet rays more active than the green in this process. Among the Metazoa light produces im- portant chemical changes in the retina of the eye, and especially in the skin, facilitating the production of pigment. That im- portant chemical changes take place in the illuminated retina follows from the experiment of placing the electrodes at oppo- site surfaces of the frog's retina. The galvanometer shows in the darkened eye a slight " current of rest " flowing from the front face to the deeper-lying part, containing the cones. If now the retina be suddenly illuminated by blue, green, yellow, red, or white light, a current, the result of chemical action, appears flowing in the opposite direction ; this continues for * Other, less important, thermal effects of light on plants are found in the formation of chlorophyll and in the quickening of transpiration, which seem, chiefly due to the red and ultra-red rays. § 3] EFFECT UPON GENERAL FUNCTIONS 171 some time, slowly diminishing, however, in intensity. Certain chemical changes in the living retina may, indeed, be studied optically. These especially concern the visual purple. This is a substance lying in the outer ends of the rods of the retina, which, under the action of light, becomes bleached, but regains its color in the dark (HELMHOLTZ, Handb., pp. 265-273). These facts serve to indicate that light may influence metabo- lism even in organisms destitute of chlorophyll. '2. Vital Limits of Light Action on Protoplasm. — We have seen above (p. 167) that the rate of assimilation diminishes in chlorophyllaceous plants with a diminution in the intensity of the light. At last a point is reached where the intensity is so low that no further assimilation can occur, and after the con- sumption of the stored-up food-stuffs, starvation and death must eventually ensue. For non-chlorophyllaceous organisms, how- ever, no such lower limit exists. Many, as parasites or cave dwellers, live in complete darkness, even through many genera- tions. A lower vital limit to the action of light exists only in the case of chlorophyllaceous plants. With the upper vital limit, it is, however, quite different. This is found in the most diverse groups. Its occurrence in bacteria being of especial hygienic importance, these organisms have been made the object of exhaustive studies. MONTE- GAZZA (see NICKLES, '65) was perhaps the first to discover that strong light kills bacteria, but DOAVNES and BLUNT ('78 and '79) were the first to study the matter thoroughly. Since their time, numerous experiments have been made upon bacteria, as well as the higher fungi. For literature, see FRAXKLAXD and WARD (*92), and WARD ('93, p. 309). Even the earliest observers found that, while cultures of bacteria reared .in the dark rapidly flourished, they not merely did not thrive when subjected to sunlight, but actually became sterilized. That the sterilization was complete was shown by the fact that when the culture was placed again in the dark, no bacteria developed in it. This result is most striking when certain bacteria, say of the species Bacillus anthracis, are mixed with gelatine or agar- agar, poured uniformly over a glass plate. If the glass plate is then covered by a black paper stencil containing some character, e.g. the letter E, and exposed to a November sun- 172 LIGHT AND PROTOPLASM [Cn. VII light for 6 hours, and if then the whole plate is placed in a dark incubator at 20° C. for 48 hours, the bacteria will be found to have developed in all parts of the plate except in the ^/-shaped area sterilized by the light (Fig. 47). Compare the earlier results of BUCHNER ('92). That in these cases it is the light and not a high temperature which induces the sterilization in the illuminated region is shown by the fact that BUCHNER ('92) obtained even more striking results when parts of the culture plate were exposed under 50 cm. of water, which cuts off the heat. FIG. 47. — Appearance of a gelatine culture of Bacillus anthracis, exposed to the light over only the area E, and then incubated for 48 hours. In the area E no colonies have developed. (From WARD, '93.) Not all rays have this bactericidal property. DOWNES and BLUNT -('78) found that only the blue rays were thus active, for behind red or yellow glass the bacteria readily developed. WARD ('94) threw a solar spectrum upon an agar film in which bacteria were developing in a dark chamber. He found that the bactericidal effect was greatest in the region of the blue- violet rays (about X = 0.43 JJL) and diminished towards the extreme violet arid the yellow, where it had almost disappeared. These facts were ascertained by incubating the bacteria for 48 hours after insolation, when certain parts affected by the spec- trum were found to remain clear (Fig. 48). When an electric §3] EFFECT UPON GENERAL FUNCTIONS 173 spectrum (obtained by the use of a quartz prism) was em- ployed, a bactericidal effect was obtained (provided no glass intervened) in the ultra-violet. That the action of the light was not in these cases primarily upon the food-film was shown by the fact that a plate of sterile agar, exposed behind a stencil plate, and then laid flat on a film of dried unexposed spores, permitted the uniform growth of the spores, in the illumined as FIG. 48. — Plate of anthrax spores, exposed for 5 hours to the solar spectrum in August, and incubated for 48 hours. The horizontal line shows the length of the spectrum. The vertical lines are not FRAUENHOFER'S lines, but serve to show the limits of the principal regions of the spectrum. The clearest area is that where fewest spores have developed in the incubation — where, consequently, the bactericidal effect was greatest. (From WARD, '94.) well as in the unillumined region. All these observations show that the bactericidal action of light is due to the action of the chemical rays on the protoplasm. Another fact of importance, first discovered by DOWNES and BLUNT, is that light has no effect upon bacteria when they are in a vacuum. This abundantly confirmed observation indicates that death only secondarily results from light. The primary cause of death is an oxidation process, — a process rendered possible by the mediation of light. As we have seen (p. 162), many organic compounds undergo oxidation in the highly refracted light rays. Probably there are in bacteria such com- 174 LIGHT AND PROTOPLASM [Cn. VII pounds, the rapid oxidation of which is incompatible with life. In any case it is clear that the bactericidal effect of light is a chemical one. Concerning the range of organisms which are thus affected, it must .be said that chiefly pathogenic species, such as the bacteria of anthrax, of typhus fever, and of cholera have been experimented with and have shown themselves most suscep- tible. Other bacteria are, however, likewise affected. Among the other fungi, WETTSTEIN ('85) found that the conidia of Rhodomyces Kochii, a human intestinal parasite, did not de- velop in the light. KLEIN ('85) found the same thing to be true for the conidia of Botrytis cinerea, and showed that the blue-violet rays were the most effective ones. ELVING ('90, p. 105) gained similar results with Aspergillus, although several days or weeks of insolation did not kill the fully ripe spores. WARD ('93) determined that insolated spores, cultivated on agar or gelatine plates, of Oidium lactis (5 cases), Saccharomyces pyriformis (4 cases), and " a 4 Stysanus ' conidial form " found as a saprophyte on the screw-pine, Pandanus, (2 cases) became injured. These are all hyaline and colorless except Stysanus, which is nearly so. Certain colored spores which WARD experi- mented with gave negative results, and WARD concluded that this is because the blue end of the spectrum is cut off before reaching the deeper protoplasm. However this may be, we actually find that in many, but not all, fungi the metabolic processes of the spores are disturbed and even death is pro- voked by intense light. Why the spores should be especially susceptible to the action of light is an important inquiry. WARD believes the answer to be that the spores contain oily substances, which are espe- cially liable to oxidation in light, as we have already seen. Finally, we have to consider the experiments which demon- strate that a strong sunlight may be injurious even to green plants. This result follows clearly from the work of PRINGS- HEIM ('81). When strong sunlight is focussed for a short time upon cells of Spirogyra, Nitella, Mesocarpus, or Tradescantia stamen hairs in atmospheric air (5 to 15 minutes), they are killed. No result occurs, however, when the same light falls upon green cells in which the atmosphere has been replaced by §3] EFFECT UPON GENERAL FUNCTIONS 175 = •= hydrogen (Fig. 49). That it is here also the oxygen (and not the carbon dioxide) of the air which is the destructive agent is shown by subjecting the plants to air freed of carbon dioxide, when they are killed by light as before. The most important results following from the conclusions of this sub-section are : a minimum vital limit of light action exists only in the case of those organisms (chloro- phyllaceous plants) which depend upon light for as- similation ; a maximum limit is found among the most diverse organisms, those with chlorophyll and those without. The rays which have the more rapid vibrations are the more active. They pro- duce chemical changes to which death is primarily clue. 3. Effect of Light upon the Movement of Proto- plasm.— Under this head we shall consider only those protoplasmic move- ments which may not be grouped under Locomo- tion, and shall discuss three classes of cases : (a) effect of low intensity of light upon movement ; (5) effect of high intensity of light upon movement ; and (c?) effect of change of intensity on contraction. a. Effect of Low Intensity of Light on Movement — Dark-rigor. — We have already seen that chlorophyllaceous plants must eventually die if kept in the dark. Some time before death occurs the plants go into a condition of immobility, which may be called dark-rigor, since return of light brings a return of movements. Dark-rigor is very marked in the sensitive plant. FIG. 49. — Piece of a sprout of Nitella mucronata which was subjected in a gas chamber to a green light in three successive experiments A, B, C. In experiment A the insolation lasted 2 to 3 minutes, the gas chamber be- ing filled with atmospheric air. In experi- ment B the insolation lasted 20 minutes in the presence of hydrogen. In experiment C the insolation occurred again in the presence of atmospheric air and lasted 5 to 6 minutes. (From PRIXGSHEIM, '81.) 176 LIGHT AND PROTOPLASM [Cn. VII If this plant is kept for several days in darkness, the usual response to touch does not occur. From some observations of .BERT ('70, p. 338), it appears that it is the absence of the blue-violet and orange-red rays which brings about this dark- rigor ; for it occurs nearly as rapidly in green light as in the dark. In these cases the absence of movement in the dark might seem to be the result of diminished assimilation. But dark-rigor occurs under conditions which destroy the general validity of this conclusion ; for example, in the reddish- purple bacteria * whose reactions have been studied chiefly by ENGELMANN ('83 and '88). It appears that in these organisms light is essential to movement ; for, after having been kept over night in the dark, they are found in the morning at first motionless ; only later, after 5 to 10 minutes of illumination, do they awaken to activity. If now, after keeping for a time in the light, the organisms are brought again into the dark, their movements gradually diminish until, in a few hours, they have ceased. We have seen above (p. 51) that oxygen is nec- essary to movement, and we know that many plants excrete oxygen in the light. We might expect that the quiescence of these organisms in the dark is a consequence of their failure to produce the oxygen necessary to locomotion, and indeed they do produce in the light a slight quantity of oxygen, by virtue of their chromophyll (bacterio-purpurin, LANKESTER). But that it is not merely oxygen which induces movement is shown by the fact that when an abundant oxygen supply is artificially furnished, no movement occurs in the dark. Thus light, in the presence of oxygen, is essential to movement ; it seems to be necessary to the irritable condition upon which locomotion depends. This irritable state of the protoplasm conditioned upon a certain intensity of light ENGELMANN calls phototonus.-\ The analysis of this matter has been carried further. It has been found that a perceptible time (latent period) elapses * This term includes bacteria known as Bacterium photometricum, Bacterium roseopersicinum, rubescens, etc., Monas okeni, Spirillum violaceum, and by other names. t The term was applied to this phenomenon by ENGELMANN on account of its resemblance to that already described for the higher plants, and to which SACHS had previously given this name. EFFECT UPOX GENERAL FUXCTIOXS 177 between the illumination of the organism and the occurrence of movement. Also, the ultra-red rays produce most rapid locomotion, next the orange-yellow, and weakest the violet-blue and violet-red. Spectrum analysis shows that the most active rays are the ones absorbed by the chromophyll (Fig. 50). This phenomenon of phototonus is not confined to the purple bacteria. Thus, FAMIXTZIN ('67, pp. 29-31) has shown that the movements of the closely related Oscillaria are dimin- ished in the dark. SOROKIN ('78) found that protoplasmic streaming in the plasmodium of Dictydium ceases at night, being awakened to movement by the light. Finally, VER- a B C FIG. 50. — a. Spectrum of the chromophyll of bacterio-purpurin, showing absorption bands at A = 0.59 ^ and A = 0.53 /n. An (invisible) absorption band has been deter- mined by means of the bolometer at \ = 0.85 M. 6. The bacteria are seen aggre- gated chiefly in the regions of the absorption bands. The accumulation of bacteria in these regions of absorbed energy seems due to the fact that the moving bacteria cannot pass from a region of high energy to one of low without a violent stimulus which impels them back again. (From ENGELMANN, '83*.) X ('89, Nachschrift, and '95, p. 393) finds that, in the dark, the ciliate Pleuronema chrysalis rests quietly in the water, only occasionally making its peculiar spring. But when diffuse daylight is focussed upon it, for instance by the mirror of a. microscope, it springs rapidly by the movement of its long cilia ; and this movement is often repeated, so long as light continues to fall. The movement is produced by blue or violet rays ; red rays have little or no effect. A latent period of from 1 to 3 seconds elapses before the response occurs. These cases serve to show that light, in the presence of other suitable conditions, is, both for some chlorophyllaceous organ- isms and plant tissues and for some organisms destitute of 178 LIGHT AND PROTOPLASM [Cn. VII chlorophyll, nearly or quite essential to movement. Phototo- mis is a convenient name for the condition induced by light. b. Effect of High Intensity of Light on Movement — Light- rigor. — We have just seen that in some organisms the most vigorous movements occur at an optimum intensity of light, which produces phototonus. At a lower intensity there is no movement. It appears, furthermore, that there is for many or- ganisms a maximum intensity of light, below that which produces death (ultramaximum), which causes a cessation of movement that may be called light-rigor. This condition is distinguished from that of death by the fact that diminished light brings return of activity. ENGELMANN ('82, p. 109) observed this condition in his Bacterium photometricum, and remarks that it is common to all bacteria. Similar light-rigor has been observed in green plants also. PRINGSHEIM ('81, p. 516) found that when, in the presence of oxygen, strong sunlight was let fall upon Nitella, the movements ceased after 1J minutes. If the insolation was now interrupted, normal move- ments