00 ?r DO OU 160807 >m OSMANIA UNIVERSnYttBRARY - . 3 f Accession No Auth . , > s book should be re. M ned o:j or beior. 1 rfit OTTO HAHN, INVISIBLE RADIATIONS OF ORGANISMS Protoplasma-Monographien Herausgogebeu von R. Chambers (New York), E.Faure'-Fremiet (Paris), H.Freundlich (London), E.Kiister (GieBen), F. E. Lloyd (Montreal), H. Schade (Kiel) f, W. Seifriz (Philadelphia), J. Spek (Heidelberg), W. Stiles (Birmingham) Redigiert von F. Weber (Graz) VOLUME 9 INVISIBLE RADIATIONS OF ORGANISMS OTTO RAHN PIIOJ'LSSOR OF JUfilKRlOLOfrr, COIIXKLL FXIVLKStTY WITH .V2 ILI.rSTKATIONS Berlin Verlag von Gebruder Borntraeger W35 Koester-Ufer 17 1936 INVISIBLE RADIATIONS OF ORGANISMS OTTO KAHN PROFESSOR OF BACTERIOLOGY, CORNELL UNIVERSITY WITH AN INTRODUCTION TO TUK PHYSICS OF RADIATION BY S1DNKY \V. IJAHNES RESEAltCH ASSOCIVTE IN PHYSICS, UNIVERSITY OF RUCUKSTRR WITH 52 II.IATSTRATIONS Berlin V e r J a g von G t b r ii d e r B o r n t r a e g e r \V :w I\ot"ter-Ufor 17 1930 insbesondero das Rroht dm- Ultt'rsetzung in fromde Sprachen, vorhehalten Copyright 193li l>y (iolirudor Borntivipger in Berlin Jiinck d^r BuchdrurkopM (let, Waisi-nhausex (1 in h H in Halli- (Saalc-i I'nntfd in fl-ormariy FOREWORD Visible biological radiations have always greatly attracted man's curiosity; fireflies and glow worms, I uniiiit went wood, phosphorescent meat, and the illuminating organs of deep sea fish are among the well-known wonders of nature. The biological importance of this luminescence seems to be in no proportion to the impression it makes upon the human mind. While it is assumed by some biologists that it has the purpose of attracting the prey, of frightening enemies, or of luring the male to the female, other investigators have contested these theories. The phosphorescent bacteria usually lose the ability to produce light when cultivated for some time on artificial media, without any apparent decrease in vitality. The emission of visible light is probably of no greater importance than color; it plays no essential role in the cell physio- logy of the organisms. Quite to the contrary, the invisible radiations of living org- anisms are of considerable physiological significance. They play a distinct part in cell division and in growth. They are evident ill the healing of wounds. Old age is accompanied by complete cessation of ultraviolet emission; perhaps this is the cause of old age. Beta-mdi'cit'um controls the heart beat. The, loss of blood radiation is used in the diagnosis of cancer; it may be that radia- tion, or some disturbance of its mechanism, is Jinked with the. cause of cancer. Its role in the metamorphosis of amphibia has been demonstrated. Mutual influences of one species upon another by radiation have been observed. Biologists and physicists have always been suspicious of radiations from living organisms, perhaps only because the average man (not to mention woman) likes to believe in human radiations. However, the principal reason for the rejection of the discovery VI FOREWORD of ultraviolet emission from living cells was the inability of some to repeat the positive experiments of others with the same results. This had led to the fallacy that negative results disprove positive ones. It is quite evident that if two experimenters obtain different results, they cannot possibly iiave made the same experiment. Both results are correct, and the important task is to find out in what points the investigations differed. With a phenomenon so little understood as these biological radiations, it is not surprising that these apparent contradictions have not as yet been explained in every ease, though several factors responsible for negative results have been discovered. The objection to biological radiations has been strongest in this country, but even here, a more conciliatory attitude has be- come noticeable since it has been shown that mitogonetic radiation is not a mysterious force, but the result of biochemical processes. Many simple chemical reactions have been found to emit weak ultraviolet rays. Another factor is responsible for the slow adoption of this new influence in biology: practically all papers on this subject are published in foreign languages, and of the very few in English, almost all happen to contain negative results. This very fact has been one of the authors' reasons for presenting the more important facts in this book. The book deals almost exclusively with mitogenetic rays w r hich exist in the ultraviolet range of the spectrum. No definite proof for the emission of infrared rays by organisms could be found (if we limit the infrared to radiations near that of the visible). #eta-ray emission from potassium is biologically impor- tant, but it is not really characteristic of the living cell; it is proportional to the potassium content, and is just as strong after death as during life. The arrangement of the subject matter is not historical, but logical. An attempt has been made to show that ultraviolet radiation from living organisms is nothing at all strange. If GOKWITSCH had not discovered these emanations 10 years ago, they would now be predicted from the results of physico-chemical investigations. An approach to historical presentation is found in Chapter IV which discusses the various methods used. FOREWORD VJf The book is not meant to represent a compilation of all literature on this subject. This would have increased its size greatly. A fairly complete list of references, up to the beginning of 1932, may be found in the book by HTKMPKLL, (1032). The literature compiled at the end of this book refers only to those papers which have been quoted in the text: we suppose that this includes the more important publications. A very brief summary of the entire book, chapter by chapter, is given at the end. This might be more useful in some respects than the customary Table of Contents. One of the authors had occasion, during a recent journey to Europe, to see many of the biologists and physicists working in this field, and he wishes to acknowledge the many valuable sug- gestions he received from those convinced of mitogeiietic radiation as well as from those who are convinced of its non-existance. The authors are further under great, obligation to Professor ALEXANDER CUIIWITHOH for sound advice 011 various points, and to Professor MACJROCT for the kindness of sending original photo- graphs of his experiments for the reproduction in this book. The authors an? further under great obligations to Mrs. MARGARET N. BARNES for her ceaseless assistance in editing tin's book, and to Miss A. J. FFJKJUSON for her he!]) in proof-reading. Fthaca, August OTTO RAHX SIDNEY W. BAKNKS TABLE OF CONTENTS FOREWORD . . V CHAPTER I PHYSICS OK KADIATION . . I A. General statements . . I B. Phenomena observed upon the interaction of radiation and mutter 2 C. The wave theory of radiant energ\ . . *J J). The <|uantuin theory of radiant energx . . 9 E. Analysis of radiation by dispeision into a spectrum IS F. Intensity measurements of visible and ultraviolet radiations . 23 CHAPTER II SOURCES OF RADIANT ENERGY. :H A. Physical sources. .... HI B. Ohemieal sources . . .... . . . .'33 0. Secondary radiation . . . . ... . . . J3 CHAPTER III EFFECT OF ULTRAVIOLET RADIATIONS UPON CELLS . . 40 A. Effect of radiations upon chemical reactions . . . . 46 B. Effect of monochromatic ultraviolet upon cells 48 C. Effect of radiation from chemical reactions upon cellb . . ."50 CHAPTKR IV METHODS OF OBSERVING BIOLOGICAL RADIATIONS . . . .H A. Mitogeiictie. rays (onion root method, yeawt bud method, increase in cell numbers, cell division in larger organisms, changes in yeast metabolism, morphological changes, physico-chemical methods, -physical methods) . . 54 B. Injurious human radiations ... ... . . 95 C. Ncerobiotic rays . . 99 I). Infra-red rays. . . . .... 10J E. Beta -radiation . . . . ... . 101 .X TABLE OF CONTENTS Pigo CHAPTER V SPECIAL CHARACTER FSTICS OF BIOLOGICAL RADIATIONS 103 A. Intermittent radiation 103 B. Influence of diffuse daylight 10S C. Secondary radiation .... I OS I). Intensity of the mitogenetic effect I U E. Retardation through radiation 115 F. Adaptation to gradual increases in intensity 117 CHAPTER VJ ANALYSIS OF THE MITOGENETIC EFFECT 119 A. The necessity of a particular physiological stage 120 B. Relation hetween the intensities of radiation and of effect ... 122 C. The minimal intensity required for an effect 123 I). The harmful effect of over-exposure 125 E. Storage of mitogenetic charges ... . . 120 F. Mechanism of the BA.KON yeast detector 127 V*. Mitogenetic effects in multicellular organisms I2S CHAPTER Vll SIGNIFICANCE OF BIOLOGICAL RADIATIONS IN BIOLOGY, MEDICINE AND AGRICULTURE 131 A. Unicellular organisms (emission at different physiological stages, reaction upon irradiation) . ... 131 B. Higher plants 138 C. Eggs and embryonic stages of higher animata . .... 142 D. Tissues of adult animals HO E. Blood radiation 148 F. Nerve radiation and conduction of stimuli 154 G. Morphological effects produced by mitogenetic rays (yoasts, bacteria, sea urchin larvae, metamorphosis of amphibia and insects, change of chromosome number, parthenogenesis) ... 101 If. Symbiosis and parasitism 170 1. Healing of wounds 173 J. The cancer problem. 177 K. Radiations other than mitogenetic 184 OUTLOOK 188 SUMMARY OF THE BOOK 193 AUTHOR INDEX 196 SUBJECT INOEX 209 CHAPTER 1 PHYSICS OF RADIATION A. GENERAL STATEMENTS (1) Radiation travels through empty space. From ^he studies of radiant energy have come several ideas about its nature. For one thing, it is known that such energy is propagated through empty spaee. Moreover, it is the only form of energy known which can flow through matter-free spaee. The vast amounts of solar radiation which maintain the earth at such a temperature that life is possible, come from the sun through millions of miles of interstellar space, which contains but an infinitesmal density of matter. . (2) Radiation energy spectrum. Radiation occurs over an extended energy range. The extremely high-energy gamma rays penetrate several inches of lead; the lower-energy jr-ray* pass through perhaps an eighth of an inch of lead ; visible light is absorbed by a metal layer only a few atoms thick; and, finally, radio waves are completely absorbed by a coarsely-woven copper wire screen. The character of radiation varies greatly, as one can see, from one part of the radiant energy spectrum to another. (3) Radiant energy travels through space with a fixed, definite velocity. Radiant energy might be given the alias of traveling energy, for it spends each instant of its existence traveling through space at its particular speed of 3xl0 10 cm. per second (speaking here of space in which the matter density is zero). This speed is entirely independent of the character of the radiation. To the best of our knowledge, radio waves, visible light and gamma rays all travel with precisely this velocity. The direction of travel is rectilinear. (4) Radiation exhibits the phenomena of inter- ference. Any form of wave motion can be made to exhibit the Protoplasms. -MonograpMen IX: Kahn ] 2 CHAPTER I phenomena of interference. The beats heard when two tuning forks of nearly the same frequency are struck, are an example. Later (see p. 128), an example of interference exhibited by light will be given. (5) Radiation may be observed when, and only when, it is allowed to interact with matter. This statement is a reminder that all recognized measurements of energy are limited to energy associated with matter. To detect or measure radiant energy, it is necessary that it be transferred into *the familiar- potential or kinetic energy of matter. This transformation obeys the law of conservation of energy, i. e. the number of ergs of radiant energy disappearing is equal to the number of ergs of potential or kinetic energy which appear. B. PHENOMENA OBSERVED UPON THE INTERACTION OF RADIATION AND MATTER (1) Kef Joe ti on. Radiant energy may be regularly reflected from a plane surface whose granular structure is small in compari- son with the wave length of the radiation. In this case the angle of reflection is equal to the angle of incidence, and the reflected energy is in the plane of the incident energy. No perfect reflectors of radiation are known; thus, always some of the radiation is transmitted or absorbed by the reflector. (2) Absorption. Matter never fails to take its toll from the radiation incident upon it. No material substance known is totally transparent to radiant energy. The mechanism of ab- sorption will be treated later (see p. 16). (3) Refraction. Matter has the property of changing the* velocity of energy which is passing through it. This results in a change of direction of the radiation (see fig. 1). The ratio of the velocity of radiation in matter to the velocity in space is called the index of refraction of the refracting substance. (4) Dispersion. The phenomenon of dispersion occurs because the index of refraction of any transparent material depends also upon the wave length of the radiant energy which is passing through it. A beam of white light is dispersed into a colored band or spectrum when passed through a prism since each wave length suffers a different refraction (see figs. 2 a arid b; also see p. 18). PHYSICS OF RADIATION 3 These are only a few of the many phenomena which characte- rize radiant energy in its passage through space and matter ; these must be explained by any theory of radiation. The question: WJiat is the nature of radiant energy ? has been nearly answered bv each of two different theories, the wave theory and the quantum Air Figure 1. Refraction. Figure 2. Refraction and Dispersion. theory. As it will be pointed out later, it seems not impossible to effect a harmonious combination of these two into one which adequately covers all uho observed phenomena of radiant energy. The so-called "mitogenetic radiation" which is the principal subject of this book is said to proceed rectiliiiearly, and to show reflection, absorption, refraction and dispersion, as will be demon- strated in Chapter TV. If so, it is a true radiation. C. THE WAVE THEORY OF RADIANT ENERGY There are three ways in which energy may be transferred with the aid of matter, namely (1) by the How or movement of definite masses of matter, such as tides in the seas, or the drive rod on a locomotive; (2) by wave motions in elastic media, such as sound in air; and (.3) by material projectiles. The wave theory of radiation is based upon the well understood principles of wave motions in elastic solids. These? may be illustrated by the following simple experiments. If a long stretched rope is given a blow at one of its supports, a rather surprising thing happens (at least so to the uninitiated) ; a hump in the rope is seen to speed along it. If, for a short period of time this end of the rope is given a regular to-and-fro motion, a disturbance as pictured in fig. 3 will travel along it with the same velocity as in the former case. This sort of disturbance is called a wave train. The length of the individual waves, or the 4 CHAPTER I wave length, A, is the distance from crest to crest or trough to trough. The frequency, v, or the number of waves passing a ^_^^ -^ Figure 3. , ' x "- ^ x A short wave train in a rope. fixed point per second, is equal to the velocity, c, divided by tho wave length: Reflection. Let this wave train be o bserved when it reaches the end of the rope. If the support there is ideally rigid, the train of waves will be reflected and will travel back along the rope with the same velocity and amplitude it had before reflection. Absorption. If the support is ideally non-rigid, the wave train will set it in motion, spend its energy upon it and completely disappear. In this event, the energy carried by the wave train has been transferred to the support. Standing Waves. There is still another phenomenon of wave motion which may be illustrated by waves in the rope. When it is fastened to the rigid support, if the free end is kept moving with a uniform to-and-fro motion, so-called standing waves will be formed (see fig. 4). The rope appears to be divided Figure 4. Above: Waves in a rope; below: standing waves in a rope. into vibrating segments called loops which are separated by points of little or no motion called nodes. These standing waves, not true waves at all, are the result of the interference of the original PHYSICS OF RADIATION 5 and the reflected waves. Their importance lies in the fact that they offer a very simple way of determining the wave length of the true waves which is equal to twice the distance between , nodes. " ' A S u PP 08e a system of ropes is strung from a central point so they lie in a plane (see fig. 5). Tf the central point is given a regular up-anil-down motion, waves will travel out along each rope and the system will present somewhat the appearance of still water Figure 5. Radiation from a central point, left: a rope system; right: waves in a rope system. into which a stone has been dropped. The wave trains traveling out- ward along each rope with the same velocity give the appearance of regularly growing or spreading concentric rings. The rings are separated by a distance equal to the length of the waves. If more ropes are added to this system so that they are stretched equally m every direction in various planes and the central point is given a regular to-and-fro motion the system will give the appearance of expanding or growing spherical shells. Here the distance between the shells is again equal to the length of the waves in the individual ropes. The mechanical rope apparatus is frequently used as an analogy to the electric field of a point (Charge. Definition of a Charge of Electricity. Electricity, according to present ideas, embodies two kinds called positive and negative. If an object has equal amounts of the two, it is said to be electrically neutral. If it has an excess of either kind it is said to be charged, and this excess is called an electric charge. Usually this charge is distributed over the surface of the object. 6 CHAPTER I In discussions of the effects of one charge upon another, and in related problems, it is convenient to ignore the object and to think of the charge as being concentrated at one point. This is called a point charge. The Electric Field of a Point Charge. We will now see why the three-dimensional system of ropes forms a rough mechani- cal analogy to the electric field of a point charge. Suppose a charge of positive electricity is fixed in space at some pornt A. If a charge of negative electricity is brought to some point B, it will experience a force which tends to draw it straight toward A as though the two were connected by an invisible stretched elastic cord. The point ft may be anywhere in space in the vicinity of ^1 and still it is drawn directly toward /I. We may then think, if we like, of the space about A as filled or made up of these stretched elastic cords (called lines of force) extending outward in every direction from the charge at point A. Now, if the charge at A were given a rapid to-and-fro motion, it would seem likely that waves should be formed and sped along the lines of force through space. This is the explanation offered by the wave theory of light concerning the manner in which radiant energy is propagated through space. We know that electric fields surround electric charges. Radiation is associated with waves traveling through these fields. The waves in the electric field about the charge which has been set in oscillation arc known to be not the only waves present. In fig. 3 is represented the shape of one of the lines of force shortly after the charge has been given a few oscillations. The train of waves which consists in variations in the direction of the line of force is traveling along this line with the velocity of light. Now it is known that the motion of an electric field through space produces an associated magnetic field. It theref on* follows that this wave train must have associated with it a train of waves of magnetic, intensity. These two wave trains lie in pianos perpend- icular to each other. The classical electromagnetic wave along one line of force is represented in fig. 6. Radiation is then nothing other than these electromagnetic waves. The wave theory predicts that the velocity of propagation of these waves should be independent of their wave length. Moreover, such waves would be expected to exhibit all the pheno- mena of interference just as radiation does. PHYSICS OF RADIATION An oscillating charge radiates energy, of course, not only along one line of force but in all directions, though the amount of radiant energy sent out in different directions varies. Zero Plane of Electrostatic Field Figure fi. Diagram ot an electromagnetic wave. ei.ergy is radiated along the line of motion of the charge and the maximum amount is radiated in a plane normal to the direction of motion. HEKTZ (1866) caused a charge to oscillate rapidly between two closely-placed points 1 ) and found that energy was being ra-dia-tcd as the result of the accelerations of the charge (sets tig. 7). He placed a metal plane some distance from the oscillating charge Mxtor Figure 7. Experiments showing the wave nature of Hertzian waves. 1 ) In fig. 7, c represents a condenser, and S a spark gap. The con- denser \\as charged to such a difference of potential that a spark occurred between the balls of the spark gap. Such an arrangement is known as an oscillatory circuit, for when a spark occurs the charge flows across the spark gap in such a way as to discharge the condenser, and it continues to flow until the condenser is recharged with the difference of potential reversed. The charge then begins to reverse its direction of flow. This process repeated many times a second amounts to a charge moving rapidly back and forth between the spheres of the gap or a charge oscillating rapidly between two closely-placed points. 8 CHAPTER I and found regularly-spaced points between the radiating charge and the reflector at which the energy was alternately of a maxi- mum and of a minimum value. These loops and nodes showed that the energy was being radiated in the form of transverse waves, the direct and the reflected beam interfering to cause standing waves. When the velocity of these waves, which arc of the wave length of short radio waves, was found to equal the experimentally- Mono - chromatic light G -* standing Surface Figure 8. Experiment showing the wave nature of light. determined velocity of light, it was immediately suggested that Jight was nothing other than such waves, only of shorter wave length. A simple experiment performed by LLPPMA^N showed that- visible light could be made to form standing waves and thus must have a wavelike nature. Light of one wave length was allowed to shine perpendicularly upon a fine-grained photographic emulsion which was backed by a reflecting layer of metallic silver. When the. emulsion was developed, cut and the edge viewed under a microscope (see fig. 8) alternate exposed and unexposed layers were found to exist throughout the depth of the emulsion At planes where the direct and reflected beams interfered to form the node's of the standing waves, the silver compound was unaffected; at planes in between, corresponding to the loops of the standing waves, the silver compound was reduced. This experiment not only showed that light was a wave motion but offered a beautifully direct way of measuring the wave length. Definition of Intensity. The intensity of radiation received from a source at a fixed point in space is defined as being the number of energy units (ergs) received per second by a square cm. of surface placed normal to the radiation at that point (see PHYSICS OF RADIATION 9 fig. 9 a). When the radiation is strictly parallel, the intensity per cm 2 will be the same at any distance from the source (fig. 9 a). With a point source, it will decrease as the second power of the distance between the radiating source and the point of measure- ment. Thus, in fig. 9b, A will receive K ergs per second while B, rr twice as far from the source, receives only -. ergs. In practice. point surfaces are rare, radiation generally being emitted by sur- faces or volumes. (This is commonly the rule* in biological radiat- Figure 9. Illustration of the definition of intensity. ions.) For these cases, the inverse square law holds only for distances so great that the source may be considered to be- a point. For shorter distances the intensity may be roughly propor- tional to the reciprocal of the distance; for points still closer to the source, the intensity may be independent of the distance. Experiment is the best means of determining the variation of intensity with distance in the region of space closely surrounding a source of finite size. The wave theory of radiation has been successful in explaining how radiant energy may bo transferred through space ; it has predicted accurately the velocity of radiant energy ; the phenom- ena of interference, reflection, refraction, dispersion, polarization and double refraction offer no difficulties; however, with respect to the emission and absorption of radiant energy, the classical theory fails and gives place to the quantum theory. D. THE QUANTUM THEORY OP RADIATION 1. Definitions. It is known that matter exhibits the curious behavior of discontinuity in processes in which it emits or absorbs radiant energy. A given atom, for example, will 10 CHAPTER 1 cgnvert, of its store of energy, only certain multiples of the unit of energy into radiation. Likewise, it will absorb radiant energy only when the energy comes in precisely the proper-sized amounts. This phenomenon is one with which the wave theory of light is unable to cope. Let us lay aside for the moment then this conception of the nature of radiation and consider the only other possible one, i. e. that radiation is corpuscular in nature. Thus we think now of radiation as consisting of small energy projectiles which travel through space with the familiar velocity of 3 X 1C 10 cm. per second. These projectiles are of course non-material; they consist simply of small units of energy. Since we have seen that the different kinds of radiation, from radio waves to gamma rays, all travel with the same speed, these differences can occur only by differences in the size of each pro- jectile, or quantum. It has been shown that the energy of a quantum can be given as the product of a universal constant, h, known as PLANCK'S constant and equal to 6.55 x!0~ 27 erg seconds, and the frequency of the equivalent electromagnetic wave. Energy of quantum E hv. In Table 1, column I are given the various wave lengths in ANGSTROM units (1 A 10~ 10 m). Column Ji gives the corres- ponding frequencies obtained from the equation where c is the velocity of light. In the third column arc the quantum energies, E, which correspond to each frequency (E -hv). Thus, an X-ray quantum is a 10" 8 erg projectile, while a quantum of visible light has an energy value of but 1C" 12 ergs, and those of the ultraviolet being about 2 to 10 times as large as those of visible light. For the understanding of emission and absorption of quanta by matter, it is necessary to discuss briefly the atomic theory. 2. Atomic Theory. Let us suppose (see fig. 10) that an electron is held at some distance, r, from a small positively - charged particle, q. From our knowledge of electrostatics we know that the electron experiences a force of attraction toward q. This force, F, is proportional to the number of units of charge possessed PHYSICS OF RADIATION 11 AXXAXXXXX I IQ t |- (^ rH CO l^ 1^ % / >. / X V N ' -' X CC rH 1-- (M CO I- 30 Oi OilOOirHOOH^OJOJ >O O HH -f H4 CO* ^1 rH 8rH q rH co 10 q q o q ^ d d d o* o* PH o d g o < FH - _ ^Q electron ,. *' *~ ~~~ ^ \ \ Electron in a circular orbit x traveling about the positive \ charge q. * ^ center (see fig. 11). Ef its orbital velocity is adjusted until its centripetal force is just equal to the force of attraction, F, exerted upon it by q, then the electron will be in stable equilibrium remain- ing indefinitely in this orbit. An example of such a system is furnished by the sun and the earth. Here it is the balance between the earth's centripetal force due to its orbital velocity and the gravitational atractiori of the sun which keeps the* earth in its orbit. If the mass of q is 1.65 X 10~ 24 grams, the distance r js 0.5 X 10~ 8 cm. and the charge q represents an electron, then fig. 11 represents a hydrogen atom, q, which is thus the unit of positive electricity, becomes the nucleus of the atom and the electron represents the hydrogen atom's one orbital electron. From elementary considerations it would seem that the nucleus with its orbital electron would form a stable system also for values of r other than 0.5xlO~ 8 cm. For any value of r, a corresponding orbital velocity can be calculated which will bring about the balance of the electrostatic attraction and the centripetal force of the electron. However, it is found experiment- PHYSICS OF RADIATION 13 ally that there are only a few orbits, out of the infinite number possible, which the orbital electron frequents. From the radius, r lt of the innermost orbit of H which we have just seen to be equal to 0.5xlO~ 8 cm. we may calculate the other possible orbits by the relation r = n 2 r l where n has any value 1, 2, 3, 4 Tlie orbital velocity of the electron decreases as ti increases, and tlie limiting case n-> oo corresponds to an atom with its electron at rest at an infinite distance from the nucleus. The energy possessed by the atom or the* nucleus-electron s\ stem, for the case when the electron is in any one particular orbit, can be easily calculated and this energy characterizes the cntrgy state of the atom. The atom lias the least energy when the electron is in the innermost orbit (state of least energy) and its energy increases as the electron exists in orbits of greater radius (states of higher energy). The innermost orbit is the preferred one, i. e. the electron inhabits this one the greater part of the time. In this case, the atom is said to be in its normal state. The usually unoccupied orbits are called virtual orbit*. We have seen that the hydrogen atom can exist in different energy states, and we will very shortly apply this idea in a discussion of the absorption and emission of radiant energy by atoms (see pp. 14 and 1C). Before considering these topics, it will be advantageous to see how other atoms beside hydrogen are constituted. The one next in simplicity is helium. Its nucleus consists of 4 simple hydrogen nuclei, called protons, and 2 electrons. Two orbital electrons complete the atom. In all atoms, the number of protons in the nucleus exceeds the number of electrons by just the number of orbital electrons; this leaves the nucleus with a positive charge 4 and the whole atom electrically neutral. For example, the carbon atom is composed of a nucleus of 12 protons and electrons about which revolve 6 electrons in the various shells. The oxygen atom consists of 8 outer electrons and a nucleus which contains 1(> protons and 8 electrons. The atomic weight of carbon is approx- imately 12 and that of oxygen is 10. It can be seen that the atomic; weight is equal, for all practical purposes, to the number of protons in the nucleus of the atom. The atomic number is equal either to the number of nuclear or orbital electrons. Table 2 shows in what way the outer electrons group themselves in the orbital shells for the first eleven elements of the periodic table. CHAPTER I Table 2. Electron Configuration for the Elements from H to Na. Element Atomic Number 7i shell _- L shell M shell H 1 He 2 2 Li 3 2 1 Be 4 2 2 B 5 2 2 1 U 6 2 2 2 N 7 2 2 3 () 8 2 2 4 F 9 2 2 5 Ne 10 2 2 6 Na 11 2 2 B 1 Jb'ig. 12 is a wholly diagrammatic representation of the A't/ atom. The central dot marks the nucleus; the first circle is called the K shell and contains two electrons. The binding energy of these negative charges is about 1000 electron volts or 1.6xlO~ 9 ergs; this amount of energy would be required to remove either one of z the K electrons from the atom. The next circle represeiitsthe L orbit, which in Na contains 8 electrons, the binding energies of which arc 1 approximately 35 elec- tron volts. In the last solid circle there is but one electron and its binding energy is about 5 electron volts. The outer dotted circles represent energy levels which are unoccupied in the normal Na atom, i. e. virtual orbits. 3. The Emission and Absorption of Radiation by Atoms. We have seen that the hydrogen atom can exist in various energy states associated with the orbit occupied by its electron. Figure 12. Diagram of the sodium atom showing the various shells. PHYSICS OF RADIATION 15 By this necessar}' mechanism can be explained the absorption and emission of radiation. If the atom changes from an energy state E m to a lower one E n , it does so with the emission of a quantum of radiant energy liv such that (1) hi> = E m E n . If the atom changes from an energy state E n to a higher state of energy E m , it can do so only by the absorption of a quantum of radiation hv of such energy value that (1) In- - Em En- In this equation h is the universal constant known as PLANCK'S constant equalling 6.547 ;< 10~ 27 erg. sees, and v is the frequency (sec p. 4). It follows that the various quanta hv may be emitted uiid absorbed by the hydrogen atom. hr Km K where m 1, 2, 3 hr - Km E! m 2, 3, 4 ... . h v = E m E 2 in - 3, 4, 5 hr -- En+, -En. Since A = - where e is a constant which is equal to the speed of light or 2.99790v 10 10 cm. per sec., the wave length of the radia- tion resulting from or producing the energy change E m K n in the atom will be given by the equation Table 3 gives the values of the first eight of the forty or so known energy states of the hydrogen atom. Table 3. Knorgy obtained by electron shifts from normal to higher orbits in the Hydrogen atom number of virtual orbit iiergb 10~ 12 corresponding wave length in A 1 16.14 1216 2 ii 19.15 1025 3 i 20.18 975 4 '! 20.70 948 ;, 20.90 940 < 7 ;| 21.05 21.16 933 928 16 CHAPTER I The wave length emitted by the hydrogen atom for the energy change E x E for example, is readily calculated from the data in this table, as _ 2.99796 x 10 10 X 6.547 x 1C" 27 __ ~~ 161.4 x 10- ia ~ ~~~ 1216 xK)" 8 cm. or ;. 1216 A. This wave length is in the far ultraviolet. Changes between other states result in the radiation of visible light and still others produce radiation in the far infra red, e. g. for the shift from the 6th to the 7th orbit, it is 178 500 A. The equation (1) states that an atom in the state E n will absorb a quantum hv and be raised to the energy state E m if the quantum is precisely equal to the difference in the energy of the two states. Supposing the energy of the quantum is slightly less than this difference will it be absorbed ? The answer is 110 ; there is no possibility that it will be. If the quantum is larger than Km En, then it may be absorbed. It will be, of course, if its energy happens to equal the energy difference between any two states provided that, at this moment, the electron is in the orbit corresponding to the lower of these states. Tf its energy is not one of these discrete values, it will not be absorbed -?- unless its energy is greater than (Eoo Kn), i.e. sufficient to shift the electron beyond the outermost orbit. In this event, it may be absorbed and the surplus, hr (K Ku), is used in shooting the electron away from the atom, or hv =E X E n + ^mv 2 where m is the mass of the electron and r is its velocity (the term 2 mv 2 represents the kinetic energy of the ejected electron). An electron so ejected from an atom is called a photoelectron, and the atom itself is said to be ionized. As has been stated before, an electron spends most of its existence in the normal state, E . When it has been raised to a state of higher energy E n by virtue of the absorption of energy, the atom is said to be in an excited state. The life of an atom in an excited state is of the order of 10~ 7 to 10~ 9 seconds. After this length of time the atom reverts to its normal state with the resulting emission of radiant energy. PHYSICS OF RADIATION 17 These icmarks on emission and absorption of radiation apply not only to hydrogen but to the other atoms as well. Only the outer electrons of the more complicated atoms behave in a manner similar to the one electron of hydrogen. In this way originate the atomic spectra. They are emitted by sources (such as the mer- cury arc or a glow discharge tube) in which the gas is at sufficiently low pressure that the atoms arc not in contact with each other but for a small fraction of the time. If the pressure is raised (also the case for solids), a contimioux spectrum is emitted which does not contain lines characteristic of the atom. This is called thermal radiation since it is the result of the temperature of the source. The atoms are so close together that the outer virtual orbits inter- mingle and are distorted. These last remarks apply equally well to absorption. An element ill the gaseous state will give a line absorption spectrum, while in the solid state it will give continous absorption. Molecular Spectra While atomic radiation results from electrons jumping from one energy level to another, molecular spectra an* assumed to nrise from the motions of atoms or, better, ions which form the molecule. Let fig. 13 represent a simple diatomic-- molecule such as NO. Such a molecule emits radiation in three wave length regions: (1) the far infra red; (2) the near infra, red; and (3) the visible or Figure 13. A simple diatomic molecule. GHZ) the ultraviolet regions. The radiation in the tirst group is ascribed, in the simple theory, to changes in the rotational energy of the dipole molecule. The second group is ascribed to simultaneous changes of rotational and vibrational energy, and the third, to simultaneous changes in rotational, vibrational and electronic? energy of the molecule. Molecular spectra are in general exceedingly more complex than atomic spectra. The great number of linos fall into groups which under low dispersion give the appearance* of bands. The absorption spectrum of a molecule is, of course, its emission spectrum in reverse, light bands replaced by dark. Pro to plasma- Mo nographien IX: Rah n 2 18 CHAPTER T In this connection, the effect of radiation upon chemical reactions should be mentioned. To realize that such an effect does exist, it is only necessary to remember the number of brown bottles that are used for storing chemicals. The most prominent effect is that of light on silver salts as in all photograph ic emulsions. The result is the reduction of the salt with the deposition of metallic silver. Without presenting the theory of the process, it is still easy to see that the absorption of radiant energy might do just this. For an absorption of energy means that^tho molecule must go into a state of higher energy-a less stable state- and this may, on the absorption of sufficient energy, become a chemically unstable state. E. ANALYSIS OF RADIATION BY DISPERSION INTO A SPECTRUM The radiation emitted by a source is characterized by the wave lengths present, together with the distribution of energy among these wave lengths. A given atom will emit only certain lines (its lme spectrum) and each one will have associated with it Atomic Molecular Jherma! IliJ moo 6000 . 1 visible- A range Figure 14. Kepresentation of atomic, molecular, and thermal radiation. a certain energy (see upper spectrum of fig. 14); a molecule may give rise to some such spectrum as that of the center strip, and an incandescent solid emits all wave lengths with varying intensity beyond a certain wave length, depending upon the temperature of the solid (see lower spectrum of fig. 14). PHYSICS OF RADIATION 19 The various wave lengths present in the radiation emitted by a source are determined by dispersing the radiation into a spectrum. This subchapter deals with the instruments and me- thods used to produce a radiation spectrum both in the visible and the ultraviolet. The next deals with the subject of measuring the intensity associated with the various wave lengths. The two instruments most frequently used to disperse light into a spectrum are the prism and the grating. When a narrow beam of parallel light falls upon a prism, the different wave, A \ S,lf prism J Figure 15. Spectrometers above: a simple .spectrometer; below: a quartz double moiiochromator. lengths present in the beam suffer different deviations in passing through the prism. As a result, each wave length emerges with a slight angular separation from its neighbors. Since, in practice, beams of light are generally divergent rather than parallel, tho prism alone gives rise to a spectrum in which there is some over- lapping of the wave lengths. The simple optical spectrometer (sec fig. 15A) prevents this by employing a lens which forms in the eyepiece a narrow image of the slit through which the light enters. By replacing the eye piece by a slit, the instrument may be used as a moiiochromator or monochromatic illuminator. For work in the ultraviolet region, the prisms and lens must be of quartz. Fig. 15 B represents the optical system of a typical quartz double moiiochromator. Tn most instruments the prisms may be rotated by some device which is connected to a drum 20 CHAPTER I calibrated in wave lengths. Turning this drum to a certain wave length reading sets the prisms so that only this wave length is permitted to pass through the second slit. Table 4 gives the relative intensities of the lines in the ultraviolet spectrum of the quartz mercury arc as transmitted by such a monochromator. 1 ) Table 4. Relative Intensities of II g Spectral Lines Wave Length Kelative Intensity Wave Length Relative Intensity A A 2907 528 2345 20.0 2925 117 2302 10.0 2894 209 2284 13.0 2804 371 2253 6.0 2754 03 2225 7.9 2700 88.0 2191 4.75 2653 735 2150 2.74 2530 J(XK) 1973 0.128 2482 218 1943 0.097 2399 51.5 1850 0.017 2378 40.0 Uratings are also used to produce spectra of visible 1 and ultra- violet light but since their application is more strictly confined to special work in spectroseopy, a discussion of their characteristics will be omitted. While monochromators are designed to give high spectral purity of the isolated light, some degree of impurity seems una- voidable. In general, some intensity of radiation of shorter wave length than that desired is transmitted by the 1 instrument. This defect may be greatly reduced by using with the moiioehromator a filter having a ''cut off' on the short wave length side of the desired radiation. By so doing, the intensity of the unwanted \vave lengths may be reduced to practically ZATO. An excellent list of such filters is given in a table (12 5) in Photoelectric Pheno- mena by HUGHES and Du BKTDCJE and the ultraviolet portion is reproduced here with their kind permission (see Table 5). The wave lengths given in the table are those at which the filter ceases 1 ) In particular, a Hilger quartz double moiioehromator. PHYSICS OF RADIATION Table 5. kShort Wave Cut-off Kilters Cut-off Material Thickness, etc. Comments WO A thin celluloid 30 to 40m/* (iradual cut-off 1230 A clear fluorite 1 to 2 mm Only very occasional specimens transmit as far as this 1450 A clear quartz 0.2 mm Different specimens have practi- (crystalline) cally identical transmission i! limits 1500 A clear quartz 2 nun Air paths have strong absorp- 1 (crystalline) tion below this point 1000 A j| dear quartz 20 mm ! (crystalline) 1700 A , oxygen in a 10 mm, at at- quartz (crystall- mos. pressure ine) cell 1750 A water in a 20 mm Steep cut-off. (Lyinaii linds, quartz (crystall- ho never, that 0.5mm of ine) cell \\ater has a sharp cut-off at 1729 A) 1750 A clear fused 0.3 nun Transmissions: IH49A, 24% quartz 1971 A, 36% 2002 A, 40% 1S50 A quartz mercury 1850 A is the shortest wave lamp length emitted by a new lamp 2000 A clear fused 3 mm Transmissions: 2000 A, 0% quartz 2100 A, 50% (different specimens vary widely in transmission) 1900 A acetic acid in 32 mm, 1 part The concentrations are merely water acid in 1000 rough estimates; it is best to parts water find by trial the desired con- centration 1>000 A acetic acid in 32 mm, 1 part uater acid in 200 parts water 2200 A code 970 1 ) 5mm Gradual cut-off; "solarizes" with short wave lengths 2200 A caleito 10 mm 2300 A rock salt 12 mm Lymaii and Pnuger, however, found rock salt to transmit as far as 1750 A *) standard filters of the Corning Glass Works. 