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The behavior of unicellular organisms in an electromagnetic field
Experimental
548 THE
BEHAVIOR
OF UNICELLULAR ELECTROMAGNETIC
A. A. TEIXEIRA-PINTO,l J. L. CUTLER,’ New England
Institute
for
Medical
Cell Research 20, 548-564 (1960)
ORGANISMS
IN AN
FIELD
L. L. NEJELSKI,
JR.,
and J. H. HELLER Research, Ridgejield,
Conn., U.S.A.
Received September 3, 1959
WHEN
radio frequency fields mere initiated as a device for applying thcrapeutic heat, questions were occasionally raised as to whether or not electromagnetic fields might have a biomedical efIect other than the simple production of thermal energy. The general consensus throughout the years has been that there is no effect other than that due to the production of heat. Since the advent of radar and microwave devices, pathological effects from exposure of individuals working near high po\ver transmitters have been noted [14]. Research in these fields again produced a consensus that thermal damage was the primary, if not the only efl’ect. To a great extent, investigators have tended to ignore some early observations of possible non-thermal effects. The first such observation was made by Xluth [lo] who described the formation of chains of emulsified fat particles in electromagnetic fields. Krasny-Ergen [6, 7, 81 analyzed these observations and explained the phenomena by the formation of electrical dipoles on each particle follo\ved by mutual electrostatic attraction. Additional analyses have been made by Liebesny [C3] and Schwan [ 111. More recently, this same phenomenon was reproduced using pulsed electromagnetic fields in place of continuous fields [12]. IJrom the standpoint the pulsed field is preferable to a continuous of biological investigations, field since the production of heat is minimized. Investigations into the possible significance of this type of alignment of particles as a nonthermal effect of high energy, high frequency fields in biological situations were reported in several preliminary papers from this laboratory [2, 3, 41. One of the phenomena noted was that motile bacteria were constrained in their motion in such electromagnetic fields. This observation led to the investiga1 Postdoctoral Fellow of the Gulbenkian Foundation (Portugal). de Histologia e Embriologia, Faculdade de Medicina, Universidade * Fellow of The National Foundation. Experimental
Cell Research 20
Permanent address: Instituto de Lisboa, Lisboa, Portugal.
Unicellular
organisms in an electromagnetic field
tion of the response of various series of interesting phenomena on these events.
higher unicellular organisms. As a result, a were observed. This presentation is a report
METHODS The experiments described herein utilized a breadboard assembly of electronic components constructed in this laboratory. It consisted of a Hartley circuit oscillating at from approximately 0.1 to 100 megacycles per second and suitably modified to deliver maximum field gradients of several thousand volts peak to peak per centimeter across the enclosure serving as the load. The oscillator was 100 per cent modulated by means of a General Radio Pulser No. 1217-A with several stages of amplification between the pulser and the oscillator. With lower field gradients, unmodulated carrier was also employed for studies principally involving non-viable material. Voltages were measured with a General Radio Vacuum Tube Voltmeter No. 1800-B.’ Wave form was monitored by a Tektronix oscilloscope No. 545.* The energy from the radio frequency source was coupled either directly or through a link from the tank circuit of the oscillator and brought into two electrodes which could be placed in a variety of configurations. One of the most commonly used assemblies was similar to one suggested by Herrick [5] and was constructed by taking a microscope slide and painting electrodes on it with silver printed circuit paint, leaving an air gap of from 2 to 5 mm between the electrodes (Fig. 1 A). The preparations to be examined were placed between two coverslips separated by a gasket of silicone grease (Fig. 1 R) and the sandwich was placed upon the electrodes. Mapping of the electromagnetic lines of force indicates that a rather homogeneous field between the electrodes existed in the horizontal plane (Fig. 7). An insulating plastic stage which could be placed on top of the metal stage of the microscope was made with a hole for optimal light transmission (Fig. 1 C). The various components in place can be seen in Fig. 2. It should be noted that there is no contact between the electrodes and the media or material exposed to the field. When larger enclosures were required to expose the root tips of plants or Drosophila, lucite chambers were constructed with metallic electrodes in their walls covered by 0.1 mm glass cemented to the electrodes. The glass-covered electrodes were separated by an air gap of from 4 mm to 1: cm (Fig. 3). Iron filings, starch particles, colloidal carbon particles of sizes ranging from 0.5 micron to 400 micra, homogenized milk, oil suspensions in water, and water suspensions in oil were studied at a variety of frequencies and field intensities. The substances studied were placed in various media, including air (in Drosophila experiments), water, oil, methyl cellulose solution, glycerol, acetone, sucrose and dextrose solutions, as well as various protozoan and bacterial media. A particularly useful substance for study was a series of excellent suspensions of colloidal polystyrene sphere9 where the particle size was quite precise with a very small standard deviation. Polystyrene spheres of 0.5, 0.8, and I.171 micra were used. Most colloids were in an aqueous 1 Kindly 2 Kindly 3 Kindly
donated by General Radio Corporation. donated by Tektronix, Inc. supplied by J. \I’. Vanderboff, Dow Chemical
Company. Experimental
Cell Research 20
550
A. A. Teixeira-Pinto, L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
Fig. I.-Microscope Fig. 2.-Microscope Experimental
cell assembly: (a) electrodes, (b) cell, and (c) plastic stage. cell assembly with all parts in operational position.