were resumed. Summing up the effects of varied intensities of light, it appears that for many organisms there is an optimum, which produces a condition of phototonus, in which the organism moves and responds regularly to stimuli. As the light inten- sity falls below, or rises above this optimum, the activity of movement diminishes, ceasing at certain points in the condi- tions of dark-rigor and light-rigor. Beyond each of these points, again, is the point of death. c. Contraction produced by Change in Intensity of Illumina- tion. — We here consider a number of cases not closely related except in this, that quick movements are produced after stimu- lation by change in the intensity of the light. The cases are found both among Protista and Metazoa. Among the sulphur-bacteria ENGELMANN ('88, p. 665 ; 88a) has noticed that a sudden diminution in the intensity of the light, produced by shading the mirror of the microscope, is followed by a spring backwards, often to the distance of 10 to 20 times the organism's length. This reaction ENGEL- MANN has called " Schreckbewegung." When the light is sud- denly increased, a forward movement takes place, but this is §3] EFFECT UPON GENERAL FUNCTIONS 179 less marked. Among the Myxomycetes, ENGELMAXX ('79) has found that the amo3boid Pelomyxa, when suddenly sub- jected to a strong light, contracts into a spherical mass. X Sudden darkening or gradual illumination produces no such contraction. Among swarm-spores, STRASBURGER ('78, pp. 575, 576) has noticed that a sudden diminution of the light puts the quiet Hsematococcus spores again in motion, and makes the Botrydium spores start as though disturbed. Such violent movements of the protoplasm indicate that a very considerable chemical change has taken place in it. Passing, next, to the Metazoa, we find that certain smooth muscle fibres are made to contract by the direct action of light ; thus, STEIXACH ('92) has offered most convincing evidence that the contraction of the iris, in the lower vertebrates at least, may occur as a direct reaction to illumination, even when the eyeball is cut out, and the iris, indeed, separated from connection with the ciliary part of the eye. Some of the higher animals react strikingly like EXGEL- MAXN'S bacteria. Thus, LOEB ('93, p. 103) found that Serpula uncinata retracts into its tube when the hand is passed between it and the light ; but sudden increase of illumination has no effect. NAGEL ('96, p. 76) finds the same thing in Spirographis, and ANDREWS ('91, pp. 285, 296) has observed the same phe- nomenon in the eyeless Hydroides dianthus. In these cases the branchiye seem to be the sensitive organs. Adult barnacles show a similar sensitiveness to light; for POUCHET ('72, p. Ill) found that momentary cutting off of the light, as by the shadow of the hand, caused arrest, for several seconds, of the rhythmic movements of protrusion of the appendages from the shell. In this case, the sensitive region has not been located. Some lamellibranchs (XAGEL, '96, p. 50) react similarly to increased light. These are examples of a phenomenon which we shall meet with again in considering growth. They serve to show that there is a wide-spread irritability of protoplasm to changes in intensity of light. Let us now review the conclusions of this section. Light — especially the thermic rays — is essential to the decomposi- tion of carbon dioxide by chlorophyllaceous plants. The only effective rays are those absorbed by the chlorophyll. The rate 180 LIGHT AND PROTOPLASM [Cn. VII of assimilation is increased by increased intensity of light. The chemical rays act to increase metabolic changes, and the output of carbon dioxide. As these rays become more intense, the metabolic changes go on with abnormal rapidity, until, finally, death ensues; thus, intense light is fatal to many, perhaps to all, organisms. Absence of light, however, is injurious only as preventing assimilation in chlorophyllaceous organisms ; but these supply the food for other organisms, so that continued darkness in any environment must likewise be eventually fatal to all life. All organisms, before succumbing to darkness or to light, enter into a condition of rigor, from which they may return to activity if favorable conditions are restored. Sudden change of intensity often produces violent protoplasmic changes, awakening quiescent organisms to activity, or causing, in the higher organisms, violent contractions. All of these effects of light, whether produced by the thermic or chemic rays, probably give rise to great chemical changes by which disturbances of metabolism, and eventually death, may be produced. Not all organisms find light immediately necessary to their existence; but very powerful light, long continued, proves fatal to most protoplasm. § 4. CONTROL OF THE DIRECTION OF LOCOMOTION BY LIGHT — PHOTOTAXIS AND PHOTOPATHY * In this section we shall (1) distinguish between false and true phototaxis ; (2) consider the observed cases of phototaxis among Protista, the parts of higher organisms, and the Metazoa as entire organisms ; and (3) discuss the general laws of photo- taxis and photopathy. * In this section we shall deal with two sets of phenomena which very likely are different, but which, in our ignorance, we cannot always distinguish. The first includes that active migration of organisms whose direction is determined by that of the rays of light. This is phototaxis. The second includes the wandering of organisms into a more or less intensely illuminated region, the direction of locomotion being determined by a difference in intensity of illumina- tion of the two poles of the organism. This is photopathy. According as the migration is towards or from the source of light, we can distinguish positive ( + ) and negative ( — ) phototaxis. According as the migration is towards or from the more intensely illuminated area, we can distinguish positive ( + ) and negative 4] PHOTOTAXIS AXD PHOTOPATHY 181 OK re] 1. False and True Phototaxis. — It must certainly be a very old observation that when small organisms are placed in a ssel in front of a window, they are soon found arranged with ference to the window ; some lying on the nearer side, some on the further side, and others swimming indifferently back and forth through the vessel. The conclusion is near at hand that this arrangement of the organisms is determined by the light. This conclusion is, however, not necessarily correct. Thus, SACHS ('76) showed that, under certain conditions, wholly passive substances — oil drops, in a mixture of water and alcohol — might exhibit a similar aggregation towards the window or away from it. These conditions are that the vessel should be cooler next the window. Then, on tlie cooler side, there will be a descending current ; on the warmer side, an ascending current ; on the surface, a current towards the win- a FIG. 51. — Vertical section through a dish showing distribution in water of passively suspended bodies, as a result of difference of temperature at the two sides of the vessel. .1, warmer side ; B, cooler side. Arrows show the direction of movement of currents in the water. The objects lighter than water are grouped at b ; those heavier than water, at a. dow ; and on the bottom, a current from the window. If the passive bodies are such as float, they will thus be carried towards the window, and will exhibit a false phototaxis (in the positive sense); if, on the contrary, they tend to sink, they will be carried from the window, and show false negative pho- totaxis. Xow this appearance, due to passive transportation by currents, may likewise, under the given conditions, be exhibited by organisms — but the phenomenon is not due to light (Fig. 51). There is at least one other kind of pseudophototaxis. This (— ) photopathy ; and correspondingly we can in this second case speak of the organisms themselves as photophil or photophob. In this nomenclature I follow GRABER. STRASBURGER used photometry for what I here call photopathy, but OLTMAXNS ('92, p. 206) has employed photometry to indicate the capacity of organisms to perceive different degrees of intensity of light. So, perhaps, the terminology here employed may lead to the least confusion. 182 LIGHT AND PROTOPLASM [Cn. VII may occur when there are in the vessel chlorophyllaceous organisms producing oxygen in the sunlight. The oxygen, more abundantly formed on the sunny side of the vessel, becomes, then, a means of attraction to other (chemotactic) organisms, whose position seems thus to be determined directly by relative brightness. A good example of this kind of pseudophototaxis is described by ENGEL- MANN ('81a): He found that the Schizomycetes in a certain drop of water, partially illuminated, were aggregated toward the illuminated side. Exami- nation revealed the presence of a chlorophyllaceous schizomycete — Bacte- rium chlorinum — in the drop, and the apparent phototactic appearances were easily accounted for as follows : Under the influence of light the Bacterium chlorinum secreted oxygen, and this acted chemotactically to attract the bacteria, which thus moved, at the same time, towards the illuminated area. That it was the oxygen produced in the sunlight rather than the light itself which attracted, was evinced by the fact that, when the supply of oxygen is abundant in all parts of the drop, or if the Bacterium chlorinum is removed, no aggregation takes place at the bright point. 2. Distribution of Phototaxis and Photopathy. — a. Protista. — We now come to the consideration of the cases of true pho- totaxis and photopathy, and shall first discuss the distribution of the phenomenon in the different groups of Protista. Of the Protista we may take up first the chlorophyllaceous forms. Flagellata and Swarm-Spores. — In no other group does phototaxis show itself more clearly than in this. The earliest studies were made here, but despite the ease of gaining results they were mostly fragmentary and uncritical. A simple ex- periment of NAGELI ('60) had, indeed, showed conclusively that swarm-spores are responsive to light. A glass tube three feet long and held vertically was filled with alga- water. When the upper end of the tube was enveloped by black paper the organisms moved to the lower end, and conversely. A diffi- culty was encountered, however, in the fact that when zoospores were placed in a plate by a window, the organisms gathered at the edge next the window, which, since the edge of the plate threw a shadow there, was the darkest part of the surface. , In consequence some authors had concluded that swarm-spores shun the light ; whereas COHN asserted, all too briefly, that they move in the direction of the rays and toward the source of light. Finally, FAMINTZIN ('67) had discovered that swarm- §4] PHOTOTAXIS AND PHOTOPATHY 183 spores which moved towards a light of a certain intensity would move from light of a certain greater intensity. That was the condition of knowledge on this subject when STRASBURGER'S ('78) epoch-making paper appeared. STRASBURGER worked with swarm-spores of various species of algoe, and with the flagellate Chilomonas and Euglena. He observed again the phenomenon that the sense ( 4- or — ) of response depends upon the intensity of the light. He also showed that the rate of movement is quicker in stronger light on account of the fact that the path taken by the organism is straighter ; and (p. 586) that phototaxis is the result of the organism putting its long axis in the axis of the inf ailing rays. STRASBURGER found also that, in general, the smaller species of swarm-spores and the smaller individuals are more respon- sive than the larger ones. Later studies have extended our knowledge of the distri- bution of phototaxis in this group. Swarm-spores have been studied by STAHL (78, '80) ; Euglena, by ENGELMANN ('82% p. 396) ; and Volvox, by CIENKOWSKI ('56), VERWORN ('89, p. 45), and OLTMANNS ('92). Especially interesting is the fact that colorless swarm-spores, like those of Chytri- diurn, which are parasitic upon chlorophyllaceous forms, respond like the green organisms. (STRASBURGER, '78, p. 568.) Desmids, especially Closterium, have been experimented with by STAHL ('78 and '79), KLEBS ('85), and ADERHOLD ('88). All are markedly phototactic in moderate, diffuse daylight. This phototaxis is the more striking since the method of loco- motion of these forms is peculiar. The crescentic Closterium moniliferum, for example, stands inclined and glides along, one extremity touching the substratum, the free extremity in ad- vance. The gliding seems to result from the secretion of a stream of mucus along the substratum. Now STAHL believed that the angle of inclination of the Closterium is dependent upon the direction of the iiifalling rays of light, being parallel thereto. This relation has been denied by KLEBS, but ADER- HOLD, by varying the direction of the infalling rays, has shown that the azimuthal position is determined by light. Under certain conditions Closterium moniliferum moves by a sort of head-over-heels motion, since the free end bends down to the 184 LIGHT AND PROTOPLASM [Cn. VII substratum and becomes attached, and the former attached end becomes free. STAHL explains this on the ground that the ends of Closterium periodically exchange their tendency to point towards the light. The appearances just described are found in diffuse daylight. In stronger light the azimuthal position is 90° from the infalling light. If direct sunlight falls upon desmids, they move from the light (negative pho- totaxis). Diatoms have been studied by STAHL ('80) and VERWORN ('89, p. 47). Locomotion is effected in these organisms as in desmids by the secretion of mucous threads. The movement towards diffuse daylight (Navicula, Stauroneis) takes place slowly, but it often affects nearly all the individuals. The long axis does not seem to be clearly oriented in the direction of the infalling rays, which may be partly accounted for by the normal zigzag method of locomotion. Under strong sun- light diatoms appear negatively phototactic. Occasionally a culture will be found whose individuals are separated into two groups — one next the positive side, the other next the nega- tive side of the vessel. Oscillaria. — VERWORN ('89, p. 50) has made experiments on the reaction of these organisms, whose method of locomotion is probably similar to that of desmids. They are markedly positively phototactic from half darkness to direct sunlight ; only in intense sunlight do they fail to accumulate at the posi- tive end of the vessel. The aggregation at the positive pole takes place by the threads assuming a direction parallel to the rays of light and creeping forward thus, side by side. VER- WORN states that after all have attained the + edge, rotation of the slide or vessel through 180° does not cause a prompt transfer of all individuals towards the light side — at least during the time of his observation only a few had crawled towards the light in its new position. According to WINO- GRADSKY ('87), Beggiatoa is generally negatively phototactic. Myxomycetes. — In its amosboid form and when subjected to strong sunlight ^Ethalium septicum retreats into the substratum, but while in the dark it comes to the surface (HOFMEISTER, '67, p. 625 ; STRASBURGER, '78, p. 620). Also, when the plasmo- dium is partially illuminated, the protoplasm tends to flow from PHOTOTAXIS AXD PHOTOPATHY 185 the illuminated region. (BARAXETZKI, '76, p. 328, and STAHL, '84, p. 167.) BARAXETZKI proceeded as follows: a glass plate was placed in a saucer so that its surface was 2 or 3 mm. below the rim. The plate was covered by filter paper which extended over the rim and here dipped into water, by which means it was kept moist. Over the saucer was ' laid an opaque cover, blackened below and provided with a narrow slit. The plasmodium was placed on the filter paper and diffuse daylight was thrown upon the slit by means of a plane mir- ror. In less than half an hour the illuminated threads of the plasmodium had become very thin, owing to the retreat of the protoplasm from under the slit to the darker region (Fig. 52). Rhizopoda. — Although, as we have seen, Pelo- myxa is irritated by a sudden illumination, a phototactic or photo- pathic response has not hitherto been certainly observed in this group. VERWORX ('89, pp. 40, 41), indeed, experimented, but with negative results, upon Amoeba limax, Amoeba princeps, Actinosphserium, and Actinophrys. Only in Polystomella crispa did he notice a slow wandering towards the source of light ; but he was uncertain whether this was due to light. VERWORX'S method was not well devised, however, for bringing out phototactic response. The Protista were placed on the slide, and, after cut- ting out heat rays by means of a plate of ice, were subjected to the light or to the EXGELMANN microspectrum, and illuminated at different intensities either over the whole body or over only a part. All disturbing influences, he says, were as far as possible eliminated : gravity, by an exact horizontal position of the microscope on a table with three screw-feet ; the action of the edge of the drop, by using a very broad drop ; and, finally, the laterally FIG. 52. — Plasmodium of ^Ethalium septicum, after having been kept in the dark for some time and then illuminated, for half an hour, over a cross-shaped area, only. The illumi- nated area is on the upper part of the figure. The protoplasm has retracted from it, leaving a partially clear region in the form of a cross. (From BARAXETZKI, '75.) 