22 CHAPTER I uut-oii 2360 A material __--- --= p-dichlor ben- JLUiCKiiess, etc. 50 mm; sat. v/ommeiiLS ""' zene solut. in \vater 2450 A tartaric acid 3.2mm; 1 part sat. wolut. in (54 parts water 2500 A tliiophcne 50 mm; sat. Steep cut-off solut. in uater * 2800 A benzol Steep cut-off 2800 A code 971 1 ) 2.] mm Fairly steep cut-off 2900 A code 971 1 ) 4.9 mm Fairly Bteep cut-ofi 3000 A pyrex 1 ) 1 .0 mm to transmit appreciably. Them* authors point out that kw a filter is seldom found in which the transmission changes from 50 per cent to 1 percent or less in 200 A". Jii some cases it is possible to rind a source giving wideh separated hues in a desired wave length region. By using a suitable filter with such a source, it frequently occurs that but one line is transmitted. Such a combination may give much greater intensity than could be obtained by the use of the monochromator. Because of the great, intensity of solar radiation and the possible effects of daylight on biological reactions, some data are given on the short wave length limit of the solar spectrum. This limit of the solar spectrum as recorded by^a photographic plate at various altitudes of from 50 to 4560 meters was found by one observer to be 2910 A. Another experiment carried out at 9000 meters showed energy present at 2897 A. The conclusion is given that the intensity, at the surface of the earth, of the solar radiation of wave length 2900 A is not more than one -millionth of the intensity at 3150 A. This sharp cut-off is ascribed to the ozone present in the upper layers of the atmosphere. Nevertheless, though the absorption approaches asymptotically 100%, we must keep in mind the possibility that extremely small intensities of the lower wavelengths exist in day light. This is important since in biological radiations, we are dealing with very low intensities, far beyond detection by photographic plates. *) standard filters of the Corning Glass Works. PHYSICS OF RADIATION 23 Table 6 gives the absorption of ultraviolet by 10.97 mm of distilled water. This, together with the absorption spectrum of oxygen, shows plainly why in biological radiations, no experiments are carried below 1900 A. Table 6. The Absorption of Ultraviolet in Distilled Water (16.97 mm) Wave Length Absorption IStiO (IX.O 1930 2t.:> 2000 1 1.2 2100 9.s 2200 9.2 2300 r>.<> 2400 r>.2 2(>00 4.2 3000 2.r l\ THE INTENSITY MEASUREMENT OF VrSFRLK AJSI) ULTRA V I OLET It A 1)1 ATION Arranged in order of increasing sensitivity, the detectors of visible and ultraviolet light are: the thermocouple, the photo- graphic plate, the photoelectric cell and the photoelectric; counter. (1) The Thermocouple: The thermocouple consists of a circuit composed of two dissimilar metals or alloys (sec fig. U>a). Figure 16. Thermocouples left: a two-metal thermo- element; right: a modern high- sensitivity thermocouple. (J If the junctions are maintained at different temperatures, a current will flow in the circuit, which is proportional to the tempe- rature difference of the two junctions. Modern high-sensitivity thermopiles are frequently built as diagrammed in fig. 10 b in 24 CHAPTER I which A is a very light bit of thin gold loaf. 1 or '2 mm 2 in area. The gold leaf is supported by two tine wires, our of a bismuth- antimony alloy, the other of bismuth-tin, which are- soldered to the heavy leads, (1. By using pieces of small dimensions, the heat capacity of the instrument is low, as is the heat loss by conduction : thus, the sensitivity is high. Used with a sensitive galvanometer, such a thermopile will give a detectable deflection when the radiation falling upon the gold leaf has an intensity of approxi- mately IJyJO 10 cal/em 2 /sec. or 1 - 10 2 ergs/cm 2/ ,sec. The ad- "'ijiiiiv 17. Spectral sensitivity cmvcs of Eastman plates i: Eastman Speeds ay; /*: Eastman 4O ; /: Eastman 35; D: Eastman Process. Log Exposure Figure IS. ( Characteristic curve photographic pla 2.0 of .1 tc. vantage possessed by the thermopile* over the other means of intensity measurement is that its response is quite independent of the wave length of the radiation. For this reason, it js often used to calibrate a source, of ultraviolet for instance, in terms of the energy in the form of visible light, emitted by a standard tungsten lam]). The disadvantage of the thermopile is its low sensitivity as compared to the other instruments for this type of measurement. (2) The Photographic Plate. Photographic plates mani- fest the defect just mentioned, namely that of giving a response which is not independent of wave length (see fig. 17 taken from C.E. K.MEES, 1931). Furthermore, the blackening of the emulsion for one wave length is proportional to the intensity only over a relatively short intensity range, perhaps 1 to 20 (see fig. IS). A paper rich in information on the characteristics of photographic emulsions is that of L. A. JONES and V. C. HALL (1926). PHYSICS OP RADIATION /. 6alvanomefcr\ Plates have been evolved which are particularly sensitive to different wave length regions; the SCHTMVNN plate, for example, in the ultraviolet. The sensitivity of the SCHUMANN plate for the ultraviolet depends upon the absence of gelatin. Gelatin has a strong absorption for wave lengths below 2800 A and becomes practically opaque even in very thin layers for wave lengths in the neighborhood of 2000 A. Recently, even better plates have been obtained by sensitizing ordinary plates. They an 1 covered with a very thin film of a substance (a car boxy lie ester of dihydro-colloidin furniture polish) which lluoresees strongly under the action of ultraviolet with the emission of longer wave lengths which easily penetrate* the gelatin. About the Bjffef>y lowest sensithity to which the photo- graphic emulsion will respond is 12000 quanta /cm 2 /'see , or 10 7 ergs/cm 2 ,'sec. at a wave length of 2.~>00 A. (3) The Photoelectric ('ell. In photoelectric cells, radiation falls upon a metal surface; the photoelectrons (see p. 10) ejected form a photoelectric current which is proportional to the intensity of the radiation. A linear relationship between the photoelectric current and intensity has been tested in properly- designed cells and found to hold over a range; of intensities of from 1 to 50 million. This is many times greater than the linear portion ot the photographic emulsion curve which extends over an inten- sity range from I to 20. Not all commercial cells give this behavior, however. In fact, it is quite unsafe to assume a linear relation between photoelectric current and light intensity for any but specially-made cells. The sensitivity of photoelectric cells is roughly that of the photographic plate. Photoelectric cells are of two kinds, vacuum and gas-filled. In principle they operate in the same way. A glass bulb (see fig. 19) has deposited over most of its inner surface a coating of the photoelectric clement, e. g. Na, K or Mg. This coating is in contact with a wire which is sealed through tho glass wall of the bulb. Another wire is sealed in a side arm, A ; a battery main- tains the coating negative with respect to the wire at A. With Figure 19. Photoelectric < 20 CHAPTER I the window covered so that no light can enter the cell, the galvano- meter reads zero indicating that no current is flowing in the circuit. If light is allowed to fall upon the metal surface, photo- electrons are ejected from the atoms of the metal film and are attracted to the central wire. These electrons flowing through the wire cause the galvanometer to deflect, which is a measure of the photoelectric current. Necessarily, in such cells, the energy, hv, of each quantum to be measured must be greater than the energy required to remove an electron from the surface, that is hv>Eoo E . This equation applies to isolated atoms. In a photoelectric cell, the photoelcctrons must not only be removed from the atoms but must be shot away from the metal surface and eventually even through it. This requires a little more energy. The energy required to remove a photoelectron from a photoelectric surface is called the "work function", W ti . The situation in the sensitive surface of a photoelectric cell is further complicated because of the* impossibility of having this surface consist of one kind of atom. With the best high vacuum technique known today it is impossible to prevent contamination with various atoms, chiefly those of the gases prevalent in the air, such as oxygen, hydrogen and nitrogen. The result is tluit 1F for a given metal depends considerably upon its history and the care with which it has been freed from gases. The purest surfaces are prepared by distilling metals in a high vacuum. Table 7 gives the photoelectric work functions, ir o , for various metals as obtained by different investigators; only a few metals being given, since these may be regarded as representative. (Further data may be found in Photoelectric Phenomena, HUGHES and Du BKIDUE). The threshold wave length in A refers to the longest wave length which will eject photoelectrons from a given surface, and this may be transformed into a value in electron volts then called the photoelectric work function by the equation 12330 W = -^ i ' vv A in A The first three columns show how the value of the long wave length limit or the work function depends upon the treatment given the surface. It is rare in this work to find the results of two investigators coming within more than approximate agreement. PHYSICS OF RADIATION' 27 Table 7. Photoelectric Work Functions of the Metals ', Threshc Metal |, no !, oiitgassing Ag j 3213 ild Wave Len partial outgassing 2888 3150 gth in A extended outgassing 2010 (20C) 2700 (ooor:) w in volts 4.73 ' 3250 : 3304 3390 Al 3400 3052 (2.5 1o 3.0) 3595 3050 4770 1 ; 5000 Cd 3050 (4.00) 1 : 3130 3140 3302 K . . -4300 5500 5800 5500 (1.70 to 2.25) 0700 5800 > ! 0200 0500 i, Ij Li 5200 7000 7000 j 4300 5400 5200 (2.1 to 2.9) 5000 Mtf 3300 5800 >3050 (-3.40) 3820 7000 Na 5830 5500 5500 5000 1.90 to 2. JO 0100 i Ni i 3050 0400 2700 3040 3182 2403 3720 3400 5.01 3.32 3.57 3305 Zn , 3010 i 3200 3425 3760 4009 (Single crystal ) 28 CHAPTER I The fourth column gives the best estimate of the value of IF that could be made for the various metals. (4) The Photoelectric Counter. The photoelectric tube counter is merely a photoelectric cell of a special geometrical shape. In such an instrument, the individual photoelectrons are recorded, making it much more sensitive than the photo cell in which photoelectron currents arc measured. The lowest intensity which is measurable with a counter is about 10~ 9 ergs/cm 2 sec. or about 500 quanta /cm -/see. Jn a counter, the photoelectric-ally active element is deposited on the inside 4 walls of a cylinder, and the collector is a fine wire Figure 20. Photoelectric tubi counter. insulated from the cylinder and stretched along its axi.s (sec fig. 20). It is filled with some gas to a pressure of about 10 cm of mercury. The metal tube is connected to the negative terminal of a battery of perhaps 1000 or 1500 volts, and the collecting wire to an amplifier, such as is found in a radio receiver. Slits may be cut in the cylinder to let in the light, or it may be allowed to shine in the ends of the cylinder. Each time a photoeleetron is ejected from the walls of the tube, it will be accelerated toward the wire (which is positive by 1000 or 1500 volts) and in its course through the gas will ionize some of the atoms with which it collides. The electrons thus freed are also attracted toward the wire and in turn form more ions. In a very small fraction of a second all these negative* ions will reach the wire. This momentary movement of charge is equivalent to a small current which when amplified , produces a "plunk 11 in the loud speaker. The number of "plunks" per second indicates the number of photoelectrons ejected per second; this number is proportional to the number of quanta striking the inner wall of the tube each second. While in use, the counter gives a few counts per minute when no radiation from the source under experiment is falling upon it. These are due to cosmic radiation, local gamma radiation and a, ft and y rays from radiocative impurities in the metal of the wire and tube ; they are PHYSICS OF KAD1ATION L>9 called "dark counts", "strays" or background radiation. The difference between tlie number of counts when the counter is e\- po 10- 2 3.00 K)- 2 1 5.20, Mr 2 j 0.57 - 10" 2 )ressed in quanta per electron 763 28 IB 134 K .Mjr \a CHAPTKR II SOURCES OF RADIANT ENERGY Radiant energy may originate under widely varying condi- tions. Warm bodies radiate heat. When the temperature rises very high, the radiation becomes visible. Practically all our sources of illumination, from the oil lamp to the incandescent light arc based on this principle. However, visible radiation may also be produced from chemical processes, without great increases in temperature, as in the slow oxidation of phosphorus or in the light of the firefly. Too, when an electric current is set up in a tube containing gas at low pressure, though the temperature remains within a few degrees of the, room, the tube will emit light. In this chapter, a division is made between physical and chemical sources of radiation. 'Chough the chemical sources of radiation are more important because they show us that we may expect radiations in biochemical processes, the physical sources arc much better known. The? discussion begins therefore with the physical sources of rays, followed by the chemical sources of such rays; in Chapter HI, the biological effects will also be first demonstrated with rays of physical origin, and afterwards with those emitted by chemical reactions. A. PHYSICAL SOURCES Thermal Radiation: Returning for the moment to the wave theory of light, we remember that the oscillations of an electric charge result in the production of radiant energy. Lot us wee how this idea may be applied to the various sources of radiation with whieh we are familiar. There is, first, the fact that all bodies emit at all times radiant energy ; they also absorb at all times radiant energy. This radiation is due to the vibrations of the atoms (built of electric charges) of which the body is composed. At ordinary 32 CHAPTER II temperatures, this radiation is of very long wave length. However, the radiation becomes visible when the temperature of the body reaeJies the neighborhood of 500 C. witness the dull red color of iron whieh is being heated. At still higher temperatures, e. g. that of the tungsten filament in an electric bulb, the color of the radiation is changed to nearly white, and in some of the hotter stars to a blue-white eolor. Radiation which arises as a result of the temperature of a body is known as black body or thermal radiation. The peculiarity of a thermal radiator is that the distri- bution of energy among the wavelengths depends not at all upon the atoms or molecules of the body, but solely upon its temperature. Table 9 gives the energy radiated by a tungsten filament at various wave lengths for the two temperatures 2500 and 3000 0. It can be seen from these data that thermal radiators are poor sources of ultraviolet. Table 9. Spectral Distribution of Thermal Radiation from Tungsten 1<\ V (A) (watt/cm 3 ) 1 ) 2500 (' ! 3000" (' Wave Length in A 2000 2500 3000 3500 4000 4500 5000 5500 oOOO (5500 7000 Atomic Radiation: Everyone is familiar with the neon signs so frequently used at present for advertising purposes. These are nothing more than discharge tubes filled with neon or a mixture of neon and other gases. They are fitted with metal 1 ) Kw(A) is the rate of omission of energy in \\atts, from 1 om 2 of surface, in a direction perpendicular to the surface, per unit solid angle, for 1 cm range of \vave lengths. ' 0.07 8.32 7 315 125 2980 8(>9 13100 33(50 35900 ' S930 73100 ! 18400 122000 ; 3MOO J 7(5000 47000 230 000 of) 200 270000 82700 315000 SOURCES OF RADIANT ENERGY 33 terminals to which electrical potentials are applied, potentials high enough to cause ioiiization of the gas atoms in the tube. The atoms lose and regain electrons many times a second with the emission of light each time an electron is regained. Such tubes are known variously as arc, glow and discharge tubes depend- ing upon the pressure of gas within them and the voltage necessary to make them function. The most practical sources of ultraviolet, namely the mercury arc and the hydrogen arc are of this type*. Other sources may depend simply upon the ioni/ation of air between two naked terminals, such as the carbon arc or the iron or tungsten spark. The latter two are strong sources of ultra- violet but suffer from the disadvantage that they are not sources of constant intensity. In this type of source the temperature is not necessarily much different from room temperature --- it is the electrical energy which causes the ionization of the atoms and the resultant emission of light. B. CHEMICAL SOURCES Most of the chemical reactions which proceed spontaneously are exothermic, i. c. they liberate energy. Ordinarily, the energy is emitted in form of heat, as the name "exothermic," implies. Occasionally, however, the reaction causes luminescence 4 , the energy being liberated as visible light. As examples may serve the light produced during the slow oxidation of phosphorus, at the hydrogen-oxygen combination, at the reaction of potassium with water, or at the oxidation of pyrogallic acid. Haber has shown that these are not cases of light produced by heat, but that part of the original energy of reaction is liberated in the form of visible light. By far more common is the emanation of ultraviolet light Recent investigations make it appear very probable that all chemical reactions emit part of their energy in the form of short ultraviolet rays. This has been proven for such simple processes as JNaOH-l HC1 = NaCl+H 2 O, and even for the solution of NaCl in water, i.e. NaCl Na + +Cl~, as will be shown later. Ordinarily, the radiations an 1 very weak, altogether too weak to be registered by the photographic plate. However, it has been possible to prove their existence by the GETGER-MULLKR counter which is essentially an extremely sensitive photoelectric cell {see fig. 21). Protoplasma-Monographien IX: Rah n 3 34 CHAPTER II Jn this way, FKAKK and RODIONOW (1932) proved that a number of common chemical oxidative reactions produced an ultra-violet radiation which could be demonstrated with a suf- ficiently sensitive 1 instrument. Table 10 gives their results. It was also observed at this time that in some of the reactions, the emanation is greatly increased in the presence of diffuse day light. This physical proof of light from chemical reactions has been verified by GERLACH (1933) who showed that this radiation appears only from quart/, vessels, not from gla?ss containers; therefore 1 , it must be of a wave length shorter than 3500 A. AUDTJBEKT and VAN DooKMAL (1933) also measured photo-elec- trically the emission of ultra-violet light by several inorganic oxidations, by the oxidation of alcohol with chromic acid, and by pyrogallic acid in air. Recently, BABTH (1934) could also prove tile existence* of ultra-violet emission from proteolysis which is known to give an immeasurably small heat of reaction. In all of 25 experiments but one, the number of photo-electrons was larger when exposed to radiation from proteolysis than without this, and in 7 experiments it was more than 3 times as large as the error. Table 10. Ultra -violet radiation from chemical reactions Time* ,t Photon impacts per Chemical Reaction Interval j. time interval Increase m impacts by chemical reaction 1 minutes exposed , conttol absolute peii'ont.-* J'yrogallic acid -f NaOH H-air 10 15 J 1.2 10 j 1.0 5 50 8 38 2.0 26 j 1.8 12 46 Sn('l 2 II#Cl 2 10 1 29.5 i 1.7 26 1.6 3.5 13 KeS( )jK 2 Cr 2 O 7 (dift. light) 6i) 18 1 1.7 13 1.5 5 38 M 4 1 ) 17.52.1 13 1.8 4.5 34 *) 18 1.7 12 1.4 6 50 ' 9 l ) 36 2.0 26.5 i 1.7 9.5 36 KeSO 4 K 2 < 1 r 2 () 7 (dark) . . 15 27 | 1.3 17 1.0 10 ! 60 - 4 1 ) 12 1.8 11 1.7 I !) 1 ) Radiation passed a nionochromator, only the rays between 2000 and 2700 A were measured. SOURCES OF RADIANT ENERGY 35 These radiations are so very weak that only the most sensi- tive counters will detect them. However, living cells under cer- tain physiological conditions react very promptly upon irradiation in the wave length range 1800 2600 A. In fact, they are so sensitive that it has been possible to use them in place of photo- graphic plates for determining the spectra of such radiations. The organisms most used in these experiments are yeasts the growth rate of which is accelerated by short ultraviolet rays under certain conditions. The biological aspects of this acceleration will be dis- cussed in Chapter TV. The original method consisted simply in plac- ing before the eollimator slit of a quartz spectro- Figure 22. First attempt to obtain a graph a quartz tube milogonctic spectrum with the reagents to bo " : T d^ 1 "^ excitod muscle: B: tho & m quartz prism; C: agar blocks with yeast on tested, and to substitute the exposed side, each block receiving rays the photographic plate ... C knou-n wave length, by a succession of tiuy blocks of nutrient agar on which yeast in the proper physio- logical condition was growing. Kig. 22 shows the first attempt,, by KFUNK (1929) to obtain tho spectrum of a frog muscle. Each yeast block was thus exposed to a definite" range of the spectrum which could be determined fairly accurately. After irradiation, the yeast was permitted to grow for a short time in order to bring out the growth rate differences, and was then compared with the controls. By this method, KANNEC HESSE it (1931) working with yeast blocks, each representing approximately 50 A, studied three types of oxidation, namely pyrogallic acid in alkaline solution by air, glucose -{- KMnO 4 , and blood serum -| H C) a It was found that the growth was stimulated only on the two blocks receiving radiation from 22202280 and 2280 -2340 A None* of the other detector blocks differed appreciably from the controls. From these results, it would appear that all three oxidations gave the same spectrum, as far as can bo ascertained with this rather crude method. 3* 36 CHAPTER II The next advancement was the division of the spectrum into separate strips of exactly 50 A each. POTOZKY (1932), by means of glass needles and heavy cellophane, prepared a chamber (fig. 23) the sections of which corresponded exactly to the 50 A divisions of her spectrograph. By this simple instrument, BBAUN- STETN and POTOZKY (1932) showed that the spectra of different oxidations possessed certain specific regions besides the general oxidation spectrum. The data of 7 separate experiments are Figure 23. Device for exposing yeast to successive ranges of the spec- trum of f>0 A each. reproduced in Table 1 J . The various spectra are not quite identical, and even the rather crude determination by 50 A strips shows differences which remained typically constant when the experi- ments were repeated. The limit of error is + 15. Table 11. Induction Efiects obtained from 50 ANGSTKOM stups of the Spectra of various Oxidations Detector: Cell Numbers in liquid Yeast Cultures s g o 8 s s 8 8 3 Oi S 7 KClOa+Hn+NaOH . . f 7 + 4 H- 8 +20 +23 + 26 + 28 o +2 FeCl 3 +NH 2 OH-IU.'l . 1- 5 _2 +3 -1- 7 - 9 +46 478 +90 +51 2 H 2 0, 1 ?t 4- 6 -3 + 13 +29 f9 +29 +20 +36 8 + 1 HgOl 2 l SnCl 2 .... + 4 + 2 + 3 f49 4 +60 (-58 4-97 436 -4 Range ob- tained by K ANNE- UIESSKK SOURCES OF RADIANT ENEKtiY 37 It was also found that diffuse daylight increases the intensity of some reactions, e. g. of K 2 Cr 2 7 j FeSO 4 , but does not affect the spectrum itself. A still more detailed analysis was finally accomplished by PONOMAREWA (1931) who used 10 A sections. It was impossible to divide the agar surface into such narrow strips and there was tilways the possibility of confusion by the spreading effect (see p. 110). Therefore, PONOMAREWA screened off all radiation except oxidation ( J'yrogallol) Hiiuar fermentation nucloase (phosphatasc) phosphate Heavauo protrolysis sum of all above rear! ions aniylnst 1 mallasc su erase re 24. Thr spoctia of SOTTIC common biological i ('actions. one narrow slit of 10 A. The position of this slit could be changed over the entire spectral range By this method, only one small part could be studied at one time, and the progress of such ana- lysis is slow. However, if the general spectrum has been investi- gated by the above-mentioned eoarser methods, the negative regions need not be examined, and the amount of work is thus greatly reduced. On account of the very low intensity, this proce- dure is usually combined with intermittent radiation (see p. 1()3). In this way, PONOMAREWA could show that the glycolytie spectrum of blood consists of only 5 regions, or, more precisely, that only ."> of the 60 spaces of 10 A each manifested mitogcnctic effects (see h'g. 24). There may be more than one spectral line, of course, in a strip of 10 A. Recently, DIOCKKR (1934) succeeded to to split the first double line of this spectrum into two different lines of 5 A each. 38 CHAPTER 11 The most important result, however, was the observation that these bands coincided exactly with those of the alcoholic fermentation by yeast, and also with those of the lactic fermen- tation by Streptococci. GURWITSCH concludes that there must be some process common to all of these sugar decompositions, giving off the same radiation. This seems probable since it is generally assumed that the cleavage of the hexo.se phosphate through glyeeric aldehyde to methyl glyoxal is common to all three types of sugar decomposition. As a consequence of these splendid findings, the method was used for a number of frequently-occurring biological reactions. Aftctro,ur;i]>li. workers for this purpose. .Kig. 24 shows that only rarely is the same line produced by two different processes. Kven then, it should be realized that we are not dealing with true spectral lines, but with relatively broad regions, and that two identical strips do not necessarily indicate two identical lines, but rather two (or more) proximate lines. There is also a spectrum shown which is the sum of all these processes. We shall see later (p. 156) that the spectra, of nerves frequently combine all of these lines, in addition to some others of unknown origin. The spectra of the action of amylase and maltase, and of suerase (invertase) have been obtained by KLENITZKV and PKO KO- FI EWA (1934). The lines of these two enzymes agree to a. much larger degree than any of the previously-mentioned processes. This is to be expected from their chemical parallelism. Another method of obtaining spectra, is that of WOLFF and RAS (19,32) who used bacteria as detectors. They made a number of vertical grooves in a glass block which fitted into the camera of the spectrograph (see fig. 25) ; the grooves were covered by a quartz plate so that they became tiny pockets into which the detector culture was placed for exposure. The wave length for each one could be determined accurately. 40 CHAPTER Jl By this procedure, they found the spectrum of neutralization of acid and alkali to consist of three lines a strong line between 1960 and 1090 A a strong line between 2260 and 2300 A a weaker line between 2070 and 2090 A. Fig. 26 shows these* regions, and also the spectra obtained by RAS (quoted from RT'VSSEN, 1933) for the Bunseiiburner flame. This latter spectrum lias also been photographed, and had many more lines of longer wave length, some of which are shown I. 1*11 11SC spectrum m^ISlMllBEii Fiyurc 26. Photographic and mito^ciietie spectra: en burner flame, milo^enetic spectrum; //. same, photographic LI; 111. Reaction tT 2 -|-(M 2 , mito^enetic spectrum; IV, Reaction Na()H + H(H, mitogenetic Kpectrum. here, but photography failed entirely at the shorter ultraviolet. The figure also shows the biologically obtained spectrum of the reaction ot hydrogen with chlorine. ft should not be gathered from this discussion that the indicated lines represent the entire spectrum of these reactions. On the contrary, it is most probable that it extends to both sides of the narrow range shown here. We arc limited, however, by our indicators. Radiation below 1900 A will be readily absorbed even by air (sec p. 23), and that above 2600 A does not produce mito- genetic effects (see fig. 28) ; therefore they can not be observed by the methods employed here. They may be capable of producing other biological effects hitherto unaccounted for, and may play an important part in chemical reactions. A good summary of the Russian studies of mitogenetic spectra, including those of inorganic reactions, and with sufficient data to obtain a conception of the error of the method, has been given by A. and L. GI T RWTTSCH (1934). The intensity of ultraviolet radiation from a chemical reaction is not proportional to the total energy liberated. This was to be SOURCES OF RADIANT KNE1KJY 41 expected because the same 1 holds true for the emission of visible light from chemical reactions. Strong mitogenetie effects can be obtained from protein digestion by pepsin or from milk coagulation by rennet while RITBNEK found the heat of reaction of proteolysis to be immeasurably small. On the other hand, the heat of neutrali- zation of aeid by alkali is so large that it can be observed even without a thermometer, yet its radiation is relatively weak. The origin of the spectra is by no means clear. LOKUNK'S criticism (1934) can be explained in a concrete example as follows: If a spectrum represents the radiant energy emitted by the reac- tion as such, then each reacting molecule must emit at least one quantum of the shortest wave length observed in the spectrum. The longer wave lengths might originate from the shorter ones by loss of definite amounts of energy. Thus, in the case of sucrose hydrolysis by invertasc (figure 24) each sucrose molecule must emit at least one quantum of the wave length 2020 A, i.e. of the energy content 9.S :< 10~ 12 ergs (see Table 1). Then* are (>.l ' 10 2:i 6 1 X 10 23 molecules in a grammoleeule, or --- t k in Ig of sucrose The minimal amount of radiant energy from 1 g of sucrose would then be : H.I > 10 23 X 9.8 < 10~ 12 - - - ergs 0.175 > 10 11 ergs -- 0.042 , 10* cal - 420 calories. The total energy liberated by this hydrolysis has been measured by RuiiNUJi (1913) by means of a BJUCKMANX thermometer in silverlined Dewar bottles, and was found to be 9.7 calories per gram. This experimental value is only one-fiftieth of the minimum amount calculated. It seems impossible that appreciable amounts of energy could have been lost by the method used. We are com- pelled to the following alternative: Either, the spectra do not originate from the reactions for which they are considered specific, but from some quantitatively unimportant side reaction; or, the calculated amounts are actually liberated, but arc at once absorbed again. This latter explanation does not appear entirely impossible. The biologist is familiar with "false equilibria", such as the stability of sugar in the presence of air, though it could be oxidized with liberation of much energy, and the 1 process might, therefore, be expected to take place spontaneously. Modern chemistry 42 CHAPTER II explains this by the necessity of ''activation" of the molecule to make it react chemically. This activation requires energy. FRICKE (1934) in an introductory summary, makes the following general estimates: "Generally, energies of activation are of the order of 100,000 gram calories per gram molecule which equals 1.5x10 19 gram calories per molecule. At ordinary temperature, the average kinetic energy of a molecule is of the order of 10~ 21 gram calorics. Only the inconceivably small fraction of 10~ 43 of Ihe molecules have energies in excess of 10~ 10 gram calories per molecule. "The quantum theory gives as the reason that activation may be produced by radiation, the fact that the energy of the radiation is carried in a concentrated form, as quanta. The energy of a quantum of radiation is 5x 10~ 16 /A gram calories where "k is the wave length in the ANUSTKOM unit. The wave length has to be reduced to the order of 3 ,000 A. before the quantum has the value L.5>, K)~ 19 gram calories which, as we saw, represents the usual value for the energy of activation per molecule." The energy of activation necessary for the hydrolysis of sucrose has not been determined. If wo assume it to be of the "usual value" as computed by FRICKTC it would bo equivalent to a quantum of about 3,000 A. wave length per molecule. When this is absorbed, the snciose molecule hydrolyscs, and the amount of energy thus released must be larger than that absorbed, because of the additional heat of reaction of I). 7 calories per gram. The free energy will be absorbed at once by a neighboring sucrose molecule which becomes activated, and hydroly/ed, and thus the reaction goes on. In this way, all the liberated energy is again absorbed, except for the difference between heat of hydrolysis and heat of activation which we measure as heat of reaction. However, then? is another small "leak". Of those molecules adjacent to the walls of the vessel, the energy may radiate into the wall rather than to another sucrose molecule, and these few quanta would leave the system, and produce a radiation if the vessel is transparent. The amount of energy thus leaving the vessel would be extremely small, and would be of a wave length equal or shorter than that required for activation. If this explanation of the mitogenetic spectra is correct, they would be an excellent means to measure the energy of activation. SOURCES OF RADIANT ENERCV 4;* C. SECONDARY RADIATION A phenomenon must be recorded here which was first believed to be typioal of living organisms, but is a property of certain chemical solutions, or systems, namely the emission of rays from a solution as a response to "primary 11 rays directed upon it. The first purely chemical effect of this kind was observed by A. and L. GfKwiTscit (1932 b) with nucleic acid. A 3% solution of nucleic acid was gelatinized in a glass trough. At one end, it was irradiated with the lines 3220- 3240 A from a copper arc, through a monochromator. At the other end of the trough, radiation of the nucleic acid could be observed, but the wave length of this "secondary" radiation w r as not the same as that- of the primary: it was between 2450 and 2500 A, which is the spectrum of nucleic acid hydrolysis (see fig. 24). This proves that it can not be merely a reflection of light, because the wave length changed, nor is it a case of fluorescence, for the secondary radiation has a shorter wave length than the primary. And most remarkable 4 of all, the induced light may be stronger in intensity than the primary HOU1VC. The best explanation is most probably the one given by OU'it \MTscjr that the primary radiation induces some kind of chain reaction (see p. 47) which is then radiating with its own spectrum. This would account for the difference in wave lengths as well as for the increase in intensity. Other examples have been given by WOLFF and KAS (193.'U>). These authors observed the same effect with sterile blood serum. They found also that sterile nutrient broth did not produce secon- dary radiation, but showed it when bacteria had grown in it, even after the bacteria themselves had been removed by filtration through a porcelain filter. A very short action of living bacteria- suffices to change the broth to a "secondary sender". These filtrates as such emit no primary radiation. WOLFF and HAS observed further that after long exposure to primary radiation, these liquids ceased to produce secondary radiation. Very intense primary light caused a more 4 rapid exhaustion. 45 minutes ex- posure of a staphylococcus supscnsion to a strong primary sender had made it unfit to produce secondary rays; however, on the next day, the suspension reacted normally again. In another ease, even 30 minutes exposure was sufficient to destroy the power of secondary radiation. 44 CHAPTER II Nucleic acid solutions also cease to function when over- exposed, and during this stage, they do not transmit initogenetic rays. Strong solutions recover again after 1 or 2 days of "rest". An important observation is that with increasing concen- tration of the acting substances, the secondary radiation becomes weaker. Table 12 represents an experiment with nucleic acid, by WOLFF and RAS. The source of primary radiation was the reaction of milk with rennet. The intensity of secondary radiation from the nucleic acid dilutions was measured by the length of exposure, required to produce a "mitogenetie effect", i. c. to accelerate the growth of bacteria. The numbers in the table represent the percentage increase in cells over the control. Table 12. Intensity of Secondary Radiation of Nucleic Acid. .solutions, measured by the time required to produce a " in 1 1 ogc ne 1 1 e e f f e c t "' Concentration of Xucleic Acid, in " .5" lito^e 30" neti ]' p ElTc 1.5' et a 2' Iter Irr< 3' 4' idiation for 5' | o' | r 8' J .000 ! 32 0.750 J i 40 26 0.500 33 55 12 0.250 () 27 21 i 0.100 M 17 j ' 1 0.020 25 32 ! i 0.004 i 0.001 o i i ! The results are somewhat surprising. The lowest efficient concentration was 0.02%, which produced a mitogenetic effect at least 5 times as strong as the 1% solution, i.e. it produced the same effect in one-fifth the time. A similar relation was observed with bacterial suspensions. The more dilute they are, the stronger is the secondary radiation they emit upon excitation by some primary source. Perhaps this is brought about by the absorjtfi-ion of rays in overexposed solutions (see above). Bacteiial suspensions and their filtrates lose the power of secondary radiation upon heating; nucleic acid solutions do not. The increase in intensity by secondary radiation enabled WOLFF and RAS to construct an "amplifier" for mitogenetic rays. sorncKs OF RADIANT KNKRCY 4r> They placed six quartz cuvettes filled with staphylococcus suspen- sion side by side, and by irradiating one side of the series, obtained a good mitogeiietic effect from the opposite 1 side in 10 seconds; the same primary source used for direct irradiation of the same detector required 4.5 minutes. This means an amplification of 27 times. The observation is added that one continuous column of the same length as all <> cuvettes together, does not give a greatly increased intensity. It must be kept in mind that the intensity of radiation lias been ascertained only biologically, by the time required to produce a mitogeiietic? effect. It has already been pointed out (p. 24) that the reciprocity law (requiring double exposure time for half the intensity) does not even hold for such simple reactions as those in the photographic plate when the intensity becomes very low. Its application to biological reactions is quite doubtful. Recently, WOI/FF and KAS (li)34a) could show that usually, secondary radiation is polarized, and they also proved that polarized mito- geiietic rays exert a very much stronger effect upon organisms. Tt is not at all certain, therefore, that the above statements really indicate an increase in intensity of radiation in the physical sense of the word. In his most recent summary, <*rK\\iTscii (1934) make>* the following statement : "It has been found that all substrates capable of enzymatic cleavage, such as glucose, proteins, nucleic acid, urea, fats etc. react also upon mitogenetic radiation and become radiant. This "secondary radiation" has certain properties of great interest . (1). it travels from the irradiated part through the liquid me- dium to distances of several centimeters with the measurable speed of a few meters per second; and (2). the radiation is resonant, i. e. the substrate reacts mostly upon those wave lengths which it emits when decomposed enzymatically." The only published experimental proof for this is that by BE KOROSI (11)34) as far as the authors have been able to ascertain. UHAFTKR III EFFECT OF ULTRAVIOLET RADIATIONS UPON CELLS A. EFFECT OF RADIATIONS UPON CHEMICAL REACTIONS Tt has long been known that energy in the form of visible light, or near this range, will produce chemical changes, which we term photochemical reactions. Just as a chemical synthesis may be brought about by heat, so may it be induced lay adding radiant enei'gy in the form of light. All organic matter is thus produced from CO 2 , 1J 2 O and nitrates, by means of radiant energy from the sun, with chlorophyl as the necessary ''transformer" of the energy. It is not necessary, however, that radiant energy be, present to increase the energy level of each reacting molecule. A number of chemical reactions are known which require some radiant energy to be initiated; however, several hundred, or even several million molecules are changed for each energy quantum which is absorbed. These reactions are exothermic. If only one mole- cule becomes activated by an energy quantum of the right size, the energy liberated by its reaction activate s another molecule. Thus, many molecules may be changed though it is quite impossible' that ono quantum can b, 1 absorbed by more than one molecule. This type of reaction, which is called a chain reaction, is usually explained in the following way (_H. S. TAYLOR, 1931, p. 1009 101 8): The quantum, in being absorbed by a molecule, ionizes it. The ion pair tlms produced initiate two independent, reactions which again produce ions. The classical example by BOBENSTETN and NJJJKNST is the photochemical reaction between the gases H 2 and C1 2 . The chain reaction can be written EFFECT OF ULTRAVIOLET RADIATIONS I'l'OX CULLS 47 01 + H 2 - HOI -j- H Cl -}- H 2 -- HOI | H H + C1 2 HC1 f (11 H i OL - HOI | Cl 01 + H 2 _= KOI |- H 01 + Ho - HOI + H H + 01 2 -,, HOI ! 01 H h 01 3 = HOI + 01 01 -I H 2 HOI -f H H ! 01 HOI. Under "chain" is understood the number of consecutive molecules entering into reaction. The chain may be as "long" as one million molecules The "length" can be ascertained by determining the number of molecules changed for each quantum of light entering the system. The chain is terminated by the reactive molecules or atoms combining with each oilier, as indicated in the above model, or by reacting with other molecules io form stable compounds. If it were not for those terminations, one. quantum would be sufficient to cause all molecules to read with one another. In fact, in the case of explosions, where the reaction liberates a large amount of energy, this is practically the result. The presence of foreign substances reacting with the com- ponents of the system is a common cause of cessation. If there were 1 only one such molecule present for every million molecules of hydrogen and chlorine, that would account for an average? chain length of one million molecules. If there 1 Here 100 times as much of the foreign substance, the chain length would be reduced to ten thousand molecules. The same reasoning holds true for ehain reactions in solutions. In the photochemical oxidation of Na 2 SO 3 to Na 2 S0 4 , the quantum yield was about 100000. This reaction was inhibited by primary and secondary, but not by tertiary alcohols. Whenever a chain 48 CHAPTER III was terminated, two molecules of the alcohols were oxidized to the corresponding aldehydes and ketones. The known chain reactions are exothermic. Jt does not seem imperative, however, that they be so if another source of energy is available. This is the ease in normally nourished cells of animals, fungi, bacteria, etc., which liberate energy constantly by meta- bolizing carbohydrates, fats, proteins or other organic, compounds. By means of this energy, they grow, i. e. they synthetize new body substance endothermically. It is not impossible, that very small amounts of energy, oven a single quantum, might produce a very noticeable effect in a living cell. This could be accomplished by releasing a complex mechanism which needs only one or several quanta of a given size to be initiated, just as the Cl 2 -molecule needed only one quan- tum of the right size to start the reaction with hydrogen. The most common, and perhaps the only reaction in the coll which can be thus released is cell division ; this results in a more rapid multipli- cation, or an increased growth rate. B. EFFECT OF MONOCHROMATIC ULTRAVIOLET UPON LIVING CELLS Ultraviolet light of any of the different physical sources mentioned in the previous chapters may have* a very distinct effect upon living cells. The best-known is the reddening of the skin by ultraviolet light. A quantitative study of the relation between the intensity of the effect and the wave length has revealed that aside from the very marked erythema produced by wave- lengths around. 