186 LIGHT AND PROTOPLASM [Cn. VII impinging rays of light, by means of a black cardboard box placed over the slide. Thus it is clear that all of his light fell upon the organism in perpendicular rays from below. This method of experimentation would clearly not show whether Amoeba is phototactic or not. I have experimented with Amoeba proteus, using methods resembling VERWORN'S, and likewise dissimilar ones, and have reached new results. In the first place, I have proceeded some- what after the fashion of VERWORN to determine whether the amoeba in a field illuminated from below, and separated by a sharp line into a light and dark half, showed any change of movement in passing from dark to light or from light to dark ; also, whether an amoeba moving in a uniformly illuminated field changed its direction when half of its body was dark- ened. Nearly all such experiments were negative. No effect resulting from the change from light to dark or the reverse could be detected. Thus far my results agreed with VER- WORN'S. In a second set of experiments, I proceeded differently. Usually one amoeba was isolated by means of a capillary tube. It was then introduced, with a drop of clear water, between two slips of glass, each about 25 by 50 mm., which were kept 2 mm. apart, and at the same time cemented together, by glass strips of equal thickness placed near the ends. By this means a broad field for movement with uniformity of conditions of con- tact was ensured. The whole space between the two glass plates being now filled with clear water, the entire apparatus was sub- merged in a vessel which contained water about 2 cm. deep, and which was slightly smaller than the stage of the micro- scope. Finally, the entire stage, but not the substage optical apparatus, was kept in the dark by means of a cone made of several thicknesses of dense black cloth fastened by a slip-noose to the objective, and folded below the stage so as completely to exclude all extraneous lateral light. Light from the mirror was cut off by an interposed card. Through a slit in the cloth on the side next the window, — a west window, — a beam of direct sunlight, or of reflected light from the morning sky, was admitted to the amoeba. The plates of glass being as nearly as possible horizontal and occasionally rotated, the directive action of gravity was eliminated. Since, so far as could be seen with § 4] PHOTOTAXIS AND PHOTOPATHY 187 the microscope, no local sources of food or oxygen occurred in the water between the plates of glass, chernotactic influences were uniformly distributed. From the conditions of the ex- periment already described, a difference in temperature or of illumination at the two poles of the amoeba is scarcely conceiv- able. The rays of radiant energy were the only directing agent. Under these conditions the amoeba nearly uniformly showed itself negatively phototactic to light of an intensity varying from strong diffuse light to direct sunlight. The absence of uniformity is to be ascribed to the accidental presence of some disturbing agent. The movements made by the amoeba were represented graphically by making at intervals a camera drawing of its outline. Two such graphic representations are repro- duced in Figs. 53 and 54. It must be said that it is difficult to get so extended a series of changes in light as is shown in Fig. 54, for the phenomenon of acclimatization comes in and the responses become irregular. But, despite such irregularities, my studies lead me unhesitatingly to conclude that Amoeba, although not at all photopathic, is strongly phototactic. This result is important, for, since Amoeba is responsive to light, it may very well be that such responsiveness is a general property of protoplasm. Ciliata. — A double action of light must be here taken into account. EXGELMAXX ('82a, pp. 391-395) states that those Ciliata which contain chlorophyll (algse) — e.g. Paramecium bursaria, Stentor viridis, Bursaria — move towards the light, but only when the oxygen tension in the water is low. Also when the water drop is illuminated by a microspectrum, instead of white light the organisms aggregate towards the red end. Here are the rays by which most oxygen is produced from the chlorophyll, since assimilation takes place fastest here. When the organisms are placed in excessively oxidized water they move from the light. The conclusions to which EXGELMAKN arrived from these and other facts were that these species have a very delicate sensitiveness to variations in oxygen tension, and that it is through this sensitiveness that light influences move- ment. Accordingly, it would seem that the apparent photo- taxis is truly a case of chemotaxis ; but this conclusion requires better evidence. 188 LIGHT AXD PROTOPLASM [Cn. VII 11:22 11:18 (2). 14:OO to 14:2O ft'" 11:12 11:02 10:55 ft 10:44 I 13:06 (3). 14:20 to 14:34 FIG. 53. — Camera drawing, showing the successive positions assumed by an amoeba subjected to light falling upon it from one side. The arrow lies in a horizontal projec- tion of the sun's rays. The amoeba retreats from the source of light. The numbers to the right of the outlines of the amoeba give the observed times between 10:28 and 11 :22 A.M. Magnified 16 diameters. FIG. 54. — Camera drawing, showing the successive positions assumed by an amoeba retreating from the light. The position of the infalling ray was successively changed from (1) to (2), (3), and (4). The arrow labelled " First direction of migra- tion " shows the direction of loco- motion of the amoeba before the light fell upon it at the beginning of the experiment. The numbers indi- cate hours and minutes. During the interval from 13:06 (=1:06) P.M. to 13:57, the amoeba was not under di- rect observation, since I was called away. Magnified 16 diameters. Cases that can be explained only on the ground of the imme- diate effect of light upon the direction of movement are cer- tainly rare. ENTZ ('88), indeed, has intimated that Opalina flees from light, but VERWORN ('89, pp. 53-57) was not able to confirm him in this point. VERWORN'S method was here, as in the case of Amoeba, not satisfactory. Instead of having the light fall from one side only upon the drop containing the Opalinas, he let the light pass vertically from below through a small hole, and could observe no tendency to avoid the illu- 10:34 ,10:28 13:00 12:54 §4] PHOTOTAXIS AND PHOTOPATHY 189 minated spot. The light in this case clearly did not act from one side, and the test of phototaxis can therefore hardly be said to have been critically made. Likewise, even with unilateral illumination, VERWORN was unable to gain a phototactic re- sponse with Stentor rceselii, St. coeruleus, Carchesium polypi- num, and Uroleptus musculus. On the other hand, we have often noticed here in Cambridge that our Stentor coeruleus is (rather indefinitely) negatively phototactic to diffuse daylight. Thus, an individual swimming free in a bit of glass tubing pointing horizontally towards the window only very slowly wanders away from the light. In conclusion, then, we must admit that Ciliata are not markedly phototactic, but more refined methods must be used before we can say of any of them that they exhibit no trace of this response. Let us summarize briefly the results obtained from Protista. Phototaxis is most marked among actively motile, chlorophyl- laceous forms. Many colorless forms are, however, also photo- tactic — Beggiatoa, Amoeba, plasmodia of Myxomycetes, and swarm-spores of Chytridium. The phenomenon is thus wide- spread, if it is not universal. b. Cells and Cell-organs. — Under this head will be consid- r* ered, (a) the rearrangement of chlorophyll corpuscles, (/3) the rearrangement of pigment in animal cells, and (7) the migration of pigment cells in the metazoan body. a. That the chlorophyll bodies of the higher plants change their position in the cell according to the intensity of the light to which they are subjected has been made known chiefly through the labors of FAMIXTZIN ('67), BORODIN ('69), FRANK ('72), STAHL ('80), and MOORE ('87). If one fastens a strip of black paper upon a leaf on which the sun's rays are falling, one will find, upon removing the paper after a time, that the darkened part is dark green whilst the brightly illu- minated part is considerably lighter, so that an image of the form of the dark paper is produced upon the leaf. This image is, however, only temporary. A few hours after the removal of the paper the leaf is of a uniform green again. Sections through a leaf thus affected show that in the dark green (shaded) part of the leaf the chlorophyll lies on those walls of the cells which are perpendicular to the incoming rays, 190 LIGHT AND PROTOPLASM [Cn. VII whilst in the light green (illuminated) part of the leaf the chlorophyll lies upon the walls parallel to the rays. When the grains are upon the superficial face of the cells they are said to be in epistrophe; when they have turned away from this face they are in apos- trophe. This apostrophic position is found under two opposite conditions of illumination : under intense light, as we have just seen (positive apos- trophe, MOORE), and upon prolonged standing in the dark (negative apostro- phe). (Fig. 55.) It appears, then, that epistrophe occurs only within certain limits of light intensity. The in- tensities included between these limits constitute what MOORE calls the epistrophic interval. The epistrophic interval varies in position and in extent in different species.* It has been found that in the case of plants which normally live in the bright sun the epistrophic * The limits were determined by MOORE, in a roughly quantitative way, by means of his photrum, constructed as follows. A room with a single window illuminated by the sun was chosen and 12 feet spaced off from the window back into the darkness. The intensity of the light diminished of course as one re- treated from the window. Plants of various species were allowed to stand, simultaneously, at varying distances from the window, and the distance back at which epistrophe began to appear, and, finally, at which negative apostrophe came in, were noted. Then a diagram ^ the actual scale was made (Fig. 56), showing the position of the points of beginning and ending of epistrophe (so- called positive and negative critical points). FIG. 55. — Cross-section through the leaf of Lemna trisulca. A. Position of the chlo- rophyll grains in diffuse daylight — epis- trophe. -B. Position of the chlorophyll grains in intense light — positive apos- trophe. C. Position of the chlorophyll grains in darkness — negative apostrophe. (After STAHL.) §4] PHOTOTAXIS AXD PHOTOPATHY 191 interval is a region of relatively high intensity (Fig. 56, 6) ; in aquatic plants the epistrophie interval occurs in a region of low intensity (Fig. 56, 1 and 2) ; and in shade-loving aerophytes in an intermediate position (Fig. 56, 4 and 5). One may say that every species is attuned to a certain intensity and range of light, in which epistrophe occurs, just as in swarm-spores there is a certain intensity and range of light in which positive phototaxis occurs, and that attunement depends upon the conditions to which the organism has adjusted itself through living in them. FIG. 56. — A diagram of MOORE'S photrum, showing for six spaces the epistrophie interval (shaded region). 1. Anacharis (Elodea) canadensis, " water- weed." 2. Lemna trisulca. 3. Saxifraga granulata (position of positive critical point). 4. Oxalis acetosella (position of negative critical point only approximate). 5. Pteris critica (positive critical point). 6. Pyrethrum sinense (garden Chry- santhemum). -+- indicates the brighter end of the photrum. Concerning the question of the mechanism of the movement of the chlorophyll grains, there is much difference of opinion. It is urged on the one hand that the chlorophyll grains move actively to attain their new positions, and, on the other, that they are passively carried by the cell currents. Of these two views analogical reasons are perhaps the strongest for preferring the second. As to the question in how far these movements can be regarded as adaptive, we may say that STAHL believed that they regulate the relation between intensity of sunlight and assimilating area, so that the quantity of assimilation shall not become too great. HIEKONYMTJS ('92, p. 466) considers them to be for the purpose of screening the nucleus. MOORE (p. 222), 192 LIGHT AND PROTOPLASM [CH. VII however, chiefly from a consideration of vegetative apostro- phe, has been led to the conclusion " that the movements of chlorophyll have no relation whatever to benefit or injury experienced by the grains, nor necessarily to the well-being of the protoplasm." /3. The Rearrangement of Pigment in Animal Cells in Response to Light. — One of the striking cases of this effect of light is seen in the pigment cells of the skin of the chameleon, as described by KELLER ('95, pp. 144, 162). He has found that the dark color of the (illuminated) skin is due to the rich ll cu. ep. FIG. 57. — Vertical section through a black dermal papilla of Chamaeleo vulgaris. ep, epidermis; CM, cutis; p, black pigment cells; p', processes of the cells con- taining pigment; yr, yellow pigment cells. (After KELLER, '95.) branching at the base of the epidermis of black pigment cells lying deep in the cutis (Fig. 57). In the dark, the pigment granules stream out of the branches into the cell body, but the branches themselves are undisturbed (Fig. 58). So long as the black pigment has this central position, the skin appears whitish. The light, on the contrary, causes the pigment, which is probably carried passively in the plasma, to move centrifu- gally. Whether the direct response to light of the pigment cells of the frog, as described by STEINACH ('91), is of the same nature, or due to contractions of the pigment cells, re- mains to be determined. Again, in the retina of the compound eyes of Arthropoda, §4] PHOTOTAXIS AND PHOTOPATHY 198 we find this capacity for rearrangement of pigment granules, as EXXER ('89 and '91, p. 104), STEFAXOWSKA ('90), SZCZA- WIXSKA ('91), PARKER ('95), and others have shown. In the higher Crustacea, for example, the pigment granules of the pigment cells surrounding the rhabdome (or " spindle ") are, in the dark, below the level of the spindle. Upon illumination, however, these granules migrate (or are carried) upwards, and partly envelop the rhabdomes: I believe it has not been deter- mined what rays are involved in producing this result. This response to light is considered to be an advantageous one, since FIG. 58. —Vertical section of a whitish-yellow dermal papilla ; lettering as in Fig. 57. p', processes of black pigment cells containing no pigment. (After KELLER, '95.) the pigment thus cuts off side rays from the perceptive organ — the rhabdome. It is interesting that we should find cells containing two so diverse kinds of pigment as chlorophyll and the retinal pig- ment responding to light in so similar a fashion. In most of the cases, if not all, this response is an adaptive one. 7. The Migration of Pigment Cells in the Metazoan Body. — It has been shown, apparently first by EXGELMAXX ('85), that the pigment cells of the retina vary their movements with the light. Thus, when a strong light is thrown upon the retina of the frog, the pigment cells send out pseudopodium-like processes between the rods and cones, whereas in the dark the 194 LIGHT AND PROTOPLASM [Cn. VII pigment lies behind all these elements. Also, among the Crus- tacea, the protoplasm of the outer pigment cells, surrounding the cones of the compound eye, migrates centripetally in a strong light, to return again to its peripheral position in darkness. These movements may also be considered adaptive. They are, in addition, movements which are discharged only by light. c. Metazoa. — We may treat of the. control by light of the movements of the higher animals somewhat more summarily than we have the preceding classes. The facts will be arranged by groups in systematic order. Among radial animals, Hydra has perhaps been for the longest time an object for photopathic study. TREMBLE Y (1744, p. 66) had noticed that Hydra viridis, and even muti- lated pieces of it, came to the light side of the vessel. When tbte light was admitted only through a chevron-shaped slit, the Hydras were later found aggregated opposite the slit in the form of a chevron. That it was not the warmth of the sunlight that attracted was shown by turning the slit towards the cooler air, whereupon the same response occurred. The observations of TREMBLEY showed that the Hydras did not move in as straight a line as possible towards the light (they must, of course, follow a firm substratum), but gradually wandered towards it. The response of Hydra must therefore be considered as photopathy. More extensive studies were made upon Hydra by WILSON ('91), who found that Hydra fusca is likewise responsive to light, and, indeed, photophil with reference to diffuse daylight, and photo- phob to direct sunlight. And it can be shown that it is an advantage to Hydra to be photophil, since many of the Ento- mostraca upon which it feeds are phototactic. Besides Hydra, I know of only one case of response by Cce- lenterata to light. The larvae of the sponge Reniera are said (MARSHALL, '82, p. 225) to flee from the light, — probably negative phototaxis. Among Echinodermata, Asteracanthion rubens (GRABER, '85, p. 155) appears to be photophil, and Asterina gibbosa (DRIESCH, '90, p. 155) to be photophob. Although, as we have seen, some radial animals may respond to light, the phenomenon is more wide-spread in the bilateral groups, — flatworms, annelids, crustaceans, insects, molluscs, PHOTOTAXIS AND PHOTOPATHY 195 and Vertebrates. The results of experiments may here be given in tabular form. Unless otherwise stated, the light is sup- posed to be diffuse daylight, and the response to be phototaetic. TABLE XVIII ORGANISM. SENSE OF RESPONSE. AUTHORITY REMARKS. Fresh-water planaria .... - LOEB, '90, p. 95 DRIESCH '90 p 155 Polygordius larva LOEB, '93, p. 90 see p. 200 Earthworm {DARWIN, '81, p. 21 GRABER, '83, p. 210 > photophob Leech HESSE, '96 LOEB, '90, p. 96 + {TREMBLEY, 1744, p. 96 BERT, '78, p. 989 1 photophil ( ?) Many marine copepoda . . Balauus, larva + -(or+) — Cor +) LUBBOCK, '82, '83 DAVENPORT and CANNON LOEB, '93, p. 96 GROOM and LOEB, '90 p. ) phototaetic see p. 200 see p. 200 Limulus, larva 160 LOEB, '93, p. 83 Idotea tricuspidata . . -j- GRABER '85 p 141 photophil Diastylis (Cuma) rathkii . Carcinus mfenas . . . + LOEB, '90, p. 91 DRIESCH '90 p 156 mud-inhabiting photophob Homarus americanus, larva Plant lice + + HERRICK, '96, p. 189 LOEB, '90, p. 55 Blatta germanica (blinded) Musca dom. (?), larva . . . Musca adult + GRABER, '83, p. 235 LOEB, '90, p. 69 LOEB '90 p 81 photophob Musca vomitoria, larva . . Musca caesar, larva DAVIDSON, '85, p. 160 POUCHET, '72, p. 113 Eristalis teuax, larva . . . Lepidoptera adult _|_ POUCHET, '72, p. 129 ( SEITZ, '90, p. 337 Lepidoptera larva -{_ ! LOEB, '90, p. 46 | LOEB, '90, p. 51 see p. 197 Ants, after gaining wings . Melolontha vulgaris. (May beetle) + f POULTON, '87, p. 315 LOEB, '90, p. 63 LOEB '90 p 86 Tenebrio molitor, larva . . Dentalium - LOEB, '90, p. 84 LA.CAZE~DUTHIERS, '57, photophob Rissoa octona -i_ p. 25 GR \BER '85 p. 144 photophil Littoriua rudis DRIESCH, '90, p. 155 photophob Gasterosteus spinachia. . . Triton - GRABER, '85, p. 148 GRABER, '83, p. 221 photophob Froo- . LOEB '90 p. 90 196 LIGHT AND PROTOPLASM [Ce. VII A study of this table reveals the fact that, in general, organ- isms which live in shady places or in the dark are negatively phototactic or photopathic, while those living in the light are positively phototactic or photopathic. Thus most fresh- water planarians and leeches are inhabitants of shady pools. Polynoe is generally found in dark retreats, the earthworm and fly larvae are lovers of the dark, and shell-molluscs are for the most part enclosed in cases impervious to light. On the other hand, Daphnia is found largely in open pools, larvae of Lepi- doptera and many adult insects live in the sun. Into this general rule there are some cases which do not so obviously fall. But we have little data concerning the habits of the races em- ployed and the absolute intensity of light used, so that these cases may perhaps be only apparent exceptions. One clear exception is that of the mud-inhabiting Diastylus, which is 4- phototactic. 3. The General Laws of Phototaxis and Photopathy. — Under this head we shall consider : (a) the sense of the response ; (6) the effective rays ; (c) prototaxis vs. photopathy ; (cT) the mechanics of response to light. a. The Sense of the Response. — In considering, now, more generally, the effect of daylight upon the direction of loco- motion of organisms, we must recognize that the sense of response (whether + or — ) depends upon internal conditions and external conditions — upon the quality of the protoplasm and the nature of the environment. Let us consider, first, the dependence upon internal conditions. We find that, under similar external conditions, different organisms respond differently. For example, many Oscillariaa (p. 184) are positively phototactic even in direct sunlight; whilst even moderately strong light will repel many diatoms. In fact, we find that a positively phototactic or photopathic organism is such only in the presence of a certain intensity of light. When the intensity is diminished below a certain point, no response will occur. When, on the other hand, the intensity is increased above a certain point, the organism moves away from the source of light. There is a certain range of intensity in which alone the positive responses occur. The position and the extent of this positively phototactic range vary for the § 4] PHOTOTAXIS AND PHOTOPATHY 197 different species, — they are closely correlated with the condi- tions of light in which the organism has been reared. As a result of these conditions, we may say that each organism is attuned to its peculiar range and intensity of light. Upon the ground of this difference in attunement may be explained the remarkable difference in behavior of butterflies and moths to light. It is well known that butterflies fly towards even the strongest sunlight, whilst moths are secluded during the daytime, but at night fly towards the candle-light. LOEB ('90, p. 46) has performed some experiments with these insects, which I will cite in detail. EXPERIMENT 1. — (a) Sphinx, Bombyx, and other moths were kept in a large glass cage in a room illuminated only by daylight. As darkness came on, the inoths began to fly towards that side of the cage which was next the window. Again (6), pupse of nocturnal moths, left in a room, emerged during the night, and were always found in the morning at the closed window of the room. Finally (c), a nocturnal moth, made to fly in the daytime, directed its way to the window. Thus, nocturnal moths are positively phototactic to diffuse daylight as well as candle-light. EXPERIMENT 2. — Hawk-moths were brought into a room with the single window at one end, and a petroleum lamp at the opposite end. It was found that, as twilight came on, the moth flew to the window, or to the light, according to the relative intensity of the one or the other at the point where the moth was liberated. Thus, there is no preference for artificial light. The conclusion at which LOEB arrived was that these moths undergo a diurnal variation in responsiveness to light, which corresponds to the change from day to night. But the fact that, in experiment 1 c, nocturnal moths flew, in the daytime, towards the diffusely lighted window, throws a doubt upon this interpretation. All the facts are equally well explained upon the following ground : Butterflies are attuned to a high intensity of light, moths to a low intensity ; so that bright sunlight, which calls forth the one, causes the other to retreat. On the other hand, a light like that of a candle, so weak as not to stimulate a butterfly, produces a marked response in the moth. We shall consider, in a moment, the cause of these differences in light attunement. We have seen that one internal condition modifying response is the racial quality of attunement. A second is that of period of life. Thus, LOEB ('90, p. 56) has found that, at the intensities 198 LIGHT AND PROTOPLASM [Cii. VI! employed, the wingless plant lice were hardly responsive to light. The winged form was markedly positively photo tactic. So, likewise, in the case of ants (LoEB, '90, p. 63), during the period of the marriage flight the males and females (1ml not the workers) are strongly positively phototactic, but after that period they show themselves neutral. The case of the house- fly, Musca, is interesting, since the larva and adult are photo- tactic in opposite senses (see table). In most of these cases, the difference in responsiveness is associated with a difference in habit. The sense of response depends, also, as we have seen, upon external conditions. In this regard, the immediately preceding conditions of light, the temperature, the concentration, and the supply of oxygen have important effects. We shall consider, in order, the action of these conditions. Light can modify the response to light; thus, GROOM and LOEB ('90) have shown that the nauplii of Balanus, as well as other pelagic animals, come to the surface of the sea during the night, but descend before the strong sunlight. This does not indicate merely a low light-attunement of the race ; for nauplii exposed to sunlight in the early afternoon are all posi- tively phototactic, and only gradually, as the day progresses, move from the sunny window, until, finally, even as dusk approaches, all are found on the side away from the window. Nor have we here to do with a diurnal change in the sense of the response. For if a culture is kept in the dark, it is found to be at first positively phototactic at whatever time of clay it is exposed ; only later acquiring the negative phototaxis. In the same way, when the young Balanus lar\ a> leave the interior of the shell of the parent, they are at first positively photo tact ic : but after being in the light for from ^ to 2 hours, they become negatively phototactic. The more intense the light, the quicker its effect. Another observation upon the nauplii is representative of a new class of light effects. When nauplii which have become negatively phototactic through exposure are covered for a t'e\\ minutes, and then suddenly again exposed to light, they move momentarily towards the light, and then begin their negative movement again. Somewhat similar are the results obtained §4] PHOTOTAXIS AND PHOTOPATHY 199 by STRASBURGER ('78, p. 574) on Ulothrix spores, which are positively phototactic in a weak light. While responding to such a light, they do not, however, turn at once when a light of repelling intensity is thrown upon them. So, also (p. 600), when Hsematococcus is responding to indigo light, the inter- position of red glass does not at once cause it to turn from its path. In all these cases, the immediately preceding condition of light continues to exert an action which modifies the response. Closely allied are the results obtained by VERWORN ('89, p. T>0) upon the diatom, Navicula brevis, which is attuned to only the faintest light. When, however, a culture had been ivured by a window for two weeks, the attunement to light had been so raised that now a slight degree of positive photo- taxis took place in diffuse light. We have in these facts examples of a phenomenon which we have observed in the art ion of other agents. It is one expression of the acclimatiza- tion of organisms to the peculiar conditions of their environ- ment. We have just seen that every organism has its optimum intensity of light for metabolism and response, and that this optimum is very varied; but, throughout, one law holds. Organisms which are accustomed to live in strong light have a high optimum intensity; and those accustomed to live in a weak light have a low optimum intensity. This relation is, indeed, so close as to raise the suspicion that the normal intensity of the light has determined the optimum. And this suspicion is confirmed by the experimental evidence just cited. Now, since the position of the optimum is usually advantageous, we may conclude that light can so modify protoplasm as to adapt it for the conditions in which it is living. We now pass to the consideration of the effect of tempera- ture upon response. This effect was noticed by STRASBURGER ('78, p. 605) in the swarm-spores of Haematococcus, Ulothrix. etc., which, at a temperature of 16° C. to 18° C., gather at the side of the drop next to the window. If, now, they are sub- jected to a temperature of 40° C., the intensity of the light being constant, they migrate to the opposite side. On the other hand, at a temperature of 35°, the + aggregation is more complete than at 16° to 18°. Control experiments with emul- sions satisfied STRASBURGER that this change is not due to 200 LIGHT AND PROTOPLASM [Cn. VII currents in the water, but is a truly vital phenomenon. That it is such is indicated also by the following curious behavior. If swarm-spores which normally aggregate at 18° towards the positive side of the drop, are suddenly brought to 18° from 30°, they appear, for a moment, negative. Conversely, if swarm- spores which normally aggregate at 30° towards the negative side of the drop are suddenly brought from 8° to 30°, they ap- pear, for a moment, positive. Thus, the immediately preceding culture-temperature affects the sense of the response. The results obtained by STKASBURGER have been in part confirmed by other authors in other species. GROOM and LOEB ('90, pp. 166, 172) state that in the case of the nauplii of Balanus — "at a higher temperature, for instance 25° C., the phenomena [of phototaxis] are run through more sharply and quickly than at a tempera- ture of about 15°"; and again, "we often succeeded in suddenly changing the sense of the heliotropism of the larva by a sudden change, of only a few degrees, in the temperature of the water." This statement is unfortunately so vague as to say little more than this, that temperature influences the response. MASSART ('91, p. 164) remarks, incidentally, that the flagellate Chromulina is + phototactic at 20° C., but — phototactic at 5° C. LOEB ('93, pp. 90, 96) obtained a result with Polygordius larvse and Copepoda which seems, at first sight, the opposite of STRASBURGER'S. Polygordius larvae, negatively phototactic at 16°, were gradually cooled to 6°, at which temperature they began to move rapidly towards the + side of the vessel. As the temperature gradually rose they became —phototactic again. Indi- viduals which were (abnormally) + phototactic at 17°' to 24°, when raised gradually to 29° became — phototactic. Sudden diminution of temperature within the limits at which response occurs did not change the sense of their response. Thus, negative individuals brought suddenly from 23° to 13° remained negative. Exactly parallel results concerning the relation of temperature and response were obtained by LOEB from Copepoda. All results may be harmonized in the expression : Diminu- tion of temperature below the normal causes reversal of the normal response ; elevation of the temperature to near the maximum accelerates the normal response. The point of light attunement varies with the temperature.