3000 A, the cells of the skin will also react upon those below 2600 A which are not found in sunlight. Between these two maxima is a zone of very weak effects. This fact is significant because the major part of this book is concerned with mitogeiietic rays which are shorter than 2(500 A. Ultraviolet light also kills bacteria and other microorganisms. Strangely, however, the intensity curve for the different wave- lengths is quite different from that of the erythema effect, it- looks almost like* the reverse. Figure 27 shows the results by COBLENTZ, STAIR and H QUITE (1932) on erythema, by RIVERS and GATES (1928) on vaccine virus and fltaphylococcus aureus and by DUGGAR and HOLLAENBEK (1934) on Bact. prodigiosum EFFECT OF I'LTRA VIOLET RADIATIONS UPON CELLS 49 and the mosaic virus of tobacco. Most of these investigations have been carried out no further than to a wave length of about 2500 A. If shorter wave lengths are taken into consideration, and especially if the intensity is very greatly decreased, it is possible Figure 27. Comparative intensities of the killing effect ol different wave lengths. The intensities are uniform for each individual organism, but vary greatly for the different curves. to obtain growtli stimulation under certain conditions which will be specified in Chapter IV. The result of the most extensive of the many experiments of this nature is shown graphically in fig. 28. 10-* 10- Figure 28. The effect of ultraviolet light of different wave lengths and different intensities upon yeast. + indicates accelerated growth; o means no effect. Protoplasma-Monographien IX: Rahn 4 50 CHAPTER III CHARITON, FRANK and KANNECUESSER (1930) used individual spectral lines, employing a monoehromator, from the sparks of aluminum, zinc or cadmium, to irradiate yeast cultures. Each culture thus obtained light of one definite wave length. By varying the intensity as well as the wave length, it could be shown that only radiation of less than 2700 A produced positive effects, i. e. increased the growth rate of yeast. A large number of similar experiments have been carried out by GITBTWTTSCH and his associates, usually as controls for organic radiations. They will be mentioned in the succeeding chapters. There seems to be very little difference in the limiting inten- sities of different wave lengths. Other experiments have shown that even at 1900 A, good effects can be obtained. Below 1900 A, absorption by quartz, water and air interferes with the experiment. C. EFFECT OF RADIATION FROM CHEMICAL REACTIONS UPON LIVING CELLS The same effect which has been demonstrated above as the result of irradiation with ultraviolet of known wave lengths, can be produced also by exposing the cells of microorganisms to the emanation from chemical reactions. One of the simplest examples is the stimulation of the bac- terial growth rate by the emanations from the neutralization of NaOH with HOI. WOLFF and HAS (1933 b) allowed these two chemicals, flowing from two tubes, to unite on a quartz plate, underneath which was the bacterial culture. After exposure, these cultures were incubated for 2 hours. Table 13 shows very distinctly in both experiments that an exposure of approximately 5 minutes to the radiation of the neutralization process has stimulated the growth; the number of cells has been increased approximately 40-50%. The same authors found that even the dissolution of NaCl in water produces a growth-stimulating radiation (Table 14). This energy emission occurs only during the act of dissolving ; it ceases completely when all the salt is in solution. No effect is noticeable when sugar is dissolved in water, or when palmitic acid is dissolved in alcohol. WOLFF and HAS concluded therefore that the process of dissociation of salt into ions is the source of ultraviolet. EFFECT OF ULTRAVIOLET RADIATIONS UPON CELLS 51 Table 13. Staphylococci exposed through quartz to the energy emanations of the reaction NaOH-(-HCl -- Na('l f-H 2 O and counted 2 hours later Exposed for (.'ells per ec . of culture 1 "..=,... minutes (control) ... 20 100 20 800 4. 20 200 31 500 4 5 27 250 37 750 39 500 ,, 30 900 each number is the average of 9-11 experiments 3- -4 experiments In the same way, bacterial growth was accelerated by ex- posure to metallic zinc in a solution of lead acetate or copper sulphate. Simple oxidation processes also emit energy which can cause an increase in the growth rate of bacteria or yeasts. This is already cited in the method of obtaining oxidation spectra, and maj be further illustrated by an unpublished experiment of Miss A. J. FERGUSON. Oxalic; acid was oxidized with permanganate in a glass vessel. Above this were fastened two small covered dishes, one of quartz and one of glass, each containing a sample; of the same culture of Bdtfcrrntu, coll. The sample in the quartz vessel grew more rapidly, having been exposed to ultraviolet light from the oxidation process; the other received no stimulus since glass Table 14. Staphylocoeci exposed through quartz to the energy emanation from dissolving substances Duration of Cells per cc. of culture Exposure 10" 20" 30" 45" r ! 1.5' 2 7 4' Control 1 | NaCl in water 32100 31400 37700 45000 -- 21750 31500 27300 - __ 35500 47500 45500 - 40000 30800 - sucrose in water 7000 6250 7250 7250 7250 palmitic acid 7500 in aloohol 7000 7000 - 8000 8500 7250 7750 NaCl after com- 1 plete solution 21750 . ___ 22000|20750 __ . . 4* 52 OH AFTER III absorbs the radiation (Table 15). There is the usual lag period of 2 hours, but after the bacteria once start to grow, the irradiated culture grows more rapidly. Table 15. Development of a culture of Bacterium eoli after ex- posure to emanations from the reaction C 2 4 H 2 + KMnO 4 =- K 2 ('O 3 + 2 MnO + 9 ( 30 7SS 001 rlifferenee 2 -4 30 32 10122 12 10 1 15 | I-V2I 7 15 187 = +23.8% Ji; same h ratal to (J0(l. Totals exposed unexposed 60 61 ti6 69 64 61 65J62 61 74 63 62 60|75 6073 81 82 71 66 01 71 65 74 73 69 73|65j61 69J6560 1066 1076 Difference jUl|-3l l|-lL_l3 ! ll ()! 2 ! -l| 5 *j 4 4 56 CHAPTER JV This technique was used by Gi'Bwrrscu and a number of associates to search for mitogenetic rays in the entire organic world. It would scarcely be worthwhile to compile a complete list of organisms found to radiate. The following list contains the more important earlier observations, compiled by GUBWITSCH (1929). Radiating organisms and tissues Bacteria Hdclcrium tuwcfacicns, Staphyloeoeci (MAGBOU) Bacterium murimorx (Acz) (SEWKRTZOWA*) JJaciUvti antHiracoidcif, Sarcina flara (BARON) Streptococcus fastis (MAGBOU) Yeast (BARON, SIKBHRT, MAUROF) Eggs of annelids Eggs of sea urchins before the 1st division (FRANK and SALKI\I>) be -fore the 2nd and 3rd divisions (SALKINP) Egg yolk of chicken, only during the first two days of incubation (SoiiiN). After establishment of circulation system, radiation ceases Embryos of amphibia hi the morn la stage (ANIKIN) Plant seedlings: root tips, cotyledons, young plumulae of Hihnn- tkiis (FRANK and SALKIND) Potato tubers: leptom fascicles only (KISLIAK-STATKEWITSCH) Onion roots connected with the bulb (GuRwrrscn) Onion base pulp (A. and L. GUBWITSCH, RIOITER and OABOII) Turnip pulp, 24 hours old (ANNA Giwwrrscif) Young tadpole heads, pulp of tad})ole heads (ANJKTN, and GABOK) Blood of frog and rat (GuitwiTscn, SORIN) Blood of man (SIEBEBT, GESENIUS, POTOZKV and Ctontracting muscle (SiEBERT, FKANK) Pulp from resting muscle |- lactic acid -\- oxygen Corneal epithelium of starving rats, but not of normal rats (L. ( J i u- \VITSCH) Neoplasms: carcinoma, sarcoma ( GUBWITSCH, SIEBERT, KEITEB and GABOB) Spleen of young frogs ( GUBWITSCH) Bone marrow (SIEBERT) Bone marrow and lymph glands of young rats (SUSSMANOWITSCH) Resorbed tissue,: tails, gills, intestine of amphibian larvae during metamorphosis (BLACKER, BBOMLEY) METHODS OF OBSERVIXC lilOMUIICAL RADIATIONS 57 Regenerating tissue of salamander and angleworm (BL \CUKK, SAMARA.TEFF) Hydra: hypostom and budding /one, not other parts. Non-radiating organisms and tissues Tissues of adult animals except brain, blood and acting muscle (most tissues have later been found to radiate slightly) Tadpoles over 2 cm. long Chicken embryo .after '2 days (blood radiates) Blood serum (becomes radiant with oxyhemoglobin) (SniiiN) (or with traces of H 2 O 2 ) (ANIKIN, POTO/KY and ZOGLIXA) Blood of asphyxiated frogs Blood of cancer patients Blood of starving rats (becomes radiant with glucose) (AM KIN, POTOZKY and ZOCJTJNA) Active tissues with chloral hydrate Active tissues with KCN Further details will be given in Chapter VIF. The greatest early support to the establishment of mito- gcnetic rays was given through the extensive and thorough work of RETTER and GABOR (192S). These two authors tested the onion root method, and verified especially the, physical nature of the phenomenon; the radiant nature of this effect was thus fortified beyond doubt (see p. 59). Considering the great claims which Gurwitseh and his asso- ciates made for their discovery, it was surprising that comparati- vely few biologists were sufficiently interested to repeat the experiments. Among these, some obtained negative results so consistently that they denied the existence of mitogcnetic rays altogether, and considered the results of the Russian workers and of REITER and (TABOR to be experimental errors. The most frequently quoted of these are SCHWA KZ (1928), ROSSMAN* (192S), and much later, in this country, TAYLOR and HARVEY (1932). Positive results were obtained by MAcuioir (1927), WARMER (1927), Loos (1930), BORODIN (1930), and recently by PAUL (1933), and many Russian workers, in several different laboratories. In order to decide whether the positive results could be considered experimental errors, SCHWEMMLE (1929) undertook a statistical investigation. He concentrated graphically all results published by GimwiTsoH and the Russian school (about 200) CHAPTER IV by plotting the percentage increase or decrease of mitoses of the exposed over the unexposed side of the root, against the total number of mitoses counted. He distinguished only between "induced" and "not induced" roots. From the "not induced" roots, he could compute the probable error of the method. The error was Jj 10% when 500 mitoses were counted, and decreased, of course, as the total number increased (fig. 30). All results Figure 30. All results with onion root as detector by UuitwrrscH and associates. Ordinate: percentage of increase over control; Abscissa: total number of mitoses counted. Circles indicate a positive induction, black dots indicate no mitogenetic effect. The lino gives the limits of error. which had been claimed to prove mitogenetic radiation were found outside the limits of error. REITKR and GABON'S experiments have a much greater error, ^ 20%, and a few experiments supposed by these authors to prove induced mitogenetic effect are really within the limits of error. The error of the experiments by WACJNER (1927), 8rHWAiiz (J928), and ROSSMANN (1928 29) is even larger than that of KEITEK and GABOH'S, and TAYLOR and HARVEY'S few experiments (1931) indicate a similar large error. All these data gave doubtful or negative results; the mitoses of two different sides of the roots varied greatly. SciiWEMMUt; did not consider it proved that the effect is caused by mitogenetic rays, because of the possibility of other physiological factors affecting mitosis during the? experiments. METHODS OF OBSERVING BIOLOGICAL RADIATIONS 59 The effect as such, however, must be considered established by a very large number of data, and the only question was its inter- pretation. That under different physiological conditions, in different countries with different onions, negative results have been obtained by some investigators is not really surprising GTJRWITSCH had always claimed that the effect of one root upon the other was caused by rays. In fact, he predicted in 1922 radiation as a factor in mitosis, and in trying to verify his predic- tion, found onion roots as the h'rst reliable indicators. The mitogenetic effect proceeds in a straight line and is reflected from glass and from a mercury surface (GrKWiTscii, SIEBERT, REITEJI and GXBOK). ft will pass through thin layers of quart/, and of water (GuKVvrrsc'ii, MAUUOU), through thin animal or vegetable membranes, thin plates of mica (UNITE it and GABOK), thin cellophane (STEMPELL), but not through thick layers of glass, such as glass slides, nor through gelatin even in very thin, layers. It seems hardly possible to account for all of this by any agent other than ultra-violet rays. A very vital question is that of the wave length of these ra t \s. and it is very interesting that two distinctly different wave lengths have been claimed, both based upon apparently reliable data. (it'KAMTSric could obtain the effect through quartz, and partly through very thin glass, but not through very thin gelatin, and concluded that he was dealing with an ultra-violet radiation of about 2200 A. PKANK and Gi/Jtwrrsen exposed onion roots to different wave lengths from physical sources, and obtained mitogenetic effects only from the spectrum between 1990 and 2370 A. Quite different were the results of RUITEK and GAIJOR (192H). They found this radiation to be transmitted through 8mm. of Joiia glass, and still noticeably through 5mm. of common glass, and also through gelatin which indicates a wave length above 3000 A. By means of special filters, they found the range 4 to In- between 3200 and 3500 A. Then, by irradiating roots with known wave lengths of the spectrum, they determined the mitogenetic efficiency of this part of the spectrum. Besides a sharp maximum at 3400 A, another smaller maximum was discovered near 2800 A. Below this, no mitogenetic effect was observed, not even in the neighborhood of 2000 A which was considered by GO CHAPTER TV and FKANK as the only efficient region. The curve resembles somewhat that for erythema (fig. 27 p. 49). The very interesting observation was made that the appar- ently inert spectrum between the two maxima will prevent mito- genetic effects by the active wave lengths, even when the inten- sity of the "antagonistic 11 rays is only one-tenth of that of the mitogenetic rays. All wave lengths between 2900 and 3200 A show this inhibition. The rays outside of the maxima were enti- rely neutral. Direct sunlight and ultra-violet arc light also inhibited mitogenetic effects. REITER and GABOR determined further the wave length of mitogenetic rays by means of a spectrograph, letting the spectrum from roots or sarcoma tissue fall upon the length of an onion root. All three experiments gave an increase in. mitoses at- the place where the wave lengths between 3200 and 3500 A had fallen on the root. These last experiments can now be explained by an error in technique. It was not known at that time that irradiation of the older parts of a root will produce- a mitogenetic effect not at the place of irradiation, but at the only reactive part, namely the meristem near the root tip. This last argument in favor of a wavelength near 3400 A must therefore, be discarded. It is considered definitely esta Wished now that mitogenetic rays range between 1SOO and 2600 A, as has already been shown in Chapters TT and 111. However, the deviating experiences of REITER and GABOR have never been accounted for in a really satisfactory way (see GURWITSOH, 1929). The publication of REITKR and GABOR'S experiments caused the .Russian workers to repeat them at once, because they con- tradicted all their own statements about the wavelength, and none of the German authors' results could be verified. The first experiment was FRANK'S spectral analysis (1929) of the radiation of the tetanizcd muscle, with a spectrograph using yeast as detector (see p. 35). Tn three well-agreeing ex- periments (fig. 31), it could be shown that no radiation above 2400 A was emitted. Then followed the detailed study by CUARI- TON, FKANK and KANNEOTESSER (1930) of the effect of mono- chromatic light from physical sources upon yeast. The results have already been shown in fig. 28. Beyond 2600 A, no variation of intensity produced any effect. Special efforts were made to METHODS OF OBSERVING BIOLOGICAL RADIATIONS 01 investigate the range around 3-400 A claimed to be so efficient by REITER and OABOR, but it yielded only consistently negative results. Considering that RJCITEK and UABOK used onion roots as detectors, the experiments were repeated with onion roots. Again, rn - 50 . , r - - W - t ~ ""r" ... -i I 30 t i i 20 I 1 10 - I _._ -- -, I 1 r r -7=- 1-- s&ra y. Ul ~"%d '...- F 1 J._ , Figure 31. Results of 3 experiments on tJu* spectrum of muscle radiation, witli the technique shown in figure 22. Since 4 the width of the agar blocks was not uniform, the results overlap partly, but show the general spectrum. the shorter wave lengths were found to be efficient, and those in the region of 3400 A gave 110 ell'ect (see Table IS). By these and other methods with a variety of indicators, tin- wave lengths of various radiations have been shown to be quite different, but all of them wen* below 2ft(M) A No records are Table 18. Irradiation of Onion Roots \\ith Monochromatic Spectral Light Efficient Wave Lengths according to (~JUKWlTRe.II RKITTCR and (\ \noit Wave Wave- Length A Relative Intensity Induction Length 1 A Relative Intensity Induction ^. i-_ i 2190 0.02 +24 | 3340 020 1 2350 2350 0.02 0.10 420 -}-30 i 3340 3340 0.40 2.00 i-5 2 2350 1.00 +25 3340 3340 80.00 O 800.00 1 3380 0.03 7 3380 4.00 i 8 3380 60.00 2 62 CHAPTER IV given of wave lengths below 1900 A. This is no proof that they do not exist, but rather, that below this point, absorption by quartz and air in the spectrograph makes aecurate determinations im- possible. It can hardly be doubted from the Lirge amount of speetra analyzed by the Russian school, and confirmed by WOLFF and KAK (p. 40), that mitogeiictio radiation consists essentially of the wave lengths between 1900 and 2500 A. Since 1928, the onion root as detector has been substituted by the time-saving yeast methods or by bacterial detectors. However, two extensive recent investigations must be mentioned which concern the question whether onion roots can be used at all as detectors. Both papers describe the technique employed very carefully, and they arrive at quite different conclusions. MOTSHEJEWA (1931, 1932) observed that roots when removed from the water show symmetrical distribution of mitoses, but after repeated removal, they do not. Pressing or rubbing of the roots will increase the number of mitoses, and after long continued pressing the opposite effect occurs. When friction and pressure were carefully avoided, no increase* of mitoses was observed upon exposure to another onion root. MOTSSEJKWA denies the existence of mitogenetic effects in onion roots and explains GUIIWITSCH'S consistent results by several assumptions: (1) One-sided pressure of curved roots in the glass tube. (2) Light applied repeatedly in centralization of roots which causes phototropic curving of the root and results in increased mitosis. (3) Selection of good roots for important experiments which results hi increased mitosis through pressure, and of less uniform roots when no effect is expected, which then leads to negative results. (4) Omission by GURWITSCH of the microtome sections showing a decreased number of mitoses when they happen to come between, sections showing an increased number. With yeast and blood as senders, this author obtained also negative results. This careful study has been considered by many critics to be the final proof against mitogcnetic radiation. Most of them do not mention the still more careful work by MAKUARETE PAUL (1933). Realizing the prompt reaction of roots to touching, PAUL fastened the small onions (hazelnut size) by means of gauze to perforated cork stoppers; these were held above the ground by simple stands; in a completely covered inoist chamber, in a dark METHODS OF OBSERVING BIOLOGICAL RADIATIONS 63 room at 20 C, the roots developed in the* air through the muslin in two to five days, while the leaves grew through the hole in the cork. The onions were never placed in water. When the roots were 1 2.5 cm. long and absolutely straight, one could be exposed to a root from another onion without being cut or even touched and without the need of glass tube holders. The exposed roots turn downwards, and a very careful investigation showed that the number of mitoses on the exposed part of the root was distinctly larger than on the opposite half, whether all mitotic stages were included or only the more conspicuous ones. The sender roots -were taken as controls, and they showed a uniform and symmetrical distribution of mitoses. Almost always, the exposed root grew more rapidly than the other roots of the same onion. When the sender root was substituted by a needle of stainless steel, the exposed root also turned downwards, but the microscopic analysis showed no increase in mitosis at the exposed side. However, the symmetrical distribution was disturbed. The object of PAIR'S investigation was the establishment of a good method for studying mitogenetie rays with onion roots. The number of examples given is not large enough to draw many more conclusions. The paper verifies GURWITHOII'S principal ex- periment, however, and it will be, the starting point for all future work with this type of detector. b) The yeast bud method BAIION (1920) suggested that the rate of bud formation of yeast could be used as indicator of mitogenetie radiation. In. his first experiments, he spread yeast over the surface of solidified nutrient agar containing glucose, allowed it to grow for from 9 to 15 hours at room temperature, and theii exposed it to the radiating source for definite short periods, usually not over 30 minutes. The yeast was then incubated for from 1 to 2 hours to permit the radiation effect to develop. After this, the yeast was spread on glass slides, dried and stained. The measure was the percentage of yeast cells showing buds. When, this percentage was higher in the irradiated culture than in unexposed controls, it was considered a proof of a raitogenetic effect. The original method has since been changed in some details by BAIION (1930) and GUBWITSCH (1932) (see p. 66). <>4 CHAPTER IV Table 19. Effect of Yeast, of Sarcoma, and of Hone Marrow upon the Budding Intensity of Yeast | Percentage of Buds in Yeast Sender Yeast Jensen Sarcoma Bone Marrow || Control 1 Exposed Control | Exposed Control | Kxposed Experiment No. 1 I 24 33 21 32 30 38 2 22 30 20 35 27 32 3 i- J3 25 20 27 ' 21 39 4|, U 25 20 26 28 38 5 I 24 33 28 35 29 39 i 23 33 30 38 20 35 7 25 3-1 25 35 23 37 8 1 27 35 25 36 22 34 9 i 25 3 25 35 27 36 10 ; 20 36 21 30 26 33 i! Average 1 22.3 32.0 23.5 32.9 25.3 36.1 The most extensive early data with this method have been published by SIEBBRT (1928a). Table 19 gives some of his ex- periments. The numbers indicate the percentages of yeast cells wit.li buds. In all his experiments, sender and detector were separated by a quartz plate. Tt is now customary to record, results as "induction' J effect, i.e., as increase in buds of the exposed yeast over the control, expressed in pereeiits of the control value. Thus, when the exposed yeast shows 33% buds, and the control, 24%, the increase is 9, and this is an increase of 37.5% over the control. The induction effect is 37.5%. __ 100 (exposed control) control SIEUEKT (1928b) used this method for a number of interesting studies in physiology. He observed the working, or excited muscle to radiate strongly while the resting, quiet muscle did not do this (Table 20). He attempted to produce radiation by changing chemically the pulp of resting muscle to that of working muscle. Finally, he succeeded by placing the acidified pulp in an oxygen atmosphere, since the addition of lactic acid alone would not produce radiation. Moreover, he obtained positive results in air by using a very dilute CuSO 4 solution as oxygen catalyst. When METHODS OF OBSERVING BIOLOGICAL RADIATIONS 65 Table 20. Effect of Electrically Excited and of Hosting Frog Muscle upon the Budding Intensity of Yeast Experiment Xo. Percentage of buds in yeast when ex- posed to muscle Experiment No. Percentage of buds in yeast when ex- posed to muscle Resting Kxcited Hosting Kxcited 1 32 45 13 23 41 LH 32 '! 14 22 33 3 23 3 15 23 32 4 2.3 3 1 l(> ! 21 30 r> 20 41 17 22 29 ti 22 30 18 22 32 i 22 31 19 22 32 s 22 2S i 20 23 30 9 2tt 35 21 25 33 10 2fi 31 22 25 33 11 12 2<> > 35 31 23 24 24 24 30 31 he finally found that JQQQTT KCN solution would prevent radiation, ho concluded that the source of radiation must be chemical. Thus started the first experiments about chemical relictions as the source of radiant energy (p. 33). ftiEBERT later (1930) concentrated his attention upon blood radiation. He verified the statement of LYDFA (lirjtwiTSOH and &ALKIND (1929) that blood of normal, healthy people radiated distinctly, while that of cancer patients did not. He found, further, that urine, radiated, and that there was a good parallelism between blood and urine radiation. Of 35 patients with cancer, the majority showed no radiation of blood or urine. The exceptions were patients after recent treatment with X-rays and isarnine blue. Anemia, luucemia, high fever (sepsis, pneumonia, scarlatina) prevented radiation of blood, as well as of urine. With syphilis, radiation varied, but blood and urine wont parallel. None of the other diseases tested caused loss of blood radiation (details see p. 153). The experiments on the metamorphosis of amphibia and insects (p. 167), and on the healing of wounds in animals (p. 173), by BLACKER and his associates, were all carried out by the yeast Protoplasma-Monoffraphien IX: Rahn 5 66 CHAPTER IV bud method. The importance of the yeast agar blocks in the establishment of biological spectra lias already been mentioned on p. 35. This method is very commonly used in mitogenetic investigations at the present time. Method: GntwiTscH gives the following directions for the yeast bud method (1932, p. 7): Beer wort agar plates are flooded with a very fine suspension of yeast in beer wort, the liquid is distributed evenly over the agar surface by careful tilting, and the surplus liquid is drawn off with a pipette. After about 5 to 6 hours, the surface is covered with a fine, delicate film of yeast, and is now sensitive, and remains so until about the twelfth hour. Neither the temperature nor the concentration oi the yeast suspension nor its ago is mentioned. The temperature is probably room temperature, and it should bo kept in mind here that on p. 14, Gunwrrsoii mentions 12 (j as room temperature. The directions are probably meant primarily for the yeast Xadsonia fulvesecus which has been used most commonly by the Russian workers, though beer and wine yeasts are occasionally men- tioned. in order to give 1 a better conception of the proper physiological con- dition of the detector plate, we quote from the same hook of GimwiTsr,n\y p. 317: "The most appropriate stage of the detector plate corresponds to a thickness of the yeast growth of about 25 to 30 layers of cells . . . (p. 31 H). \Vocan be certain that the lowest layers of cells which arc in immediate contact with the nutrient medium consist essentially of young cells in rapid multipli- cation . . . The cells of the middle layers arc not in optimal condition, and are no more capable of developing spontaneously the maximal energy for development, which was found in the lowest layers ... It can hardly be far from the truth to deny any appreciable multiplication in the topmost layers. However, they arc not real resting forms as yet." The method of making smears to count the buds is not given in GTK- WITSCII'S book in any detail. It consists simply in smearing the yeast cells on a glass slide, drying and staining them. Only those buds are counted which arc smaller than half of the full-grown cell. Some authors limit their counts to even smaller buds. CUTCWITSCH recommends that the 1 person counting the buds should not know which slide or experiment he has under the microscope; this prevents subconscious arbitrary decisions. Attention should be called here to a leaflet published by Jng. G. TKRZANO & C., Milano, manufacturers of the "hemo- radiometer" of Protti's. All conditions are quite precisely standard- ized, and this may account for the good results of Italian in- vestigators. This method has been varied by other authors. TUTHJLI^ and RAHX (1933) studied the mode of bud formation of Burgundy METHODS OF OBSERVING BIOLOGICAL RADIATIONS 67 yeast on raisin agar at 30 C. A typical result including all sizes of buds is shown in fig. 32. The culture immediately after being transferred contains but very few buds, and new buds are not formed at once. The old yeast ceils which had ceased to multiply require some lime before their reproductive mechanism is working normally. During this rejuvenation process, commonly called the lag phase, cells" respond most promptly to mitogenetie rays. It is also seen that they soon reach a maximum percentage of buds. ]f, at this latter stage, the rate of cell division were accelerated by mitogenetie rays, it could not increase the per- centage of buds (see also p. 69). The most opportune time for using such a plate as detector is evidently about an hour or two before bud formation begins. If such a detector is exposed for 30 minutes, and then incubated for one hour (so that the mito- genetie elleet might manifest itself by an increase in buds) we should have bud formation beginning on the exposed plate while the control has not yet become quite ready for budding. The "lag period", i.e. the time interval between the seeding of the plate and the first active bud formation, depends upon the age of the culture used for the seeding. With the Burgundy veast employed by TimnLL and liAHN, a plate seeded with a 24 hours old culture was a good detector immediately after seeding When a (> days old culture was used, it was advisable to incubate the plate for about 2 hours before exposure (woe Table 21), or to incubate for several hours after exposure if exposure had taken place immediately after seeding. In liquid cultures, the old yeast cells retained their buds for many days while old cultures on agar surface lost them readily. Since a low initial percentage of buds is very desirable, inoculation with yeast from agar surface cultures is recommended. This detector is really quite different from BARON'S or GUK- WITSCH'S described above. All yeast cells are at the same stage Figure 32. The development of buds in an agar surface culture of wine yeast. 68 CHAPTER IV Table 21. Kffeut of Irradiating Yeast Surface Cultures after Incubation for Different Times Length of Exposure : 30 minutes Incubation after Exposure: 30 minutes Age of culture hrs 10.5 hrs; 1 hr when exposed 1.5 hrs: 2 hrs [2, ,5 hrs Percentage of Buds i| exposed area 25.0 i 29.2 ! 4.0 | 4.2 , 9.8 10.1 control area 3.0 3.5 4.4 4.0 9.5 18.4 increase 21.4 25.7 ; 0.2 0.4 1 0.3 -2.0 1 I nduction effect V.^.O 724.0 5.0 -1.0 4.0 1 1.0 i 1 exposed area 7.0 0.0 ; 8.5 22.5 i 20.2 44.4 ! control area 0.0 7.0 7.0 19.0 17.0 27.8 j increase 1.0 -1.0 0.9 3.2 U.2 16.0 } Induction effect 17.0 17.0 12.0 17.0 54.0 <>2.0 .Age of Parent I 1 ( Culture ! 24 hours . 6 days . of earliest rejuvenation when exposed, and the cells are far enough apart not to influence each other. The "mitogeiictic effect" is much greater than in the other method because there are no old, inactive cells to "dilute'' the counts. In fact, these are probably the largest mitogeiietie effects ever recorded. It is quite per- missable to count all buds because the percentage at the beginning is very low, and a limitation in size is not necessary. This should make the counting easier. On the other hand, this type of detector is so different from the BAKON type that it may react differently in certain experi- ments. As long as they are used merely as detectors to prove the existence of radiations, both types are good. Probably, with very weak radiations, the BARO.N type is more sensitive because the old cells act as "amplifiers" (see p. 127). Method by TUTIIILL and KAHN (designed for Burgundy yeast): The yeast is kept throughout the experiment at 30 C. A 24 hours old culture in raisin extract 1 ) is flooded over a solidified, sterile raisin agar l ) Raisin extract: 1 pound of chopped, or seeded raisins is heated with 1 liter of water in steam for 45 minutes., the extract is pressed off, made up to 1 liter, 5g. KH 2 PO 4 and 5 g. yeast extract (or meat extract) are added; the resulting medium is sterilized at 100 (\ The pH is about 4 to t.5. Raisin agar: Melted 6% water agar is mixed with an equal volume of the above raisin extract and sterilized by heating for 20 minutes at METHODS OF OBSERVING BIOLOGICAL RADIATIONS 69 plate; the surplus liquid is poured off, and the culture permitted to develop lor 24 hours. This surface growth is washed off with 5 cc. of sterile water, the suspension is then diluted 1 : 100 with sterile water, and with this dilution, some sterile solidified plates of raisin agar are Hooded, the surplus liquid is drained off at once, and the plates should be exposed within half an hour. Half of the plate should be shaded to servo as control. The length of exposure will depend upon the intensity of the sender: 30 minutes proved a good time with young yeast cultures. One to two hours incubation, counted from the beginning of the exposure, was the most suitable time to bring out the differences. In these plates, the yeast cells are so far apart that the buds can be counted directly on the agar surface. The organisms art* killed by placing a cotton wad with tincture of iodine in the Petri dish. Soon after that, a coverglass can be placed on the agar surface, and the slightly-stained yeast is observed in situ, eliminating all possibility of breaking oil buds by smearing on glass. Tn all methods where growing cells in glass or quartz containers are used as detectors, radiation from these growing cells may be re- flected by the glass or quartz walls, and may thus produce radiation effects in controls as w*! 1 as m the exposed cultures. Protection against reflection is advisable in all such cases (see p. SO). LKJ uid cultures have also been used successfully. Here, too, the rule applies, that increases in the bud percentage can be expected only during the lag phase (see fig. 32). When all cells are out of tho lag period, and produce buds at a constant rate, the percentage of buds cannot be changed by a change in the growth rate. Since this has been overlooked by some experi- menters, e.g. by RICHARDS and TAYLOK (1932), it may be ad- visable to explain this important point in more detail. Let us, for this discussion, distinguish five equal periods in the complete coll division of the yeast, 4 with buds and one without. Fig. 33 shows a first approximation of a ''cross section" through a yeast population growing at a constant rate. It requires 5 time units for each cell to complete the cycle, i.e. to produce two cells of the same developmental stage. The percentage of buds is not constant, but fluctuates between 67 and 80%. This fluctuation is due partly to the arbitrary selection of 5 stages, but mostly to an error in tho cross section. In a growing culture, there must 100 C. On account of the high acidity, the agar becomes hydrolyzed under pressure, and foils to solidify; the same is true with prolonged heating at 100. 70 CHAPTER TV necessarily be more cells of the young stages than of the old. If we take all (jells of the first 5 units as a more appropriate cross flection, we obtain the following picture: Yeast Culture at a Constant Growth Rate Time units I 9 no buds 9 tiny 9 small 9 medium 9 large 18 no buds 18 tinv 8 tiny 8 small 8 medium 8 large 16 no buds J6 tiny 10 small 7 small 7 medium 7 large 14 no buds 14 tiny 14 .small Hmedim 6 medium 6 large 12 no buds 12 tiny 12 small 12 medium 12 larjro 5 large 10 no budsj 10 tiny 10 small 10 medium 10 large 20 no hud with bads. 26 ' 30 34 39 45 52 60 without . . . 9 10 12 14 16 18 20 Total 35 40 46 53 1 01 70 80 i / buds. . , 74.4 /. 176.0% '73.9% 73.7 % 73.9 % 74.4% 75.0% There is a fluctuation of only about 1/ . This percentage depends only upon the variety of yeast used. Whether it grows rapidly or slowly, whether the time unit is 30 to 00 minutes (at cellar temperatures) or 10 minutes (under optimal conditions), the percentage of buds remains 74 75%. A change of the growth rate would not affect the bud percentage at all. However, a change would become noticeable if only one certain stage of the cycle should be accelerated. If niitogenotic radiation should speed up the very first stage so much that the "tiny" buds never appeared, the number of buds at the period would drop from 26" out of 35 to 18 out of 27, or from 74.4 to 66.8%, and this lower level would continue as long as radiation accelerated the one particular stage. It is rather probable that only a certain stage of the cell cycle is affected by mite-genetic rays. Many observations suggest this, and our present conceptions of the mechanism of cell division do not contradict it. This would offer a good explanation for the "false mitogenetic depression" of (rUBWiTScn (1932, p. 210) and especially of SALKTND (1933) where the percentage of buds decreases while the total cell count increases under the stimulation of mitogenetic radiation. The reliability of the BAKIXN method has been doubted by NAKAI DZUMI and HCHKEIBKK (1931) who claimed to have followed BARON'S method explicitly. However, they have kept their de- tector cultures for 9 12 hours at 25 C while BARON used room METHODS OF OBSERVING BTOUMJICAL RADIATIONS 71 temperature which may be as low as 13 C in .Russia. That their cultures were far too old, can be seen from the fact that in most of their experiments, the percentage 1 of buds decreased distinctly in 2.5 to VII 1.4 7.9 2.9 28 VIII 2.3 10.9 4.1) 35 1929 TabU I 1.1 13.4 2.() 67 The increases resulting from irradiation are very much larger than, the error, regardless by what method it is computed. 72 CHAPTER IV c) Detection by increase in cell number To the investigator not familiar with yeasts, it may seem that there should be no essential difference between the total number of cells or the percentage of buds as a measure of t he- growth rate. For this reason, the preceding pages were written. There is a difference so fundamental that in a number of ex- periments, the yeast bud method indicates a decrease in the growth rate while the total cell count shows an increase (e. g. Table, 34 p. 116). The number of cells is the best measure of the growth rate, better than any other of the* methods described in this book, because it ascertains cell divisions directly while the mitoses in onion roots, the buds of yeast cells, the respiration, etc. are indirect measurements of growth, and may be influenced by secondary effects. This method is used with yeasts as well as with bacteria. While they are identical in principle, they differ in the method of measuring since yeasts arc so much larger than bacteria; they shall bo treated separately in this chapter on Methods. (1) Yeasts: The number of yeast cells in a liquid may be ascertained by plate count, by hemacytometer count, by measuring the volume of all cells, or by comparing the turbidity by means of a nephelometer. The plate count method has rarely been used. It is the same as that used for counting bacteria except that more preferable media are beer wort agar or raisin agar. The hemacytometer has been used by the Russian workers as well as by HEINKMANN (1932) in his studies on blood radiation. HEINEMYNN'S method for testing the radiation of blood consists essentially in thr exposure of a 12-houi culture of beer yeast (temperature not mentioned) in liquid beer wort, to the radiation of blood diluted \\itli an equal amount of a 4% MgS() 4 solution to prevent coagulation. The exposure lasts 5 minutes, and it is made discontinuous by moving some object between blood and yeast at intervals of 2 seconds (see p. 103). 0.5 ee. of the exposed yeast suspension is then mixed with an equal amount of beer wort and incubated for three hours at 24 C. The total number of yeast cells (counting each smallest bud as an individual) is determined with a hemacytometer at the start, and again after three hours' incubation, both in the control and in the exposed culture. All cultures are preserved for counting by the addition of H a SO 4 . The control is counted twice. An example 1 of the results and of the method of calculation is given in Table 22. METHODS OF OBSERVING BIOLOGICAL RADIATIONS 73 Table 22. Yeast Cells Counted in Homaeytometer be/ore and after Irradiation by Blood 1 i 1 stait' Control aft or 3 hours Irrad- iated ! Incro Control ISO Of Irrad- , iated ,,lnduktion" lOO(Irr.-Con) 1 actual increase in growth rate ' a b i /. 0/ , Uontrol /0 ii Healthy person . 41.5 62 <>3.0 84 1 4 1 102 ; f-108 82 Carcinoma patient iti.O 08 GK.f> 56 48 22 | -54 42 Chronic tonsilitis . 29.0 48 48.5 49 , (i ' r > 65 1 HEIJSJKMANJN[ emphasizes t-liat this method depends upon the physiological condition of the yeast, and that no effect can be expected when the- control grows too rapidly, i. e. when the control increases to more than double* during the incubation, period. This means, in othor words, that the yeast must he in lag phase, else it would double in less than ,*J hours. To avoid errors from this source, he tested each blood sample with two different yeast strains The results by this method verified all former experiences with blood radiation, especially in regard to cancer patients, an will be shown in Chapter VI 1. He also added somo important new facts regarding radiation of the blood of old people and of patients with chronic tonsilitis. The measurement of the growth rate of yeast by cell volume has been studied in detail by LITAS (1924). The accuracy is not greater than with plate counts, and the curves and data published by this author show so little deviation only because they are presented in logarithms instead of actual numbers. However, the volume is sufficiently accurate to prove mitogenetic radiation, and the method requires less time and less eye strain than the hernacytometer method, also giving quicker results than the plating method. BUAINKSS (quoted from Ontwrrscu, 1932, p. 17) has adapted the method for the small vnhimina available 1 in mitogenetic work. Volumetric Method: KALUNDAROFF (1932) used beer yeast in a strong wort (18 22 Balling); only cultures ]5 20 hours old (temperature not given) which arc actively fermenting, are suitable as detectors. After exposure, the yeast is distributed evenly, and a definite} amount of the exposed culture is measured by means of a micropipette, e. g. 0.2 ec. This is added to 1 ee. of fresh wort, and incubated for 4 hours at 2s C. The 74 CHAPTER IV Table 23. Height of yeast column of centrifuged yeast cultures, exposed to the various spectral regions of the radiation produced by gastrie digestion of serum albumin, and of their controls \Vave Length |l mm ( ij eol j| control >f yeast nnii exposed Induction Effort Wave Length !' mm o eol control 'i f yeast mm exposed Induction Effect 2320 1 15 15 i 23(>0 IS 17 2330 | I* ir> O 2370 17 IS 15 15 O , i 14 14 2330 1 20 23 lf> 2380 | 12 l<> 33 23f>0 ! 20 ! 20 24 25 20 25 2390 9 i 1 9 12 12 33 33 i 20 20 30 ; 20 2. r > 25 20 20 30 2390 : 15 JS 20 2400 15 20 33 23f>0 - I 10 ID 21 2(> 24 23(10 ; ' ID O 20 2J 20 yeast cells are killed l>y adding O.2 cc. of 20% ir 2( SO 4 , and arc centrifuged in pij>ettes commonly used for measuring the volume of blood corpuscles (the illustrations of the Russian workers appear to be VAN ALLEN hematocrit tubes). The yeast column of the exposed sample is compared uith that of the control. 