* Not only light and heat, but also the concentration of the medium affects light attunement. We are indebted to LOEB * It follows from these experiments that it is necessary in any phototac- tic investigation to regard not only the intensity of the light, but also the temperature. §4] PHOTOTAXIS AND PHOTOPATHY 201 ('93, pp. 94, 96) for information on this subject. Negatively phototactic Polygordius larvae were placed in sea water to which 1% to 1.3% XaCl had been added. They now appeared positively phototactic. Positively phototactic individuals, on the other hand, placed in sea water diluted with 40% to 60% fresh water became negatively phototactic. Similar results were obtained with Copepoda. Thus increased concentration rendered -f phototactic (raised light attunement), while dimin- ished concentration rendered — phototactic (lowered light at- tunement). Increased concentration works, therefore, upon Polygordius and Copepoda, according to LOEB, like diminished temperature. Finally, the chemical condition of the medium has an impor- tant effect on photopathy, as can be judged from certain observations of EXGELMANX ('82% p. 391). Various chloro- phyllaceous Ciliata, e.g. Stentor viridis and Paramecium bur- saria, are photopathic only when the oxygen supply in the medium is below the normal. In such media they are strongly photophil. This case is clearly not a case of phototaxis, as we have just seen (p. 187). The response is advantageous since it brings these organisms into the sunlight, where chlorophyll can produce oxygen. To recapitulate : the sense of response in phototaxis is modi- fied by previous subjection to light, by temperature, and by concentration. These agents modify the attunement of the organism. Any quantitative experiments upon phototaxis must therefore take all of them into account. Certain chloro- phyllaceous organisms exhibit + photopathy, but only in an insufficiently oxygenated medium. From the foregoing considerations we conclude that for every phototactic organism there are three ranges of intensity to be distinguished : the positively phototactic range in which the organism moves towards the light ; below this, the indiffer- ent range extending to darkness ; above it, the negatively phototactic range extending up nearly or quite to the point of light-rigor. The limits of these ranges vary with both external and internal conditions. b. The Effective Rays. — We have hitherto considered chiefly the action of white light, merely referring casually to the 202 LIGHT AND PROTOPLASM [Cn. VII action of the different rays of which it is composed. We must now answer the question : What different effects do the different rays have ? The effect of the different rays in phototaxis is very clearly seen in the various groups of Protista and among the Fla- gellata and the swarm-spores, there is entire uniformity of response according to the testimony of COHIST ('65, p. 36), STRASBURGER ('78, pp. 593-599), ENGELMAXN ('82% p. 398, in Euglena), and VERWORN ('89% p. 49, in Navicula). Here the more actinic rays with shorter and more rapidly vibrating wave, act exactly like white light, whilst the rays from the opposite end of the spectrum have no more effect than darkness. More precise determinations were made by STRASBURGER ('78, p. 597), who found that the swarm-spores of the alga Botridium responded to the blue and violet, but especially to the indigo, whilst the green and ultra-violet were alike without effect. And ENGELMANX, by means of his microspectral apparatus, was able to determine that Euglena responded chiefly to the rays X = 0.47//. to X =0.49 /-t ; that is, rays very near FRAUEX- HOFER'S line F. The colorless Myxomycetes agree with the chlorophyllaceous forms, according to BARAXETZKI ('76, p. 332), in responding to blue rays only. Among the higher organisms, Hydra, according to WILSOX, accumulates especially behind blue glass, to a small extent behind green glass, and is entirely indifferent both to the upper violet rays and those below the green. The photophil starfish Astracanthion rubens, even when deprived of its eyes, was found by GRABER to be "cyanophil"; even, though in slight degree, to a low intensity of light. Among Mollusca, GRABER found that the photophil Rissoa moved towards the blue even when the intensity of the blue light was less than that of the red, and DRIESCH asserts that the photophob Littorina rudis shuns only blue rays. Thus, without multiplying cases, the results of experiments may be summed up as follows : positively pho- totactic or positively photopathic organisms are such only in the presence of the blue rays. There are some few observations which are in apparent dis- cord with this conclusion. Whether the ultra-violet rays are ever active is a fairly debatable question. LUBBOCK ('82, §4] PHOTOTAXIS AND PHOTOPATHY 203 p. 127 ; '84, p. 137) showed that Daphnia and some ants are very sensitive to the violet rajs, and GRABER ('83, p. 214) found that the photophob earthworm withdraws from ultra- violet rays. This result is unusual, however, for most experi- menters have agreed with this much of BERT'S ('78, p. 989) conclusions, that " the animals see . . . only those rays which we ourselves see," or, better, that the range of irritability of the protoplasm of our retina is as great as that of any other protoplasm. Below the blue, some authors have believed the yellow rays, the brightest of the spectrum, to be prevailingly photopathic. Thus both BERT ('68, p. 381) and LTJBBOCK ('83, p. 214) find that Daphnia accumulates especially in the yellow and green parts of the spectrum. Regarding these results I have only the comment that they need further confirmation. c. Phototaxis vs. Photopathy. — We have hitherto assumed the existence of two dissimilar sorts of locomotor response to light — phototaxis and photopathy. Phototaxis we defined as migration in the direction of the light rays, and photopathy as migration towards a region of greater or less intensity of light. Are we justified in making this distinction? The chief ground for this distinction is the existence of two sorts of phenomena which, not having been generally recog- nized as different, have led to extensive discussion. The best- established of these phenomena is phototaxis, which was proved to exist by certain crucial experiments of STRASBURGER on Protista, and of LOEB on Metazoa. Mr. W. B. CANXON and I have used STRASBURGER'S methods on Daphnia, and con- firmed his results. Figure 59 gives a view of our apparatus, which was essentially the same as STRASBURGER'S. It con- sisted of a hollow prism P, containing a dark solution and placed over the trough T, with its organisms. STRASBURGER ('78, p. 585) put swarm-spores of Botrydium and Bryopsis into the trough, and reflected the light perpendicularly through the prism upon the trough. There was now a perfect gradation in intensity from the thick end to the thin edge of the prism. Yet the organisms showed no tendency to aggregate at the clearer end. The light was now permitted to enter the trough obliquely, the thicker end of the prism being next the source T OF THE UNIVERSITY )) 204 LIGHT AND PROTOPLASM [Cn. VII of light, as in the figure. The spores now moved towards the source of light, i.e. in the direction of the inf ailing rays but constantly into a region of less intensity of light. FIG. 59. — Diagram showing the position of apparatus and the direction of the rays in an experiment in phototaxis. T, trough of water containing organisms, A and B its two ends, M its middle. P, a prismatic box containing a solution of India ink. S, screen to cut off extraneous light. L, gas-lamp having a WELSBACH burner. Drawn to scale. LOEB'S ('90, p. 32) results were obtained by the use of quite different methods. In one case he employed a chamber made of two test tubes placed with their mouths together. One of the tubes was darkened except for a clear streak at one end, c ; and this darkened tube was pointed towards the light, so that the rays fell through its axis. Although the clear chamber was evidently the brighter, the Porthesia larvae with which he experimented moved into the darkened chamber and thus towards the source of light (Fig. 60). Again, a clear test tube containing larvae (Fig. 61) was placed so that its closed end b was directed towards the window FF. A bundle of sun's rays SS struck nearly perpendicularly the mouth of the tube a, when the larvae were aggregated at the beginning. Neverthe- less the larvae, since their progress in the direction of the per- pendicular rays was soon interrupted by the walls of the tube, moved towards the window, from the region of greater intensity of light in the direction of rays which passed more nearly in the axis of the tube. That this is not negative photopathy to strong light is indicated by the fact that the Porthesia larva is attuned to a high intensity of light. The evidence would thus seem satisfactory that the direction of migration of certain PHOTOTAXIS AND PHOTOPATHY 205 organisms is determined by "the direction of the light rays. There is, then, such a thing as phototaxis. But is the direction of locomotion ever determined by a dif- ference of intensity of light in adjacent regions, without refer- ence to the direction of the light rays ? Whole series of obser- vations make this probable ; for a migration to a definite part of the trough has followed unequal illumination by rays perpendicular to the trough. Thus LUBBOCK found that JF FIG. 60. — Two test tubes a and b, containing Porthesia larvae c, which move towards the window FF, although in doing so they pass from a brighter to a darker region. (LOEB, '90.) FIG. 61. — Diagram to show how Porthesia larvae move in the test tube ab towards the window FF, although in doing so they leave the part of the tube more brightly illumined by the sun's rays SS. (LOEB, '90.) Daphnias, placed in a trough nearly perpendicular to the rays dispersed by a prism, moved towards the brighter part of the spectrum. GKABEB employed screens of diverse translucency and color, which were placed adjacent to one another, and found that the organisms tended to aggregate opposite the one or the other. OLTMANNS ('92, p. 195) has offered certain new experiments pointing in the same direction. These experi- ments were made upon Volvox minor and Volvox globator, which were placed in a trough between which and the source 206 LIGHT AND PROTOPLASM [Cn. VII of light a vertical screen was interposed. This screen formed one side of a wooden box and consisted of two glass plates making an angle of 2° with each other, the interspace being filled with a solution of India ink in gelatine. When the sun- light was let through this screen, the individuals in the trough behind it sorted themselves into two groups ; the partheno- genetic individuals, which collected opposite the clearer part of the screen, and the female individuals, with fertilized eggs, which collected behind the darker part of the screen, each suit- ing itself to the intensity of light to which it was attuned. When the intensity of the light was changed, the organisms also changed their positions. Finally, LOEB ('93, pp. 100-103) has found that fresh-water plaiia- rians (Planaria torva) gradually accumulate in the darker parts of the vessel, since the light con- stantly stimulates them to move- ment, and in their wanderings they gain the dark places by accident and there are at rest. So it comes about that when these Planaria are in a shallow cylindrical vessel (Fig. 62, #, 5, f)4 p Frog, adult (summer) . . Frog, adult 42° to 43° 43.8° water ; death at once Death-rigor in 7 to 14 minutes 374 MORIGGIA, '91, p. 385 SPALL A.XZ \vi 41° Raised in from 5 to 10 1787, p. 55 See p 253 Kabbit 1 44° to 45° minutes Death point when OBERNIER, '66, p. 22 Dog I Man 45° convulsions at 42° In water * giddiness in EDWARDS '^4 p Human spermatozoa . . . Vertebrate muscle .... Vertebrate muscle (frog) 50° 40° to 50° [ 45° to 50° | 35° a few seconds Died in 10 minutes Raised in 30 seconds Raised in 18 minutes 374 MANTEGAZZA, '66, p. 186 KUHNE, '59, pp. 784-804 GOTSCHLICH, '93, p. 123 The determinations given in the above table may be compared only with caution, for diverse conditions give results which cannot be directly correlated. Thus individuals of the same species, but reared under diverse environment, have a different resistance period to heat. The lethal temperature varies accord- ing as the organisms are suddenly or gradually subjected to the high temperature. Also, the individuals of the same species will die at different temperatures according as they are rap- idly subjected to the high temperature or gradually accus- tomed to it, and, as we have seen, a lower temperature, long continued, often produces the same result as a higher tempera- ture during a brief period. Finally, too little care has been exercised in most cases to determine the temperature of the water immediately upon, and a few minutes after, placing the animals in it, — an operation which lowers the temperature of the water. In the experiments cited, unless otherwise stated, the conditions were gradual subjection continued for a short time only. The quality of the water in which the 238 HEAT AND PROTOPLASM [On. VIII experiments were carried on is supposed to be, except for its temperature, normal for the species. Summarizing the table, we find that the Protista have the highest maximum tempera- ture of any group, it being in extreme cases about 60° for active organisms, but generally between 40° and 45°. Among the Metazoa, the highest maxima recorded (excepting Rotifera and Tardigrada) are 44° to 45° for Turbellaria, Anguillula, Naididse, Nepa (water-scorpion), Notonecta (water-boatman), Cloe larva, Salamander, and mammals. A water-mite (Hydrachna) is said to have withstood up to 46.2°, and some vertebrate tissues resist up to 50°. For the great majority of Metazoa, the maxi- mum temperature lies below 45° and, in the case of marine species, below 40°. The low maximum temperature of marine species is probably due to the low maximum temperature of the sea as compared with ponds. We may consequently con- clude from the foregoing that the maximum temperature for protoplasm lies generally between 35° and 50°, the lower limit being characteristic of organisms living in a medium of low temperature (the sea), the latter, of organisms reared in warm pools or of organs (vertebrate muscle) in a body kept at a high temperature. The question now arises, what is the cause of the death of protoplasm at high temperatures? To get some insight into this matter, let us examine the phenomena accompanying death of protoplasm from overheating. KUHNE ('64, p. 44) thus describes the appearance of an Amoeba subjected for a moment to a fatal temperature (45°). The structure is entirely altered since it has become transformed into a mass of knobbed, opal- escent, solid lumps, which, even in transferring to the slide, become easily broken apart. This appearance is clearly due to a coagulation of the protoplasm. A similar coagulation takes place in Actinophrys eichhornii (KtJHNE, '64, p. 67) at 45°. " The sphere shrinks into a flat, hardly transparent, cake, no longer reacts to the strongest induction shocks, and breaks up after 24 hours into a heap of small granules and irregular pieces." Likewise, in muscles a change is produced by heat which is evidently a kind of coagulation. A coagulation then seems to be the immediate cause of death at high temperatures. But just what is the component of protoplasm which co- §3] TEMPERATURE-LIMITS OF LIFE 239 agulates at the death point of organisms? As KUBGSTE ("64, p. 1) pointed out, it cannot be ordinary egg albumen ; for, excepting the contractile substance, we know of no native albumen which coagulates between 35° and 50° C. It was KUHNE'S great service to show that there is a substance which can be pressed out of frozen, triturated, and then thawed muscle, which becomes quickly opalescent at 40°, through the separation of the muscle plasma into myosin and a serum. This serum, in turn, contains an albuminoid which coagulates at 47° (DEMANT, '79 and '80). Now, since there are proteids in muscle which coagulate at about the point at which muscle goes into permanent heat-rigor, and since these proteids can no longer be squeezed out of rigid muscles, the conclusion seems justified that permanent heat-rigor in muscle is due to the coagulation of these proteids. Related, easily coagulable proteids occur in widely dissimilar organisms. For example, myosin has been found in vegetable protoplasm (\VEYL, '77, p. 96), and HALLIBURTON ('88) has described a globulin from blood corpuscles which coagulates at 48° to 50°. Their distribution in protoplasm is, therefore, probably general, and so we are justified in concluding that the death of protoplasm by heat is, in general, the result of the coagulation of a proteid (globulin). Death occurs because the vital machinery has been broken down.