'Fable 23 shows some results obtained with tins method by BILLH;, KANJVEUIESSEK and HOLOWJEFF (1032) who used it in the determination of the spectrum of gastric digestion. A still more rapid method for estimating the amount of growth is the measurement of the turbidity of the culture by means of a nephelometer. This method lias been used occasion- ally by bacteriologists for several decades; the methods are reviewed and analyzed by STRAUSS (1929). The nephelometer can be used for bacteria as well as for yeasts, while the cell volume of bacteria is too small to be measured with sufficient accuracy in the earlier stages of growth. Attention may be called to the description of a simple nephelo meter by RICHARDS and JAHN (1933). More complicated is the differential photoelectric, nephelo- meter described by (U'RWTTsriT (1932, p. 17). METHODS OF OBSERVING BIOLOGICAL RADIATIONS 75 Table 24. Bacillus mescntericus Irradiated Continuously by Yeast at 12 mm. .Distance, through Quartz Time (Jells per on bio millimeter start ontrol irradialed Effect 2 lira 3108 11 410 18290 2.5 528 , 7 920 8 970 3 4048 12490 10 010 3.5,, i 1584 105)12 13552 00 13 ; 28 24 Generation Times r |'j mo i control irradiated niin. inin. 1 2 hrs 63.7 40.8 2.5 , 38.1 37.7 3 : 110.8 90.7 3.5 i 75.5 07.8 increase 30 o 22 II Another way of estimating the amount of growth in \east cultures has been suggested by BARON (1930) who compared the size of yeast colonies in hanging drops. This method has been slightly modified by BORODIN ( 1 934) who photographed the colonies and measured their area with a planimeter. (2) Bacteria: The stimulation of bacterial growth by mitogeiietic radiation had already been observed by BARON (192(>) and by SKWTCRTZOWA in 1929. Table 24 gives some of the results obtained by the latter. The data were verified by Ars (1931), who irradiated liquid cultures of Kacillns murvmors with agar cultures, either of the same species, or of yeast, and found that the effect by "miito-induetion", i e , by the same species, was the greater. BARON and Ars did not recommend the growth rate of bacteria as a universal indicator for mitogeiietic radiation. This was done most successfully by WOLFF and K\s (1931) These authors worked with different species, and the number of cells was determined by the customary method of bacteriological technique, the agar plate count Instead of making dilutions in water, they took their samples with a WKKJHT pipette which 76 CHAPTER IV delivers - of a cc. using the slide cell method. FERGUSON and ^oU i KAHN (1933) obtained good results with the ^ cc. pipettes used in tlie standard (Breed) method for the mieroseopie count of bacteria in milk. The plating of such minute quantities is necessary because, only very small amounts of the culture (about 1 cc.) can be, exposed, 011 account of the strong absorption of ultra-violet light by the customary bacteriological media. WOLFF and RAS (1931) allowed that a layer of standard nutrient broth 0.5mm. thick transmitted only rays above 2500 A; if broth is diluted with 9 parts of water, a layer of 1 mm. still transmits some rays as low as 2200 A. WOLFF and HAS irradiated their bacteria in standard broth in a layer of 0.6mm.; even then part of the bacteria \\ere shaded. FwimrsoN and KAHJS (1933) verified this observation. I cc. of a standard broth culture 1 of Bacterium coli in a quartz dish in a layer of (U> mm. irradiated from below showed no increase over the control, while a culture in broth diluted 1 : 10 showed a good mitogenetic effect. WOI/FF and HAS pointed out that only during the lag phase, definite results could be obtained. During rapid growth, there was no effect. A most interesting observation was the, exhaustion of bacteria by continued irradiation, resulting in a decreased growth rate. The two experiments in Table 25 show a lag period of about 2 hours in the control Irradiation decreases this period very distinctly, and with increasing intensity, or decreasing distance, the lag becomes shorter and shorter. However, continued irra- diation after the lag phase retards the growth for some time, and at 5 hours, the control shows more cells per cc, than any of the cultures whose growth was distinctly stimulated. This retarda- tion of growth is only temporary ; most of the irradiated cultures almost doubled their number during the 5th hour, indicating a return to the normal growth rate (fig. 34). ATetliod (the most recent method by WOLFF and HAS, 1933a). A fresh suspension of staphylococci in broth, with about 20 000 cells per cc., is placed in a glass dish in a very thin layer, covered with quartz, and exposed (e. g. to milk \- rennet in a quart/ tube). After exposure, the dish with the bacteria is incubated at 37 (' for 1/5 to 30 minutes; so is the control. Hy means of a capillary pipette, samples of the exposed culture and control are either plated on ugar, or brought into "slide cells" according to WREGHT. METHODS OF OBSERVING BIOLOGICAL RADIATIONS 77 Table 25. The Effect of Different Intensities of Coiitinous Radiation through Quartz upon the Rate of Crowlli of Niaphy- IOMCCHS aweus Sender: Agar surface culture, of Ntapliylocoerits nitrcim, at various distances Distance* between Cecils per cubic ccntimetei sender and detector ! ,, . , Control 12.5 cm. [ 5 cm. 2 cm. ' start 31 200 31 200 31 200 31 200 Experiment r after 1 hour 31 400 32 200 ;w 700 :o 200 1 j ,,2 hours 32 100 55 700 54000 48 800 !i 3 .. 45 000 57 000 51 700 40 800 1 - 4 ,. 133000 128 500 117000 51 200 j! r > 202 000 237 000 135000 79 200 ! start 14 700 1 1 700 14 700 14700 Experiment . ,, I hour 14050 14 200 10 400 24 500 1 L |, ,, 2 hours 15 100 10 000 32 400 20 250 ] 3 ,. 17 100 28 700 32 700 22500 i ., 4 ; 50700 80 800 40 700 21 400 j! * I2300O 108 500 70300 44 100 The umrradiated controls never show oro\\th in this shojt time A\hile the irradiated cells do. WOLFF and HAS consider over-exposure the most common cause of failure; over-exposure either produces no effect at all, or eventually decrease of growth rate (see p. 115). ItSMO Figure 34. Developpemcait of two liquid staphylococcus cultures of which one was exposed continuously to the radiation from a staphylococcus agar plate. 78 CHAPTER IV The error of the method is mentioned in 1934; four detailed series are given, the counts being 73.1 ^ 3.6; 1)1.2 ^ 3.3; 133.8 : 4- 4.5 and 171.1 i 7.3. The errors are between 3.4 and 4.9% of the total count; the increase by irradiation amounted to 25% and 28%. Method: FEUGUSON and RAHN (1933) studied the best conditions for a good mitogenetic effect with bacterium cult, and found that it depended primarily upon the age of the culture, and tho transmission of ultraviolet by the medium. 24 hours old cultures never reacted; cultures 4K hours old or still older always responded. The best medium was 1 part standard broth plus 9 parts water. Tho simplest procedure is to irradiate 1 ee. of an old culture in dilute broth (either in a quartz dish of 4 5 em. diameter, through the bottom, or in a quart /-covered glass dish, from abo\c), dilute this 1 cc. after exposure 1 : 10000 with dilute broth, incubate, and plate at least every 2 hours for 6 to . The induction effect calculated from the numbers directly is in one cast 1 (]5 minutes exposure) 183, while the effect in tho same culture, computed from the generation time, is only 27. However, it must be considered that the number 183 has no real biological significance while the other indicates that during the four hours, this culture had a growth rate 27% higher than the average of the two controls. METHODS OF OBSERVING BIOLOGICAL RADIATIONS 79 Table 26. 3 days old culture of Bacterium cult , irradiated for various lengths of time by an agar surface culture of the same bacterium (The numbers are cells per cc. after diluting the irradiated cultures 1 : 10 000 with broth) 1 ( Control Duration of Irradiation Control No. 1 60 min. 30 min. 15 mm. 7.5 min. No. 2 start 5 050 5 (>5< > 800 7 250 250 (5 550 after 2 hours . . . r> 700 5 HOO 7 950 7750 5 850 7000 ,,3 11 000 14350 14 400 8 300 8 500 ,,4 2',} 7f)0 24 250 29 000 35 350 19 150 19550 1 88 000 223 000 434000 4f>7 000 139000 137000 Induction Effect 37 168 I83| Generation Times, 111 minutes, for the time interval from 2 --6 hours Generation Times Induction Effect . 47.6 45.5 ! 41.5 14 40.8 f-27 52.5 I This procedure has also been used in Table. 24. It could be used with yeasts as well, e. g. in Table 22. However, the error becomes very large if the increase is small. In the tables given by iScuitEJBKR (1933), the generation time of yeast for the first 2 hours when the mitogenetic effect is strongest is mostly more than 2 hours. This means that not all cells had divided in this time. Though HCHK.EIIJER does not give the actual numbers of cells from which he calculated the generation times, it seems from his curves that they were computed from less than 100 cells. This makes the error very large 1 , and a comparison of the growth rates must necessarily result in enormous percentual differences. Thus, SCHKKIBEII found the variations in duplicate plates of tiaee/Miromycefi cMipxoirfcuti commonly to read) -40%, and occasion- ally more, and in one case even 103%. With Nadxonia, the deviation of duplicates went as high as 237%. With such a large error, there is little hope of detecting mitogenetic effects. The "Induction Kffect" as usually calculated (p. 64) has no definite meaning. It permits no comparison, with the probable error of the method. It is very unfortunate, therefore, that many investigators, especially the Russian scientists, record, the 80 CHAPTER IV obtained effects merely by giving the "Induction Effect". The critics point out very justly that such relative numbers are not convincing. It would add a great deal to the general recognition of biological radiation if the actual data obtained (numbers of cells, of mitoses, percentage of buds etc.) were given for the exposed culture, the control and also for the same culture before the beginning of the experiment. d) Detection by cell division in larger organisms The three detectors mentioned above are the only ones that have been commonly used to prove the existence of mitogenetic radiation, a few others have been employed occasionally, but not often. Mention is made of the reaction of mold spores upon mito- genetic rays. The first publication of actual effects is probably that by SCHOUTEN (1933). WOLFF and HAS (1933 c) mention that they react slowly and require about ten times as long an exposure as staphylococcus cultures. FERGUSON and RASN, in some unpublished experiments, observed that the radiation of the detector culture may be reflected, and may thus give a mitogenetic effect even in the controls which received no radiation from outside. Spores of Aspergillus niger were spread on an agar surface. When the dish was covered with a glass cover or a quartz plafc, germinat- ion was more rapid than when the cover consisted of black paper or sterile agar. The effect varies in magnitude, and is not always present, but must be guarded against in this tech- nique and probably in most others. This reflection may be the cause of many failures to observe mitogenetic rays. The strong effect can be explained by polarisation of the rays through reflection (see p. 45). Percentage of germinating mold spores Exper. No. Reflecting surface Non-reflecting surface Glass Quartz Agar 1 Black paper 13 36.0 35.0 19.8 19.6 14 33.3 35.1 14.5 23.3 15 42.8 39.0 22.4 35.2 16 33.2 37.4 17 45.8 18.4 14.8 METHODS OF OBSERVING BIOLOGICAL RADIATIONS 81 In the animal kingdom, the eggs of the smaller animals have been used occasionally to demonstrate the mitogenotic effect. BEITER and GABOB (1928) showed that frog eggs when irradiated with the spectral line 3340 A developped more rapidly into tad- poles than the controls. Too long an exposure retarded the de- velopment. The wave length is unusual as in all publications by REITEK and GABOB (see p. 60). In a short paper, WOLFF and RAS (1934b) showed that eggs of the fruit fly Drosophila melanogaster hatch more rapidly after having been exposed to the radiation from bacterial cultures (see also p. 144). The eggs of sea urchins were found to be quite good detectors. The rate with which they divide, can be easily seen under the microscope, and the percentage of eggs in each of the different stages is a good indication of the growth rate. SALKTND, POTOZKX" and ZoGLfNA (1930) were the first to show that biological radiation from growing yeast or contracting muscle will increase the rate of development of the eggs. The Italian school of mitogcnetioists has also used sea urchins repeatedly. Not all species are equally well adapted as detectors, some being much more sensitive than others. Table 27 shows some data by ZIBPOLO (1930). Table 27. Mitogenetic Effect upon the Eggs of the Sea Urchin Paracentrotus lividus Percentage of Eggs Sender Control Irradiated Bacillus Pierantonh unchan. 82.6 2 blast. 4 blast. unchan. 10.7 2 blast. 87.6 4 blast,. 1.7 13.9 3.5 > 74.5 25.5 35.0 65.0 70.0 30.0 8.4 88.4 3.2 Penieillium (in dark) (diffuse light) 97.8 70.4 2.2 25.7 3.9 7.9 91.5 0.0 The morphological changes of the larvae brought about by irradiation of sea urchin eggs will be discussed later (p. 164) since a different principle is involved. Tissue cultures would appear to be an interesting subject for the study of this radiation. The first investigation was started without the knowledge of GUBWITSCH'S discovery. Protoplasma-Monographien IX: Rahn 6 82 CHAPTER IV GUILLEEY (1928) observed during some experiments on the growth -promoting agents for tissue cultures, that two or three cultures in the same dish influenced one another. While the most rapidly-growing culture usually maintained its growth rate, that of the more slowly-growing ones was distinctly increased. Fig. 35 shows the relative daily increase of three cultures from the heart of chicken embryos, which were transplanted at different ages of the embryo, and therefore had different characteristic growth rates. These remained constant as long as the cultures 6 8 10 12 ft days Figure 35. Daily growth increments of three cultures of chicken embryo, grown separately for 8 days, then united in the same dish. were grown in separate dishes, but were changed when all throo were continued in the same dish. Incisions in the solid medium which separated the cultures and prevented diffusion from one to the other did not prevent the mutual stimulation. Even when a strip of solid medium was completely removed between two cultures, the effect sometimes continued. When a glass slide was used to separate the cultures, the influence ceased, even if the slide did not touch the bottom, and permitted diffusion. Further experiments showed that the effect spreads re ctili nearly ; by placing several cultures in the same dish, and inserting small glass strips between some of them, the effect of the glass was found to be that of shading. This can be explained only as radiant energy which stimulates growth. It could also be shown that the radiation was reflected from metal mirrors. These observations suggested to GUILLEBY the possibility that the embryo extract which is necessary for the growth of tissue cultures, acts essentially as a source of radiation. He irradiated a number of cultures in a dish, from one side, with embryo extract, but the result was negative. However, with a METHODS OF OBSERVING BIOLOGICAL RADIATIONS 83 head of a chicken embryo, ho obtained in all 4 experiments a more rapid growth in the culture nearest to the radiating substance. The second paper on this subject was that of CHRUSTSCHOFF (1930) who observed that growing tissue cultures began to radiate some little time after transplantation from the original tissue. With a spleen culture of Amhystoma tigrinum, radiation began after 60 hours ; with a fibro blast culture from the heart of a chicken embryo, after 12 hours. CHRUSTSCHOFF believes that radiation is due to autolysis of nccrotic parts in the culture. Next, CHRTTSTSCHOFF used the tissue culture as detector. A fibroblast culture (chicken) was divided into halves, both were cidtivated in separate drops on the same quartz cover glass, and one of the two was irradiated for 48 hours by a beating embryo heart, which was replaced when necessary. At this time, the irradiated culture appeared much denser than the control; after 3 days (the last day being without radiation), the exposed culture was far in advance of the control. JAEGER (1930) observed that blood radiation retarded the growth of tissue cultures. Here, as with all other detectors, some investigators obtained negative results. LASNITZKI and KLEE- RAWIDOWICZ (1931) as well as DOLJANSKI (1932) could find no stimulation by mitogciictic radiation. DOLJANSKI used cultures which reacted promptly upon addition of embryo extract, but they did not respond at all to organic radiations, even if tbo distance was only % mm. Very recently, JULIUS (1935) obtained definite growth stimulation of chick fibroblast cultures, but only on poor media. By placing one half of a culture on glass, the other half on quartz, and exposing both to radiation from stapbylococcus culture, those on quartz grew more rapidly as measured by means of a planimcter. The induction effect was recorded as the ratio of increase in the quartz culture over that in the glass culture. In 56 such pairs, the average effect was 1.92 0.15. Another set of 48 parrs, but without irradiation, gave the ratio 1.01 _j_ o.o^ proving no chemical effect from glass or quartz. The actual stimulation by radiation from bacteria was therefore 0.91 0.17, the effect being 5.3 times the probable error. It may be that a certain stage of development is necessary to make the cells sensitive to the mitogenetic stimulus, as was shown for yeast and bacterial cultures (pp. 61) and 120). 6* 84 CHAFTER IV Table 28. Mitogenetic Effects produced in the Oorneal Epithelium of vertebrates by 3 4 minutes exposure of the left eye to the spectral line 2030 A Number of Mitoses Triton Frog Rat left eye exposed 54 right eye control % incr. left eye exposed right eye control o/ /o incr. left eye- exposed right eye control 1944 o/ /o incr 7fc 27 100 277 108 158 3086 134 66 100 3200 2070 50 3312 2050 6( 130 50 53 51 30 38 150 08 40 615 695 885 333 205 221 85 240 300 1644 118*7 4( 440 1 ) 2593 1 ) 196 1578 12J 7( A very good detector is the corncal epithelium of verte- brates, according to LYDIA GURWITSCH and ANIKIN (1928). It is the only easily accessible tissue of the grown animal showing frequent mitosis. The number of mitoses varies with the physio- logical state ; it increases rapidly with good nourishment, dropping in rats during starvation from approximately 2000 to about 50. Fortunately, the two corneae of the same animal always show very nearly the same number of mitoses, so that one eye can be irradiated, and the other used as control. Method: For physical light sources, an exposure of 3 4 minutes was sufficient, the animal's head being held in the hands of the experimcntor. With biological sources, exposure had to be continued for 20 minutes, which necessitated the tying down of the head into an immovable position. Ordi- narily, after irradiation, 3 4 hours time was given for the manifestation of the effect. The cornea was fixed for 40 minutes in 70% alcohol 4 5% acetic acid, stained with hamalaun, and clarified in glyeerol. The results in Table 28 show very strong mitogenetic effects. e) Detection by changes in yeast metabolism G-ESENius (1930 a) concluded that such decisive changes as the acceleration of the growth rate of yeast must be accompanied, or perhaps preceded by changes in metabolism. He studied, therefore, the influence of irradiation upon the rate of respiration and of fermentation of yeast. The technique employed was *) The source of radiation was a yeast culture. METHODS OF OBSERVING BIOLOGICAL RADIATIONS 85 essentially that of WABBUKO (1923), or of RUNNSTROM (1928) for measuring respiration of tissues or tissue pulps. The organism used as detector was a wine yeast, which was exposed in quartz- bottomed dishes to yeast radiation for 4 hours before being tested. The result was a stimulation of fermentation, but a retardation of the oxygen uptake. The results can be briefly summarized in the following way: \ f68 increase Fermentation in N-CO, I ]02 cxporlmelltg 4 t 102 experiments < 4 decrease atmosphere J | 3 Respiration (oxygeii-up- j |40 decrease take) in 2 atmosphere, > 54 experiments [ 1 increase without sugar J [l3 within t he limits of error The number of yeast cells in these tests was very large, 7 to 10 billion cells per cc. This is 10 to 100 times the maximal population which can develop in the medium used. The mutual irradiation of the cells must play an important role in thew experiments, and probably accounts for the depression of respiration. GESENIUS tried further the iniluencr of radiation upon macerated yeast, free from cells, i. e. upon zymase. While yeast radiation produced no effect, blood radiation decreased the fermentation in 28 out of 30 experiments, the average depression being 14%. The same retardation of respiration also could be obtained with sea urchin eggs during the early stages of cell division. GESENIUS (1930b) applied this test, after the improvement of the technique, to blood radiation and found that normal blood always radiated 1 ). He observed further that from patients with most diseases, the blood radiated, and that the consistent ex- ceptions were only with cases of pernicious anemia, leucemia, carcinoma and severe sepsis (see fig. 40 p. 153). His results agree very well with those of L. GLTEWITSCH and SALKIND and of SIEBERT. The role in cancer diagnosis of this loss of radiation will be discussed in Chapter VII. *) "Healthy blood never fails. If a failure occurs, it is time to test either the yeast or the apparatus." (GESENIUS, 1932.) 86 CHAPTER IV f) Morphological changes by biological radiation Quite different from the previously described manifestations is a decided morphological change in the irradiated organisms. The first observations of this kind were those by J. and M. MAGKOU (1928) who exposed the eggs of the sea urchin Paracentrotus lividus to radiation from bacteria, yeasts or other organisms, and even from chemical reactions. They obtained quite abnormal, more or less spherical larvae while the normal larvae possess a very characteristic conical form (see fig. 49 p. 165). Upon various criticisms, the greatest care was taken in later experiments (1931) to prevent any chemical influences. In each case, the effect was transmitted through quartz, but not through glass. Table 29 shows a summary of the results of these experiments, and the purely physical nature of the effect cannot possibly be doubted. While the MAGROTJS originally explained this morphological change through mitogenetic rays, later experiments, together with REISS (1931), offered another possible explanation. They found that though a layer of glass prevents the effect, two layers of glass with a coat of paraffine between them permit the effect to pass. From this and similar experiments, these authors conclude that they are dealing with an electric effect. The larvae become abnormal if the source of radiation is separated from the medium of the sea urchin eggs by a very good electric insulator, and if further the difference in oxidation-reduction potential between the two liquids is very great. When the electric insulation was prevented by a metallic connection, the larvae remain normal. This explanation is tentative. In their recent publications, the MAGKOTJS do not give it preference to the ultraviolet radiation theory. In 1929, CHRISTIANSEN observed very strange morphological changes in yeasts and in bacteria brought about by menstrual blood. The effect passed through quartz coverslips, and must therefore be considered as the result of biological radiation. The yeast cells either became large and spherical, with enormously distended vacuoles ; or, they elongated and produced hyphae ; or, they did not grow at all, but died. During the last two years, the author and his associates have regularly observed similar morphological changes. METHODS OF OBSEKVING BIOLOGICAL RADIATIONS 87 Table 29 MAGROU'S Results with Biological Irradiation of Sea Urchin Larvae I No. of ,1 11 . , 1 Source of Had in turn Experiments Larvae Separat- ed from Source of Radiation by % of Experiments Showing Ab- normal Larvae 7(5 none ^lass 4 : dead bacteria glass 7(> i J^rudotHOHdx t'Uituc- /(If 1 {'US quartz 75 7 same j-lasH 43 30 , none jjjlass 25 titnphylococt'HH uumis quart/ 75 3 same, agglutinated quartz lf> none ^lass 12 Rtri'ittocwrux lactix (jiiartz 93 2 : same ^lass 4 baeteria-f'ree seruni of Streptococcus culture quartz 25 7 none, olass 5 Saeeliaroniyces and J)ebaryomyees quartz 100 30 none i^lass 18 HKKTU BLOT'S eullure medium (juartz 10O 6 same ftlass O 5<> none p;lass 3fi glucose oxidized by fer rieyani d e , perman - ganate or bichromate quartz 78 14 1 same jrJa,ss S 11 in m a r y 221 none US 9 J microorganisms \ ^;la.ss quartz 33 78 54 20 J chemical reactions | quartz glass 85 88 CHAPTER IV The effects in beer and wine yeasts produced by saliva, were essentially identical with those observed by CHRIHTIANSKN. When irradiated by plants, however, the tendency was not a shortening but rather a lengthening of the cells; this was so pronounced with some Mycoderinas that their growth strongly resembled that of mold mycelium. However, we could never observe the true branching of cells which OJIKISTTANNEN has described. Of the various parts of plants, the roots, young seeds, seedlings and pollen were the most effective, while leaves had little or no effect (see fig. 48 p. 102, and Chapter VII). g) Physico-chemical detectors LIESEGANU II ings: It was recognized by NT KM TELL that a physico-chemical detector would carry much more weight than biological ones for the proof of mitogenetie radiation, lie observed that the LiESEdANcj rings an* disturbed by biological radiation. These rings appear when, on a gelatin gel containing certain salts, a drop of another solution is placed causing a precipitate with the salts in the gelatin. The precipitant diffuses gradually into the gelatin, and the* precipitate is deposited in concentric rings. Method: 2 ec. of eliminate gelatin (12g. gelatin, KiOcc. water, 0,4 g. ammonium bichromate, being mixed, immediately before use, with 1 co. water -| I drop of 3" aqueous p\Togallic acid) are poured hot upon a clean glass plate 3.. r ) 4 inches. After solidification, 2 drops of a 20% AgN<) 3 solution are placed on tin* center of the plate by means of a fine pipette. Silver cbromate is gradually precipitated in concentric rings \\icii spread over the entire plate in about 24 hours. The uniformity of these rings is disturbed by radiating material placed in quart/, tubes as closely as possible over the gelatin surface. STEMPELL'S first experiments (1929) with onions were not accepted since, it could be shown that the ally 1 -mustard oil of the onion caused a chemical disturbance of the LIESKUANU rings. In later publications, however, STEMPKLL could prove disturbance of the rings when chemical influences were completely eliminated. The situation is rather complicated. Strong ultraviolet light from a quartz mercury vapor lamp thrown through a narrow slit upon the gelatin intensifies the ring formation at the, exposed places, while weak light, at the edge of the slit, decreases it. Onion oil acts in the opposite way ; a large dose of onion, oil gas lessens the ring formation while small doses intensify it. METHODS OF OBSERVING BIOLOGICAL RADIATIONS 89 STEMPELL (1932, p. 46) states that the disturbance of CIANG rings is usually brought about by a combined action of radiation and chemical effect of a "gas", i. e. a volatile substance produced by the sender. He considers this chemical effect to be very important, biologically, and states that the LIESECJANCS rings at present are the only detector for this substance, since none of the other detectors for mitogenetic radiation react to it. Since this book is meant to be limited to biological radiation, the interesting speculations of STEMPELL (1932, p. 40) regarding Figure 36. The effect upon LIES EG A NG rings of onion base pulp in metal tubes with a slit whose position is indicated by the black line left: effect through cellophane; right: effect through 0.5 mm of quartz. the possible biological meaning of the chemical emanations will be omitted. Decomposition of Hydrogen Peroxide : Another, quite different detector, has been found by STEMPELL (1932, p. 5/3). It Ls based on the deterioration of H 2 O 2 into H a O -f- O, under the influence of ultraviolet radiation. One onion root, or a bundle of several roots, is fixed so as to touch the underside of a thin quartz plate. On the opposite side of the quartz plate is placed a drop of H 2 O 2 , just over the root tips. After long exposure in a moist chamber, the peroxide concentration in this drop is less than that of the control. Flocculation of Colloidal Solutions: A promising method has been worked out by HEINEMANN (1934, 1935) who observed that inorganic sols flocculate more readily when exposed to mitogenetic rays. Gold sol was found to be more satisfactory than iron hydroxides. The gold sol was prepared in the following way: To 1000 cc. of a slightly alkaline solution of 0.8% glucose, 90 CHAPTER IV 150 cc. of a neutralized solution of 0.1 g. AuCl 3 are added. Upon heating, the gold salt is reduced by the glucose to a bright-red, clear gold sol. A small amount of Nad solution is added just before the beginning of the experiment to make the gold sol unstable. This is done in complete darkness. The solution is then distributed between two beakers, and each is covered with a quartz dish. They are placed into a very sensitive photoelectric differential nephelometer, and the suppos- edly radiant substance is placed into one of tho*quartz dishes. The difference in turbidity between the two beakers is read every minute, by means of a galvanometer. Radiation causes a more rapid flocculation, and therefore a change of the galvanometer reading while, in the absence of radiation, the readings remain fairly uniform. The following readings were obtained in 15 con- secutive minutes, the* arrow indicating the moment when the radiating material was applied to the quartz dish: control: 0001 1.5 1.5 1 2 2 2 22 blood: 0^2 3 5 7 7 8 10 11 IG 20 19 19 25 2G control: 0000001 1.5 1.5 2 2 2 2.5 3 3 NaCl, dissolving: 2 1 1 0$ 5 10 10 11 11 15 21 h) Measurement by physical instruments It may be surprising that radiation by organisms has not been recognized and proved conclusively long before this. The reason must be sought in its very weak intensity. The* inten- sity is so slight that the most sensitive photographic plates and the most elaborate physical instruments have in most cases failed to record this radiation. The best detector is still the living organism. This is unsatisfactory since we must allow for con- siderable individual variation of the detector organisms, and as a rule, the measurements are not as accurate as with physical ex- periments. The only photographic records are the ones reproduced in fig. 15 of KEITER and GABOR'S monograph, and those by BRF- NETTI and MAXTA (1930) and by PROTTI (1930). None of them are absolutely convincing, and GUBWITSCH has refused them all as proofs of the physical nature of mitogenetic radiation. TAYLOH and HARVEY (1932) could obtain no effect by exposing plates to frequently renewed fermenting yeast for ninety days. METHODS OF OBSERVING BIOLOGICAL RADIATIONS 91 Table 30. Measurements of Mitogcnetic Radiation by Means of the GETGER Counter Experiments of RAJKWSKY Time Impacts per interval Radiator interval Control Exposed Onion root .... 5 minutes 42.0LO | 51.07.0 Onion base pulp . . 10 minutes 42.2+1.2 50.0^1.5 Onion base pulp . . 10 minutes 39.8.1 0.3 49.3J : 2.2 Carcinoma of mouse 10 minutes 23.4.10.0 30.3 J : 0.5 Onion root .... 9 minutes 33.3 1.4 3.7 ] 0.5 Experiments by FRANK and RODINOW Frog musclo . . . 6 minutes 12-| 1.3 40.| 2.0 Frog muscle . . . 8 minutes 12,11.3 20J.1.7 Frog heart .... 11 minutes 1911.1 23 r j 1.2 Frog heart .... 5 minutes 34-J-.2.4 46 J 2.8 Mu self * pulp .... 4 minutes 13 i 1.8 20 J 2.2 In 1929, RAJEWSKY succeeded in obtaining direct physical proof of this radiation by means of a photo-electric counter (see p. 28). His data with onion roots and carcinoma are shown in Table 30. These experiments were repeated successfully by FRANK and RODJONOW (1930) with working muscle (see Table- 30 and Fig. 21, p. 29). Later experiments of this nature are those by BARTH (1934) and SIEBERT and SEFFEHT (1934). However, these results have not been accepted generally, at least not by physicists. LOKENTZ (1933, 1934) could show that bringing the "radiating material near the counter, or opening a shutter between the counter and source, may definitely change the counting rate even if there is no radiation present. To enumer- ate : (1) If the biological material is not in a closed quartz container, the water vapor from the material, even though slight, will con- dense upon the quartz of the counter, changing its resistance and thus altering the counting rate. Some experimenters have even placed their moist material directly upon the quartz window which will certainly change the counting rate. (2) If the water vapor is carefully kept away from the counter, the charges which are inevitably present on the outside of the quartz tube containing the biological material are almost sure to change the counting rate. (3) In one case, at least, muscle was tetanized by means of an induction coil directly in front of the window. 92 CHAPTER IV Table 30a. Photo-electric yields obtained for ultraviolet light Authors Photoelectric material Yield in quanta per electron wavelength A RAJEWSKY, 1934 . . SCHRKIBER, 1930. . GREY and OUELLET, 1933 Cd K Pt about 1 X 10 2 2x10* 6xl0 3 2650 2540 2540 FRANK and RODIO- NOW, 1932 . . . LORENZ 1933 . . . KREUCHKN, 1934 . Cd, Al Cd Cd, Al, Zn 2xl0 3 3X10 8 25x10* 2540 2540 2540 4 To separate the effect of extremely low intensity from these other effects is very difficult even when their existence is realized. In none of the physical measurements of mitogenetic rays is it absolutely certain that these errors have been excluded, and it seems well to view with caution the positive results claimed for the physical detection experiments so far carried out. A more careful description of the method of exposure, and a number of experiments with water blanks or non-radiant or- ganic materials will be necessary to produce evidence which is physically irreproachable. The experiments of SIEBBBT and SEFFERT (1934) who obtained increased counts with several hundred normal blood samples, but no increase with blood from carcinoma patients, are a step in that direction. Most convincing is the experiment that a counter gave a definite increase when exposed to normal blood, but showed 110 increase when this was removed and replaced by blood | KCN. Many more extended experiments of this general nature will be necessary to establish finally the radiant nature of the biological effects. In a recent paper, KREUCHEN and BATEMAN (1934) reviewed the field of physical detection and present their results in a table (see Table 30 a) which gives the photoelectric yield (see p. 29) of the surfaces used by the various investigators. In all cases except the first, more than 2000 quanta are required to eject one electron. Though some early workers indicated higher sensi- tivities, it seems from later work that this value has never been sur- passed if, in fact, it ever was reached. In his latest paper, KREUCHEN (1935) obtained yields of from 1C 6 to 10 4 quanta per electron from hydrogen-activated zinc and cadmium surfaces. METHODS OF OBSERVING BIOLOGICAL RADIATIONS 03 It would be of great advantage if some surface having a higher efficiency for the ultraviolet could be obtained. Pre- liminary experiments by one of the authors using magnesium surfaces sensitized by oxygen seem to offer encouraging results. RAJBWSKY estimated from lu's experiments the intensity of this radiation, and found it for onion roots and for carcinoma tissue to be of the magnitude of 10~ 10 to 10~ 9 erg/cm 2 /sec. (10 to 100 quanta/cm 2 /sec. for the wave length 2300 A). FRANK and RODINOW observed higher values; they obtained with pulp from muscle, with the working frog muscle and heart, values up to 2000 quanta/cm 2 /sec. For the reproduction, of the various niitogenetic phenomena with physical sources of light, much larger intensities are required (about 6.6 xlO 5 quanta, according to STEMPELL, 1932). i) Unaccounted failures in proving radiation It must be stated with perfect frankness that biological detectors sometimes fail for unknown reasons. Probably all investigators working with biological detectors have been worried by such failures. Some of them have published short remarks. GOLLSHEWA (1933) mentions that out of 373 experiments with blood radiation, in OUKWITSOH'S laboratory, 54 failed on account of a poor quality of the yeast culture which was used as detector. This cause became evident through the fact that all other associates using the same culture on the same day obtained negative results. No reason for the abnormality of the yeast culture is mentioned. WOLFF and RAS (1933 c) working with Staphylococci had a similar experience. Twice it happened that all the experiments of one day proved to be negative though the culture had reacted promptly on the previous day. It was found that the sensitivity of the culture had changed, and that a longer exposure was necessary. These authors believe that a change in the opposite direction may also take place. Acs (1932) claims to have in- creased sensitivity by selection. Most of the sudden failures of cultures to react have, not been published, but by discussing this point with the various investi- gators in this field, practically all seem to have had the same ex- perience. Professor GUKWITSCH has told the author that in his experience such a condition usually remained for several days, or even for a number of weeks, and it was impossible to produce even 94 CHAPTER IV the simplest mitogenetic effect. Eventually the culture reacted normally again. Doctor HEINEMANN, after a very successful diagnosis of cancer by the absence of blood radiation (see p. 181) in Frankfurt and in London, with yeast as detector, suddenly experienced a complete lack of reaction, and none of the various attempts to obtain normal reactions proved successful, not even the testing of a large number of different yeast cultures. This failure induced him to look for physico-chemical methods of detec- tion (see p. 89). Professor WERNER SIEBERT'S many succesful experiments with a yeast detector have been mentioned in practi- cally every chapter of this book. But with him, too, the yeast suddenly ceased to react, and he resorted to the GEIGER electron counter as a more dependable detector (see p. 92). The author himself has also had long periods of negative results in his labora- tory, and they come and go at irregular intervals. As a rule, the investigators do not discuss these periods of failure because there is still considerable doubt among physicists and some biologists concerning the existence of the mitogenetic phenomena, and the emphasis of such periodical failures might increase this doubt. While this can not be denied, it does not seem wise to belittle this experience. On the contrary, by calling attention to it, it may help to explain the cause of these failures, and thereby may bring about a better understanding of mitogenetic effects. Whether it is due to disturbance by short radio waves (suggestion by GURWITSCH), to a change of sensitivity of the detector culture ( WOLFF and HAS), to a retarding effect by human radiation (RAHN), to climatic changes or some other cause, is not known. The cause of this disturbance might be more readily traced and overcome by the cooperation of various laboratories. At the present, we do not know whether the culture, the experimenter or the environmental laboratory conditions have changed, in fact, it is only an assumption that the change has influenced the detector. If the cause should prove to be of such general nature as e. g., weather, cosmic rays, terrestrial magnetism, sunspots, it might be that the senders do not function under the prevailing condition. These occasional failures have nothing to do with the error of the method. When mitogenetic effects are observed, they are outside the limits of error. The failures might be compared to METHODS OF OBSERVING BIOLOGICAL RADIATIONS 95 the experience of expert florists that sometimes, certain plants refuse to bloom in the greenhouse. This was not caused by poor seed nor wrong soil, but remained unexplained for a long time until it was found that the number of hours of light per day decided this. The consistent observation of this disturbance by most (if not ah 1 ) investigators who have obtained large series of positive results, points out a common error in some criticisms. It has been claimed that many simultaneous parallel experiments prove more* than similar experiments spread over a longer period of time. E. g. KRETJCHEN and BATEMANN (1934) state that one series of theirs is equivalent to 140 single experiments by GURWITHCH. That is a mistake. As long as it is not known why the occasional failures occur, no great stress can be laid upon the results obtained on any one day. It might be a day where the detector fails, and the multiplication of experiments on such days would only reveal the error of the method as such, but would not increase the proof or disproof of biological radiation. B. INJURIOUS HUMAN RADIATION It is an old "superstition" that a harmful emanation comes from the body or the hands of menstruating women. It is believed that bread dough kneaded by them will not rise, that food preser- ved by them will not keep, that Hewers in their hands wilt readily. Experiments to prove this have been successful with some in- vestigators (ScHiOK, 1920; MACHT and LUBIN, 1927 ; BOHMER, 1927) and gave 110 results with others (&ANGKU, 1921; FRANK, 1921; POLAND and DIETL, 1924). Still, the belief in this effect seems to have been rather prevalent among medical men, and the term "menotoxin" for the hypothetical compound causing it is in general use. CHRISTIANSEN, as bacteriologist of a dairy laboratory in Germany, observed that the pure cultures used for dairy starters occasionally developed poorly and abnormally. After eliminating all other causes, it was ultimately found that this abnormality occurred during the menstrual period of the woman technician in charge of the cultures. A detailed investigation by CHRISTIANSEN (1929) led to the discovery that the effect from menstrual blood passed through 96 CHAPTER IV quartz, and must, therefore, be considered a radiation. Aside from this proof, CHRISTIANSEN did not go into the nature of the effect. He worked with menstrual blood and saliva, but not with radiations from the body. The blood produced cither abnormal morphological changes in yeasts and bacteria, or killed them. The effect was much stronger in summer than in winter, and since one woman who had been treated with ultraviolet light in winter, continued to show July 1931 August 13M February March June 1331 July Figure 37. Alternation of growth and no growth of yeast- in droplet cultures transferred hy the same person. H- signs indicate growth, o signs in black bars indicate no growth. strong radiation, he thought it very probable that the seasonal difference was brought about by the variation in solar irradiation. He called attention to the fact that the above-mentioned investig- ators obtaining positive results had made their experiments in summer, while the negative ones had been obtained in winter. Further, he showed that wine made with a pure culture yeast by a menstruating woman fermented feebly, while the control showed a vigorous normal fermentation. FERGUSON (1932) during an investigation of morphological changes in yeast by plant radiation, observed occasionally that for short periods, covcrglass cultures of one yeast did not grow when made by one certain woman student. The periods of no growth alternated with those of growth through summer and fall, while in winter, the controls nearly always grew normally (fig. 37). The droplet culture on the coverglass requires that the coverglass be held in the fingers while the droplets are made with a pen dipped into the yeast suspension. METHODS OF OBS FRYING BIOLOGICAL RADIATIONS 97 The above-mentioned experiments of CHRISTIANSEN made it probable that this failure was due to human radiation, and some experiments proved that an emanation from the fingertips of this person killed yeast in 5 minutes while others making the same test with the same culture produced no marked effects. In this experiment, the fingertip was held closely over the yeast by the support of a glass ring (fig. 38). In another experiment, a quartz plate of 2 mm. thickness was placed between finger and yeast (fig. 38); by this method, it re- quired 1.5 minutes to kill the yeast. This student menstruat- ed rarely and irregularly, and the ease suggests an abnormal i; ^g, yea/t'7 drops of parallel to CHRISTIANSEN'S oKser- / raisin vations. This test has been applied to a number of people, and it \ ; was found that this radiation ^^J^ $* rir >9 occurs only very rarely. It was ^ $d f ^overglaL most pronounced with a man Fimro38 who had recently recovered from Methods of testing finger herpes zoster of the face; for radiation, about fi months after recovery, he frequently killed yeast through quartz in 15 minutes, but this was not always the case. During this timo, he did not fool quite woll. After a summer vacation, he felt perfectly normal, and his power of radiation was gone. With this person, radiation also occurred from the tip of the nose and from the region of the eye. 1 ) A third case was a hypo-thyroid patient tested only once. A fourth case was one of the authors, during 3 successive