* 2. Temporary Rigor and Death at the Lower Limit of Tem- perature, Minimum and Ultraminimum. — Whilst towards the upper limit of ordinary terrestrial temperatures (35° to 40° C.) molecular changes in organic compounds are hastened, towards the lower limits (—40° to — 50°) molecular changes are slow, being principally confined to the transformation from the liquid or gaseous to the solid or liquid condition. This transforma- tion does occur in the water of protoplasm, but the colloids, * At this place reference may be made to the fact that protoplasm subjected to a high temperature sometimes breaks to pieces with the suddenness and com- pleteness of an explosion. Thus STRASBURGER ('78, p. 611) found that Chilo- monas curvata was uniformly killed at 45° C. by the explosion of the body, and Dr. W. E. CASTLE tells me that he has observed the same phenomenon under like conditions in Stentor. An investigation of this profound change in pro- toplasm would be sure to throw valuable light upon the nature of the living .substance. 240 HEAT AND PROTOPLASM [Cn. VIII which constitute the living part, are not modified by even the lowest of the terrestrial temperatures, except that the molecular changes which they undergo are very slow. This being true, protoplasm which contains no water, or very little, ought not to be changed by low temperatures, — that is to say, the machine will not be injured. With the activities of the machine — with the vital processes — it is, however, quite different. They are essentially chemical processes, and hence we should expect them to be diminished at a low temperature. If, as PICTET ('93) maintains from an extensive and highly important series of experiments, no chemi- cal processes take place at temperatures below — 100° C., then protoplasm ought to exhibit no vital processes at this tempera- ture, and, indeed, experience shows that, as we have already seen, as the temperature is lowered below the optimum, all manifestations of activity diminish. It is clear that at a certain point they must entirely cease. And at that point deathr following the usual definition of the word, would ensue. But does the cessation of the vital processes, without injury to the mechanism, necessarily preclude the possibility of a return to activity ? Let us examine the experimental evidence on this point. SCHUMACHER ('74, p. 179) subjected yeast to cold and found that, at the lowest temperature produced (— 113.7°), the yeast cells were not completely killed. More recently, PICTET ('93% cf . also C. DE CANDOLLE, '84) has sub- mitted various dry seeds and spores of bacteria to a temperature of nearly — 200°, at which temperature the atmosphere becomes liquefied, but without fatal effects. Other results were still more remarkable : vibratile cilia from the mouth of the frog were cooled to — 90°, and recovered their movement upon raising the temperature. Some Rotifera and Infusoria were frozen in their native water at — 60°, and kept at that tem- perature, apparently, for nearly 24 hours. Most have subse- quently regained their activity. Eggs of the frog, lowered slowly to — 60°, can revive. Eggs of the silk-worm can resist to —40°. Other experiments of PICTET* will be referred to * This general criticism of PICTET' s paper is, I believe, valid. He does not give us data enough upon time of subjection to the low temperature, time em- ployed in reducing the temperature, and other details. Thus, concerning his § 3] TEMPERATURE-LIMITS OF LIFE 241 later. As a result of these and others' investigations we may conclude : Protoplasm may under certain circumstances, of which one of the most important is the absence of water, resist, uninjured, the lowest temperatures. TJiere is no fatal minimum temperature for dry protoplasm. We must now turn our attention to those cases in which the phenomena of cessation of activity or death appear, and seek to determine their causes ; and first concerning temporary cold-rigor. We have already seen that as the temperature is lowered, the rate of metabolic processes and protoplasmic movements is lowered. What happens at the lower limit of activity, and where does this lie? The chlorophyll granules of Vallesneria move (according to VELTEX) only about 1 mm. per minute at 1° C. and not at all at 0°; the rotation of Nitella ceases (\AGELI, '60, p. 77) at 0°C.; in Tradescantia hairs, movement is wholly arrested on freezing the cell sap (KtJHXE, '64, p. 100, and DEMOOR, '94, p. 194). Even in seeds and bacteria, which are not killed by the lowest temperatures, all vital activities have probably ceased at 0°, for DE CANDOLLE ('65) found that in only one species out of ten could he get a seed kept at 0° to germinate, and even then germination was so retarded that it took from 11 to 17 days as opposed to 4 days at 5.7°. Likewise, bacteria do not multiply below +5° to -f- 10° (BOXARDI and GEROSA, '89). Among animals, KTJHNE ('64, p. 46) found Amoeba cooled to near 0° almost motionless. PURKIXJE and VALENTIN ('35) first noticed that the ciliated experiments on Scolopendra, lie merely says : "I have frozen to — 40° three Scolopendras which perfectly resisted the treatment and lived after thawing out. Submitted to — 50°, they have also resisted. Frozen a third time to — 90°, they are all three dead." Now, in the absence of further data, it is quite possible that the heat of metabolism kept the internal body temperature considerably above that of the chamber, the thick cuticula preventing rapid loss of heat, very much as a man's clothing enables him to withstand the — 40° of an arctic winter. Another experiment of PICTET'S lends greater probability to this explanation of some cases of great resistance. Three snails were subjected to a temperature of from — 110° to — 120° during several days. The operculum of two of these was not intact, so that it did not close the orifice. These two individuals died ; but the third, which was completely sealed up, survived. Those which were not sufficiently clad, so to speak, lost so much internal heat that their internal fluids were frozen. Of course this criticism cannot apply in the case of those organ- isms mentioned in the text which are without a thick cuticula. 242 HEAT AND PROTOPLASM [Cn. VIII epithelium of the frog ceased its movements at 0°. Muscles of the frog were found by KUHNE ('64, p. 3) to become at _ 3° to — 7° a solid lump which did not, however, wholly lack irritability. The evidence of all these cases shows that activity nearly ceases in protoplasm at or near 0° C. Another effect produced on protoplasm by cold — an effect which often immediately precedes quiescence — is violent con- traction. This has been repeatedly observed. The protoplasm of Tradescantia hairs, which has been in cold-rigor, was found by KLJHNE ('64, p. 101) to lie in separated rounded drops and lumps, — an appearance like that resulting from excessive stimu- lation. The rapid freezing of muscle gives rise, according to HERMANN ('71, p. 189), to violent contractions. The sciatic nerve of the frog's leg when cooled to — 4° to — 8° causes clonic contractions of the muscle, lasting two minutes. (AFFAXA- SIEFF, '65, p. 678, and others.) It is clear, then, that cold acts as a violent stimulus to protoplasm. The final result of temporary rigor is thus clearly brought about by the cooperation of two causes : (1) the diminution in the chemical processes upon which metabolism and movement depend, and (2) the directly stimulating effect of the cold, which acts like contact or excessive heat. Both causes work to produce a quiescence which may be replaced by activity when the causes are withdrawn. The fact that cold-rigor usually occurs close to the zero- point indicates that the activities of protoplasm are closely determined by the fluid state of water. This fact is not to be explained on the ground that freezing prohibits all chemical change — many chemical changes take place below the freez- ing point of water, but, apparently, few of those which are involved in metabolism. Nor is the rigor due to the change which the freezing of the protoplasmic fluids brings, because as the temperature approaches the zero-point, but while the water is still perfectly fluid, metabolism diminishes ; and it diminishes at such a rate as to cease just where water begins to freeze. The critical point for vital activity has been adjusted to this critical point of water. So, too, the composition of protoplasm is such that at a tem- perature, lying below the normal and above the freezing point §3] TEMPERATURE-LIMITS OF LIFE 243 of water, those chemical changes rapidly occur which we desig- nate response to the stimulus of cold. This composition of protoplasm, upon which cold can work such important modifi- cations, is a quality of immense importance in the economy of the organism, as the changes of each autumn testify. Below the point of temporary cold-rigor lies that of death, if death point there be. The position of the death point is, however, very diverse in different organisms. Part of the diversity in the death points assigned by different authors is, however, due to the fact that in the methods of determining the death points there has been a lack of uniformity. Five elements ought always to be regarded in experiments on the ultrarninimuin temperature. (1) History of the tem- perature conditions in which the individual or its race had lived before experimentation ; (2) rate at which the organ- ism has been cooled, — if possible, the temperature of the organism itself rather than that of the medium ; (3) inten- sity of cold just sufficient to kill ; (4) duration of applica- tion of the cold and the kind of medium in which the erganism is subjected to the cold ; * and (5) the rate of thawing out.f These elements have been too much neg- lected in the past. I shall now present in tabular form some of the more reliable determinations of the death point of organisms, pref- acing with the caution that the results are not closely comparable. * The duration of application and intensity of the fatal cold stand in an inverse relation, so that organisms which resist a temperature — A° for X min- utes will resist a lower temperature, —(A + a)°, for a shorter time, X — x. Thus, Clepsine complana resists — 8°C. for 15 minutes ; — 5°C. for 90 minutes. So Planorbis corneus resists — 7° for 5 hours, but — 5° for 48 hours. Again, Musca domestica can resist — 12° for 5 minutes ; — 8° for 20 minutes ; and — 5°C. for 40 minutes. (ROEDEL, '86.) Since many authors have little re- garded the duration of action of the cold, their determinations have little scientific value. t The importance of this is illustrated by some experiments of SACHS ('60, p. 177), who found that the leaves of the beet or cabbage frozen at from - 4° to - 6° died if they were thawed in air at 2° or 3°, or in water at 6° to 10° ; but lived when slowly thawed in water at 0°. In general, the more gradual the thawing, the lower the fatal temperature. 244 HEAT AND PROTOPLASM [Cn. VIII TABLE XX* DETERMINATIONS OF THE ULTRAMINIMUM OF ORGANISMS REARED UNDER NORMAL CONDITIONS SPECIES. MINIMUM TEMPEBATUEE. CONDITIONS OP EXPERIMENT. AUTHORITY. Plant cells. Trad.esc3.ntia — 14° + In water, rapidly i — 14° — frozen In air, rapidly fro- f KUHNE, '64, p. 100 — 1° zen Fully frozen cautious- STRASBURGER '78 Swarm-spores (Proto- coccus) 0= to — 1° ly thawed p. 612 COHN, '50, p. 720 Protozoa. Amoeba 0° — Rapidly frozen on KUHNE '64 pp 46-47 Animal tissues. White blood corpuscles : of Amphibia . f — 2° to — 3° 1 slide over ice and salt • During 8 hours; warm- ed rapidly SCHENK, '69, p. 26 of rabbit -r - 3° For a short time; warmed rapidly During 15 minutes " 26 * « « Qg Saliva corpuscle .... Red blood corpuscle . Spermatozoa i of Amphibia -6° to -8° -15° + _ 40 to _ 70 Over 60 minutes " 27 POUCHET, '66, p. 18 SCHENK '69 p 29 of Mammalia .... of frog — 6° — _ 8° to — 10° — Returned to activity on thawing " 30 of frog — 10° to 12° of man — 17° Gradually thawed p. 353 Eggs of Amphibia . . . Ciliated epithelium of Anodonta .... - 7° , , i — 3° During 1 hour Subjected a very short time 183 SCHENK, '69, p. 28 ROTH, '66, p. 189 « « ion [ minutes * Temperatures all in degrees Centigrade. — before a number indicates below zero, - or + after a number indicates that the true lethal temperature lay slightly below or above that number. (A) indicates that the organism was in air ; (W), in water. §3] TEMPERATURE-LIMITS OF LIFE 245 SPECIES. MINIMUM TEMPERATURE. CONDITIONS OF EXPERIMENT. AUTHOKITY. Platy helminths. Dendroccelum lacteum ^fo^usca. Helix hispida 0° to — 1° — 8° Suddenly or gradually subjected, till ice forms During 30 minutes ROEDEL, '86, p. 207 " " 191 Helix pomatia Helix pomatia . — 10° (— 14° to— 18°)+ During 600 minutes Gradually frozen for " 192 POUCHET '66 p 28 Helix hortensis .... Helix aspera . . . (—14° to —18°)+ (—14° to —18°)+ 180 minutes, and then thawed During 180 minutes " 180 " <« «« « «« Planorbis - 7° " 300 " (A) ROEDEL, '86, p. 212 Limnaja — 7° " 180 " (A?) " " 212 Pulmonate embryos . Limax (Pto — 1° — 17°+ " Died upon freezing " (WandA) During 2 hours (A) " 212 POUCHET '66 p 26 Annelida. Aulastomum gulo . . . Clepsine complanata . ' ' Leech ' ' 2° — 5° — 6° During 12 to 15 hours (W) During 90 minutes ( W) * ROEDEL, '86, p. 206 " 213 DOENHOFF '72 p 725 Inseeta. Apis mellifica — 15° utes" (W) During ^10 minutes ROEDEL, '86 p 212 Apis mellifica — 1 5° DOENHOFF '72 p 7^4 Formica rufa Pelopaeus (chrysalis) . Lema sp -1.5° -28J- — 6° " 180 minutes Out-of-doors, with- stood this tempera- ture During 30 minutes ROEDEL, '86, p. 196 WYMAN, '56, p. 157 ROEDEL, '86 p. 197 Psederus riparius . . . Phytonomus sp Alelolontha - 4D -12° — 18° + " 45 " 90 " 1°0 " (A) " 197 ii 197 POUCHET '66 p 26 Melolontha (larva) . . Cetonia Hydropbilus j Dvtiscus -15° + -17° + - 4° " 180 " (A) " 120 " (A) " 60 " (A) " 26 " 26 KOCHS, '90, p. 682 Vanessa cardui, larva Vanessa io, larva . . . Smerintbns populi . . Ocneria dispar — 15° -173 + -10° - 4D " 600 " 120 " 150 " 30 " ROEDEL, '86, p. 212 POUCHET, '66, p. 27 ROEDEL, '86, p. 212 " " 212 Culex pipiens, larva . Musca | 3 _ 50 to — 10° " 60 " 180 " " 212 DOENHOFF, '72, p. 725 Alusca dom 5° " 00 " ROEDEL '86 p 201 Various insects .... 0° " 2 to 30 minutes (on ice) PLATEAU, '72, p. 98 246 HEAT AND PROTOPLASM [CH. VIII SPECIES. MINIMUM TEMPERATURE. CONDITIONS OP EXPERIMENT. AUTHORITY. Arachnida. Phalangium opilio . . Tegenaria domestica . Argyroneta aquatica . Hydrachna cruenta . . - 9° — 6° . - 4° - 4° — 2° to — 3° During 60 minutes " 60 " 180 " 30 " 480 " ROEDEL, '86, p. 201 " 201 " 201 " " 201 DOENHOFF,'72,p.724 Crustacea. Cyclops quadricornus . Cyclops spirillum . . . Daphnia pulex .... 0° - 6° 0° 1 "(TF) " 120 "(IF) f OF) •! PLATEAU, '72, p. 300 ROEDEL, '86, p. 201 " 201 Gammarus pulex . . . Asellus aquaticus . . . Astacus fluviatilus . . Vertebrata. Rana esculata 0° 0° — 11.5° — 4° to — 10° " 30 "(TF) (TF)| " a day (A) " 180 minutes (A) PLATEAU, '72, p. 300 ROEDEL, '86, p. 205 PLATEAU, '72, p. 299 ROEDEL, '86, p. 205 POUCHET, '66, p. 32 « « 19 Summarizing these conditions, we find that the following organisms, even when thawed out carefully, are killed by sub- jecting to a temperature of between 0° and — 5° for 60 minutes: Amoeba, swarm-spores, white blood corpuscles of Amphibia, planarians, pulmonate embryos, small leeches, certain entomos- tracans, and some insects and spiders. These organisms are all either soft bodied or of small size, and, excepting animals which, like the bees, live in protected situations, they do not winter over in our northern temperate countries. The following species, on the other hand, resist — 10° for at least 60 minutes : some snails (possibly), the beetles Melolontha (perhaps) and Phytonomus, the Vanessa larva, Pelopseus chrysalis, and the crayfish, — all protected by a thick covering, or of rather large size. The reason why large size and thick covering should increase resistance is not far to seek, — both conditions tend to prevent the rapid loss of heat, — to defend the body from freezing through and through. Another cause of variation in resistance to cold is, doubtless, the amount of water in the protoplasm. I have already referred to this cause as explaining the fact that dry seeds and spores § 3] TEMPERATURE-LIMITS OF LIFE 247 can withstand almost any temperature.* MULLER-THURGAU ("80) found that the " succulent labellum of Phajus freezes at — 0.56°C. ; the succulent leaf of Sempervivum, at —0.7°; the potato tuber, at — 1° ; the leaf of Tradescantia mexicana, at — 1.16° ; the ivy leaf, at — 1.5°; the leaves of Pinus austri- aca, at — 3.5°; young shoots of Thujopsis, at — 4°." (ViXES.) In this series of plant tissues, we see that the more succulent the tissue, the higher its ultraminimum. Possibly the reason why spermatozoa have so low an ultramaximum, despite their small size, is on account of the denseness of their protoplasm. Not all variations in ultraminimum temperature are, however, explicable upon the ground of difference in size, body-covering, or density of plasm. The interpretation of the difference in sensitiveness to cold of the honey bee (Apis melifica) and the red ant (Formica rufa), between Bombyx on the one hand and Smerinthus and Vanessa on the other, must wait for further knowledge. The question now arises, what is the cause of death in organ- isms and protoplasm which succumb to low temperatures ? With the higher animals the immediate cause is doubtless in part asphyxia resulting from a stoppage in the flowing of the frozen blood plasma, and in part the destruction of the red blood corpuscles, as well as the white. f With the simpler organisms, like planarians, Protozoa, or Tradescantia hair-cells the case is different. An insight into the changes which pro- duce death in such organisms may be gained from KUHXE'S ('64, p. 101) description of the effect of a temperature of — 14° on Tradescantia hair-cells. The frozen hairs were placed in water and observed under the microscope. " The appearance," * Striking cases are on record of the resistance of gemmules, or "animal spores," to cold. Thus, WELTNER ('93, p. 276) saw gemmules of Spongilla fragilis frozen in an aquarium from December 26 to January 24, from the end of January to February 5, from February 20 to March 6, and from March 12 to 24 ; the intervals being occupied by thawings. Yet these gemmules produced young sponges. In other cases, a certain amount of freezing favors the subse- quent development of gemmules, e.g. those of fresh-water Bryozoa (BRAEM, '90, p. 83) and the eggs of the silk-worm (DUCLAUX, '71). t POUCHET ('66, p. 18) found that when blood of the frog was let fall into a capsule at a temperature of - 15° few of the red corpuscles were uninjured. In most the nuclei had been cast out into the plasma. 248 HEAT AND PROTOPLASM [Cn.VIII says KUHKE, " was very remarkable, for there was no trace of the protoplasmic network; but the violet cavity of the cell con- tained, in addition to the naked nucleus, a large number of separate round drops and lumps." In this case, the separate pieces eventually became active again, so that the protoplasm, though nearly killed, was not quite so. The phenomena seen by KUHKE so closely resemble those produced in the same kind of cells by the galvanic current and other strong irritants as to indicate that cold acts as an intense irritant. We cannot, however, conclude that cold acts in no other way. It is clear that the expansion of forming ice in the vacuoles of the protoplasm must seriously disturb the structure, and, since the whole matter has received little attention, it is possible that a molecular change of some sort takes place when there is much water in the freezing protoplasm. To summarize : Death by freezing results in the higher animals largely from asphyxia, and in the simpler organisms from excessive irritation, mechanical rupture, and, perhaps, other causes. I shall now sum up this section on the effect of extremes of heat and cold. As the temperature is elevated above the opti- mum, molecular changes occur in the protoplasm leading to its contraction. The contraction becomes more violent as the tem- perature is still raised, until, finally, a new series of molecular changes occur by which the protoplasm begins to coagulate. At this point the protoplasm begins to lose its irritability. If this process has not proceeded far, the vital activities may, under favorable conditions, return (temporary heat-rigor). Beyond a certain point (death point) recovery is impossible. The death point varies with the species, but lies not far from the maximum natural temperature attained by the medium in which they live. On the other hand, as the temperature is diminished from the optimum, the chemical processes of metabolism decrease in vigor and come to a standstill at about the freezing point of water. Violent contractions accompany the cooling process, concomitantly with which the protoplasm breaks down. From this condition of temporary cold-rigor recovery is still possible ; but a little below, at a point dependent upon the size of the body and the diathermous qualities of its §-4] ACCLIMATIZATION TO EXTREME TEMPERATURES 249 covering, the water of the body begins to freeze, and in that process, or the subsequent thawings, the protoplasm undergoes a (partly mechanical) change resulting in death. If the body, however, contains no water, freezing cannot kill it. Thus the effect of high temperatures is principally chemical, involving the living plasma ; that of low temperatures is prin- cipally mechanical, involving the water of the body. Both raising and lowering the temperature act also as irritants.* Finally, the positions , of the maximum and minimum stand in most intimate relation to the inorganic environment of the organism and have been molded to that environment. § 4. ACCLIMATIZATION OF ORGANISMS TO EXTREME TEMPERATURES The phenomena to be discussed in this section fall naturally into two subsections: (1) acclimatization to heat and (2) accli- matization to cold. They will be considered in that order. 1. Acclimatization to Heat. — Our study of the maximum temperature which organisms reared under ordinary circum- stances can withstand, led us to the conclusion that few active organisms can resist a temperature of over 45°, and for whole groups like Coelenterata, marine Mollusca, and Crustacea, and the fishes, 40° is a point of death. Yet, on the other hand, it has long been known that there are organisms living in certain hot springs in waters of considerably higher temperature. I shall now give in tabular form some cases which I have collected of organisms living at or above the normally lethal temperature of the species, f * Whether sudden change of temperature has an especial effect upon the movement of protoplasm is a disputed question, which has been answered posi- tively by DUTROCHET ('37, p. 777) and HOFMEISTER ('67, p. 53) for Nitella, and DE VRIES ('70, p. 394) for root hairs of Hydrocharis, but has since, as a result of careful experiments, been denied by VELTXER ('76, p. 214) for Nitella and other plant cells. I It is desirable that accurate data concerning the temperature of organisms in hot springs should be made, and we have, in this country, unusually favorable conditions offered for this study, especially in Arkansas, California, and the Yellowstone National Park. It is to be hoped that persons who have had the proper training should, when contemplating a visit to hot springs, provide them- 250 HEAT AND PROTOPLASM [Cn. VIII TABLE XXI LIST OF SPECIES FOUND IN HOT SPRINGS, WITH THE CONDITIONS UNDER WHICH THEY OCCUR No.* SPECIES. TEMP. C. LOCALITY AND CONDITION OF LIFE. AUTHORITY. 1 Chroococcus 51° to 57° Benton's Hot Springs, Cal. WOOD, '74, p. 34 2 Nostocs or Pro- 93° Geysers, Lake Co., Cal. ; not BREWER, '66, p. 391, tococcus abundant at this tem- also WYMAN, '67, perature p. 155 3 Nostocs 51° to 57° Benton's Hot Springs, Cal. WOOD, '74, p. 34 4 Anabsena ther- 57° Dax, warm springs SERRES,'80,pp.l3-23 malis 5 Leptothrix 44° to 54° Carlsbad Springs COHN, '62, p. 539 6 Oscillaria or 54° to 68° Yellowstone Nat. Park, WEED, '89, p. 399 "Confervse" U.S.A. 7 " 54.4° Springs, Bernandino Sierra, BLAKE, '53, p. 83 Cal. 8 ii 57° Algeria, Constantine prov- GERVAIS, '49, p. 12 ince, waters of Hammam- Meskhoutin 9 «< 57° Hot Springs, Taupo, New SPENCER, '83, p. 303 Zealand 10 « 60° to 65° Geysers, Lake Co., Cal., BREWER, '66, p. 392 U.S.A. 11 " 60° to 65° Hot Springs, Ark., U.S.A. JAMES, '23, II, p. 291 (Long) 12 " 71° Hot Springs at Banos Luzon, DANA, '38-'42, p. 543 Philippines 13 " 75.5° Soorujkoona Hot Springs HOOKER, J. D. '55, 1, p. 24 14 « 81° to 85° Ischia EHRENBERG, '59, p. 493. 15 i< 98° Iceland FLOURENS, '46, p. 934 16 " ? Outlet of Lake Furnas, DYER, '74, p. 324 Azores. selves with a hand lens, bottles of alcohol for preserving organisms for further study, and an accurately calibrated thermometer. A source of error to be guarded against lies in the precise determination of the temperature of the water immediately surrounding the organism observed; for in some warm springs or their outlets the surface water is said to be much warmer than the deeper layers in which the organisms are found. Finally, if possible, it would be desirable to determine on the spot, experimentally, the maximum temperature which these organisms can withstand. For this determination some of the methods referred to on p. 220 should be used. ' * Notes on each of these cases will be found at the end of this chapter, pp. 263-267. §4] ACCLIMATIZATION TO EXTREME TEMPERATURES 251 No. SPECIES. TEMP. C. LOCALITY AND CONDITION OF LIFE. AUTHORITY. Diatoms Frequently associated with other algae in hot springs 17 Physa acuta 33° to 35° Sources of Dax, St. Pierre, DUBALEN, '73, p. iv France 18 Paludina sp. 50° Thermal waters, Abano, DE BLAINVILLE, '24, Padua p. 141 19 "Bivalve testa- ? Hot Springs, Ark. MITCHILL, '06, p. 306 ceous animal" 20 Rotifera and An- 44° to 54° Carlsbad Springs, Bohemia COHN, '62, p. 539 guillulidse 21 Anguillulidae 45° Aix, springs DE SAUSSURE, 1796, V, p. 13, § 1168 22 " 81° Ischia, in hot springs EHRENBERG, '59, p. 494 23 Cypris balnearia 45° to 50.5° Hammam-Meskhoutin MONIEZ, '93, p. 140 24 Stratiomys larva 69° In hot spring, Gunnison GRIFFITH, '82, p. 599 B Co., Col. 2.-. " ? In hot spring, Uinta Co., BRUNER, '95 Wyo. 26 " Water beetle " 44.4° In warm spring, India; HOOKER, J. D. '55, p. abundant 24 27 « ? Hot spring, Port Holier, DALL, W. H. (per- Alaska sonal letter) 28 Barbels 34° ? BERT, '77, p. 169 29 Frogs 38° " Baths of the Pise " SPALLANZANI, 1787, Tom. I, p. 55 To summarize : Protista are stated to have been found in nature in water at temperatures far above 60° C. The most striking cases are of Oscillaria and " Conferva " from several localities, which resist nearly up to the boiling point of water. The closely allied Nostocs are, perhaps, next most abundant and resistant, reaching 93° (possibly Protococcus) in the Cali- fornia geysers. Metazoa are stated to live at temperatures far above 45°. Although some doubt has been cast on No. 22 by HOPPE-SEYLEK'S inability to confirm EHRENBERG'S observa- tions, the case seems established of Cypris thriving at 45° to 50.5°. Very extraordinary are the observations Nos. 24 and 25 on Stratiomys Iarva3, which, however, are sadly in need of confirmation by competent observers. Leaving out of account, for the moment, the less well established cases, there still remains abundant evidence that organisms can live and thrive 252 HEAT AND PROTOPLASM [Ck. VIII in hot springs at a temperature near or above that which proves fatal to their close allies. No one doubts that in all the cases cited above the individuals living in hot springs have been derived from ancestors which lived in water whose temperature rarely exceeded 40° C. The race has therefore become acclimatized, and the question arises : How has that acclimatization been effected ? Now experiments have shown that organisms, when gradually accustomed thereto, may resist a temperature which would have killed them if they had been suddenly subjected to it. There- fore it seems probable that the acclimatization of organisms to hot springs has been a slow, long-continued process, during which they have become gradually accustomed to higher and higher temperatures, probably attaining the hot springs by slowly advancing up their effluent streams. This adaptation may have taken place without selection, purely by the capacity of individual adaptation which organ- isms possess. That individual adaptation is sufficient to account for the vitality of organisms in hot springs has been shown by experiment. DUTROCHET ('37, p. 777) observed, long ago, that an organism which at first seemed injured by a high temperature gradually regained activity while still subjected thereto. Thus, he found that the current of Nitella was at first diminished by raising it to 27° C., but it soon became rapid again ; raised, now, to 34°, the circulation began to fall off again, but in a quarter of an hour, the same temperature continuing, the circulation became very rapid. This phenome- non was repeated, also, at 40°. Similarly, HOFMEISTER ('67, p. 53) brought Xitella flexilis suddenly from -f- 18.5° to 4- 5°C. The streaming movements ceased. After staying 15 minutes in the cooler room, however, the rotation of protoplasm recovered. Much more important, however, are the remarkable experi- ments of DALLINGER ('80). He kept Flagellata in a warm oven for many months. Beginning with a temperature of 15.6° C., he employed the first four months in raising the tem- perature 5.5° ; this, however, was not necessary, since the rise to 21° can be made rapidly, but for success in higher tempera- tures it is best to proceed slowly from the beginning. When the temperature had been raised to 23°, the organisms began § 4] ACCLIMATIZATION TO EXTREME TEMPERATURES 253 dying, but soon ceased, and after two months, the temperature was raised half a degree more, and eventually to 25.5°. Here the organisms began to succumb again, and it was necessary repeatedly to lower the temperature slightly, and then to advance it to 25.5°, until, after several weeks, unfavorable appearances ceased. For eight months, the temperature could not be raised from this stationary point a quarter of a degree without unfavorable appearances. During several years, pro- ceeding by slow stages, DALLINGER succeeded in rearing the organisms up to a temperature of 70° C., at which the