days of sinus infection. Never before or afterwards did this person show any effect upon the yeast. *) This has been interpreted by imaginative but uncritical newspaper reporters as a scientific proof of the "Evil Eye", regardless of the fact that this radiation does not reach further than a few inches, and that only one especially sensitive species of yeast could be killed in this way; the authors are in no way responsible for this kind of publicity, but have not been able to prevent it. Frotoplasma-Monographien IX: Rahn 7 98 CHAPTER IV The experiments were always made with droplet cultures through quartz, as shown in fig. 38. In all more recent work, the droplets were prepared by the same person who had been found over a 2-year period never to radiate. The only organism which reacted upon this radiation was Saccharomyces mycoderma punctisporus GUILLIERMOND, isolated from the scum of a fruit juice. This yeast does not cause fermen- tation. Other yeasts showed slight retardation, but never wan complete killing observed. This radiation is quite likely duo to a skin excretion, as will be shown later (p. 185). Another killing effect was frequently observed with saliva. The saliva of many persons changes the oval or elliptical, granu- lated cells of beer and wine yeasts to spherical cells of increased size, with greatly distended vacuoles and homogeneous cell con- tents. Often, the organisms either do not grow at all, or cease to do so after a few cell divisions. It is not certain, however, that the effect is one of radiation. It was observed in droplet cultures which were mounted imme- diately above the saliva; this did not exclude a chemical effect. When saliva was mixed with the raisin extract in which the yeast was cultivated, it produced no abnormal cells, not even with half saliva and half raisin extract. This seems to exclude any chemical effect. On the other hand, the typical saliva reaction could not be produced through quartz; with yeast on one side and saliva on the other, only partial effects could be obtained, such as absence of granulation, or tendency to become spherical. The pictures were never convincing, and it must be left to a later investigation to solve this problem. This harmful effect is not typical for all saliva. It is typical for the individual, and pratieally independent of the diet. An investigation of the saliva reactions of several members of a family showed the above-mentioned injurious effect with male members of the family, and one female member. Two other females stimulated yeast growth, produced elongated forms, and, with Saccharomyces mycoderma punctisporus, a manner of growth strikingly resembling mycelium. METHODS OF OBSERVING BIOLOGICAL RADIATIONS 99 C. NECBOBIOTIC BAYS LBPESCHKIN had observed (1932 a) that the stability of living matter (plant cells, erythrocytes) is increased by irradiation with weak ultraviolet light, while strong intensities made cells less stable. The two effects are independent of each other. Ho con- cludes (1932b) that weak intensities of ultraviolet must help in the synthesis of cell constituents, and that vice versa, when cell constituents break down, the energy absorbed during synthesis must be released again, emitted, partly at least, as ultraviolet radiation. The experimental proof is given in considerable detail in a later paper (1933). The test organism was nearly always yeast, though parallel experiments were made also with leaves of Jtilodea, with petals of flowers, e. g. of Pajmver, and with suspensions of Bacillus subtilis. When silver nitrate was added to a living yeast suspension in the dark room, the yeast died within 12 minutes, and the suspension was gray. When the yeast had been killed by ether or by heat before the silver salt was added, the suspension remained white, but turned gray upon exposure to light. The gray color of the silverprotein precipitate with living yeast was supposed to be brought about by ultraviolet rays emitted by the dying colls. A similar difference could be observed when yeast was suspended in a mixture of solutions of KBr and AgNO 8 . Living yeast with ether caused a dark discoloration, but when the yeast had been killed by ether before the AgBr mixture was added, the mixture remained light-colored. Since these experiments did not exclude chemical effects, AgBr suspensions in small quartz tubes were inserted into tubes with dead yeast, and also into those with living yeast plus ether. After exposure with continuous shaking in the dark room, the AgBr suspensions were removed and mixed with a photographic developer. In all experiments, the suspension exposed to dying cells proved to have received some radiation. Then, very sensitive photographic plates (EASTMAN SPEED- WAY) cut in small strips, were submerged directly into the yeast suspension. Part of the plate was covered with filter paper which excluded physical, but not chemical effects by soluble substances. After 10 to 25 minutes exposure to yeast previously killed by ether, the plates upon development remained light. With living 100 CHAPTER IV yeast, they were also light. However, when ether was added to the living yeast, the dying cells affected the plates so that during developing they turned dark, except for the little strip shaded by the filter paper. This proved to LEPESCHKIN'S satisfaction that the effect was physical and not chemical. From the absorption of these rays by glass and by gelatin, LEPESCHKIN estimates their wave length to be largely between 1800 and 2300 A, with a very weak emission of greater wave lengths. This agrees fairly well with the range of mitogenetie rays. From the above experiments, it seems as if the necrobiotie rays were stronger than those from actively fermenting yeast cells. LEPESCIIKIN could obtain an indication of an effect upon AgBr suspensions if he used living beer yeast instead of baker's yeast, and added 10% sugar. However, though there was a slight effect from the fermentation upon the AgBr, the effect from the same mixture with ether i. e. with dying cells was much stronger. LEPESOHKIN then ventures further to state that many of the mitogcnetic phenomena are in reality due to necrobiotie rays. He emphasizes the radiation of necrobiotie processes e. g. auto- lysis and of wounds as proof for his contention. Evidently, LE- PESCHKIN was not familiar with the latest literature on this subject. In the case of wounds, it is not the injured cells which radiate, but the uninjured cells next to the wound. The radiation spectra of the various chemical processes are ample proof that dying cells are not necessary for the production of mitogenetic rays. LE- PESCHKLN apparently feels this; he believes it possible that "necrobiotic rays" might also be emitted from a decomposition of vital compounds in living cells which might be imagincable during very rapid physiological processes. He mentions respira- tion as a possibility. However, it would be necessary to consider almost all exothermic reactions as neerobiotic processes in order to combine the two types of radiation. SUCHOW and SUCHOWA (1934) have perhaps found the link between LEPESCHKIN'S and GURWITSCH'S explanation. They conceived the idea that the "necrobiotic rays" were emitted from the coagulation of proteins, and they tested it by coagulating egg white by alcohol in quartz or glass vessels which were placed over AgBr-suspensions as in LEPESCHKIN'S experiments. The experi- ment was carried out in the dark, and after exposure, the two METHODS OF OBSERVING BIOLOGICAL RADIATIONS 101 suspensions were brought into light. In 25 experiments, the suspension which stood under the quartz vessel uniformly darkened sooner than the other. D. INFRA-RED RADIATION It might appear rather probable that some organisms would emit infra-red rays since some are capable of producing visible and ultraviolet rays. The only case of near infra-red emanation known to the authors is, however, a series of observations by STEMPELL (1931) that sprouting peas will increase distinctly the rate of spontaneous decomposition of a saturated solution of H 2 O 2 . The effect passed through glass, and was not visible, it must therefore be of an infra-red nature. The temperature of the peas rose 0.5 by their own respiration, but an artificial increase of 5.5 was necessary to bring the rate of peroxide de- composition to that produced by peas. Equally rare are observations of an effect of infra-red rays upon living organisms. The only one known to the authors is an experiment by NELSON and BIIOOKS (1933). They exposed the unfertilized eggs of 2 sea urchin species and of one worm to infra- red rays of 8000 to 12 000 A, obtained from a Mazda lamp by means of a monochromator. After 15 to 45 minutes exposure, the eggs were fertilized by the usual method. The irradiated eggs showed, in each of the 9 experiments a distinct decrease in the percentage fertilization. The decrease varied between 18.5% and 87.1%. The temperature difference between control and exposed eggs was not more than 0.06 C. The authors believe therefore that the reduced fertilization is caused by a photochemical effect. E. BETA-RADIATION An entirely different type of radiation should be mentioned only in passing, namely the bet a -radiation of potassium. Though it has been proved experimentally, and is of importance to life processes, it will not be discussed extensively here because this type of radiation is utterly unlike the mitogenotic and related rays. It originates from the radioactive fraction of potassium, and is really not characteristic of the living cell, but of its potassium content. The intensity of this radiation is the same whether the cell is alive or dead. 102 CHAPTER IV. METHODS OP OBSERVING BIOLOGICAL ETC. Nevertheless, this radiation is biologically important, and may be the chief reason for the indispensibility of potassium in living organisms. ZWAABDEMAKEB (1921) was the first to test experimentally the physiological importance of potassium radia- tion. He succeeded (1926) in keeping isolated frog hearts beating by substituting the potassium of RINGER'S solution by radioactive equivalents, rubidium, uranium, thorium, radium or ionium. In 34 experiments, he could show that frog hearts which had ceased to beat in RINGEB'S solution minus potassium, started again in the same solution after about half an hour's irradiation by mesothorium (in glass) or radium (through mica). After removal of the radioactive substance, heart beat soon ceased again and could frequently be brought back a third time by new exposure to beta rays. SCOTT (1931) determined the total energy from potassium of the average human heart to be 9.64x 10~~ 6 ergs per second. He states that "one may readily conceive that the free energy of the beta particles can be cumulative and, reaching a maximum, transform the potential energy of the heart muscle in response to node and bundle impulses into the enormously greater mani- festation of kinetic energy, the systolic contraction". CHAPTER V SPECIAL PROPERTIES OF M1TOGENETIC RADIATION The preceding chapter has revealed that the socalled mitogen- etic radiation is a real radiation according to tho strict physical definition of the word. It travels through space rectilincarly ; it can be reflected (pp. 59 and 80) ; it can be absorbed (p. 59) ; it shows refraction and dispersion (p. 35). The present chapter describes some peculiar properties of initogenetic rays which are not common to all types of radiations. A. INTERMITTENT IRRADIATION Very early in the history of initogenetic radiation, it was discovered that the effect could ho intensified by irradiating intermittently instead of continuously. The customary method for this purpose is the insertion, between sender and detector, of a rotating disk which contains one or several openings or windows. The width of these, their number, and the rate of rotation of the disk allow one to vary the three items concerned in intermittent processes, i.e., the frequency of exposures, the duration of each, and the total time of actual irradiation. The most striking result with intermittent irradiation is the much shorter total time of exposure necessary to bring about distinct mitogenetic effects. OrmwiTSCH (1!)32) determined the threshold value in mutual yeast irradiation by means of disks rotating at approximately 3000 revolutions per minute. The disks contained one or several windows of varying width. The width of the window is measured by the central angle (fig. 39). From this angle and from the number of revolutions, the duration of each interval and the frequency of interruption can be calculated. Table 31 gives the results obtained. It indicates that the minimal 104 CHAPTER V C O Op O ppp o p o L-" ooo" i-H G^ C^l i~H rH r-l SPECIAL PROPERTIES OF M1TOGKNET1C RADIATION 105 actual exposure must be more than 10 seconds, 12.5 to 13 seconds being sufficient. When the same experiment was tried with uninterrupted irradiation, it required 6 to 8 minutes to produce a distinct effect. The rhythmic interruption of radiation had decreased the threshold time to about l/30th of the amount required with continuous exposure. The frequency of interruption has some bearing upon the threshold value. When the frequency is between 800 and 100 per second, 13 seconds are sufficient for induction. However, Figure 39. Rotating disk for in- termittent radiation, at right: side view showing the position of the two agar blocks with the yeast sides facing each other, for intermit- tent muto-induction. when it falls to 50 per second, 15 and 10 seconds of total exposure are usually insufficient, and 30 seconds are necessary to show a mitogenetic effect. This is to bo expected, since it signifies an approach to continuous irradiation. ZOGLINA (quoted from GUKWITSCH 1932, p. 256) repeated the same experiments with a half -disk rotating at 1 Or. p.m. This meant uniform intervals of exposure and irradiation of 1 / 2 o^ second each. The following per- centages in increase of buds were obtained with a total exposiiro time of 60 seconds: 7 15 5 +4 60 +28 -|-56 +64 +28 +36 The increase in efficiency of a photochemical reaction by intermittent irradiation has its analogy in the increase of photo- synthesis of green plants by intermittent exposure. WARBURG (1919) measured the amount of CO 2 absorbed by an alga, CHLO- BELLA, during 15 minutes of actual exposure, either continuously or discoiitinuously. A rotating disk was used which was in principle like that of fig. 39, but the times for light and dark were made equal. The results are given in Table 32. An increase up to 106 CHAPTER V -d so CO CO CO -t CO >O iO - 00 OS SPECIAL PROPERTIES OF MITOGENETIC RADIATION 107 practically the double amount was observed with strong light, but no difference with weak intensities 1 ). WARBURG considers two possible explanations: either assi- milation continues for some time after darkening (e. g. through some short storage of energy); or, assimilation is more rapid at the first moments of exposure because more substances have accumulated ready for photosynthesis while later, their concen- tration is only comparatively small. His intention to investigate in more detail the latter, more probable case seems never to have materialised. It is not at all certain that this observation is really anal- ogous to the increase of the mitogeiietic effect. WARBURG could double the amount of photosynthesis by distributing the same total radiant energy over twice as long a period. GURWITSCH, however, found a 30-fold increase in the threshold value while the greatest difference between light and dark periods was only 1:0. Besides, there seems to be no difference between strong and weak intensities. On the other hand, we cannot be certain that these threshold times are reliable measures of intensity of radiation (see p. 114). The greatly intensified susceptibility of tho living detectors by rhythmic discontinuity of radiation shows that by this method, radiations can be detected which otherwise produce no effect whatever when applied continuously. This holds true not only with very weak senders, but also with very strong sources which produce either no effects or depressions (see p. 115). The greater susceptibility also permits transmission over longer distances. While mutual induction of yeast has its limits at 3 -4cm. with continuous exposure, very good results can be obtained over 15 cm. with intermittent irradiation (GuRwrrson. 1932, p. 200). These facts differ greatly from WARBURG'S observations with algae, where weak intensities of radiation could not be induced to produce stronger photosynthesis by intermittent exposure (Table 32). A rhythmical interruption of the radiation seems to be essential for mitogenctic induction. Parallel experiments were made with l ) After the manuscript was finished, a paper by EM EKSON and ARNOLD (1932) came to our notice. They were able to increase photosynthesis 400% by intermittent irradiation whereas WARBURG (1919) could only double it. 108 CHAPTER V two disks rotating at the same speed, both with a total window width of 75. In one disk, the 75 were distributed uniformly; the other disk contained openings, varying in size from 2.5 to 30, in irregular distribution. Only with the regular spacing were positive results obtained (GuRWiTSCH 1932, p. 261). This, could hardly be accounted for by any of the two explanations of WARBURG'S. B. INFLUENCE OF DIFFUSED DAYLIGHT Many of the common "senders" of mitogenetie radiation affect other organisms only when in daylight. Diffused lignt is entirely sufficient. This has been observed for onion roots cut off from the onion bulb, and for the pulp of the onion base, as well as for the pulp of a number of plant tissues. In 1930, POTOZKY gave several series of experiments showing that the same holds true also for yeast. The experiments were made by the Baron technique, measuring the percentage increase of buds on yeast grown on agar blocks. All experiments wero made by muto- inductioii of yeast, i. o. by exposing yeast to yeast. Table 33. Influence of Daylight upon the Yeast as Sender and as .Detector of Mitogeiietic Effects Obtained by Muto-Induction jl Sender: || Dark Yeaht Daylight Yeast Dark Yeast Daylight Yeast Detector : Dark Yeast Dark Yeast Daylight Yeast Daylight Yeast exposed in daylight | dark daylight j dark daylight dark daylight | dark Percentage '' 0.7 16.0 21 ! 8.0 4.5 35 ! 9 Increase 5.4 3.3 22 --8.6 3.2 1.8 30 j --8 in Buds 5.5 4.3 20 2.9 3.1 3.6 30 -9 11.0 2.2 23 i 3.9 1.0 1.8 25 1.8 2.1 30 1 11.0 24 15 1.8 30 j 9.4 29 H.3 3.3 20 9 i 2.3 22 7 Average . . + 1.4 +6.5 24.0 5.9 +4.6 | 2.9 27.0 1 +0.2 Three factors wore varied: the sender yeast, the detector yeast, and the light during exposure. Yeast grown in the dark had no effect upon yeast, whether grown in the light or the dark, and whether tested in light or dark. Yeast grown in daylight SPECIAL PROPERTIES OF MITOGKNETIC RADIATION 109 showed distinct radiation and mitogenetic effect upon the detector yeast, regardless of whether this had been grown in light or dark, but only when the exposure was made in daylight. This accounts probably for a number of negative results by some experimentors. Yeast grown in the dark regained the property of radiation after remaining in daylight for about two hours. FKANK and RODIONOW have shown, by means of a GEIGEK counter, that light affects greatly radiation from some chemical oxidations, as o. g. K 2 Cr 2 O 7 f FeSO 4 (p. 34). C. SECONDARY RADIATION The original experiments by GTJKWITSCH had shown that only the meristem, i. e. the growing tissue near the tips of onion roots radiated while the older parts of the root, where the cells had waned to multiply, were inactive. Even the root tips radiated only when connected with the bulb, or at least with part of it. They lost their radiation completely when severed from the bulb. In search for an explanation, GURWITSCH discovered that a root, after being cut from the bulb, will emit a radiation when it is exposed to ultraviolet light. This ''secondary" radiation of the root ceases when the "primary" radiation docs. It seemed quite impossible that the original rays as such could have- been transmitted through the root by reflection without having been absorbed completely. A chemical effect could hardly be passed along so rapidly. There was only one alternative loft : the radiation Irom the outside induced the exposed cells to produce some radiation of their own ; these "secondary rays" again induced the neighboring colls to radiate, and so the effect was passed along the root without losing in intensity. This explanation was proved by many variations of the original experiment. For some time, it was believed to bo a phenomenon characteristic of the living cells only, until in 1932, A. arid L. GTJRWITSCH found it to occur also in nucleic acid solut- ions, and WOLFF and HAS (1933, 1934) proved it to be primarily a photochemical phenomenon. Many illuminating details have been worked out by POTOZKY and ZOCLTNA (1928), ALEXANDER, ANNA and LYDTA GUUWITSCH and others. Secondary radiation was observed in muscle, in liver, in nerves, in suspensions of yeast, of bacteria, of protozoa. In 110 CHAPTER V ail these cases, secondary radiation seemed to be glycolytic. The fact that livers from starving animals which are free from glycogeii, did not radiate, supports this view. However, this cannot be generalized because the secondary radiation from nucleic acid is not glycolytic. By this mechanism, primary radiations can be spread and transmitted to distant parts of the plant or animal body. It will be shown later that the tips of onion roots are only secondary senders; the primary rays are produced in the onion bulb, by oxidation. This spreading of the mitogenetic effect can be plainly shgwii with densely grown agar surface cultures of yeast. GTTRWITSCH irradiated such a culture through a slit 0.1 ram. wide. When tho percentage of buds was counted, there was a distinct increase not only in the irradiated region, but as far as 10mm. distant. The increase in buds was 65% at the irradiated zone, and at a distance of 1 mm. 2 mm. 3 mm. 4 mm. 5 mm. 6 mm. 7 mm. 8 mm. 9 mm. 10 mm., it was 100% 87% 86% 79% 80% 80% 55% 43% 33% 25% respectively Experiments with larger irradiated surfaces gave the same amount of spreading, 9 12 mm. from the border of the irradiated area. The ability of roots to produce and conduct secondary radiation is limited to a short time after the severing of tho root from the bulb; POTOZKY and ZOOLINA (1928) found a positive effect after 30 minutes, but not after 40 45 minutes. Tho same authors could also show that the production of secondary radiation exhausted the plant rapidly. Freshly-cut roots which gave strong secondary effects during the first 5 minutes of irradiation showed no reaction after 10 more minutes of ex- posure to monochromatic light of 2020 A. Another experiment with starving yeast cells may help to throw some light on this phenomenon. Yeast cells radiate imme- diately after being washed, but not 30 minutes later. Even then, the organisms will still produce secondary radiation under the influence of an arc light spectrum. This exhausts the yeast so much, that one hour later, it has produced 41% less buds than the unirradiated control. Exhaustion has also been demonstrated with chemical solutions (see p. 44). A very recent illustration for such exhaustion has been given by LATMANLSOWA (1932) on the secondary radiation from nerves SPECIAL PROPERTIES OF MITOGENETIC RADIATION 111 after mitogenetic irradiation. The sciatic nerve of a frog was irradiated by a yeast culture. Another yeast block, serving as detector, was placed near the irradiated part of the nerve so that it was exposed only to secondary radiation from the nerve, but not to primary radiation from the yeast. This block was changed every 5 minutes. Fig. 40A shows that the induction effect of secondary rays is very strong at first, but decreases after 5 minutes,, Figure 40. Secondary radiation from a nervo exposed to con- tinuous yeast irradiation. A: the first 40 minutes, showing exhaustion of the nervo; B: a nerve, ex- hausted after 35 minutes, is given 10 minutes rest after which irradiation is minutes minutes continued. and after approximately .30 minutes, it has disappeared, and does not appear any more upon continued irradiation. If, however, the nerve is given a ''rest" for 10 minutes, by removal of the source of irradiation, it will recover sufficiently to react again upon renewed irradiation (fig. 40 B). But the nerve is still "tired" and will become much more readily exhausted than the first. This phenomenon, too, is not characteristic of nerves only. It can be duplicated with cell-free solutions (p. 43). The observation that secondary radiation could be passed on over considerable distances, suggested measuring the rate of travel. After some preliminary experiments by ALEXANDER GUKWTTSCH, ANNA GTJKWITSCH (1931) made some accurate measurements with onion roots, by means of a rotating disk (fig. 41). This had two windows, one nearer the center through which the primary rays (from a yeast culture) fell upon the older part of the root, and another towards the periphery of the disk through which the radiation from the meristem of the root fell upon the detector. These two slits were so arranged on the rotating disk that after the primary rays had fallen upon the root, the disk had to be turned through 50 before the secondary rays from the meristem could fall upon the detector. This central angle was varied from 20 to 85. With a definite speed of rotation of 3000 r. p.m., only the angles between 25 and 50 gave positive. 112 CHAPTER V results. This signifies that a certain time (0.0022 seconds) must pass before primary radiation falling upon one part of the root, is conducted to another part and is emitted there. These values refer to a condition over 2.5 cm. Then, the distance was increased to 5 cm. The central angle for positive effects was hereby increased to between 40 and 70, which means an average increase of 15. This corresponds, at 3000 r. p.m., to 0.00083 seconds, and this is Figure 41. Kotating disk for measur- ing the rate of travel 4 of secondary radiation in onion roots. At right, side view showing position of primary sender S, onion root, and detector D. the time required for the secondary radiation to pass the additional 2.5 cm. The rate of conduction is therefore about 30 meters per second. Allowing the same time for transmission through the other 2.5 cm. of root, the total time required for transmission through 5 cm. of root is 0.00166 seconds. The total time corresponding to the average angle of 55 is 0.00306 seconds. The difference, 0.00140 seconds, was required for processes other than conduction, such as local reactions at the points of absorption and emission. Recently, LATMANISOWA (1932) has measured the rate of conduction of secondary radiation in the sciatic nerve of the frog. The method was exactly the same, except that the 2200 A area of the copper arc was used as primary source. This was sufficiently intense to permit the reduction of the window for primary radiation to 3 and that for secondary radiation to 1. The accuracy of the method was thus greatly increased, and the rate of conduction in the nerve was found to be 303 meters per second. This is in good agreement with physiological measurements on the rate of conduction of nerve impulses. In the experiments with secondary radiation of onion roots, radiation was observed from the same side of the root which had been exposed to primary radiation. Further experiments showed SPECIAL PROPERTIES OF MITOGENETIC RADIATION 113 that the radiation effect was transferred longitudinally with great ease, but that no conduction occurred transversely across the root to the opposite side. In the nerve, however, strong radiation has been obtained from the unexposed side (LATMANISOWA 1932). An important phenomenon for the explanation of mitogenetic effects is the observation (GuRWiTSCH 1932, p. 300) that radiating cells or tissues lose this power rather readily when they are them- selves exposed to mitogenetic rays. Even young, actively radiating cultures lose their power when exposed to their own wave lengths, ^our yeast agar blocks were placed so as to irradiate one another. The first mutual induction was plainly noticeable. After 15 minutes, they were separated and tested as senders ; one was tested at once, the others after 16 and 30 minutes. None produced an increase in budding, as the following data show: Mutual induction during 15 minutes .... 37% increase over control After 15 minutes rnuto-induction, effect upon new detector 2% After 15 minutes muto-induction and 15 mi- nutes recovery 0% ,, After 15 minutes muto-induction and 30 mi- nutes recovery 1-8% ,, Perhaps 30 minutes is too short a time for recovery of yeast, the generation time of which must have been at least one hour under the condition of the experiment. This phenomenon itself can bo at least partially explained by the experiences with secondary radiation (see p. 45). Practically all these facts had been discovered, before it was found that in certain cell-free solutions, the same effect can be obtained (see p. 44). This does not alter the explanations mate- rially, however. It only means that secondary radiation need not be connected with life processes. It is brought about by some unknown influence of ultraviolet rays which induce certain chemical reactions in cells or complex organic substances. In their latest publication (1934 a), WOLFF and RAS point out that mitogenetic rays become polarized by reflection like common light does, and that apparently, polarized mitogenetic rays have an enormously greater biological effect. When mito- genetic rays fall upon any cell, it seems highly probable that at Pro to plasma -Monographien IX: Rahn . 8 114 CHAPTER V least part of this radiation will be reflected from the cell walls, and thus will become polarized. It is not possible, at the present moment, to foresee all the consequences of such polarisation. D. INTENSITY OF THE MITOGENETIC EFFECT It has already been stated repeatedly that the intensity of the effect is not proportional to the intensity of the radiation. One very simple reason for this is the usual method of recording the results. The "induction effect" as the increase in the exposed yeast over that of the control, expressed in percentages of the latter cannot possibly be used as quantitative measure, as ex- plained on p. 79. The error of this method of recording resu]ts becomes most evident when applied to physical measurements. If the number of impacts induced by biological radiations is expressed in per- centage of the stray radiations of the surroundings, it is utterly meaningless from a quantitative viewpoint because this stray radiation (the background radiation) can be greatly altered by shielding the instrument with iron or lead. This does not affect the intensity of the biological radiation at all. The "mitogenetie effect" as used especially by the Russian investigators has no quantitative value whatever. It is not surprising that it was never possible to use it for measurements of intensities. The customary way of comparing intensities is to compare the minima] time of exposure (threshold time) required to give definite mitogenetie effects. This method has been used repeatedly by the Russian workers, and recently also by WOLFF and RAS. Examples may be found in Table 12 p. 44, Table 46 p. 145, and Table 49 p. 157. However, there are physical reasons to warn against quantitative conclusions from threshold times. It has been pointed out above (p. 24) that, with photographic plates, the reciprocity law (double intensity means half as long exposure) does not hold with very low intensities. All previous measurements of intensities have become practically meaningless since WOLFF and RAS (1934 a) showed that mitogenetie rays may easily become polarized, and that polarized rays have an enormously much stronger biological effect than the ordinary radiations of this type. SPECIAL PROPERTIES OF MITOGENETIC RADIATION 115 E. RETARDATION THROUGH RADIATION It seems quite probable that an overdose of radiation might produce the opposite effect of mitogenesis, and prevent or retard mitosis. GUKWITSCH called this phenomenon mitogenetic de- pression which, really, is a self -contradictory term; it should be "depressed mitogenesis". Observations of this kind have been recorded rather frequently, but the circumstance that different detectors sometimes give opposite results, warns against hasty conclusions. As early as 1928, STLSSMANOWITSCH irradiated onion roots biologically for 12 hours and longer, together with control roots exposed only during the last 2.5 to 3 hours. She observed a decrease of mitoses in the exposed side of the root as compared with tho opposite, shaded one. This was interpreted as "ex- haustion" by too much radiation. Strong physical light produced tho same depression in a few minutes. It must be remembered, however, that with roots as detectors, we have 110 real controls; a difference between tho two sides of the root may mean stimulation on one side, or retardation on the other side, or both. In this case of over-exposure, there may have been retardation through over-exposure, or it may mean no effect through over-exposure, and stimulation (through secondary radiation) on the shaded side. We must therefore turn to other detectors which permit absolute controls, i. e., to unicellular detectors. The yeast bud method appears to be the one by which "depression" is observed most easily. But it is just these retardations by the yeast bud method which are frequently contradicted, in the same experi- ments, by parallel measurements of the actual cell increase. Table 34 shows SALKIND'S experiments (1933) with rat blood radiation. With exposures of 2.5 minutes and longer, the percentage of buds showed a decrease against tho controls, the actual number of cells, however, is larger than that of the controls. This can only mean that the yeast bud technique fails to indicate the true growth rate (see p. 69). Real retardation by biological radiation can be measured only by decrease in the growth rate. The measurement of the actual number of cells permits of only one interpretation; a smaller increase than in the control can only signify a retardation 8* 116 CHAPTER V Table 34. Induction Effects from Intermittent Radiation of Rat Blood, as Measured by the Relative Increase in Yeast Buds, and by the Increase in Total Cells Length of Induction Effect obtained Exposure by yeast buds | by yeast cells 7 seconds . . 3; 3 15 23; 41; 30 1; 7? 12; 13 30 17; 20; 28; 30; 35 47; 69 1.5 minutes . . 5; 9; 13 2.5 . . 31; 41 46; 50; 57; 69; 74; 123 6 18; 20; 23; 24; 22; 22; 40; 43; 109; 120 24; 25; 28; 28; 33 10 30; 33; 33; 35 73 20 52 of the growth rate. Such cases are also reported. WOLFF and RAS (see p. 77) consider it the normal reaction after continued irra- diation. In his analysis of this phenomenon, SALKIND (1933) observed that with prolonged irradiation, the depression did not increase. A more detailed investigation revealed a certain periodi- city; after stimulation followed depression, but after depression, if radiation continued, again stimulation could be observed, (Table 35). This was the case with physical as well as biological senders. In each instance, as well in the experiment of Table 34, irradiation was applied intermittently. No definite periodicity could be found in SALKIND'S data. GUKWITSCH as well as WOLFF and RAS (1933c) have verified this observation of several maxima at widely different exposure times while between these maxima, no mitogenetic effects were obtained. A striking parallel exists between this effect and that of the photographic plate, as may be seen by the following quotation from NEBLETTE (1930). "Reversal by Light: With a short exposure to light we get a latent image which on development yields a negative. If the exposure is lengthened considerably, the image becomes positive instead of negative when developed, while still further exposure will produce a second negative, and it is probable that the cycle may be repeated indefinitely, although SPECIAL PROPERTIES OF MITOGENETIC RADIATION 117 Table 35. Periodicity of the Mitogenetic Effect Measured by the Increase in Cell Numbers with Yeast Percentage Increase over Control Cultures Line 2350A Serum Albu- Source of Radiation Carbon(orCu) Arc Light Agar Culture of Yeast min in gastric juice Experiment No I II I II I II Exposed for 18 seconds . . +48 8 2 minutes . . 8 18 +20 +27 +43 5 + 1 +94 + 6 + 85 +80 8 + 8 +28 6 + 2 23 10 +38 +39 + 77 38 12 13 19 11 + 17 32 16 +37 2 +37 + 7 +37 18 -^0 +30 16 +88 30 +79 40 +40 owing to the enormous exposures required, no one has been able to go past the second negative stage. The reactions which result in reversal are still obscure." GURWITSOH (1932, p. 219) gives some examples whore, after too long an exposure, the effect was not at once harmful, but was delayed for a short time, acceleration being noticeable followed by a distinct retardation. He calls this "secondary depression". F. ADAPTATION TO GRADUAL INCREASES IN INTENSITY When the intensity of radiation is gradually increased from below the threshold to a value which would produce a strong effect under usual conditions of exposure, no induction takes place. This has been demonstrated most simply in experiments on mutual yeast irradiation (GuRWiTSCH 1932, p. 263). An experiment was started with two yeast agar blocks mounted 6 cm. apart, on the movable substage of a microscope. This distance is too far to produce a mitogenetic effect. Very slowly, the two blocks were made to approach one another, until after 5 to 8 minutes, they were very close together ; they remained in this position for some 118 CHAPTER V. SPECIAL PROPERTIES ETC. time. The total irradiation time corresponded to that of another set with the same yeast culture which had been placed in the final position at the start. While this latter set showed increases of 40 to 50% over the controls, the yeast of the equally long exposed, but gradually nearing agar blocks paralleled the controls. The time required for this slow approach must be about 5 to 6 minutes. When it is reduced to 3 minutes, the regular mitogenetic effect is observed. The same phenomenon was obtained when an elliptical disk was rotated between two yeast agar blocks. This disk was mounted so that in rotation, it gradually exposed the two agar blocks to each other, and gradually shaded them again. This was sufficient to prevent induction. CHAPTER VI ANALYSIS OF THE MITOGENETIC EFFECT At the present time, the mechanism by which short ultra- violet rays affect living cells is not understood. This chapter does not offer one theory, but presents a number of attempts to account for the various phenomena observed. These rays were not discovered by chance. From a certain rhythm observed in the division of the sperm cells of amphibia, and in plant roots after special treatment, GURWITSCH (1922) predicted a factor which controlled cell division. From the mode of action, he concluded that this factor could not be chemical, but must be of a physical nature, and in 1923, he succeeded in proving it with onion roots. Mitogcnetic radiation was considered at first merely from the cytological viewpoint, as an emanation produced somehow in the very complicated process of cell division. Only after 1928, when SJEBEJK.T proved this radiation to be emitted also from purely chemical oxidations, the physico-chemical viewpoint entered into consideration. The theories which were developed during the "biological stage" of the discovery have never been fitted completely into the physico-chemical facts observed later. Hence, we lack a clear conception of the working mechanism of these rays. GURWITSGH has always distinguished between the "Ureffekt", the primary effect which is the increase in the number of mitoses, and all other effects of secondary importance. All of GURWITSCH'S speculations and explanations start with the results obtained with onion roots; these were the first detectors, and since he made a large number of experiments with them, they are to him probably the most familiar of all detectors. However, they have the great disadvantage of not offering perfect controls. The number of mitoses in different roots oven from the same bulb varies greatly. The customary method is to 120 CHAPTER VI use the shaded, unexposed side of the same root as control; but we cannot be at all certain that irradiation of one side does not influence the cells on the opposite side as well. In fact, REITER and GABOR claim tha,t they are affected. The authors of this book who have made no experiments with onion roots, but are familiar with yeasts and bacteria, prefer to start with these simple, unicellular forms as the first objects for an attempt to interpret the primary mitogenetic effect. The best method for this purpose is the yeast bud method* by TV THILL and RAHN (p. 68) where all cells are of the same age ; no secondary radiation from older cells complicates the analysis, and the per- centage of buds is a true measure of the rate of cell division. Any interpretation of the mitogenetic effect should account at least for the most remarkable facts observed. The following have been selected as the most important: (1) The necessity of a particular physiological stage of the cell. (2) The relation between the intensities of ra-diation and of effect. (3) The minimal intensity required for an effect. (4) The harmful effect of over -exposure. A. THE NECESSITY OP A PARTICULAR PHYSIOLOGICAL STAGE This neces&ity will not appear improbable to a cytologist. The cells of growing tissues are morphologically and chemically quite different from the old, resting cells of the same tissue. This holds not only for the larger plants and animals, but also for cultures of yeasts and bacteria (see e. g. HBNKICI, 1928). When any cell changes from the stage of active cell division to the resting stage, this is caused by some external or internal factors. These factors must be removed, or changed, before old cells can divide again. With unicellular organisms, rejuvenation is brought about by transferring the old cells to a fresh medium. The old cells need from one to several hours before they are "rejuvenated", i. e. able to multiply at the normal rate. This period of adjustment is called the lag phase (see p. 67). In multicellular organisms, old cells can be induced to cell division by wounding, or by unknown outside stimuli as in the case of gall formation in plants, or neoplasma in animals. ANALYSIS OF THE MITOGENETIC EFFECT 121 It would not appear probable that a resting cell can be induced to a new cell division by a short, weak irradiation. Though we do not really understand physico- chemically the ageing process of a cell, it does not seem likely that the factors which induce ageing could be removed by irradiation. This is borne out by experiment. Mitogenetic effects are not, as a rule, observed with old cells left in an old environment. The stage of active cell division, does not appear very favorable either for mitogenetic effects. WOLFF and RAS (1932) make the unrestricted statement that mitogenetic effects can be obtained only during the lag phase, but not later, i. e. not during the phase of constant growth rate. Their experiments support this claim. They explain it by the assumption that the rapidly multiplying cells irradiate one another, and being so close together, their own radiation is stronger than that from any external source, which is necessarily weakened by distance and absorption. If this explanation were correct, such cultures should rcart to outside irradiation at low temperatures where the rate of metabolism, and consequently the intensity of radiation, is weak ; they should also react to an external source when they arc widely dispersed so that the cells are far apart. The latter was tried without success by FEKGUSON and RAHJS (1933). Cultures of Bacterium coli, 24 hours old, never reacted upon irradiation, whether exposed as such or diluted 1 : 10 000, while older cultures gave very pronounced effects. The fact that actively dividing cells do not respond readily to mitogenetic rays is thus verified, but the explanation by WOLFF and RAS is doubtful. With yeasts as well as with bacteria, the stage of rejuvenation, the lag phase, is one of strong response. Very striking are the results of TUTHILL and RAHN (Table 21, p. 68) where the yeast produced buds very rapidly when exposed within half an hour after being transferred to the fresh nutrient medium, but failed to respond an hour later, though the control had not as yet started to produce buds. There seems to be one vstage during rejuvenation when the cells are most susceptible. The explanation may be cytological, chemical or physical. It may be that but one mitotic stage can take advantage of the energy introduced into the cell by this radiation. Perhaps, a certain chemical process in the rejuvenating cell is greatly stimu- lated ; e. g. the ultraviolet, by means of a chain reaction, might 122 CHAPTER VI set up the reduction potential necessary for normal cell functions (light the candle which then keeps on burning as long as the cell feeds normally). Or, possibly, the cell wall becomes transparent to these rays only at one certain stage of development. Whatever be the explanation, it must be kept in mind that so far, the later stage of active cell division does not seem to be greatly influenced by these rays. It appears that the difference between rejuvenation and active cell division might give us the clue for the mitogenetic effect. There seems to be another stage where cells respond, namely immediately before entering the resting stage. The description of the physiological condition of BAKON'S yeast plate (p. 66) suggests this strongly. The volumetric method as described by KALENDAROFF (p. 73) appears to make use of this stage, and so does HEINEMANN'S hemacytomcter method (p. 72). Apparently, the cells, at the point of going to rest, are stimulated to at least one more cell division by irradiation. This may also bo the cause of the mitogenetic effect in onion roots (see p. 129). This need not necessarily involve a mechanism different from that assumed in the rejuvenation process. It may well be that in the ageing cell, the additional, properly dosed energy from the sender prevents a certain phase of the ageing process, for a short time, sufficiently long to permit one more cell division. This may be the same mechanism which, under the more favorable conditions of re- juvenation, is stimulated so greatly. B. RELATION BETWEEN THE INTENSITIES OF RADIATION AND OF EFFECT It has been one of the most annoying puzzles of mitogenetic experiments that there seemed to be no proportionality between cause and effect even when polarisation is excluded. We could not expect this with detectors involving secondary radiation by old cells, such as the onion root, BAKON'S yeast plate, or the volumetric yeast method. But even with the yeast plate of TUTHILL and RAHN, where mutual cell influences are practically excluded, the percentage of buds was not at all proportional to the length of irradiation time. When freshly prepared detector plates were exposed for different lengths of time, the following percentages of buds were found (after 2 hours of incubation) : ANALYSIS OF THE MITOGENETIC EFFECT 123 minutes exposed for . . 