experi- ment was ended by an accident. In this case it is plain that the high temperature acted upon the same protoplasm at the end of the experiment as it did at the beginning. But while the protoplasm at the beginning of the experiment was killed at 23° C., at the end it withstood 70°. It will be seen that, by gradual elevation of the tempera- ture, Flagellata may become acclimated to a temperature of water far above that which they can withstand when taken directly from out of doors, and approaching that of the hottest springs containing life. A series of experiments, less extensive than that of DAL- LIXGER, was carried on by Dr. CASTLE and myself ('95, pp. 236-240) upon the tadpoles of our common toad, Bufo lentigi- nosus. Recently laid eggs were divided into two lots : one lot was kept in a warm oven at a constant temperature of 24° to 25°, others at about 15° C. Both lots developed normally, but the former much the more rapidly. At the end of 4 weeks the point of heat-rigor was ascertained for each lot, by gradually heating (in from 5 to 10 minutes) the water contain- ing them, until the tadpoles showed no response to stimulus, but. upon cooling, regained activity. The result was that the toad tadpoles had gained an increased capacity to heat. For when they were reared at a temperature of about 15° C., every tadpole went into heat-rigor at 41° C., or below; whereas, when they were reared at 24° to 25°, a temperature 10° higher, no tadpole died under 43°, the average increase of resistance being 3.2°. This increased capacity of resistance was not produced by the dying off of the less resistant indi- viduals, for no deaths occurred in these experiments during 254 HEAT AND PROTOPLASM [Cn. VIII the gradual elevation of the temperatures in the cultures. The increased resistance was due, therefore, to a change in the protoplasm of the individuals. The question now arose : In how far is this change in the protoplasm permanent? Will a return of the, individuals to cool water cause a return to the old point of heat-rigor? We made a few experiments on this subject which showed that tadpoles which during 33 days in warm water have acquired an increased resistance of 3.2° lose part of that acquired resist- ance during 17 days' sojourn in cooler water. But the loss is a very slow one. The effect of the high temperature on the tadpoles is not, therefore, transitory, but persists — we have not been able to determine how long — after the cause has been removed. So we may conclude : Individual organisms have the capacity of becoming adapted to a high degree of temperature, so that a temperature which normally is fatal may be withstood. This adaptation of the individual accompanies the subjection of organisms to temperatures higher than those to which they have already become accustomed. This capacity exists among both Protozoa and Metazoa. The effect of the elevated tem- perature persists (though in diminished degree) a considerable time after the individual has been restored to a lower tempera- ture. Acclimatization may show itself not only in the change of the maximum temperature, but also in the elevation of the optimum. This is shown by the following experiments of MENDELSSOHN ('95, p. 19). When Paramecia are placed in a trough whose temperature is 24° to 28° at one end and 36° to 38° at the other, they are found to collect at the cooler end, which indicates that the temperature of that end lies nearer their optimum. If, however, the Paramecia, while uniformly distributed in the trough, are subjected to a uniform tempera- ture of 36° to 38° for from 4 to 6 hours, and then, in the same trough, to a temperature varying from 24° at one end to 36° at the other, they no longer collect at the usual optimum of 24° to 28°, but at 30° to 36°. Thus in 4 to 6 hours, by the action of a temperature of 36° to 38°, the optimum has been raised 6° to 8°. §4] ACCLIMATIZATION TO EXTREME TEMPERATURES 255 Since experiments have proved the fact of acclimatization, it now remains to determine, if possible, its cause ; to answer the question, by virtue of what property can organisms which, like Flagellata, normally perish at 45° C. come to live at 70° or even higher temperatures ? We have seen that death at high temperatures is apparently due to coagulation of certain proteids in the protoplasm which undergo a chemical change at between 45° and 50° C. Now, although the matter has not yet been studied in these proteids, it has been shown for egg albumen that in proportion as it is dried its coagulation point rises, as the following table from LEWITH ('90) shows : — EGG ALBUMEN. COAGULATION TEMPERATURE. In aqueous solution With 25% water With 18% water With 6 % water Without water 56° C. 74° to 80° C. 80° to 90° C. 145° C. 160° to 170° C. Since the coagulation point of egg albumen is raised by dry- ness, it is very probable that a similar cause may act to raise the coagulation point of protoplasm in organisms of hot springs. Experimental studies are much needed upon this point. Mean- while it can be said that one of the qualities which gives ca- pacity of resistance to high temperatures is dryness. I shall now cite some cases that I have collected, which prove this point. It has been found that while moist yeast is killed at a tempera- ture below 60°, dry yeast may be heated to 100° C. without losing its vitality (SCHUTZENBERGER, '79, p. 162). Damp uredo-spores are killed at 58.5° to 60° C., but dry ones with- stand up to 128° (HOFFMAN, '63); and dry spores of some molds up to 120° (PASTEUR, '61, p. 81). According to DAL- LIXGER ('80, pp. 11-14), the dry spores of various Flagellata are capable of withstanding a temperature from 10° to 27° C. higher than that which these spores can resist in fluid. Accord- ing to DOYERE ('42, p. 29), various animalcules (Rotifers, Tarcligrades) which cannot in water withstand a temperature of 50° C. may, after long drying, be heated in air to 120° C. 256 HEAT AND PROTOPLASM [Cn. VIII (rarely to 125°) without all dying. The foregoing cases show clearly that increased resistance capacity is frequently gained by subjecting the protoplasm of the organism to dryness. But there are other conditions under which the living sub- stance shows extraordinary resistance capacity. In general, as is well known, the spores of organisms withstand higher tem- peratures than the motile stage, when both are in water. This rule holds for many cases : The spores of some bacteria may be heated for a time above 100° C. without killing them, although their motile stage is killed by 50° to 52° (LEWITH, '90). DALLINGER and DRYSDALE ('74, p. 101) and DALLINGER ('80, pp. 13, 14) have determined maximum temperatures for several Flagellata and their spores in water. While none in the motile stage could withstand a temperature higher than 61°, the spores in water withstood maximum temperatures varying between 65.5° and 131° for the different species. Have the high resistance capacity of dry protoplasm and that of spores a common cause ? Or, in other words, is the proto- plasm of spores especially free from water ? Many observations make it appear probable that this is so. Thus in the case of bacteria, the protoplasm of the spore stage is optically denser and occupies less space than in the motile stage. (Cf. LEWITH, '90.) In the case of the ciliate Infusoria, the larger size of the protoplasmic mass makes the comparison of the condition of the protoplasm in the two stages easier. We glean the facts from BUTSCHLI ('89, pp. 1652-1654). As the process of encyst- ment proceeds, the contractile vacuole continues to function, the intervals between its contractions gradually increase, and finally it disappears some time after the encystment is com- pleted. Hand in hand with these changes goes a gradual con- densation of the protoplasm. This condensation BUTSCHLI believes to be due to an excretion of water from the proto- plasm. In Actinosphserium the change from the richly vacuolated motile form to the encysted condition is even more marked. As BRAUER ('94, p. 193) has shown, the protoplasmic mass becomes, during the process of encystment, smaller and denser. The loss of water from the protoplasm is without doubt due to §4] ACCLIMATIZATION TO EXTREME TEMPERATURES 257 the continued activity of the contractile vacuole at a time when 110 fluids are being taken into the protoplasmic body. From the foregoing considerations it appears probable that one of the important characters of " spores " is the diminished amount of free water held in the protoplasm; or, in other words, its dryness. This dryness of the coagulable substance would seem to be cause of its higher resistance. So far the evidence seems complete. Whether, however, loss of water is the ultimate cause of the high resistance capacity of hot-spring organisms or of those gradually acclimatized is still uncertain. Analogy renders it highly probable that such is the case. 2. Acclimatization to Cold. — Just as organisms may become acclimatized to high temperatures, so also may they live in very cold regions. I cite a few examples : Several species of Pro- tista are said to live in the Alps above the snow line, coloring the snow red. (SHTJTTLEWORTH, '40.) A tardigrade is found in the same locality. Certain insects live on or in the snow or ice. Thus Desoria glacialis (or glacier flea) lives on the Swiss glaciers, and on the snow live Podura hiemalis, Trichocera brumalis (when the temperature is "below the freezing point," FITCH, '46, p. 10), and other species of Trichocera and Podura. Cf. also Boreus hiemalis and B. brumalis (FiTCH). Although swarm-spores are usually extremely sensitive to cold, STRAS- BUEGER ('78, p. 613) cites a case of a marine alga in which they were being formed and thrown out when the temperature of the water was between —1.5° and —1.8° C. Increased resistance to cold seems often the result of the action of cold on the organism. Thus, while SCHWARZ ('84, p. 69) found that Euglense gathered in the summer time were not responsive below + 5° to -f- 6° C., ADERHOLD ('88, p. 320) found that Euglense gathered in the winter would respond even at 0°. We may say, the winter cold had in some way lowered the heat attunement of these Protista. In seeking for an explanation of acclimatization to cold we should recall that the cause of death from cold is chiefly the freez- ing of water in the protoplasm, and the irritation of excessive cold. Accustomed to great cold, protoplasm would doubtless be no longer irritated by it ; whether under these circumstances 258 HEAT AND PROTOPLASM [Cn. VIII it would contain less water is a question which lacks an experi- mental answer. The conclusion from the results offered in this section is this : protoplasm may become so modified through the action of excessive heat or cold that it is no longer killed at the ordi- nary fatal temperatures. This result is partly due to the fact that it is then not so strongly irritated by these extreme tem- peratures, and partly owing to the fact that the coagulation and freezing points have been shifted, possibly through loss of water. § 5. DETERMINATION OF THE DIRECTION OF LOCOMOTION BY HEAT — THERMOTAXIS Our knowledge of this subject is still in its infancy and de- pends chiefly upon the observations of STAHL ('84), VER- WORN ('89, pp. 67-68), GRABER ('83 and '87), LOEB ('90, p. 43), DE WILDEMANN ('94), and MENDELSSOHN ('95). The first two and the last two mentioned have employed Protista, and we may consider their work first. STAHL'S studies were made upon Myxomycetes. He used two beakers, of which one was filled with water at 7°; the other with water at 30°. These were placed near each other, and a strip of filter-paper, on which lay the plasmodium of JEthalium septicum, was stretched between them. The two ends of the strip with the corresponding ends of the plasrno- dium hung into the two glasses. The result was that the plasmodium moved from the colder water toward the warmer, although before the experiment it was moving in the opposite direction. WORTMANN ('85) added the observation that when the warmer temperature rose above 36° a repellent action of the warmer water was discernible. VERWORN experimented chiefly with Amoeba. The difficulty in this operation depended upon the necessity of warming only a part of the body of so small an animal. He used a glass plate of 5 sq. cm. area, to the upper surface of which was glued a piece of black paper, in which had been cut a rectangular open- ing, 3 sq. mm. large, and with very sharp edges. This plate was placed on the stage of the microscope so that the hole lay § 5] THERMOTAXIS 259 in the rays of the infalling, concentrated light of midsummer, reflected from the mirror. Upon the black paper was placed the cover-glass with the amoeba in a drop of water. The light from the mirror was cut off until the amoeba, in its migrations, lay half-way over the edge of the orifice. Then concentrated light was let through the slit. A small part of the body was still moved across the line of demarcation; then for a moment movement ceased and a few seconds after the protoplasm of the amoeba began to flow backwards. In from 10 to 30 seconds the amoeba was wholly in the dark again. Similarly, when the cover-glass was moved so that the amoeba was brought half-way over the open orifice it retreated into the dark. Direct measure- ment showed that the temperature at the illuminated part was 40° to 50° C., whilst over the black paper it was 15° to 20° less. That the movement was not due to the light was shown first by cutting out, by means of ice, the heat rays only. No reac- tion occurred. Secondly, by cutting out the light but not the heat, by passing the light through a solution of iodine in CS2 so that only the ultra-red rays (which act like darkness to all organisms) went through; the typical reaction occurred when the temperature over the slit was 35°. From all of these ex- periments the conclusion seems justified — Amoeba is positively thermotactic towards that temperature. Similar results were obtained by VERWORN with the shelled Rhizopod Echinopyxis aculeata, and later (see JENSEN, '93, p. 440) with Paramecium. More complete studies on the latter were, however, made by MENDELSSOHN, who worked in VERWORN'S laboratory. MENDELSSOHN devised an excellent method of study. A brass plate 20 cm. x 6 cm. and 4 mm. thick is properly supported in a horizontal position, and to its under face are affixed, transversely, tubes through which hot or cold water may be run from a reservoir placed at a high level. In the middle of the plate a space 10 cm. x 2 cm. and 2 mm. deep is cut out and into it is fitted a glass or ebonite trough. Special thermometers whose bulbs are coiled in the plane of the trough, and hence perpendicularly to the stem, serve to measure the temperature of the water in the trough at any point. By means of water running through the transverse tubes either end of the trough may be heated or cooled as 260 HEAT AND PROTOPLASM [Cn. VIII desired. Starting now with the trough filled with infusion water, the Paramecia are seen to be uniformly distributed (Fig. 71, a). Hot water is run through tubes under the right end of the trough. After 10 minutes the thermometers show the temperature of the water at the right end to be 38°, at the left end 26°. At this moment, all Paramecia are in the left third of the trough (Fig. 72, b). If now the hot water be passed through the left tube only, the temperature rises at that end to 36° or 38°, falling to 27° or 28° at the other, and 13-° 26s 385 .'•• v~'.;^8| •• tttM 10 Q 25? FIG. 71. — Distribution of Paramecium in a trough of water with variable temperature at the ends. (From MENDELSSOHN, '95.) the Paramecia swim to the right end. Thus, with reference to a temperature of 38°, Paramecia are negatively thermotactic. If, now, cold water be passed through the left tube so that the temperature of the left end of the trough falls to 10°, while the right end is at 25°, the Paramecia migrate to the right end. Towards a temperature of 25° Paramecium is thus positively thermotactic (Fig. 72,