10 20 40 % o/ /o o/ /<> / /o through quartz . direct 21.0 22.5 25 23 36.5 37.5 24.5 27.5 The sender was a 6 hours old yeast surface culture. The exposure of 20 minutes produced a strong effect, either directly or through quartz, 15% more than the control. If there were proportionality, the 10-minute exposure should have produced an increase of approximately 7%, and the 40-minute exposure a 30% increase. Neither of these other times showed any great effect, however. This may be explained by the recent discovery of WOLFF and HAS (p. 43) that nutrient media produce secondary radiation when they have been in contact with microorganisms. The entire detector plate begins to emit radiation as soon as it is exposed to a sender. The intensity of secondary radiation does not depend so much upon that of the primary source as upon the chemical composition of the medium. All hope for proportion- ality must be given up in this case. Only with a medium which does not produce secondary radiation, does a biological measure- ment of intensity seem at all possible. tJ. THE MINIMAL INTENSITY REQUIRED FOR AN EFFECT All measurements of the intensity of mitogeuetic rays are very inaccurate, but the order of magnitude of the strongest senders appears to be about 100 to 1000 quanta per cm 2 per second. The detector plate by BAKON is completely covered with cells, but that of TUTHTLL and RAKN has single cells. The pro- bability that a yeast cell of 6x7 JLI is hit in one second, assuming 1000 quanta/cm 2 /sec, is P = 0.00000042 x 1000 = 0.00042 The probability of being hit in one minute is 0.0252. It will require 40 minutes of continuous, uniformly dispersed radiation before each of the yeast cells is likely to be hit by one quantum of ultraviolet light. Since we find the strongest effect under this 124 CHAPTER VI arrangement at 20 minutes (see above), it would seem that one quantum per cell is sufficient to produce the mitogenetic effect. There has been a good deal of speculation as to the mechanism by which one single quantum could affect the cell so greatly. However, since certain solutions such as blood serum, or broth in which bacteria have lived or are living, will produce secondary radiation, the assumption of a single quantum acting upon the cell has become unnecessary, even improbable. The raisin agar upon which the yeast is spread will gradually become transformed into a secondary sender by the very presence of the yeast. Irradiation will then set the entire mass of agar radiating, and the number of quanta thus produced, or absorbed by the cells, cannot be estimated. It is known that yeast cells, or onion root cells, or pulp of tissues, yield secondary radiation; it seems safe to assume that living protoplasm generally will respond in this way. Then, if the cell absorbs one or a number of quanta of ultraviolet, the entire cell begins to radiate, not visibly, but measurably. This induces a state of excitement, and it is quite probable that a more rapid cell division may be brought about, provided that the cell is at the proper cytological stage. Possibly, the synthetic powers of the cell work to a certain morphological and physiological culmination which can be released only by a very accurately measured impulse, i. e. the absorption of one quantum of ultra- violet of fairly definite wave length. Considering the systematic arrangement of all molecules in the cell, it can be well imagined that such a release will start many wheels turning, many processes going on automatically and exothermically, until cell division is completed. This is, in slightly different terms, GURWITSCH'S original conception of the mechanism of the mitogenetic effect. He claimed, and seems to assume oven now that no cell division is possible without this external, ultraviolet stimulus. It would appear that single -cell cultures of bacteria and yeasts were a proof against this assumption, but they may be explained in some other way. One fact, however, makes the above explanation too simple. Every fermenting yeast cell liberates, within the cell, energy of definite mitogenetic wave lengths, namely of 1900, 1910, 1930, 1950 and 2170 A (p. 37). If all yeast cells produce this wave length, how can cells be stimulated by the same wave lengths from ANALYSIS OF THE MITOGENET1G EFFECT 125 an external source ? We may return to the first of our fundamental facts that only at a certain cytological stage, cells will react to mitogenetic radiation. No reaction has been observed at the stage of most active multiplication which is also that of most active metabolism. A very definite and strong response was obtained at the first stage of the rejuvenation process. If we could make the assumption that during the period of sensitivity, the cells show no metabolism, or at least emit no ultraviolet, then the mitogenetic effect could be easily explained. But the assumption is not justified. RAHN (1928) and RAHN and BARNES (1932) found that old yeast cells, compressed baker's yeast as well as beer yeast stored for several weeks at low temperature, fermented strongly within 10 minutes after being placed in sugar solution. Whatever the explanation, it is certain that the mitogenetio effect does not occur merely through the increase in energy content of the cell. I). THE HARMFUL EFFECT OF OVER-EXPOSURE The harmful effect of over-exposure is more easily under- stood by the secondary radiation of the cell and of the medium. Before this was found, the stimulating effect which the first quantum had produced, appeared to be counteracted by the ab- sorption of a second quantum. Now we realize that the first as well as the second quantum are probably multiplied manyfold within and outside the cell. It has been shown (p. 43) that too long exposiu-e destroys the ability of a solution to produce secondary radiation. After a day or two of rest, this property returns. Nothing is known about the chemistry involved, but the assumption of an equili- brium, disturbed by irradiation and slowly re -established after discontinuance, fite best into our present conceptions of life functions. Something similar to these effects may happen in the cell. We have already seen that very likely they are all capable of secondary radiation. They also will become exhausted upon long -continued exposure (p. 110). Moreover, it has been shown that recovery is slow. If we assume that all cells are brought to a state of radiation, or excitation, but that only those cells which 126 CHAPTER VI are at the proper cytological stage can respond to this stimulus by dividing more rapidly, the cells of other stages will soon become exhausted. This would mean at first a normal rate of cell division, and eventually a temporary interruption of mitosis, on account of exhaustion of certain chemicals in the cells, by the prolonged secondary radiation. Since exhaustion of solutions lasts for a day or two (p. 43) and exhaustion of cells for hours (p. 110), it would be difficult to explain the periodical alternation of stimulation and depression observed with long- continued irradiation by SALKIND (p. 116). The data of WOLFF and RAS (Table 25, p. 77) also seem to indicate recovery from depression though irradiation is continued. The time during which mitogenetic effects can be observed seems to vary with the detector. The sharpest limitations observed aro those by WOLFF and RAS (Table 12, p. 44) : strong positive effect with 5 minutes exposure, none whatever with 4, 6, 7 or 8 minutes, etc. This is doubtless caused by the uniform age of all cells in this kind of detector while BARON'S yeast plate with cells of many different physiological stage*, has a more gradual range of response and tolerance. E. STORAGE OP MITOGENETIC CHARGES FERGUSON and R\HN (1933) observed that bacterial cells could be kept in their old environment for 2 hours after exposure to mitogenetic rays, and still show stimulation of growth when transferred to a fresh medium (Table 36). Table 36. 3-day old culture of Bacterium coll, irradiated by an agar culture of Bacterium coli for 30 minutes transplanted immediately after irradiation transplanted 2 hours after irradiation Control Exposed ( Control Exposed start 4950 5050 4350 3950 after 2 hours . 4950 5750 3700 5900 3 . 4 5100 5600 6500 8700 3650 5200 6 24500 83500 10500 44000 , 8 . 234200 1500000 _ ANALYSIS OF THE MITOGENETIC EFFECT 127 The storage of energy as such appears out of the question. A continued internal secondary radiation is also impossible. We can only assume that the cells were changed chemically, that the unknown process of rejuvenation was released, but could not materialize under unfavorable environmental conditions ; as soon as this situation was altered, rejuvenation set in at once. This observation may eventually help to locate the exact process released by the mitogenetic impact. F. MECHANISM OF THE BARON YEAST DETECTOR In his monograph, GUEWITSCH devotes 50 pages to the analysis of mitogenotic effects in the BAKON yeast plates. The limited space of this book does not permit detailed quotation, especially since the complexity of this detector leaves too many possible explanations. However, since it is a good parallel to multicellular detectors, we shall at least give a brief summary here. The complex structure of the BARON yeast plate, with old cells, beyond the stage of cell division, on the surface, and with normally-dividing cells at the bottom, has been described in detail on p. 66. The old surface cells react upon irradiation by producing secondary radiation. They can emit only a definite (though unknown) amount of radiation. After that, they are exhausted and remain inactive, though absorbing ultraviolet, for the duration of the experiment. The number of these secondary senders decreases therefore gradually during exposure. The absorption of one quantum is sufficient to induce secondary radiation in a surface cell provided that the cell is not too old. Secondary radiation consists in the emission of a number of quanta. Since the emission takes place in all directions, the intensity decreases very rapidly with the distance from this cell. However, since in this detector, cells are lying closely side by side, a quantum emitted through secondary radiation may be absorbed by another reactive cell which then, on its part, emits new secondary quanta. On p. 110, it has been shown that in these yeast plates, mitogenetic effects may be observed 10mm. removed from the exposed cells. Some of these secondary quanta will penetrate into younger cells, and stimulate them to bud formation. 128 CHAPTER VI GURWJTSCH considers the "mitogenetic field" similar to an electro-magnetic field. He believes that a uniform stream of quanta striking a cell from all sides will not produce a mitogenotic effect. The mitogenetic stimulus consists in the one-sided discharge (release) of a neighboring secondary sender. GUBWITSCH assumes that if two or more quanta strike the surface of the detector at the same moment, the peripheries of the streams of secondary quanta may be partly superposed and thus by interference, pro- duce no effect ; though the total energy is increased, the potential necessary to initiate cell division is lacking. Such "equalization" will occur more commonly with physical sources of ultraviolet because the light is more uniform, while in biological sources, radiation comes from many cells unevenly distributed. Con- sequently, there is less equalization, and therefore a relatively stronger mitogenetic effect must be expected from biological senders. By irradiating a liquid bacterial culture in quartz from above and below at the same time, Miss FERGUSON, in an unpublished experiment, obtained a strong mitogenetic effect. The two radi- ations did not cancel. Such interference seems rather improbable if we look at the mitogenetic effect as a photochemical one. We would not expect the reaction between hydrogen and chlorine (p. 46) to be suspended if the gas mixture were irradiated from two opposite sides. Such interference is imagineable in a one- dimensional system, e. g. a nerve fiber, but hardly in three- dimensional media. G. MITOGENETIC EFFECTS IN MULTICELLULAIl ORGANISMS There is one essential difference between the cells of unicellular and multicellular detectors which must be kept in mind to prevent misleading generalizations. Bacteria and yeasts in the detectors mentioned have a very large amount of food at their immediate command, whereas in tissues, e. g. in onion roots or in the cornea, the food supply is limited. Thus, in the latter case, there may not be enough food readily available to permit rapid cell division, even after adequate stimulation; or after a premature mitosis, the food may be insufficient for continuing at a subsequent normal rate of cell division. ANALYSIS OF THE MITOGENETIC EFFECT 129 The one multicellular detector that has been studied cyto- logically is the onion root. The interpretations of the onion root effect by GURWITSCH (p. 65) and by REITER and GABOR do not agree. The latter have based their intrepretation upon a cyto- logical analysis of the onion root which deserves attention because it suggests a close analogy of the root with the BARON yeast plate. The root can be divided into transverse cross sections which may be designated by the number of successive cells from each section to the tip. This number is fairly uniform whether Figure 42. Distribution of various cells in the onion root. Below: cross section through the root, with its vascular bundle ; the shaded zone indicates the sensitive part of the root. Above: distribution for the four cell types in different parts of the root; the abcissa represents the distance from the tip, measured by the number of cells. ft -- elongated, resting cells; a = short dividing colls. a t newly- born cells, a 2 ripe nuclei, a 3 mitotio stages. the successive cells are counted in the center of the root or along the sides. The most sensitive region in respect to mitogeiietic rays is approximately 50 to 70 cells from the tip. REITER and GABOR studied the distribution of cell types along the root. They distinguished the a-type, short actively- growing cells, and the /?-type, elongated, resting cells. The first type could be subdivided into 3 nuclear stages : a^ = newly-born nuclei; a 2 ripe nuclei; 3 = various mitotic stages. The distribution of these types is shown in Table 37 and fig. 42. In the root tip, as far as about 25 cell layers upwards, no colls of the /J-type, i. e. no resting cells are found. All cells arc in active division. In the region which is 125 or more cells distant from the tip, practically all cells are resting ; dividing stages arc very rare. The zone of mitogenetic sensitivity contains cells of all types, the resting cells amounting to less than half of the dividing types. To analyze the effect, the authors raised two questions: How muoh does the exposed side of the root differ from the Pro to plasma - Monographien IX : R a h n 9 130 CHAPTER VI. ANALYSIS OF THE MITOGENETIC EFFECT Table 37. Distribution of the Nuclear Stages a i a a 8 P newly- born ripe dividing resting nuclei nuclei nuclei nuclei 21 64 15 50 cells upwards from the tip 16 56 11 17 80 5 21 3 71 opposite, shaded side ? and How much does the exposed side differ from the normal ? The latter question could be answered only by analogy. The conclusion was that "under the influence of mitogenetio irradiation, all cells born during the experiment remain in the actively-dividing stage, and produce again ripe nuclei while normally, without irradiation, a certain percentage would go into the resting stage. On the opposite side of the root, more cells go into the resting stage than would normally do so". GUKWITSCH (1932) does not agree with this interpretation. He doubts the possibility of accurately distinguishing between nuclei of resting and dividing cells. He criticizes the method of counting "ripe nuclei" only, and points out that a decrease of mitoses in the opposite side of the root has been observed occasion- ally, but only in about half of all his (GuRWiTscn's) and also of ROSSMANN'R experiments, and this can be accounted for by the method of sectioning and counting. There is a certain similarity between the onion root thus described, and the BARON yeast plate. Both consist of many closely-packed cells; both have the oldest, non-dividing cells on top, and very young, dividing ones at the bottom. Jt will be seen later that the onion root is irradiated continuously from above, by the bulb, and this stimulus is transmitted by secondary radiation of the old cells to the young, growing cells in the root tip. CHAPTER VII THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS IN BIOLOGY, MEDICINE AND AGRICULTURE A. UNICELLULAR ORGANISMS (1) Emission at Different Physiological Stages: It had been emphasized throughout this book that intense mito- genetie radiation lias been observed almost exclusively in young, aetively growing tissues or cell cultures, while fullgrown ones do not radiate at all, or only rather weakly. According to GURWTSCII, we must distinguish between two sources of radiation, the one emitted at the moment of nuclear division, and the other resulting from general cell metabolism, such as oxidation, proteolysis, glycolysis. A culture of yeasts or bacteria should be a good sender as long as either the cell division is rapid, or the metabolism remains active. Under optimal condi- tions of food and temperature, both these functions should cease almost entirely within 24 hours after transfer. Fig. 43 shows the development of a culture of Streptococcus lactis at 21 C (RAira, 1932, p. 401). The loft-hand curves represent the multiplication of the bacteria and the gradual accumulation of lactic acid from sugar, by glycolysis. The right-hand curves show the increase* in each 3-hour interval. These increases must be proportional to the intensity of radiation of the culture, as it is apparent that there can be no other important source of radiation. The bacteria starting with 38 000 cells per cc., can have emitted a noticeable degree of mitogenetic radiation only between 12 and 24 hours. Before that time, the radiation per cell might have been quite as strong or stronger, but the number of cells was too small; while afterwards the cells, though more than a billion per cc., have ceased to produce acid, and therefore to radiate. 9* 132 CHAPTER VII With a heavier inoculation, e. g. by flooding the surface of an agar plate with a suspension of yeasts or bacteria, radiation begins sooner because the "active mass", i. c. the number of cells, is greater. At lower temperatures, the intensity is less, but the phase of active radiation is prolonged. With processes which do not result in an inhibiting product like acid, the period of active radiation may also be longer. This a priori deduction is made doubtful by the secondary radiation of the medium in which the bacteria grow* The actual radiation of the bacteria them- selves must follow the lines of fig. 43; the emission by the entire culture may not. In- tensity of secondary radiation may not be proportional to primary intensity, and be- Figure 43, Development of a culture of Streptococcus lactis in milk. Age of culture in hours. Full line: number of cells per cc.j dotted lino: mg of lactose decomposed in 100 cc. left: total cells and total lactose fermented; right: cell increase for each successive 3-hour period, and lactose consumed during these 3 hour periods. sides, since the nutrient medium is continuously changed by the bacteria, its property as secondary sender may change. However, the theoretical deductions are essentially verified by the observation of WOLFF and RAS (1932) that an agar surface culture of Staphylococcus aureus remains actively radiating until approximately 18 hours old. When the cultures are lull- grown, i. e. when there is no further appreciable increase in cells, nor in metabolic products, the culture ceases to radiate. Whether metabolism without cell division can produce mitogezietic rays, was decided in the affirmative by a simple experiment of the authors with bakers' yeast suspended in sugar THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 133 Table 38. Mitogenetio Effects from Protozoa in Glucose Solution Without Sugar i imme- diately \fber Addil 15 minutes later ion of Su$ 20 minutes later +36 +24 $ar SOmintues later Opalina +4.4 2.6 +2.5 +40 +20 Paramaecium +2.7; +2.0 2.8; +1.0 1.0; +3.0 7 + 7 +3.7 + 11 + 12 +27 3.8 + 14.0 solution. This yeast cannot grow under the experimental condi- tions because too many cells are present; however, even if slight growth were to occur, many hours would be required to overcome the lag. During the first hoiir, therefore, no radiation from cell division is possible while alcoholic fermentation starts at once. An interesting set of data on the radiation of Hydra fiisca, the well-known fresh-water polyp, has been given by BLAOHER and SAMARA JEFF (1930). They found that a pulp of the entire organism radiates. If dissected, the hypostom and the budding zone radiate while the other parts do not. No increase in emission could be detected during regeneration. The common infusoria do not appear to radiate under normal conditions, but produce a secondary radiation. According to Zoglina (quoted after GURWITSCH 1932, p. 64) the following mitogenetic effects were, obtained: from Opalina 0; +4; +3; +6; +12 irradiated by blood +47 frog heart .... +21 spectral light . . . +15; +19; +38; +50 from Paramaecium +2; +1; +3; 3 , irradiated +20; +22; +13; +40 Radiation could also be obtained from protozoa when glucoso was added to the culture; it required about 15 minutes before radiation became noticeable. The mitogcnotic effects observed are given in Table 38. A very interesting claim has been made by Acs (1932). Ho succeeded in increasing the mitogenetic effect of muto-induction of bacterial cultures (Bacterium typhi-murium) by using the detector of one day, i. e. the irradiated culture, as sender for 134 CHAPTER VII the next experiment. His data show a very consistent increase in the mitogenetic effect through 6 to 7 such transfers. The proof is not complete, however. It is well known that old bacterial cultures require a longer time to start growing than young ones. The rate of growth of old cultures can be increased for several successive transfers, without any irradiation. Without accurate statements concerning the number of cells in the sender and in the detector, no definite conclusions can be drawn. (2) Reaction Upon Irradiation: Bacteria* and yeasts, when transferred from an old culture to a fresh medium, do not start growing at their maximal growth rate. The old cells undergo a rejuvenation process, morphologically and physiologically (see p. 120). This results in a very slow growthrate during the first hours after transfer. The bacteriologist calls this the lag phase. It has been observed by RAHN (1907), PENFOLD (1914) and others that bacteria after being transferred from an old culture rejuvenate more rapidly if many cells have been trans- ferred while with a few cells in a large volume of fresh medium, rejuvenation is slow, and single-cell cultures frequently die. This observation had been very puzzling to biologists since tho opposite should be expected, until it could be explained as a simple stimulating effect by mutual irradiation of the cells. The closer they are together, the stronger must be the effect, the quicker the recovery. The best example is very likely that of HENKICT (1928, Table I) with yeast. He inoculated increasing amounts of yeast into flasks of sugar -peptone solution, so that all the decimal gradations from 2095 to 20949750 cells per cc. were present. These different inocula resulted in very different lag periods. The times required to reach the maximal growthrate were: with an inoculum of Lag Period Average cell distance 1 ) 20 949 750 cells per cc. 2 8 hrs. 0.036 mm. 2094975 ., 4 8 0.086 209498 612 0.192 20950 1224 0.422 2095 2436 0.914 1 ) The average cell distance is computed from the equation distance = cell diameter ||/~~ 74.04 ~ J \ cell volume in 100 cc. / Jt is absolutely correct only with spherical organisms. The volume of one yeast cell is taken as 1 18 ^ 8 , the average diameter as 6.4 //. THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 135 Table 39. Growth Acceleration by Mutual Irradiation with Saccharoniyces ellipsoideus Number of Buds per 100 Cells Experiment No. 1 Experiment No. 2 Inoculum : 8,000 cells 80,000 cells 8,000 cells 80,000 cells start | after 3 hours . '' after 5 hours . 3 16 after 6 hours . ' . 5 49 after 7 hours . ' 14 60 after 10 hours . 1 17 91 after 12 hours . ... -- 35 91 To these proofs that the lag phase is shortened by the mutual irradiation of (jells, BAKON (1930) added another. After having shown that bud formation in fresh yeast cultures begins sooner when the inoculum is larger (Table 39), he showed that this difference disappears when gelatin is added (Table 40). ftelatin Table 40. Prevention of Growth Acceleration by Absorbing the Mitogenetic Kays by Means of Gelatin Number of Buds per 100 Cells Inoculum start after after after after 3 hours 5 hours 7 hours 8 hours after 10 hours after 12 hours Experiment A Experiment R 8,000 cells 80,000 cells 8,000 cells 80,000 cells ! - i 2 2 18 21 12 15 30 32 __ . 35 33 41 42 48 52 62 61 absorbs mitogenetic rays very completely (see p. 59), and while all other conditions of life remained the same (gelatin contains no nutrients for yeast), muto -induction was prevented, and, as a result, the lag phase became independent of the cell concen- tration. 136 CHAPTER VII S - H O S TJ ^ 0) a | *-* o a ^ 2 1 l o F t o d W o t oo S O !| -go rH rM O rH O i Hi 5 ^3S ic i- S8 00 rH "8 ,? ^ r} A A f Mitoses Exposed Induction Effect % living morula stage, animal hemi- sphere 750 931 +24 living morula stage, vegetative hemi- sphere . . 428 410 4. living embryo, before hatching ; dorsal side 230 268 + 16 living embryo, before hatching; ventral side 492 471 4. pulp of medullar plate 324 409 +26 pulp of embryo without medullar plate pulp of embryos' brains I 482 627 505 832 + 5 +33 pulp of embryos' brains 11 pulp of embryos' brains III .... pulp of entire embryo without brain I pulp of entire embryo without brain 11 pulp of embryo's liver 865 920 724 689 631 1042 1076 728 696 635 +20 + 17 + 1 + 1 pulp of brain of grown animal I pulp of brain of grown animal II 617 848 632 847 + 2 Of the insects, only the larvae of Drosopkila have analyzed. They do not begin to radiate until shortly before pupation, and cease to radiate 90 hours after this (see also p. 169). Quite different is the behavior of the chicken embryo. SOKIN and KTSLIAK-STATKEWITSCH (1928) working with entire embryos two days old and also testing the brains of older embryos could find no evidence of radiation. However, during the second and third day of incubation, positive results were obtained with the liquefied zone around the embryo. (d) Embryos as Detectors. The only example of embryos or larvae as detectors of mitogenetic radiation is that of BLACK wit and associates who could make the fore leg of a tadpole grow more rapidly by exposure to radiation (see p. 168). The morpho- logical changes in sea urchin larvae observed by MAC; ROT: may also be mentioned here (see p. 165). Pro to plasma - Monographien IX : R a h n 10 146 CHAPTER VII D. TISSUES OP ADULT ANIMALS Tissues as Senders: Until recently, most of the tissues of adult animals had been thought to be non-radiating. If we consider radiation to be produced largely by the chemical reactions in the cells, it would seem that all tissues should radiate quite strongly. GUBWITSOH (1934) has given some convincing ex- planations for the differences between radiating and non-radiating tissues. The very strong absorption of these short-waved rays has already been emphasized repeatedly. GURWITSCH found that extremely thin films of oil or related substances, films of practi- cally only one molecule thickness, suffice to absorb completely a mitogenetic radiation of normal intensity. On the other hand, equally thin films of substances capable of enzymatic cleavage, such as lecithin, are also capable of secondary radiation (see p. 45), and they will pass on radiation not as a beam, but in all dimensions of the film. Thus we observe strong radiation in blood which contains no membrane, and in nerves which contain plenty of lecithin. These two will be treated separately in the next two chapters, on account of their importance. The cornea of the eye is also a good sender, being a con- tinually renewed tissue. WOLFF and RAS (1933c) consider it to be one of the strongest sources, about 10 times as strong as blood. GTJBWITSCH has recently favored the peptic digestion as a strong and fairly constant source. Other actively reacting digestive enzymes are likely to be good senders, but would hardly be considered as tissues. POTOZKY, SALKIND and ZOGLIKA (1930) studied the tissues of two crabs, Carcinus ma-enas and a species of Pachygrapsus ; they found the pulp from gills and from testicles inactive, while the hepatopancreas radiated distinctly. This organ is the seat of active proteolysis. Probably, autolyzing muscle would radiate. With vertebrates, negative results had been obtained by all earlier workers with lymph glands, testicles, ovaries, skin, liver, and with the resting muscle. The working muscle, however, proved to be very active. Negative results, particularly when obtained with tissue pulp, are now considered of little significance since better methods, especially with organs in situ, showed distinct radiation. THE SIGNIFICANCE OP BIOLOGICAL RADIATIONS ETC. 147 On p. 65, SIEBEKT'S results have been given, showing that the muscle radiates only during work, and that pulp from resting muscles does not radiate, while that from active, tired muscle does. According to recent experiments by FRANK and KBBPS (quoted from GUKWITSCH, 1932, p. 149), this result was caused by an irritation of the muscle during grinding. By dropping the organ into liquid air and grinding it while frozen, the results were the opposite. In pulp from working muscle, the pH changed from 5.96 to 5.82 during the experiment, and radiation was absent ; while with rested muscle, the pH changed from 6.33 to 5.85, and radiation was present. This refers only to the pulp, however; the muscle in situ radiates strongly while working, but weakly also when resting. The spectral analysis indicates that the main source of muscle radiation is not glycolysis; partly it is an oxidation process, partly of unknown origin. It must be remembered that there is no quantitative relation between the total energy liberated by a chemical reaction, and the ultraviolet radiation emitted (see p. 41). It may well be that some proteolytic process whose energy output is negligible for the muscle work emits most of its energy in radiant form, while glycolysis, with a much larger total energy output, emits only a very small fraction of it as mitogenetic rays. A good example of applied spectral analysis should be men- tioned in this connection. GUKWITSCH had been greatty puzzled in his earlier work by the observation that the very distinct radiation from the rabbit's eye disappeared during starvation, but reappeared after 8 days of continuous starving. Several years later, spectral analysis brought a simple explanation (GuRWiTSCH 1932, p. 67). The normal radiation is glycolytic, and ceases during starvation because of lack of sugar. After prolonged starvation, proteolysis of the tissues becomes necessary for the continuation of life processes, and this produces a proteo- lytic spectrum- Tissues as Detectors: Only a few instances are known where fullgrown animals or their tissues reacted upon mitogenetic rays. The role of radiation in wounds will be treated in one of the following chapters. With multicellular organisms, the stimulation of the growth - rate is not so readily proved. In growing organs, the cells lie so 10* 148 CHAPTER VII closely together that the optimal intensity of radiation may already be furnished by the organ itself. Emanations from outside, if they are not absorbed completely before they reach the region of growth, very likely can only be harmful. A few instances arc known, however, where the rate of cell division is accelerated. The onion root is usually considered the classical example though the data have been interpreted somewhat differently by REITER and GABOB- (see p. 129). Another example is the cornea (see p. 84). Further illustrations am the effects of resorbed tissue in different stages of amphibian metamorphosis (see p. 167). These latter results together with those obtained with em- bryos (p. 144) suggest very strongly that the developmental mechanism controlling size and form makes use of ultraviolet radiations as well as of purely chemical means to achieve its purpose. This and the possibility that neoplasms, especially cancers arise through mitogenetie radiation will be discussed in later chapters. The only animal tissue that has been actually used as detector is the cornea. According to GURWITSCH, it yields vory good results (see p. 84). E. BLOOD RADIATION A comprehensive review of all work on blood radiation has been given by W. SIEBER.T (1934) in the second volume of ,,Hand- buch der allgemeinen Hamatologie". Blood radiates quite strongly, even in adult animals and men, the only exception being extreme old age and a very few diseases which will be discussed later. The blood of various mammals, birds and amphibia, and also the hemolymph of the crabs Care in us and Pacliygrahsus and of the clam Mytilt/s edulis has been tested, and strong mitogenetic effects have always been observed. Ac- cording to KANNEUJESSER and KAZWA (quoted from GTJRWTTSCH, 1932, p. 124), dog's blood which possesses only vory weak gly- colysLs gives very fluctuating results. Blood radiates within the veins or arteries as could be shown by removing the tissues around the veins or arteries, and exposing a detector to the blood radiation through the inner wall of the blood vessels which is transparent to these rays (spectrum see fig. 44). THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 149 Outside the blood vessel, blood loses its radiating power within 10 to 15 minutes; this holds also for the hemolymph of crabs. In hemolyzed blood, radiation can be restored by adding glucose. For radiation experiments, blood is mixed with an equal amount of a 4% MgSO 4 solution to prevent clotting, or it is hemolyzed with distilled water (PoTOZKY and ZOGLINA, 1929). It is possible to dry blood on filter paper and thus preserve its radiating power for 2 to 3 days. Since this might be of practical importance, especially in diagnostic medicine, GUBWITSCH'S method (1932, p. 121) shall be mentioned here: The blood should be drawn without previous disinfection of the skin (iodine, alcohol) because traces of disinfectants appear to inhibit glycolysis. The drops are spread widely on the filter paper to prevent coagulation and thick layers. Drying must be accomplished very rapidly to preserve full radiating power; the dried sample should be kept dark. In making the test, detector and everything else must be prepared before water is put upon the dry blood. The spot is cut into very small pieces, and softened in a tiny shallow dish with 6 to 6 drops of distilled water, under continuous stirring. As soon as the water becomes dark red, it is drawn off with a pipette and used at once for irradiation. From the adding of the water to the beginning of exposure, not more than 1 to 1.5 minutes should elapse. HELNEMANN'S method for detecting blood radiation has been mentioned on p. 72. With starving mammals, the blood does not radiate, but can be brought back by adding glucose. POTOZKY and ZOGLINA starved rats until they had lost about 30% of their body weight. The mitogenetic effects obtained with the blood of those animals were without addition 2.5 5 2 with 2% glucose 21 44 36 39 These strong effects could be obtained only by adding much more sugar than is normally contained in the blood. A very important observation is the disappearance of blood radiation after continued work. BBALNESS (quoted from GUR- WITSCH, 1932, p. 132) investigated a number of laborers before and after the day's work. Table 47 gives a few of his data which show consistently no radiation immediately after long- continued work, but normal radiation after 2 hours of rest. This must be considered when making blood tests for diagnostic purposes. 160 CHAPTER VII Table 47. Mitogenetic Effects from the Blood of Factory Workers before and after Work Person Month before work at the end of the day's work 2 hours later K October 25 6 K K January December 24 45 -! 36 26 B November 38 3 * 31 B January 28 6 22 M January 32 __ 5 11 , E S S January January January 33 35 21 2 13 12 27 10 27 D January 26 1 30 WASSJLIEFF (1934) repeated the investigation with mental work (calculation). The; first results showed a decrease in radiation, but this happened before psychic analysis showed mental fatigue, and it could be demonstrated that muscular work connected with calculation caused the decrease in radiation while the mental work as such does not affect it. The spectrum of the radiation of rabbit blood from the streaming blood in the vena saphena is shown in fig. 44 as measured by GOLISCHEWA (1933). It has practically all the lines charac- teristic for glycolysis, proteolysis, creatin hydrolysis, phosphatid hydrolysis, and oxidation, and, in addition, some new lines. Of these latter, the lines 192030, 194050, 196070 and 2000 2010 have also been found in nerve spectra (fig. 47). The main source of blood radiation with mammals appears to be glycolysis. In addition to the starvation experiments just mentioned, KANNEGIESSEK and KAWZA made extensive experi- ments with dog's blood, measuring the glycolytic power and the radiation of each sample. There was a good, though not quanti- tative, relation between the two. Intravenous insulin injection reduced glycolysis and radiation to zero. In some eases, addition of glucose (probably in overdose) interfered with radiation. Rat blood treated with heparin loses its radiation by NaF, but not by KCN. This suggests glycolysis, which is affected by THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 151 NaF, but not by KCN. Hemolymph of crabs, however, loses its power promptly in the presence of 0.0001 molar KCN, and so does hemolyzed rat blood. This suggests that in normal rat blood, glycolysis is the dominant source, but in hemolyzed blood, and in the blood of crabs (which contains no hemoglobin) oxidation is the deciding reaction. Of the various fractions of the blood, the serum radiates immediately after the blood is taken from the animal, but loses this power rapidly. It continues, however, to produce secondary 1 I n i n \m\ w \mt\m u Figure 44. Above: spectrum of streaming blood in the artery of a rabbit. Below: the combined spectrum of 5 common biological processes (oxi- dation O f glycolysis -^ G, creatinin hydrolysis C 9 protcolysis -- A r , phosphatid hydrolysis =_ 7*). radiation, even after several months (p. 43). The leucocytes, polynuclear as well as lymphocytes, radiate distinctly but not very strongly, according to KLENJTZKY (1932). The spectrum indicates glycolysis, oxidation and phosphatase action, and pro- bably proteolysis. The intensity of blood radiation is greatly changed after wounding (see p. 174). During the metamorphosis of amphibia, it varies enormously in the different stages of development. PROTTI (1931) observed a great decline or complete absence of radiation in blood during senility, and this result was confirmed by HEINEMANN (1932). In consequence PROTTI injected blood from young persons into old ones. Distinct clinical evidence of "rejuvenation" by this treatment has been claimed, and the blood of old people thus treated radiated again. Table 48 gives some of PROTTI'S results. A somewhat different way was used by HEINBMANN and SEYDERHELM to renew blood radiation in old persons. In his effort to find the reason why ultraviolet light was beneficial to animals, SEYDERHELM (1932) succeeded in isolating a compound 152 CHAPTER VII Table 48. Mitogenetic Effects Produced by the Blood of Old Persons After Injection of Blood from Young Persons Mitogenetic Effects Blood Group young blood old blood old blood after injection Clinical Results A 70 5 30 very good A 20 3 4 none A 60 29 38 *good A 70 15 38 very good A 20 10 10 little A j 80 30 53 very good B 72 16 32 good 81 24 40 very good O 62 30 37 good 22 17 20 little from the blood corpuscles which he called cytayenin. When this substance was injected into healthy persons, it increased blood radiation (fig. 45). With patients suffering from anemia, it washed the newly-formed blood corpuscles out of the bone marrow and produced a normal blood picture (HEINEMANN, 1932). This was possible in all cases of secondary anemia, but not in pernicious anemia in which the bone marrow no longer produces blood cells. When injected into patients 70 to 80 years old, without blood radiation, cytagenin produced either no effect or a slight depression effect during the first days, but after 1 to 2 weeks, the blood began to radiate, and repeated tests showed no decrease. How long these experiments were continued, HEINEMANN did not state. The blood of asphyxiated animals does not radiate. It loses this power before the animal is dead, even when the process is still reversible. Quite remarkable is the persistence of blood radiation during illness. L. GUBWITSCH and SALKIND (1929) obtained radiation from the blood of tuberculous guinea pigs until almost imme- diately before death. Diabetes, lues, osteomyelitis and ulcer of the stomach did not decrease blood radiation. The only diseases which showed this conspicuous absence were leucemia, and severe septicemia with high fever (also poisoning with nitro-benzene), THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 163 and cancer. This was verified by SIEBERT who observed absence of radiation in severe cases of sepsis, pneumonia, and scarlatina, Injections 11 / 2 3 Jim / 83 10 11 Figure 45. Increase of blood radiation by injection of cytagenin. At loft: black indicates radiation before injection, white after injection. / and // are normal healthy persons, III is a very old person, IV is a carcinoma patient. At right: the effect of repeated injections upon a carcinoma patient. and by GESENIUS (1930). HEINEMANN (1932) added chronic; tonsilitis to this non-radiating group; in some cases, radiation appeared again soon after the removal of the tonsils. l^ig. 46 shows the results obtained by GESENIUS (1930) who measured blood radiation by the decrease of yeast re- spiration (see p. 84). Since only very few easily recognized diseases prevent blood radiat- ion, it can be used for the diagnosis of cancer (see p. 180). A very comprehensive re- view of his extended research blood radiation has been on given by PBOTTI (1934 a). All measurements refer to appar- ently healthy persons, and are Decrease of Respiration Figure 46. Decrease of respirat- ion of yeast irradiated with the blood of healthy and sick persons. 154 CHAPTER VII made by the yeast bud method by means of the hemoradio- meter (p. 66). En a large number of tables and graphs, he demonstrates the decrease of radiation with age, the stronger radiation of tall, slender people as compared with short indi- viduals with a tendency for stoutness, and the slight increase of male over female blood. In the same individual, radiation in- creases 1 to 2 hours after each meal, and decreases with physical fatigue. During the winter months, it is slightly lower. It is increased by high altitudes, also by a trip to the sea shore, further by inhalation of oxygen, though in this latter case, the effect does not last more than one hour. Menstruation and pregnancy have characteristic curves. PROTTI states that even among healthy individuals, "normo- radiant, hypo-radiant and hyper -radiant" types can be disting- uished, all reacting in a parallel manner upon the same changes. P. NERVE RADIATION AND THE CONDUCTION OF STIMULI IN ORGANISMS The results obtained with secondary radiation led GUR- WITSCH to the idea that possibly secondary radiation might be a controlling factor in the conduction of stimuli in plants and animals. Let us recapitulate briefly the facts obtained with onion roots (see p. 140). Irradiation of the older, upper part of the severed root produced radiation from the tip. Irradiation of the tip produced radiation from the older tissue. The impulse was conducted longitudinally at the rate of approximately 30 meters per second, but there was no conduction to the diametrically opposite side of the root. Later, FRANK observed that the same held true for muscles. The resting sartorius muscle of a frog when irradiated biologi- cally at a definite place for 10 seconds emitted a strong mito- geiietic radiation from a place 20 mm . away. In fact, at a distance of 20 mm., the effect was much stronger than at 10 mm. ; the intensity had increased by conduction. These results, together with the discovery of secondary radiation in nerve tissue, inspired in GURWTTSCH the bold thought that conduction of stimuli in nerves and muscles may be ac- THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 155 complished or aided by secondary mitogenetic radiation. This statement is possibly too blunt, but it expresses in a few words the ultimate aim. GUKWITSCH himself (1932 a) says that this theory may appear bold, but it seems justified to approach it experimentally, on account of several suggestive facts. One of them is the possibility of studying, by moans of the mitogenetic spectrum, the metabolism of the nerve in the resting and in the excited stage. It is evident that the chemical analysis can give only a rather incomplete picture of the chemistry of nerve stimulation, because it involves destruction of the nerve, while radiation can be observed without injury. The result of the spectral analysis of nerve radiation has, as a matter of fact, revealed some chemical processes which had not as yet been discovered by chemical analysis. The views concerning nerve radiation have undergone considerable change with the improvement of the methods. The older experiments by ANIKINT with the brain of adult sala- manders (see p. 145) and those by KEITJCR and GABOK (1928) with the sciatic nerve of the frog gave negative results. They were confirmed by WASSJLIEW, FRANK and GOLDEN BEKO (1931) who obtained positive results only with the olfactory nerve of the pickerel. By using the yeast volume (p. 73) as detector, KAJLENDAUOFF (1932) not only proved that the sciatic nerve of the frog radiated distinctly, but he could also study its spectrum by using intermittent exposure. The spectrum was determined with resting as well as with irritated nerves. Irritation of the nervo was accomplished either by cutting into it (traumatisation), or by electrical tetanization with platinum electrodes 3 4mm. apart, 8 12 shocks per minute (faradisation), or mechanically, by hitting with a light hammer, intermittently. All experiments were conducted with intermittent exposure by the rotating disk (see p. 105). This not only increased the intensity of the effect, but also eliminated the possibility of secondary radiation from the nerve induced by the yeast detector. The minimal total exposure required to bring about mito- genetic effects was approximately 6 minutes with the resting nerve and 2 3 minutes with the excited one. The detailed spectra are shown in fig. 47. The rather surprising fact was revealed that different kinds of irritation produce slightly different spectra. 156 CHAPTER VII Further, it was found that radiation at the point of irritation differs from that of the same nerve a short distance away. The spectra (of which each 10 A strip is the result of at least three determinations) show five well-known chemical pro- cesses: oxidation, glyeolysis, phosphatase action, cleavage of resting nerve pulp of resting nerve nerve, mechanically excited nerve, electrically excited nerve, excited by incision 20 mm. from point of electrical excitation 20 mm. from point of mechanical excitation combined spectrum of 6 common bio- chemical reactions (fig. 44) optical nerve, excited by illumination of the eye Figure 47. The spectra of the sciatic nerve of the frog under different con- ditions. The lowest line represents radiation from the optical nerve in situ. creatinm phosphate, and de-aminisation of amino acid A few lines were observed also which cannot at present be accounted for. 1 ) These five chemical processes agree with chemical investi- gations on nerve metabolism. Strange is the absence of certain lines. Fn mechanical and electric irritation, most of those con- cerned with do-aminisation are missing, and in the electrically- stimulated nerve, the glycolytic regions are absent while* in the spectrum of the same nerve, 20 mm. removed from the zone of *) Two lines of the glycolytic spectrum, 1930-40 and 195060, are missing in most of the nerve spectra while they all have the neighboring lines 194050 and 196070. This may be due to a slight shift in the cali- bration of the instrument. J^ine 2410 20 might also be accounted for in this way, as a shift of the preceding line to the right, or rather an error in the calibration to the left. However, the line 2000 10 in more than half of the spectra and 2370 80 in 3 of the spectra cannot be explained by the same error of calibration. THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 157 irritation, they are present. The creatinin lines are absent in mechanical stimulation. The spectra of nerve conduction lack entirely the phosphatase lines. This difference in the spectra raised the question of ' 'ad- equate" nerve stimulation. A good example of this has been furnished by ANNA GURWITSCH (1932) with the optic nerve of the frog. After decapitation and removal of the lower jaw, the* optic nerve was laid bare by dissection from the roof of the mouth. A small window, 1.5x2 mm., is sufficient to permit access to the chiasina and tracti optici with part of the optic nerve. The radiation from this was tested in the darkroom while directing a beam of light on one of the eyes. Increase in number of yeast cells by direct microscopic count was the method used to prove the radiation. The re- suits were always positive while controls without illumination of the eye showed consistently 110 effect. The spectrum is shown in the lowest line of Figure 47. It agrees essentially with those by KALENDAROFF. It has also some unknown lines agreeing with KALENDAROFF'S. Those corres- ponding to proteolysis (de-aminisation) are present only in part. These experiments have revealed that there are differences in the metabolism of the resting and the excited nerve, and that even the type of excitation may change the spectrum. It might be well to remember here GURWITSCH\S statement that these spectra represent "minimal spectra". The established lines are doubtless correct, but there may be other lines too weak to be recorded by the detectors used. These fine results induced ANNA OURAVITSOH (1934 a) to approach the more fundamental question whether this radiation of the optic nerve after illumination of the eye was only a simple secondary radiation, or represented an important functional part of nerve conduction. She laid bare the optic nerve, the optic lobes, the medulla oblongata and the spinal cord of living frogs, and kept these parts covered with the skin. For several hours after the operation, all nerves radiated strongly, but 24 hours later, lobes and hemispheres did not radiate while medulla and spinal cord usually continued to radiate weakly. As soon as light was directed onto the eyes of the frog, the lobes arid hemispheres radiated strongly while the medulla and spinal cord did not change. 158 CHAPTER VII When the optic lobes were still in the excited stage from the operation, they did not react promptly upon illumination of the eye. This suggested an interference between traumatic radiation and normal reaction upon optic irritation. The explana- tion could be verified by irritating electrically the sciatic nerve. Before the circuit was closed, the lobes reacted strongly upon illumination of the eye ; when the current was applied, the reaction upon light was weak and irregular; after ceasing the irritation, but continuing illumination, radiation ceased completely for a short time, and after that, the normal strong effect appeared. It had been shown at this time (p. 45) that secondary radiation usually is a resonant radiation responding only to the wave lengths characteristic for it. ANNA GUKWITSCH studied the reactions produced by various spectra applied to the chiasma of the optic system. The emission from an electrically irritated nerve made the lobes radiate weakly, while the hemispheres showed no effect. Yeast radiation, however, induced strong radiation in both. Addition of the glycolytic component (1900 1920 A from a monochromator) to nerve radiation produced also good radiation in lobes and hemispheres. The spectrum of the radiation of the lobes contains always glycolytic lines even when radiation has been brought about by exposure of the chiasma to rays from the, oxidation of pyro- gallic acid which are very different from those of glycolysis. It is, therefore, not a mere secondary radiation, but indicates the release of an unknown independant chemical reaction in the optic lobes. The extent of radiation in a nervous system during illu- mination permits thus the experimental approach to the problem of localisation of functions in the brain. In another paper, ANNA GUBWJTSCH (1934b) obtained differ- ent spectra from the hemispheres of the frog brain when the eyes were illuminated with different colors. Green light produced the lines of glycolysis, proteolysis, oxidation and phosphate cleavage. With red light, the last mentioned part was missing, and with blue light, there were no proteolytic lines. Probably, the metabolism of the nerve was not changed completely, and all lines were present, but their relative intensity was varied. The intensity of the colored lights used and the wave lengths of the colors are not mentioned. THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 159 The effect of the intensity of light was tested by ANNA GUBWITSCH (1932) by varying the distance between light and eye from 6 to 18 cm. (i. e. varying the intensity from 9 to 1) and determining the necessary exposure time. The result was as follows : Length of Exposure . . 1 3 5 10 15 20 25 30 35 seconds Induction Effect at 6cm. 5 19 22.5 4 -- Induction Effect at 18cm. 14 4 4 13 16 6 The weaker light (18 cm. distance) requires a longer time to pro- duce the effect, but there is no appreciable difference in the intensity of the effect. It could further be shown that continued exposure to light produces a long after-effect if the head is cut from the animal; in the living animal, however, radiation ceases when the eye is darkened (see Table 49). Table 49. Mitogenetic Effect of the Optic Nerve after illumination and darkening of the eye Seconds of Mitogenetic Effect Exposure dark 1 light 1 second darkening Isolated Head 20 2 22.0 21.8 20 2 14.5 12.0 20 6 20.1 14.6 Living Animal 15 4.5 22.5 2.0 10 15.5 2.5 Another important advancement in the effort to correlate nerve conduction with mitogenetic radiation is the accurate measurement of the velocity of progress of secondary radiation in the sciatic nerve. LATMANISOWA (1932) found this to be 30^3 meters per second (see p. 112) and this agrees, within the limits of error, with the rate of conduction of nerve stimuli. Very suggestive, though not positive proof, are also the experiments by LATMANISOWA on the "fatigue" of the nerve by continued irradiation with strong light (see p. 111). Later publications by LATMANISOWA (1933, 1934) have added another impressive fact to prove the role of mitogenetic 160 CHAPTER VII rays in nerve conduction. By exposing the sciatic nerve, with the attached muscle, in a moist chamber for about 2 hours to the radiation from protein digestion, the nerve showed all symp- toms of parabiosis. Two electrodes touched the nerve, and the place between them was irradiated. For about 2 hours, the nerve reacted nor- mally upon electric impulses, causing a stronger muscle contraction with a stronger impulse. After this time, the nerve became at first more excitable, but soon the muscle reactions became weaker and weaker, and then, the typical parabiotic stage set in, the contraction being stronger when the impulse was weaker. Finally, the nerve ceased to react altogether, and it took two hours after the removal of the mitogenetic source before the nerve had re- covered, and its reactions became normal again. This experiment has been repeated more than 40 times with the same success. Controls with gastric juice without protein were not affected. The nerve outside of the irradiated zone reacted normally at the same time when the exposed part of the same nerve showed parabiosis. The author quotes a paper by LAPITZKI who obtained the same effect by rays from a mercury vapor lamp in a few minutes. The nerve at the parabiotic stage has not ceased to radiate ; on the contrary, the emission seems to be much stronger than that of the normal nerve. It has not been possible as yet to produce muscle contraction, or a corresponding eifect, by irradiating a nerve. This may be due to the absence of an * 'adequate" stimulation. Perhaps, a combination of definite wave lengths is necessary to produce such effects. The one example of true adequate stimulation are the above-mentioned experiments by ANNA GrrrtWiTSCH with the optic nerve. It seems too early to speculate on the relation between the mitogenetic radiation of nerves and the action current. Though a number of physiologists oppose this idea, it does not seem impossible that the two observed facts may be some time be com- bined to produce a more comprehensive explanation of the nerve mechanism. THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 161 G. MORPHOLOGICAL EFFECTS While the classical example of morphological effects produced by biological radiation is that of sea urchin larvae 4 , they shall be preceded, for the sake of logical arrangement, by a short note on unicellular organisms. 1. Yeasts and Bacteria: CHRISTIANSEN (]{)28) observed striking morphological changes in yeast cells as well as bacteria under the influence of radiations emanating from menstrual blood. It seems that most of these experiments were* carried out without exclusion of chemical effects from volatile substances of the blood, but the same effects could be obtained when a quartz coverglass protected the test organisms perfectly against vapors from the blood or from outside. At times, the blood was strong enough to kill microorganisms ; more commonly, it affected their cell forms. With Bacterium coli, the non -motile cells immediately above the drop of blood were 3 to 5 times as long as the farther removed cells; Bacterium vulgare lost its pellicie formation ; Laclobacillus bvlyaricus grew to long threads without cell division ; Oidium formed no oidia. Streptococcus luctis and cremoris did not appear to be changed morphologically. Just as striking were the changes in yeasts. Frequently, retardation of growth was accompanied by an enormous expansion of vacuolcs, leaving the protoplasm only as a very thin layer around the cell membrane. In other cases, there was a decided tendency to grow into hyphae. Still other cultures showed for- mation of giant cells. Similar morphological changes could be produced by exposure* of yeast to saliva of apparently normal persons (FERGUSON, 1932). Particularly the large spherical cells with tremendously extended vacuoles and with complete loss of granulation are considered typical "saliva cells". It has not been possible, however, to produce these same forms by interposing a quartz eoverglass between saliva and yeast. Thus, tho physical nature of this phenomenon is not proved. On the other hand, it is evidently not a purely chemical effect of some saliva constituent because the yeasts grew quite normally in mixtures of equal volumes of saliva and raisin extract; these cultures must have obtained more saliva constituents than could possibly distil over from the Protoplasma-Monographien IX: Rahn 11 162 CHAPTER Vll Figure 48 a. Normal growth of tiacclwrowycett Mycoderma puttctisporui Above: 100 times; below: 500 timca. THE SIGNIFICANCE OJB 1 BIOLOGICAL RADIATIONS ETC. 103 Figure 48 b. Growth of irradiated tiaccliarontyces Mycodcnna -punctisporus. Above: 100 times; below: 600 times. 11* 1(>4 CHAPTER VII saliva droj) to the yeast culture. These experiments, as well as those by CHRISTIANSEN, were made in hanging droplets on a coverglass which was sealed to a moist ehamher slide with vaseline or lanolin; the "sender" was at the bottom of the cavity. By the same method, the effect of plants upon the morphology of yeasts was studied. Tt was found that most parts of the various plants stimulated yeast growth considerably, and also produced great morphological changes. The most sensitive yeast was tiactfiaro'tnyces M ycoderma putu'tisporus GuiLLfEimoND which was frequently greatly elongated so that it appeared under the low power like a mold colony. However, true branching was never observed, and the growth resembled more that of a MoniJia Fig. 48 shows the change produced by exposure to a young agar culture of the same species. Home of the wine yeasts also showed changes of cell form and size. Seed embryos, pollen, and roots produced the strongest effects ; leaves, the weakest effects. These results could not usually be repeated with the inter- position of a quartz coverglass between yeast and sender. "Only the one or the other symptom appeared occasionally (see fig. 48), but scarcely ever the complete set of morphological changes. However, the steam distillates of carrots or potatoes, when added to the culture medium in large amounts did not affect the mor- phology; nor did the juice of crushed carrots produce any change. When the sender was poisoned chemically, killing all cell activity, no effects were observed. Perhaps we are dealing here with a combined chemical and physical effect, such as STEMPELL (1932) assumed to be rather common in nature. In the aiithor's laboratory, Mrs. BARNES has isolated a ba- cillus which through a quartz coverglass, will change morphologi- cally the yeast cells, giving them the appearance of "saliva types". 2. Sea Urchin Larvae* It has already been discussed on p. 86 that sea urchin eggs, when exposed continously to mito- genetic rays from various sources, develop into very abnormal larvae. It has also been stated there that recently, it has been suggested that this is not due to a real radiation, but to an electric effect. Mcrophotographs of these forms are shown in fig. 49. THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS F/1V. 165 J. and M. MAUKOU (1931) have studied the abnormal larvae histologically, arid have come to the conclusion that they are primarily due to overproduction of the mesenchyme, while the ectoderm and endoderm appear normal (fig. 50). This #iu's a simple explanation of the morphological changes by the inito- * // Figure 49. larvae of the sea urchin. Paracentrotm lividus. I. Control, untreated. 11. Same origin and age as /, but exposed continuously through quartz to an acid solution of phenosaffranino reduced by KHSO 3 . 111. Control, fertilized with normal sperm. IV. Eggs of the same origin as ///, but fertilized with sperm exposed for 45 minutes to the same solution as 11. 166 CHAPTER VII genetic effect. However, there is considerable difference in the reaction of ectoderm and endoderm cells on one side, and mesen- chymatic cells on the other. The former are not essentially affected (there may be some retardation eventually), while the mosenchyrno grows entirely out of bounds though it is really protected against radiation by the outer cell layers. blast. Figure 50. Cross section through sea urchin larvae. Left: the normal larva. Right: larva exposed to the radiation of ttact. tumefadens. J = mouth; Hast blastophore; ect = ectoderm; Mid endoderm; est stomach; int intestine; mes -= mestmchyme ; oca -^ oesophagus. The most remarkable fact is perhaps the observation that the irradiation of the sperm alone or of the unfertilized ovum alone will suffice to bring about the same morphological changes. Thus, the radiation received by the single egg or sperm cell affects the entire future development of the larva, and the sti- mulus received by such a cell affects only one part of the offspring, i. e. the mesenchymatic cells. THE SIGNIFICANCE OP BIOLOGICAL RADIATIONS ETC. 167 3. Metamorphosis of Amphibia. The role of mitogenetic radiation in the metamorphosis of amphibia has been studied extensively by BLACKER and his associates. The most detailed investigation concerned the development of tadpoles. The following developmental stages are distinguished: Stage I : hind legs differentiated II : hind legs scarcely motile Ilia: hind legs actively motile, belly rounded, forelegs not visible through the skin. Illb: belly becomes loan, elbows of forelegs distend the skin. IV : forelegs arc out; tail has full length Va: about one-fourth of the tail is resorbed Vb: about one-half of the tail is resorbed Vc: about three-fourths of the tail is resorbed VI : the entire tail is resorbed; metamorphosis completed. Radiation arises from the resorbed tissues. The riewly- forrned fore or hind legs do not radiate. The intensity of radiation was estimated by the amount of the mitogenetic effect (yeast bud method) which is not a particularly good measure. The results are shown in fig. 51. All organs radiate only during their resorption. The gills initiate the process, followed closely by the intestine which is shortened considerably, the main shortening occurring at the time the forelegs develop. When the gills are almost completely resorbed, the tail begins. Other parts of the tadpole were tried, e. g. the back of the skill, but they did not radiate. In a later paper, BLACHER and LIOSNER (1932) estimated the intensity of blood radiation of Jiana ridibunda during meta- morphosis. They ascertained the minimal time necessary to bring about a distinct mitogenetic effect. The results were at Stage II : 5 minutes; relative intensity: normal Ilia: 30 minutes one-sixth normal Illb: 15 seconds 20 times normal IV : 5 minutes normal Vb: 30 seconds 10 times normal VI : 5 minutes normal The strongest blood radiation coincides with the stage where gills, intestine* and tail are all near the maximum of radiation. Otherwise, there is no parallelism. The second pronounced 168 CHAPTER VII maximum of blood radiation occurs after gills and intestine have completed their metamorphosis. BLACHER and associates (VII) further showed that there was a relation between the resorbed tissue and the developing limbs, and that transplantation of gills increased the growth rate of the corresponding foreleg. Removal of the gill, or the freeing of the foreleg from the gill cavity where resorption of the gills occurs, retarded growth. Finally, it could be shown that a tadpole lying in a quartz -bottomed vessel will show a more rapid growth of Figure 51. Intensity of radiation of the resorbed organs of frog tadpoles, during successive stages of metamorphosis. the legs if the bottom is placed over pulp of tissues in the process of resorption. The foreleg to be irradiated was freed from its cavity by a cut in the covering skin, while the other leg which served as control remained covered, and therefore shaded against radiation. The result of the operation as such was that in the vessels with glass bottoms, the freed leg grew a little more slowly than its mate in the cavity: 6.5% i 0.8 less in one series of 22 tadpoles, and 4.5% i 1.3 less in a second series with 44 tadpoles. In the quartz -bottomed vessels, however, the freed legs grew more rapidly; 4.1% ^ 1.1 more in one series of 33 tadpoles and 7.4% it 1.2 in the other series with 51 tadpoles. The actual THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 169 gain by irradiation was therefore 10.6% 1.4 in the one set and 12.1% i 1.8 in the other set of experiments. The gain is 7 times that of the average error. The triton Pleurodeles Wallii shows similar radiations (BLACKER etc., HI), though less intense; and the axolotl Am- bmtoma tigrinum which retains gills as well as tail throughout its life radiates still less. Experiments were made also with Drosophila larvae (BLACKER etc., V). They do not radiate until about ready to pupate. The maximum intensity is reached 24 hours after pupation, and radiation nearly ceases after 30 hours. There is no direct proof that in these cases, the radiation of the resorbed parts had any distinct form-giving effect. Posit- ively established is only the radiation of the resorbed tissue, and an accelerating effect of this radiation upon the rate of development of the organs concerned. 4. Change of Chromosome Number. It has been repeat- edly observed that polyploidy occurs fairly frequently in some individual branches of tomato plants infected with Bacterium tumefaciens. KOSTOFF and KENDALL (1932) conceived the idea that polyploidy might be somehow linked with the strong rnito- geiietic radiation which is known to be emitted by this bacterium. To test this, 120 tomato plants were inoculated with Bact. tumefaciens. After the development of the tumor, the plants were cut off 3 to 5 cm. above the tumor in order to induce the development of new shoots. Seven shoots arose directly in the tumor and of these, 5 could be rooted. One of them proved to bo tetraploid. While these data are, of course, too scant to permit tin* drawing of conclusions, they are suggestive of a new cause for polyploidy. 5. Parthenogenesis by Radiation. RKITEB, and OAROK after having verified GURWITSCH'S statement that there is always radiation around a wound, refer to the theory of BATAILLON that the developmental mechanism of an egg is released by the wound produced through the entrance of the spermatozoon. He had succeeded in causing cell division in unfertilized frogs' eggs by very fine needle pricks; only a very few percent of the eggs thus treated initiated development, but in some cases, BATAILLON obtained tadpoles and even frogs by this method. 170 CHAPTER VII REITEB and GABOB speculated that this effect was in all probability due to the mitogenetic radiation of the wound (see wound radiation p. 173), and they reasoned that in this event, radiation alone, without wounding, should produce the same result. They exposed unfertilized eggs of salamanders and frogs, obtained with great precautions from the females, to various sources of ultraviolet. Of all the wave lengths tried, only those of 3340 A proved efficient (Attention may be called again to the circumstance that REITEB and GABOR have always claimed that only the rays around 3340 A are mitogenetic, see p. 60). Even with this wave length, only 2 unfertilized eggs of the salamander could bo induced to develop, the first division being completed 3 hours after the 5-minute period of irradiation. A repetition caused only one such egg to develop. In. the controls, as well as in all experiments with longer or shorter irradiation, or with different wave lengths, the eggs died in a short time. The number of eggs exposed is not given, except that it was stated that each group contained about 5 eggs. Of these "induced eggs' ', one developed to 32 cells, one to the 8 -cell stage. In a later experiment with "a few hundred" unfertilized frogs' eggs, 3 could be induced to start cell division by irradiation with monochromatic light of 3340 A. H. SYMBIOSIS, PARASITISM AND ANTAGONISM It seems very likely that biological radiations play an im- portant role in the mutual relationship not only of cells, but also of cell groups and multicellular organisms. This field of mitogenetic radiation has as yet scarcely been entered. A few isolated facts stand out plainly. From the material mentioned in the preceding chapters, biological radiations appear to be largely stimulating, or bene- ficial. Possibly, this conception is wrong. It may be that only for reasons of technique, we have been able to observe for the most part this one type. It has been shown that as a rule, unicellular organisms further each other's growth. The radiation from yeast stimulates the cell division of many bacteria, and the rate of germination of mold spores. Similar effects with multicellular organisms are imagineable, but not proved. The radiation from an onion root will stimulate cell division in another root under laboratorv THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 171 conditions, but that can hardly occur under normal conditions of plant growth, with the soil absorbing all radiation. The role of radiation in parasitism is strongly suggested, but not proved in the case of plant tumors caused by ttact. tmm>- faclens. MAUROU (\U27c) observed that cell division in the tumor was not limited to the immediate presence of the bacterial cells. However, it was not attempted to prove that a tumor would be formed even if chemical effects by bacteria were excluded from action through a wall of quartz. The abnormal proliferations on the leaves and stems of plants due to insect eggs (commonly called galls) are usually attributed to chemical irritation by the larva, but it does not seem impossible that the young larvae radiate strongly during their early period of growth, and cause abnormal proliferation of tissue 1 by irritation through ultraviolet rays. Parasitic existence is limited to a very few species of plants or animals. It is customary to assume that for chemical reasons, all other plants and animals are prevented from living as parasites. The assumption that .specific; radiatioiis also play a part offers itseK naturally. However, this assumption would imply very specific radiations: either beneficial, which favor one speeies, but none of its nearest relatives, or, more probably, antagonistic radiations which prevent growth, or cell division, of all species except the parasite; for some reason, the parasite is not affected by the harmful radiation. The assumption, of specific radiations has very little experimental evidence for its support. While we are still far from knowing all sources of mitogenetic rays, all well-known radiations arise from entirely unspecific biochemical processes such as proteolysis, glycolysis, oxidation, etc., processes which are common to most plants and animals. The pathogenicity of the typhoid bacterium to man, but not to rats could not be ex- plained by different radiations from the two hosts, at our present state of knowledge. But it must be admitted that we have no chemical explanation either. On the 4 other hand, there has never been observed any specific action of definite wave lengths upon definite organisms. All experiments so far point to the conclusion that any radiation between 1900 and 2600 A can stimulate the division of any cell, plant or animal, unicellular or part of a tissue, providing that the physiological conditions permit (see e. g. fig. 28 p. 49). 172 CHAPTER VII Jt is known, of course, that an overdose of rays will not stimulate, and might even retard growth, but then* is no evidence that parasites are more tolerant to radiations of this kind than related, non-parasitic species. While the 1 assumption of specific radiations would be a very convenient explanation for some problems in parasitism, it has as yet no experimental foundation. Antagonistic Radiations: The only good case of specific antagonistic radiations is the investigation of Acs (1933) who experimented with microorganisms known to antagonize each other when grown simultaneously in the same culture, such as Bacillus anthracix and Psc udow onax pyocyanca, or yeast with staphylococci or streptococci. Acs used for his experiments cultures which were 6 S hours old, i. e. at the stage of rapid growth, and exposed the one pure culture to the radiation from the other, hi this way, he obtained distinct retardation of growth. Irradiation of B. finlhrricift for 1 to 2 hours by Ps. pyocyanca gave growth retardations of 22 to 136%, in 14 experiments. In two experiments, he reversed the arrangement, using B. ant/tracts as sender, and found Px. pyocyanca 42 and 48% retarded. The same organism was used simultaneously to irradiate Racillus rnfhnors, which was not retarded, but stimulated 42 and 50%. Yeast radiation was found to retard staphylococci very distinctly, in 5 experiments, and also streptococci in the one experiment made. Since such selectivity of antagonistic radiation cannot be explained by our present knowledge, a more detailed speetro- seopic investigation of such types might add greatly to our under- standing of the significance of biological radiations. Different from the specific harmful radiation which injures one species, but stimulates other species, are the generally harmful human radiations observed by CHRISTIANSEN and by BAK^ES and RAHN (see p. 184) which are linked with certain pathological conditions. The radiation may be truly specific ; only one species of yeast, ftaccharomyces Mycodcrma jmnctisporus, could be killed, while other yeast species were retarded, or not at all affected. The example of the sea urchin larvae (p. 86 and 164) is quite characteristic of a harmful effect produced by a primarily beneficial radiation. In this case, the harm is probably done by an overdose and not by any specific wave lengths since such widely different sources as chemical oxidation, glycolysis by THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 173 yeasts or streptococci, and protcolysis by staphyloeocci gave the same results. The main cause of the harmful effect in this case is the difference in sensitivity or reactivity, the mesetich vine cells being the only ones which were stimulated out of proportion. I. THE HEALING OF WOUNDS When wounds begin to heal, this is usually accomplished by mitosis of the cells nearest to the wound. In most cases, as with grown animals or plants, this would mean that cells which have already come to a resting stage, revert to multiplying cells. This resembles somewhat the situation of old yeast or bacterial cells transferred to a fresh medium. Something like a rejuvenation process of the old resting cells is necessary before they can divide again. Tt was precisely at this stage that we found mitogenetic radiation to be most effective upon bacteria or yeasts. The first discussion concerning the possible role of biological radiation in the healing of wounds occurred in 1929. HABJOKLAND had found that full-grown, but young leaves of fledum s-pectahile and Esrhcverta sec nmla can be torn without rupturing the cells of the mesophyll. They separate, and leave a dry surface. Such a surface will not "heal", i. e. the cells show no signs of division if the torn leaf is kept in a moist chamber. They will begin to divide, however, if smeared with juice of crushed leaves. They will also reproduce if the leaf is cut instead of carefully torn, leaving the wound surface wet with cell debris. GURWTTSCH (1929) expressed his opinion that such new di- vision of old cells could not take place without a mitogenetic stimulus, and that very probably it came from the cell pulp. HABERLAND (1929) attempted to discover whether radiation by sedum leaf pulp would cause a healing or renewed mitosis of a dry, torn leaf. He found that leaf pulp did not induce any division of cells when held at short distances from the injured leaf and concluded that the wound hormones act chemically and not physically. GURWITSCH (1929) found, using yeast as detector, that pulp of scdum leaves did not radiate when fresh, but would do so after 18 to 24 hours, losing this power again after 48 hours. He states that in his opinion, radiation alone will not cause the healing process in this case ; that there are chemical requirements 174 CHAPTER VJ1 besides the physical ones ; but, that radiation is one of the necessary factors. While the argument concerning the sedum leaf wounds lias never been settled experimentally, BROMLTGY (1930) has proved that the tail of the salamander Anibi/tttonui tigrimim radiated strongly after being amputated. It is not the new tissue which radiates, but the old cells immediately under the newly-formed tissue. Exposing yeast for 18 minutes to the ground tissue, the following "induction effects" were obtained: 1 23 5 6 12 24 hours after amputation 6.2% 2.1% 29.(>% 41.1% 39.9% 48.4% 50.4% induction It requires 3 hours for radiation to be established. The radiation of the injured tissue reaches a maximum after 1 2 days, decreases decidely and reaches a second maximum after 5 days. This agrees with the two maxima in pll of healing wounds as observed by OKUNBFK (1928). The same double maximum could be observed with wounded tadpoles of the frog Pdobatcs futic.utt, by BLACIIER, FIUCHIMO- WITSCII, LTOSNKR and WORONZOWA (1932 b). They cut 10mm. from the tip of the tail, and after different times, removed the old tissue which produced the regeneration, ground it up and used the pulp to irradiate yeast plates. The threshold time necessary to produce distinct mitogenetie effects was the measure 4 of intensity. In Table 50, two distinct maxima are seen at 12 and 36 hours after wounding, and a minimum at 24 hours, and a great drop in intensity after 4 days. Very interesting was the observation by the same authors that the blood of the tadpoles changed its radiation decidedly upon wounding of the tail. The blood of normal, uninjured tadpoles produced a marked effect in 5 minutes. 12 hours after injury, 1 minute of exposure to blood sufficed for a similar effect ; 24 hours after injury, 2 minutes were required, and on the fourth day, an exposure of 15 minutes became necessary. This means that at this stage, the blood radiated less than half as strongly as that of uninjured tadpoles, and it stayed at this low level during the entire 18 days of the experiment. By this long-continued effect of the wound upon blood radiation, the entire body is affected and brought into play. THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 175 Table 50. Induction Effects Produced in Yeast by Irradiation with Old Tissue Bordering the Regeneration of Wounded Tadpole Tails (Pdobates fuscus) Time after Irradiated for Wounding 10 sec. 15 sec. 30 sec. 1 miu. 2 niin. 5 min. 10 min. 15 min. 6 hours + 28 . . 12 +2 -his + 29 + 7 28 29 + 7 _ 24 __. --- 5 + 18 - 36 -h 4 + n __ __ 2 days _ + 2 + 2f Sugar in mg, after 3 hrs 67.6 69 66 78 70 69 77 Mitogenetic Induction +33.4 * 4-26 + 13 + 7 + 7 * 4 4- 5 6 davs 8 davs 10 days 13 days 16 days cells through an incision of the skin of the back; after 6 days, a tumor the size of a barleycorn had developed. In short intervals, they determined the decrease in blood sugar, and the mitogenetic radiation (Table 53). While the latter decreased distinctly from the fifth day, glycolysis and sugar content were not affected. There is really much more to this negative induction effect of cancer blood than merely the absence of radiation. HEINEMANN (1932) very definitely found growth retardation when he used the actual rate of cell increase as a measure (Table 22, p. 73 and fig. 45, p. 153). As early as 1920, LYDIA GURWITSCH and SAL- KIND observed that the blood of cancer patients suppressed the radiation of normal blood upon mixing the two. Rats, injected with cell-free tumor extract, showed no blood radiation after 2, 3, and 4 days, but were normal again after 9 days. Injection of cytagenin (see fig. 45) caused a temporary increase of blood radiation. When cytagenin is discontinued, emission of rays drops to its negative level in 1 to 3 days. Cancer Diagnosis: Blood radiation is so conspicuous even with patients suffering from many kinds of severe illnesses that its absence in cancer has been considered, since the earliest observations as a diagnostic means. GESENITTS reports (1932) that in pernicious anemia, radiation begins again after liver diet, while in carcinoma, even the complete removal of the tumor and radium treatment will not restore normal blood radiation. THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 181 Recently, KLENITZKY (1934) observed the return of blood radiation when every last trace of cancerous tissue had been removed. Since practically all diseases producing decreased mitogenetic radiation in blood are readily recognized, cancer diagnosis by this method has been studied systematically. The best results obtained are very likely those recorded by HEINEMANN from one of the hospitals in Frankfurt, Germany. "On the one side, further observations with carcinoma suspects justified the conclus- ion that a positive mitogenetic effect excluded the possibility of a tumor with certainty. On the other hand, a negative effect changing promptly to positive after cytagenin injection, caused us repeatedly to search for a tumor and to prove it beyond doubt, though the preceding, most careful clinical investigation had given no reason to suspect tumors (e. g. a small carcinoma in the upper rectum which caused no direct pain)." Very encouraging is further the summary by GESENIUS (1932) of 3 years experience in the Berlin University clinics. The method consisted in the decrease of yeast respiration by blood radiation (see p. 84). During the last year, only such cases in which diag- nosis was uncertain were investigated. Occasional exceptions have been observed; blood radiation has been found infrequently connected with severe carcinoma, and has rarely been absent in patients where autopsy revealed absence of carcinoma. Nevertheless, blood radiation is a valuable diagnostic means when used as a part of the clinical examination. Loss of radiation is independent of the size and location of the tumor. It remains absent after removal of the tumor, and cannot be used as a test for the success of treatment. OriginofCancer:It has been stated repeatedly that normal adult tissues radiate very weakly while normal blood radiates strongly; in cancer, the opposite takes place, the new growth radiating strongly while the blood ceases to do so. These facts plainly suggest that the growth-stimulating source of radiation is removed from the blood and concentrates in the neoplasm. However, this simple assumption does not agree with our explana- tion of these radiations as originating from biochemical reactions. It has just been shown that sugar content and the rate of glycolysis in the blood of cancer patients are not greatly altered. For this 182 CHAPTER VII reason, this explanation of the origin of cancer is not usually stressed. Somewhat different is PROTTI'S conception (19346) who assumes that a cellular disorder may arise when blood radiation becomes very low, eventually resulting in a neoplasm. He proved his point by injecting a cell suspension of adeno-carcinoma into two lots of 12 mice each; one lot was being fed normally, while the other was on a starving ration. For the first 15 days, the tumors grew more rapidly in the starved mice whose blood radiation had decreased, on the day of injection, from about 50 to about 10. It must be remembered that cancer is most frequent in old age when blood radiation has a tendency to become very low. PBOTTI observed further that a mixture of neoplasmic cells and yeast cells in vitro resulted in a destruction of the neoplasmic cells while cells from normal tissues were not influenced by yeast. The same happened when the neoplasmic cells were separated from the yeast cells by a quartz plate. PROTTI calls this "cyto- photolysis". Repeated injections of yeast suspensions into the Galliera- Sarcoma of rats caused a liquefaction of the tumor, without pus formation, leaving finally a cavity with thin fibrous walls. Intravenous injections produced no effect upon the tumors. With the adeno-carcinoma of mice, injections of small amounts of yeast into the tumor stimulated its growth while large amounts retarded it. The same was the case with intravenous injections. Yeast heated to 60 produced no effect which suggests, together with the abovemcntioned "cytophotolysis", that the results are not due to enzymes, but to radiation. Cancers have been produced by frequent application of certain chemicals to the skin. Since the compounds found so far are not normal or pathological products of the human body, as far as is known, the discovery of their effect does not really explain the formation of cancers, but may give valuable suggestions towards the solution of the problem. More important is perhaps the discovery that cholesterol metabolism is in some way connected with cancer. It has been claimed by SHAW, MACKENZIE, MORAVEK and others that chol- esterol stimulates the growth of malignant tumors. ROFFO (1933) found the cholesterol content of the skin near cancerous or pre- THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 183 cancerous lesions to be much higher than the normal skin of the same patient. He found the frequency of skin cancer to be distributed in the following way, as average of 5000 cases: skin of tho face 95.61% skin of the back of the hand 3.07% scalp 1.02% skin of the foot 0.50;, KAWAGUCHJ (1930) had shown that the cholesterol content of the skin increases when it is exposed to sun light. According to MALCYNSKI (1930), ultraviolet irradiation of healthy persons increases the blood cholesterol while with cancer patients, there is a decrease of 25 to 40%. ROFFO (1934 a) studied the "heliotropism" of cholesterol very thoroughly. He proved it to take place only in living or- ganisms. With white rats, sunlight as well as ultraviolet light increased the cholesterol content of the exposed parts of the skin. The wavelength of the ultraviolet was above 2300 A. X-rays or radium rays produced the opposite effect. Then, ROFFO (1934b) studied the tendency for cancer devel- opment in white, rats. 700 rats were kept in sunlight daily for not more than 6 hours, avoiding tho hottest sun. After 10 to 11 months, 52% of all rats had developped cancer, which was ex- clusively on the naked parts of the body (ears and eyes mainly, also twice on the hind feet and once oil the none). 150 rats wen; exposed to ultraviolet light, of an intensity of 14 erythemal units, beginning with 5 minutes per day and gradually increasing the exposure to 6 hours. Within 4 months, every one of these rats showed tumors, and many of them had several tumors. Of the control rats receiving light from a tungsten filament lamp, not a single animal had developped a tumor. This agrees very well with the above-mentioned frequency of skin cancer on different parts of the human body. The mode of development and the histology of the rat cancers corresponded exactly with that of human cancers. The author has not been able to ascertain whether ROFFO has tested the mitogenetic range of rays by itself, i. e. the range between 1800 and 2600 A. It would be a splendid agreement if this range which is known to stimulate cell division could also be shown to produce abnormal cell division of the epithelial cells. The fact that sunlight can do this speaks very much against 184 CHAPTER VII it since sunlight contains no wavelengths shorter than 2700 A. However, very small amounts, such as have been shown to pro- duce mitogenetic effects, might possibly be present (at least in Buenos Aires where these experiments were made). The much stronger effect of artificial ultraviolet containing also the shorter waves suggests that the shorter waves are more efficient in bringing about abnormal cell division. Attention may be called here to the effect of radiation of oxy- cholesterol upon yeast cells. It is impossible to sa^y whether any relation might exist between oxy- cholesterol and the cancer problem. K. RADIATIONS OTHER THAN MITOGENETIC In Chapter IV, three types of radiation from organisms have been mentioned which are not identical with mitogenetic radiation. One was entirely different, and does not really belong in this group, namely the TJeto-radiation of the potassium in the cells. Then, there was necrobiotic radiation. This type, though apparently stronger than mitogenetic rays, is emitted by dying cells, and does not have any definite biological purpose or effect as far as has been ascertained. It is an emission of energy typical for the chemical changes connected with death, but it is absorbed by the surrounding medium, and should it produce biological effects, it would be purely accidental. The third group are the harmful human radiations, and they produce distinct biological effects. Quite commonly known are the reactions brought about by menstruating women : flowers wilt readily in their hands; menstruating women are excluded from collecting flowers for the perfume factories of France; mushroom beds are said to be utterly ruined by their mere presence; pure cultures of yeasts and bacteria become abnormal when handled by them (see p. 95), and the common belief that bread dough will not rise normally, and that pickles and sauerkraut packed by them will not keep, does not sound improbable, since CHRISTIANSEN proved that wine fermentation was distinctly affected (see p. 96). The facts as such can be considered fairly definitely establish- ed. Details may be found in the paper by MACHT and LX^BIN (1923 1924). But it cannot be considered proved that the effect THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 185 is of physical origin. The more common conception among medical men is that it is chemical, produced by a compound called meno- toxin. The intensity of this effect varies greatly with the individual. We have but rarely been able to obtain good results with wilting flowers. CHRISTIANSEN found the effect to be much stronger in summer than in winter. Attention should be called here to the observation by HILL (1933) that certain persons kill bacteria on agar plates when placing their fingers right upon the seeded plate. In this case, it has not been decided as yet whether the effect is physical or chemical. Probably the same type of harmful radiation has been observed in a very few cases of illness (p. 97) ; at least, the yeast was killed by emanation from the finger tips, and even from the face, through a thick quartz plate excluding chemical influences. The Source of Harmful Human Radiations: MACHT and LUBIN (1923 1924) came to the conclusion that the u meno- toxin", i. e. the compound in the blood during menstruation responsible for the various phenomena just mentioned, must be either oxy cholesterol or a closely related compound. This induced BARNES and RAHN (1933) to test whether this compound radiated. The results were quite definitely positive. Out of 5 batches of oxycholesterol made at different times, 4 killed Myco- derma during the first day when exposed continuously through quartz, but not on the second day. With Batch No. Ill which had no effect upon the yeast, an error was made in the separation of the oxycholesterol from the other reagents; on account of this, the exposure could not be started until one day later, and by this time, this product usually has lost its power. The colori- metric tests mentioned for oxycholesterol can be obtained long after radiation has become too weak to be detected biologically. Since it is not probable that a compound as such radiates, it appears more likely that the radiation comes from some reaction of the cholesterol or oxycholesterol, and we would think above all of oxidations. In the above experiments the oxycholesterol was emulsified with a little water to make oxidation possible. Why this radiation is harmful instead of stimulating, cannot be stated at present. The simplest assumption would be that the death of the Mycoderma cells is due to over-exposure because in 186 CHAPTER VII Table 53a. Effect of Oxy-cholesterol upon Mycoderma through fused Quartz time after manufacture of Batch J Batch II Batch III Batch IV Batch V ozy-oholesterol 1 day dead dead normal mostly dead (stimulated) dead 2 days normal retarded 3 days stimulated 4 days greatly greatly retarded retarded 5 days slightly normal retarded 6 days normal 7 days normal normal - 8 days normal 14 days normal all experiments the yeast was exposed continuously to this ra- diation. However, complete killing of all cells is not usually ob- served even in over-exposure, and only rarely was stimulation found. The other alternative that we are dealing with different wave lengths does not seem very probable either. All wave lengths between about 1800 and 2600 A give the same mitogenetic effect (see fig. p. 49), and no exceptions are known; the oxycholesterol radiation passes through quartz but not through glass, and can therefore be hardly anything but ultraviolet. An analysis of the spectrum may give the explanation. Oxycholesterol in the Body: A study of the distribution of cholesterol and oxycholesterol in the fats of the human skin, by UNNA and GOLODETZ (1909) shows that oxycholesterol is not present in the normal cellular fats but only in secreted fats (Table 54). An exception is the fat in the finger nails and toe nails. This agrees quite well with our observation that radiation was obtained from hands (and feet) and from the face where sebacious glands exist, but not from the chest where they are very rare. While a good deal of attention has been paid in recent years to cholesterol metabolism, very little is known about oxycholesterol in the body. Even its chemical composition is not certain. The only more recent investigation on oxycholesterol in the body is that by PFEIFFEK (1931). According to his analyses, the largest THE SIGNIFICANCE OF BIOLOGICAL RADIATIONS ETC. 187- Table 54. The Cholesterol and Oxycholesterol Content of Human Skin Fat melting point Unsap- onifiable fraction In % of free Chol- the unsa] Chol- esterol >onifiable fraction Oxycholesterol % esterol Esters Comedon fat (black- 1& head) 53.0 30.50 9.16 very much ll hand sweat . . . 46.5 28.40 5.40 0.79 very little 8 foot sweat .... 36.5 22.30 18.70 1.60 fairly much surface skin fat 48.0 32.20 50.00 2.06 Js horny skin fat . . 51.0 36.30 54.00 8.91 3 "ta nail fat 38.0 41.60 43.70 fairly much 1 **" vernix caseosa . . 38.0 36.00 45.00 8.38 subcutis liauid 1.15 15.80 0.04 amount of oxy cholesterol, on the basis of total solids, is found in the brain (0.44%), next are bone marrow, adrenals and bile (about 0.29%), followed by the liver (0.09%). Lowest are the erythrocytes with 0.003%. While previous authors, e. g. UNNA and GOLODETZ, estimated the oxycholesterol colorimetrically, PFEIFFER determined it by the difference in melting points between cholesterol and oxyeholesterol. The results may therefore not be comparable, on account of the uncertain chemical composition. It has been stated already that oxyeholesterol radiation ceased when the colorimetric tests were still very strong. The relation between this substance in the tissues and radiation is therefore not definitely established. In the preceding chapter, attention has been called to the close relation between cholesterol and cancer. The above results suggest that an investigation about the role of oxy- cholesterol in connection, with the cholesterol metabolism in cancer might yield interesting results. OUTLOOK The bioJogical part of this book has been written by a biolo- gist who is convinced, from his own experiences as well as from the study of literature, that mitogenetic radiation exists. He has realized that it is difficult to prove it because we are dealing with an extremely weak effect, and with very sensitive detectors. Above all, we are dealing with an entirely new phenomenon, and consequently cannot predict which changes of technique might increase or decrease the effect. It does not speak well for the present status of science that it has not been possible to settle definitely, in the course of 12 years, the question of the existence of this radiation. The fault lies equally with the two groups of contestants, those for and those against radiation. The facts are these : GXJBWITSCH and a number of his pupils and also many other investigators have presented a very large amount of experimental data to show that mitogenetic radiation exists. Many others have repeated these experiments, following directions as exactly as they were given, and obtained no mito- genetic effect. Several of the latter group have claimed therefore that they have disproved biological radiation. Such claim is unscientific as has already been pointed out in the foreword. The only way to disprove any theory is to obtain the same results, and to show that they are due to another cause. Some such attempts have been made (e. g. MOISSEJEWA, LORENZ) but they have not been carried far enough, or have been contradicted by other, more recent investigations. Most of the critics dismiss the question with the simple statement that all so-called mito- genetic effects are within the limits of experimental error. OUTLOOK 189 Let us realize from the beginning that the differences of opinion center around two essentially different points; one is the existence of the biological effect, and the other is its inter- pretation as an ultraviolet radiation. A different interpretation will not make the effect less important for biology. The effect is the important thing; the explanation is secondary. After all, only the facts remain permanent in science, while the theories come and go. The difficulties in deciding the existence of the mitogenetic effect are to be sought largely in the sensitivity of the methods. It is evident that this point can be settled only by biological experiments. Physical measurements can tell only whether or not it is caused by radiation, but the absence of radiation does not disprove the biological effect. Biological measurements are not at all simple. When higher organisms are used as detectors, the controls are not perfect. Several authors have questioned the use of one side of the onion root as control for the other side. It may be that both are affected. This objection may also be made to other tissues, e. g. the cornea. With unicellular organisms, where large numbers are used, the controls are as reliable as they can possibly be in any biological experiment. While it is easy to make yeasts or bacteria grow in the customary culture media, it requires a thorough under- standing of their physiology to interprete differences of growth rate, and errors have been made in this respect by those opposing biological radiation as well as by those convinced of it. The error in biological experiments is not as absolutely fixed as in physical or chemical methods, e. g. in an analysis where it can be stated reliably for all laboratories that the accuracy of the method is 0.005 g. In biology, it depends very much upon the choice of the organism (e. g. the variety of onion), the uni- formity of the material, the treatment of the organisms before the beginning of the experiment, the uniformity of environment before and during the experiment, the ability of the experimentor to recognize and avoid disturbing or secondary influences. A fine example is the painstaking work of M. PAUL (1933) with onion roots. As a result of the various factors creeping in, the error of the same method may be widely different in different laboratories 190 OUTLOOK or even in the same laboratory with different investigators. This explains the difference of error in the onion root experiments which was 10% with some investigators and 50% with others (see p. 58). When it comes to such delicate instruments as the GEIGER counter, even physical measurements show great diffe- rences (see Table 30a p. 92). The frequently made statement that the biological investi- gators do not state the error of their methods is not in accordance with the facts. When the error or the reliability of the method is not stated as such, it is usually possible to compute the error from those experiments where no mitogenetic effects were obtained. This has been done by SCHWEMMLE for all earlier onion root experiments (p. 58) and the authors gave some similar cal- culations for the yeast bud method on p. 71. TUTHILL and RAHN have also published two sets of counts of yeast buds from many different parts of the same culture. GIJRWITSCH and his asso- ciates have stated repeatedly that with the yeast bud method, they consider any increase less than 15% over the control to be experimental error. The reliability of the yeast measurement by volume can be estimated from the data by BILUG, KANNE- GIESSEK and SOLOWJEFF. HEINEMANN has stated that in his method of counting yeast with the hemacytometer, the mitogenetic effects were more than 3 times the experimental error. WOLFF and RAS have frequently given all individual counts of bacteria for one experiment (p. 78). Other error limits can be found on pp. 83 (JULIUS), 34 (BAETH) and 168 (BLACKER). It is somewhat surprising to find that in critical summaries, some weak papers are quoted extensively while some of the best proof in favor of mitogenetic radiation is completely omitted. NAKAIDZUMI and SCHBEIBEB, working with the yeast bud method, omit the work of SIEBEJRT who published more detailed experiments than any other investigator in this field. RREUCHEN and BATEMAN also mention neither SIEBERT'S papers nor the extensive work of WOLFF and RAS with bacteria which is the best material with this detector. It is only natural that any new development in science will attract speculative minds who generalize from a few experiments and come to conclusions which are not shared by the more conservative workers in this field. Any serious criticism should start with the most reliable and best founded papers. OUTLOOK 191 On the other hand, the critics have good reason to disregard rapers which give no precise account of methods or results. Probably the main reason why mitogenetic effects are still doubted las been the recording of results by merely giving the "Induction Effect" without mention of the experimental data from which >he effect was computed. The actual number of mitoses in a sornea, the percentage of buds, or the yeast volume measured, /ell a good deal about the performance of the experiment. Even ;he chemist whose methods are really standardized publishes lot merely the formula of his new compound, but also the actual inalytical data. Since the error in biological experiments varies yith the investigator, the publication of the complete records vould give the reader a conception of the reliability of any >bserved effects, even when the standard deviation has not been Computed. Another justifiable argument against the weight of some jublished papers is the lack of precision in the description of the nethod. Since the biological detectors do not respond at all imcs to mitogenetic rays, but only in a definite physiological state, it is of greatest importance to describe all details. Such statements as "8 10 hours at room temperature" are too inde- inite; the term "yeast" means very little, and even such terms is "onion root" or "cornea of a frog" should be specified in much nore detail since there are many different kinds of onions or rogs, and the roots as well as the number of mitoses in the ;ornea are affected by the season. As an example may be mentioned the paper by SALKIND ind PONOMARBWA (1934) which might be very important for the physiological explanation of the mitogenetic effect. However, ;he authors do not mention the age of the yeast culture, nor he medium on which it was grown ; they give only the induction ffect so that the reader does not know whether the controls lad 3% or 25% of buds which would have given a suggestion it least of the condition of the yeast. Therefore, the value of the mper is largely lost. It might be argued that the burden of the proof lies with he opponents since the mitogeneticists claim to have proved heir point, but such an attitude would not be very helpful in lolving the real problem. We are dealing with a very complex 192 OUTLOOK phenomenon, and both sides should do all they can to bring about a real understanding of the facts. The complexity is greatly increased by the occasional failure of the phenomenon for unknown reasons (p. 93). Errors have been made by the defendants of both sides of the argument, and the authors hope sincerely that by pointing out the mistakes and misunderstandings, a settlement of the question of mitogenetic radiation will be accomplished in a very short time. AUTHOR INDEX 197 Page II. BLACHBK and N. W. BROMLEY, Mitogenet. Ausstrahlungen bei der Regeneration des Kaulquappenschwanzes. 121, 79 IT I. and 0. G. HOLZMANN, Mitogenetischc Ausstrahlungen wahrend der Metamorphose der Urodelen. 128, 230 IV. and N. W. BKOMLEY, Mitogenetische Ausstrahlungen bei der Schwanzregeneration der Urodelen. 128, 240 V. O. G. HOLZMANN, Mitogenetische Ausstrahlungen wahrend der Metamorphose bei Drosophila melanogaster. 128, 266 VI. N. W. BROMLEY, Der EinfluB der primaren Verheilung der Wunde auf die Entstehung mitogenetischer Ausstrahlungen in ihr. 128, 274 VII. L. J. BLACKER, M. A. WORONZOWA, L. D. LIOSNER and W. N. SAMARA JEW, Die mitogonetischen Ausstrahlungen als Stimulus des Wachstums der Vorderbeine bei der Meta- morphose von Rana temporaries. 124, 138 BLACKER, L. J., and L. D. LIOSNER, 1932, Mitogenet. Blutstrahlung der Amphibien wahrend der Metamorphose. Roux' Archiv 127, 364 167 , A. I. IRICHIMOWITSCH, L. D. LIOSNER and M. A. WORONZOWA, 1932 a, EinfluB der mitogenetischen Strahlen auf die Goschwindig- keit der Regeneration. Roux' Archiv 127, 339 175 , , , -, 1932 b, Die mitogenetische Strahlung des Regenerate und des Bluts der Kaulquappen wahrend der Regeneration. Roux' Archiv 127, 363 174 and W. N. SAMARAJEW, 1930, Die Organisationszentren von Hydra fusca als Quelle mitogenetischer Ausstrahlungen. Biol. Zentr. 60, 624 133 BOIIMER, K., 1927, Menotoxin und Hefegarung. Deutsch. Z. f. d. ges. gerichtliche Medizin 10, 448 95 BORODIN, D., 1930, Energy emanation during coll division processes. Plant Physiol. 5, 119 57 , 1934, M-rays macro-effect and planimetric drop culture method. I. Internat. Congress of Electro-Radio-Biology, Venice 75 BRAUNSTEIN, A. E. and P. A. HEYFETZ, 1933, Die Glycolyse und die mitogenetische Strahlung dos Bluts bei experimentellem Carci- noma. Biochem. Z. 259, 175 179 and A. POTOZKY, 1932, Oxydationsreaktionen als Quelle mito- genetischer Strahlung. Biochem. Z. 249, 270 36 and B. A. SEVERIN, 1932, Der Zerfall der Kreatinphosphorsaure als mitogenetische Strahlungsquelle. Biochem. Z. 255, 38 38 BROMLEY, N. W., 1930, Der EinfluB der primaren Vorheilung der Wunde auf die Entstehung mitogenetischer Ausstrahlungen in ihr. Roux' Archiv 128, 274 174 see also BLACKER. 198 AUTHOR INDEX Page BROOKS see NELSON. BRUNETTI, R., and C. MAXIA, 1930, Sulla fotografia a la eccitatione delle radiazione del Gurwitsch. Atti Soc. fra il Cultori delle Science Med. e Nat. Cagliari 2 90 CHARITON, J., G. FRANK and N. KANNEGIESSER, 1930, tJber die Wellen- lange und Intensitat mitogenetischer Strahlung. Naturwisson- schaften 18, 411 50, 60 CHRISTIANSEN, W., 1929, Das Monotoxinproblem und die mitogene- tischen Strahlen. Ber. d. deutsch. hot. Ges. 47, 367 86, 95, 161, 184, 185 CHRUSTSCHOFF, G. K., 1930, XJber die Ursachen des Gewebewaclistums in vitro. I. Die Quellen der mitogenetischen Strahlen in Gewebe- kulturen. Archiv f. exper. Zellforschung 9, 203 83 COBLENTZ, W. W., R. STAIR and J. M. HOGUE, 1931, A balanced thermocouple and filter method for ultraviolet radiomotry, with practical applications. Bureau of Standards, Journ. of Research 7, 723 48 DECKER, G. E., 1934, t)ber die Scharfe mitogenetischer Spektrallinien. Archiv Sciences Biol. (Russ.) 36, 145 37 DIETL see POLANO. i>u BRIDGE sec HUGHES. DOLJANSKI, L., 1932, Das Wachstum der Gewebskulturen in vitro und die GuRWiTsCH-Strahlung. Roux' Archiv 126, 207 83 VAN DOORMAL SCO AUDUBERT. DUGGAR, B. M. and A. HOLLAENDER, 1934, Irradiation of plant viruses and of microorganisms with monochromatic light. Journ. Bact. 27,219 48 EMERSON, R., and W. ARNOLD, 1932, A separation of the reactions in photosynthesis by means of intermittent light. Journ. Gen. Physiol. 15, 391 107 FELDMAN, W. H., 1932, Neoplasms of Domesticated Animals. Phila- delphia, Saunders 177 FERGUSON, A. J., 1932, Morphological changes in yeasts induced by biological radiation, Thesis, Cornell University 96, 161 and OTTO RAHN, 1933, Zum Nachweis mitogenetischer Strahlung durch beschleunigtes Wachstum der Bakterien. Archiv f. Mikro- biol. 4, 674 76, 78, 121, 126, 137 FRANK, G., 1929, Das mitogenetische Reizminimum und -maximum und die Wellenlange mitogenetischer Strahlen. Biol. Zentr. 49, 129 35, 39, 60 and M. KUREPINA, 1930, Die gegenseitige Beeinflussung der Seeigel- eier als mitogenetischer Effekt betrachtet. Roux' Archiv 121, 634 138 and S. RODIONOW, 1932, Physikalische Untersuchung mitogene- tischer Strahlung der Muskeln und einiger Oxydationsmodelle. Biochem. Z. 249, 321 29, 34, 91, 109 AUTHOR INDEX 199 Page FRANK, G., and S. SALKIND, 1926, Die Quellcn der mitogenetischen Strahlung im Pflanzenkeimling. Roux' Archiv 108, 696 ... 141 and S. SALKIND, 1927, Mitogenetische Strahlung der Seeigeleier. Roux' Archiv 110, 626 142 FRANK, M., 1921, Menotoxine in der Frauenmilch. Monatsschrift fur Kinderheilkunde 21, 474 95 FRICKE, HUGO, 1934, The chemical-physical foundation for the biologi- cal effects of x-rays. Cold Spring Harbor Symposia IT, 241 . . 42 FRIEDRICH see SCHREIBER. GABOR see REITER. GATES see RIVERS. GERLACH, W., 1933, Physikalisches zum Problem mitogenetischen Strahlung. Gesellsch. f. Morphol. u. Physiol. Miinchen 42, 1 . 34 GESENIITS, H., 1930a, t)ber Stoffwechselwirkungen von GURWITSCH- Strahlen. Biochem. Z. 225, 328 84 , 1930 b, tTber die GuRWiTSCH-Strahlung menschlichen Bluts und ihre Bedeutung fur die Carcinom-Diagnostik. Biochem. Z. 220, 257 85, 153 , 1932, Blutstrahlung und Carcinom-Diagnostik. Radiobiologia 1 (2), 33 85, 179, 180, 181 GILTNER, W., and L. R. HIMMELBERGEK, 1912, The use of lactic acid cultures in combating infections of mucous membranes. J. Comp. Pathol. and Ther. 25, 312 177 GOLISCHEWA, K. P., 1933, Die mitogenetische Spektralanalyse der Blutstrahlung am lebenden Tier. Biochem. Z. 260, 52 . . 93, 150 GOLODETZ see UNNA. GREY, J., and C. OUELLET, 1933, Apparent mitogenetic inactivity of active cells. Proc. Roy. Soc. B 114, 4 92 GUILLERY, II., 1929, tiber Bedingungen des Wachstums auf Grund von Untersuchungen an Gewebskulturen. Virchows Archiv 270, 311 82 GURWITSOH, ALEXANDER, 1922, t)ber Ursachen der Zellteilung. Roux' Archiv 52, 167 59, 119 , 1923, Die Natur des spezifischen Erregers der Zellteilung. Roux' Archiv 100, 11 54, 119 , 1929 a, Die mitogenetische Strahlung aus den Blattern von Sedum. Biol. Zentr. 49, 449 141, 173 , 1929 b, tlber den derzeitigen Stand des Problems der mitogene- tischen Strahlen. Protoplasma 6, 449 66, 60 , 1932, Die mitogenetische Strahlung. Berlin, Springer. 380pp. 63, 66, 70, 73, 74, 103, 107, 108, 113, 117, 127, 142, 147, 149 , 1932 a, Die mitogenetische Strahlung des markhaltigen Nerven. Pflugers Archiv 231, 234 155 , 1934, Der gegenwartige Stand des mitogenetischen Problems. I. Internat. Congress of Electro-Radio-Biology, Venice . . . 45, 146 200 AUTHOR INDEX Page GURWITSCH, ALEANDER, and L. GURWITSCH, 1925, ttber den Ur- sprung der mitogenetischen Strahlen. Roux' Archiv 105, 470 . 55 , , 1929, .Die mitogcnetische Strahhmg des Carcinoma. II. Z. f. Krebsforschung 29, 220 178 , , 1932 a, Die mitogenetische Sjpektralanalyse : IV. Das mito- genetischo Spektrum der Nukleinsaurespaltung. Biochem. Z. 246, 124 38 , , 1932 b, Die Fortleitung des mitogenetischen Effekts inLosungen und die Beziehungen zwischen Fermentatigkeit und Strahlung. Biochem. Z. 246, 127 \ 43, 109 , , 1934, L'analyse mitogenetique spectrale. Vol. IV of Exposes de Physiologie, Paris, Hermann and Cie., 39 pp 40^ GURWITSCH, ANNA, 1931, Die Fortpflanzung des mitogenetischen Er- regungszustandes in den Zwiebelwurzeln. Roux' Archiv 124, 357 111 , 1932, Die mitogenetische Strahlung der optischen Bahn bei adaquater Erregung. Pfliigers Archiv 231, 255 157, 159 -, 1934a, L'excitation mitogenetique du systemo nerveux central. Ann. de Physiol. et Physicochem. biol. 10, 1153 157 , 1934b, L'excitation mitogenotique du systeme Tierveux pendant Teclairago monochromatique dc 1'oeil. Ann. de Physiol. et Physicochem. biol. 10, 1166 158 GURWITSCH, LYDIA, 1931, Die mitogenetische Spektralanalyse : II. Die Spektren des Carcinoms und des Cornealepithels. Biochem. Z. 236, 425 38 and N. ANIKIN, 1928, Das Cornealepithcl ats Detektor und Sender mitogenetischer Strahlung. Roux' Archiv 113, 731 84 and S. SALKIND, 1929, Das mitogenetische Verhalten des Bluts Carcinomatoser. Biochem. Z. 211, 362 65, 152, 180 HABERLANDT, G., 1929, t)ber ,,mitogenetische Strahlung". Biol. Zentr. 49, 226 173 MALL see JONES. HAUSSER, K. W., and K. H. KREUCHEN, 1934, Quantenausbeuten bei Lichtzahlern. Z. Techn. Physik 15, 20 92 HARVEY see TAYLOR. HEINEMANN, M., 1932, Cytagenin und mitogenetische Strahlung des Bluts. Klinische Wochenschr. 11, 1375 72, 122, 151, 152, 180, 181, 190 , 1934, Physico-chemical test for mitogenetic ( GURWITSCH) rays. Nature 134, 701 89 , 1935, Physico-chemical test for mitogenetic ( GURWITSCH) rays. Acta Brevia Neerland. 5, 15 89 HENRICI, A. T., 1928, Morphologic Variation and the Rate of Growth of Bacteria. Springfield, C. C. Thomas. 185 pp 120, 134 HERLANT, M., 1918, Periodische Anderungen der Permeabilitat beim befruchteten Seeigelei. Compt. rend. soc. biol. 81, 151 .... 142 AUTHOR INDEX 201 Page HEYFETZ see BRAUNSTEIN. HILL, J. H., and E. C. WHITE, 1933, Action of normal skin on bacteria. Archives of Surgery 26, 901 185 HlMMELBERGEB See GlLTNER. HOLLAENDER 866 DlTGGAR. HOLZMANN see BLACKER. HUGHES, A. L., and L. A. DU BRIDGE, 1932, Photoelectric Phenomena. New York, McGraw-Hill, 531 pp 20, 26 IRICHIMOWITSCH see BLACKER. JAEGER, C., 1930, L)er Einflufi der Blutstrahlung auf die Gowebs- kulturen. Z. f. Zellforschung 12, 354 83 JAHN see RICHARDS. JONES, L. A., and V. 0. HALL, 1926, On relation between time and inten- sity in photographic exposure (IV). J. Opt. Soc. Am. 13, 460 . 24 JULIUS, H. W., 1935, Do mitogenetic rays have any influence on tissue cultures ? Acta Brevia Neerland. 5, 51 83 KALENDAROFF, G. S., 1932, Die Spektralanalyse der Strahlung des markhaltigcn Nervcn im Ruhezustande und bei kiinstlicher Erregung. Pfliigers Archiv 231, 239 73, 122, 155 KANNEGIESSER, N., 1931, Die nu'togenetische Spektralanalyse I. Biochem. Z. 236, 415 35 see also BILLIG and CHARITON. KARPASS, A. M., and M. N. LANSCHINA, 1929, Mitogenetische Strahlung bei Eiweifiverdauung. Biochem. Z. 215, 337 52 KAWAGUCIII, S., 1930, tJber den EinfluB der Lichtstrahleii auf don Gesamt-Cholesteringehalt der Haut. Biochem. Z. 221 , 232 . . 183 KENDALL see KOSTOFF KISLIAK-STATKEWISCH, M., 1927, Die mitogenetische Btrahlung des Kartoffeleptoms. Roux' Archiv 109, 283 141, 145 , 1929, Die mitogenetische Strahlung des Carcinoms. Z. f. Krebs- forschung 29, 214 178 see also SORIN. KLEE-RAWIDOWICZ sec LASNITZKY. KLENITZKY, J., 1932, Die mitogenetische Strahlung der weifien Blut- elemente. Biochem. Z. 252, 126 151 , 1934, Le retablissement de la radiation mitogenetique du sang apres 1'extirpation d'une tumeur cancereuse. Arch. Sciences Biol. (Russ.)35, 218 181 and E. PROKOFIEWA, 1934, L'analyse spectrale de la radiation mitogenetique de la desaggregation des polysaccharides. Arch. Sciences Biol. (Russ.) 35, 211 39 KOROSI, K. DE, 1934, L'action a distance de la levure sur les sucres. I. Internat. Congress of Electro-Radio-Biology, Venice 46 202 AUTHOR INDEX Page KOSTOFF, D., and J. KENDALL, 1932, Origin of a tetraploid shoot from the region of a tumor on tomato. Science 76, 144 169 KREUCHEN, K. H., 1935, Messung geringer Lichtintensitaten mit Hilfe von Zahlrohren. Z. f. Physik 94, 549 92 und J. B. BATEMAN, 1934, Physikalische und biologische Unter- suchungen uber mitogenetische Strahlung. Protoplasma 22, 243 92, 95, 190 see also HAUSER. KUREPINA see G. FRANK. LANSCHINA see KARPASS. * LATMANISOWA, L. W., 1932, Die mitogenetische Sekundarstrahlung desNerven. Pfliigors Archiv 231, 265 110, 112, 113, 159 , 1933, Parabiose der Nerven als Folge mitogenetischer Bestrahlung. Naturwissenschaften 21, 330 159 , 1934, Sur la parabiose mitoge'n&ique du nerf. Ann. de Physiol. et de Physicochem. Biol. 10, 141 159 LASNITZKI, A., and E. KLEE-RAWIDOWICZ, 1931, Zur Frage der mito- genotischen Induktion von Warmbluterzellen. Z. f. Krebs- forschung 34, 518 83 LEPESCHKJN, W. W., 1932a, Influence of visible and ultraviolet rays upon the stability of living matter. Am. J. Bot. 19, 547 ... 99 , 1932 b, Nekrobiotische Strahlen. Ber. d. deutsch. bot. Ges. 50, 367 99 , 1933, Nekrobiotische Strahlen I. Protoplasma 20, 232 99 Loos, W., 1930, Untersuchungen iiber mitogeiietisehe Strahlen. Jahrb. f. wissensch. Botanik 72, 611 57 LIOSNER see BLACKER. LORENZ, E., 1933, Investigation of mitogenetic radiation by means of a photoelectric counter tube. U.S. Health Reports 48, 1311 . 91, 188 , 1934, Search for mitogenetic radiation by means of the photo- electric method. Journ. General Physiol. 17, 843 41, 91 LUCAS, G. H. W., 1924, The fractionation of bios. Journ. physical Chem. 28, 1180 73 LUBIN see MACHT. MACHT, D. I., and D. S. LUBLIN, 1923/24, A phyto-pharmacological study of menstrual toxin. Journ. of Pharm. and Exp. Ther. 22, 413 95, 184, 185 M \GROU, J., and M. MAGROU, 1927a, Recherches sur les radiations mitoge*n6tiques. Bull. d'Histologie appliqu6 4, 253 . . . . 57, 178 , , 1927 b, Radiations emises par le Bacterium tumefaciens. Rev. path. vf 150 during methamorphosis 107 in old age 151 in disease 152 intensity 151 methods 72, 1 19 - oi cancer patients 17!) - - dogs 148, 150 man 148 rats 110 staiving animals I (!) spectrum 15O serum radiation 151 bone marro\v radiation 04 brain radiation 140. 158 of tadpoles 145 C Cancer, blood radiation in 17t> caused by irradiation 183 - cells destroyed by \east 182 definition 178 diagnosis 18O origin 1st problem 177 , radiation of tissue 178 , spectrum 179 carcinoma see cancer cell concentration and mitogcnetic. effect 134, 13S 14 210 SUBJECT INDEX chain reactions, definition 47 - - and radiation 43 chemical radiation 33, 50 chicken embryo, radiation 145 cholesterol and cancer 182, 187 - heliotropifctm 183 chromosome number affected by radiation 169 coagulation omitting radiation 89, 100 colony si/e method 75 colloid coagulation as detector 89, 100 conduction of radiation J 1 1 _ --- [u muscle 154 - in nerve 112, 150 ---- in roots 111, 154 - in yeast cells 110 cornea as detector 84 --, radiation intensity 146 - - - , spectrum 147 cotyledon radiation 141 crown gall origin 171, 178 - and polyploidy 169 cytagenin 152, 180 cytophotolysis 182 day light affecting mitogenetic rays 108 death through irradiation 49, 97, 161, 185 depression of mitogenesis 115 ~- false 70 - secondary 117 detectors, failure of 93 see also methods discontinuous irradiation 103 disks, rotating 103, 105, 112 dispersion 2, 19 dissolution of salt as source of radiation 50 dog blood radiation 148, 150 drosophila eggs as detectors 81, 143 - larvae, radiating 145, 169 K eggs as detectors 81, 142 as senders 142 electric, field 6, 128 embryo radiation 143, 145 Enchelys radiation 137 enzyme radiation 38* error, expcri mental, general 190 with bacteria 78 & Geigcr counter 34, 91 onion roots 58 - tadpole legs 109 - tissue cultures 83 yeast buds 71 - yeast volume 74 -, .sources of in bio- radiation 69, 73, 76, 77, 80 erythema 48 exhaustion by irradiation 43, 76, 113 over-exposure J15, 125, 160 -- of nerves 111, 160 of radiating power 110 eye radiation, see cornea and human radiation r failure in mitogenetic experiments 93 - , see also errors false mitogenetic depression 70 fermentation affected by radiation 85 causing radiation 124, J31 spectrum 37 filters for light 21 finger radiation 97, 184 flocculation of colloids by radiation 89, 100 fruit flies, see drosophila frog eggs as detector 81, 142 larvae see tadpoles - muscle radiation 29, 65 f spectrum 35, 61 SUBJECT INDEX 211 galls and radiation 171 gas effect from organisms 89 Geiger counter measurements 29, 35, 91 gelatin opaque for ultraviolet 25, 59 preventing mitogenetio effects 59, 135 generation time formula 78 glucose causing radiation in blood 148, 149, 180 in protozoa 133 glycolysis spectrum 37 gradual increase in intensity 117 growth rate computation 78 stimulation, see mitogenetic effect H H 2 O a -decomposition by mitogenetic rays 89 by infra-red rays 101 healing of wounds 175 ^ heart beat controlled by beta- radiation 102 Helianthus roots radiating 139 seedlings radiating 141 heliotropism of cholesterol 183 hemoradiometer 66, 154 human radiation 95, 184 hydra, radiating 133 induction effect and intensity 79, 1 14 , formula 64 , significance 79, 191 infra-red rays affecting organisms 101 from organisms 101 injury, see wounds through over-exposure 49, 97, 115, 125, 165, 185 intensity of radiation, definition 8 , increase by selection 134 intensity of radiation, measure- ment, physical 23, 93 f biological 79, 114, 123 , minimal, see threshold of optic nerve 169 of secondary radiation 46, 123, 124 interference 1, 128 intermittent irradiation 103 lag phase 67, 120, 121 larvae radiating 145, 169 leucemia decreasing blood radiation 152 leucocyte radiation 151 light affecting mitogenetic rays 108 filters 21 Liesegang ring method 88 liquid yeast culture method 69 M malignant tumors, see cancer maltase spectrum 37, 39 mechanism of mitogenesis 119 menotoxin 95, 185 menstrual blood, radiation 86, 95, 96, 161, 185 metamorphosis affected by radiation 167 methods of detection: bacterial increase 76, 78 Baron 63, 66 blood drying 149 colloid coagulation 89 cornea 84 finger radiation 97 flocculation 89 egg development 81 Geiger counter 90 Liesegang rings 88 metabolic changes 84 mold spores 80 morphologic changes 86 14* 212 SUBJECT INDEX methods: necrobiotic radiation 99 onion root 54 peroxide decomposition 89, 101 photoelectric counter 90 photography 90, 99 tissue cultures 81 yeast buds 66, 68 yeast growth 72 - yeast metabolism 84 mitogenctic depression 70, 115 effect, analysis J 19 and cell concentration 134 , cause of 122 , formula of 64 from bacteria 87, 131, 161, J77, 178 blood 65, 72, 148 bone marrow 64 - cancer 178 chemical reactions 50, 87 cornea 146 eggs 142 enzymes 37, 52 embryos 143 infusoria 133 larvae 143 menstrual blood 86, 96 mo lds 82, 142 muscle 65, 147 neutralisation 50 onion roots 55 oxidation 29, 51, 64 plants 138 polarized light 45, 80 proteolysis 52 protozoa 133 sarcoma 64 tissue cultures 81 tissues 146 yeast 64, 87 , physical nature of, 59 intensity, see intensity , properties 59, 103 , wave length 49, 59, 61 mitogen?tic effect, spreading of 110 field theory 128 radiation, discovery 55, 59, 119 stimulation, see mitogenctic effect range 49, 61 mitotin 140, 142 monochromator 19 morphologic changes by radiation 86, 96, 161 muscle, conducting radiation 154 radiation 65, 147 - , spectrum 61 N necrobiotic; rays 99 nephelometer measurements 74 nerve conduction 112, 156 exhaustion 111, 160 metabolism 156 , optic, radiation 157 radiation 154 spectra 156 neutralisation, radiation from 50 , spectrum 40 nuclease action, spectrum 38 nuclei in onion roots 55, 129 nucleic acid, secondary radiation of 43, 44 onion root, nuclear stages 129 method 63 radiation 54 , detector mechanism 129 , discovery 55, 59, 119 , sender mechanism 139 , secondary radiation 109, 140 spectra 140 opalina radiation 133 over-exposure 43, 70, 77, 110, 113, 115, 125, 160 SUBJECT INDEX 213 oxidation causing radiation 29, 51, 64 reduction potential 86, 122 spectrum 36, 37 oxy cholesterol radiation 185 , distribution in human body 187 P parabiosis through radiation 160 Paracentrotus, see sea urchin Paramecium radiation 133 parasitism and radiation 171 parthenogenesis through radiation 169 potato radiation 141 phosphate cleavage spectrum 38 photoelectric cell 25 - - tube counter 28, 91 work function 27 yield 29, 92 photoelcctron, definition 16 measurement 28 photographic detection 90, 99 plate, reversal of effect 116 , sensitivity 24 photosynthesis 105 physical nature of mitogenetic effect 59 plant radiation 138 - - affecting morphology 96, 164 tumors 169, 171, 178 polarization by reflection 45, 80 polarized mitogenetic rays 45, 80, 113, 114 polyploidy through radiation 169 potassium radiation affecting heart beat 102 proteolysis radiation 34, 52 spectrum 38 protozoa radiation 133, 137 Q quantum energies 11 , single producing effects 46, 124 theory of radiation 9 U Radiation, atomic 32 , chemical 29, 33, 50 , definition 1 , human 95, 184 , infra-red 101 , mitogenetic (see also mitogen- etic effect) 54 causing antagonism 172 cancer 183 healing of wounds 175 morphologic changes 86, 161 _- parasitism 171 - parthenogenesis 169 polyploidy 169 tumors 171, 183 , discovery of 55, 59, 119 , resonant 45, 158 , secondary, see secondary rad- iation -, solar 22 , thermal 32 , ultraviolet, see ultraviolet rad- iation radium rays and organisms 101 rate of conduction in nerves 1 1 2 in roots 111 recovery from over-exposure 43, 113 reduction potential 86, 122 reflection, definition 2, 4 of mitogenetic rays 45, 80 as source of error 80 refraction 2 rejuvenation of cells 67, 120, 121 persons by blood grafting 151 resonant radiation 45 in brain 158 resorption of tissues 167 respiration of yeast and radiation 84 retardation through irradiation 70, 115, 125 reversal of effect with organisms 1 15, 160 214 SUBJECT INDEX reversal of effect with photo- graphic plates 116 rhythm in cell division 119 rhythmic interruption of radiation 107 rotating disks 103, 112 saliva affecting yeasts 96, 98, 161 sarcoma, definition 178 , radiation 64 sea urchin eggs as detectors 81 aa senders 142 retarded by infra-red 101 larvae changed by irradiation 86, 164 secondary depression 117 radiation, definition 43 , effect on intensity measure- ments 123, 124 of cells 108 of solutions 43 , rate of travel in nerves 112 in r0 ots 111 sedum leaves 141 sensitivity, see intensity, and thres- hold, and photographic plate septicemia decreasing blood rad- iation 152 solar radiation limits 22 spectrometers 19 spectrum, atomic 17 , molecular 17 , mitogenetic 35 , thermal, of tungsten 32 of amylase 37, 39 blood 150 bunsen burner flame 40 cancer tissue 179 chemical reactions 37, 40 cornea 147 fermentation 37 frog muscle 35, 61 glycolysis 37 spectrum of mercury arc 20 maltase 37, 39 - muscle 61, 147 -- nerves 156 neutralisation 40 nuclease 39 -- - onion roots ]40 oxidation 36, 37 - phosphate cleavage 38 proteolysis 38 * -- sucrase 37, 39 - solar 22 a storage of radiation 126 sunflower, see Helianthus symbiosis and radiation 170 T tadpoles affected by radiation 167 , radiation of different parts 145 - , wound healing with 175 thermocouples 23 threshold time as measure of inten- sity 44, 114, 123, 175 for production of effect 104, 123 tissue cultures as detectors 81 as senders 83 radiation 146, 167 tonsillitis decreasing blood radiation 152 tumors caused by radiation 171 of plants 178 , see also cancer turbidity measurements 74 U ultraviolet rays, absorption 23, 25 , affecting organisms 48 photographic plates 24 -- causing cancer 183 from chemical reactions 29, 33 * from organisms, see mito- genetic effect, and radiation, mitogenetic Ureffekt 119 SUBJECT INDEX 215 volumetric method 73 W work function, photoelectric 27 wound healing by radiation 175 , blood radiation 174 radiation 173 treatment with bacteria 177 fly maggots 177 x-rays 10 1 yeast bud method 63, 66, 68, 69 changed by radiation 86, 95, 161 , liquid culture method 69 metabolism affected by rays 84 morphology affected by blood 96, 161 _., by plants 164 by saliva 96, 161 radiation 64 affecting cancer cells 182 volume measurements 73