Cell Research 20
Unicellular
organisms in an electromagnetic field
Fig. 5.-Lucite
sample enclosure.
suspension, but other materials were substituted for the suspending phase where this was feasible. The biological substances used included mammalian erythrocytes, macrophages, E. coli, Cl. botulinum, E. typhi, B. sutilis, the root tips of common garlic (Allium sativum), Drosophila melanogaster (wild type), various species of Paramecia, Amoeba proteus, Amoeba limax, Euglena gracillis, and other Euglenoidina, Actinophrys sol, T’olvox, Pandorina, Eudorina, and Chlamydomonas.
EXPERIMENTAL One of the first phenomena observed when particulates were placeci in the electromagnetic field described above was the orientation of asymmetric with their long axes along the lines of force followed by chain formaparticles tion (Fig. 4). Symmetric spheres can show no preferential orientation since there is no long axis, and, hence, only chain formation can be seen in this situation (Fig. 5). Early observations indicated that the orientation and chain formation phenomena were frequency dependent as well as voltage dependExperimentul
Cell Research 20
552
A. A. Teixeira-Pinto,
L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
ent. Thus, if one takes a suspension of polystyrene and explores the effect of a variety of frequencies, there is one frequency band where optimal chain formation occurs with a minimum of voltage between the electrodes. As one departs from this optimum frequency band to either lower or higher frequencies, the amount of voltage required for chain formation, as judged by chain length and time required for their formation, increases by a considerable factor. In several cases these frequency optima \vere found to be within a range where the dielectric constant for both particles and suspending media do not vary. For instance, with polystyrene and water there is no significant change in dielectric constant between 1 and 100 megacycles [l ], yet optimum frequencies for chain formation are found within this range. As biological materials were studied, the frequency and voltage dependency became even more obvious. In one of our previous publications [a] it has been pointed out that motile microorganisms, when placed in the field, were constrained to travel along the electromagnetic lines of force instead of in random directions (Fig. 6). These lines were portrayed by taking microphotographs of the alignment of inert particles in the field (Fig. 7). Such lines of force also occurred around a single electrode with the other electrode at a virtually infinite distance. As various frequencies were explored, it was found that most motile organisms traveled “east-\vest” 1 along the lines of force at frequencies in the lower megacycle range for as long as the field was maintained. As the frequency was increased, however, the organisms made a pivot of 90” and moved “north and south” across the lines of force (Fig. 8). Different organisms took an east-west or north-south orientation at different frequencies. Thus, in a preparation of mixed Colpidium, Rhabdomonas incurva, and Astasia Klebsi at a frequency of 8.5 megacycles, an inter-electrode peak to peak voltage of 309 volts per centimeter was required to have all of the organisms traveling in an east-west direction. =\t 11.5 megacycles it was necessary to use a voltage of 1016 per centimeter to have Rhabdomonas traveling east-west and Astasia north-south, while Colpidium was almost random. At 27 megacycles, only 582 volts per centimeter were required to have all of the organisms travelling in a north-south direction. Hence, it was possible to choose the frequency 1 “East-west” denotes an axis along the lines of force, as portrayed in Fig. 7. “North-south” is the axis at right angles to the lines of force and parallel to the edges of the electrodes.
Fig. 4a.-Potato Fig. 4b.-Same Experimental
starch grains in random preparation
distribution.
as Fig. 4~7, with
Cell Research 20
applied
field at a frequency
of 10 meg.
Unicellular
organisms in an electromagnetic field
Experimental
Cell Research 20
554
A. A. Teixeira-Pinto,
L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
with a sample of mixed organisms where one type is going east-west and another type is simultaneously going north-south. Efforts to define specific east-west and north-south frequencies for different As an example, some Euglena were placed organisms were unsuccessful. betlveen two microscope corerslips sealed with silicone grease. At 5 megacycles the Euglena all went east-west, and at somewhat under 6 megacycles they made a 90” pivot and went north-south. Twelve hours later the organisms still responded by an east-west orientation at 5 megacycles, but the frequency now had to be raised to 18 megacycles to make the cells pivot and orient north-south. The only possible explanation for this response \\-as the fact that the metabolites, either in the cells or media, had been produced and destroyed in the intervening hours. Obviously, this was an extremely small change which caused major diiferenccs in terms of frequency response. This type of varying optima for orientation has been seen in virtually all living cells, depending upon difl’erences in the age of the preparation or in the composition of the media. The difrerence in frequency response was probably due to the change in dielectric constant of ions in the solution as a function of frequency [13]. In view of such sensitivity to very minute amounts of ions, the orientation frequencies could be determined only for a specific sample. In a given situation one can observe, in addition to east-west and northsouth frequencies, “dead” and “confusion” frequencies. The “dead” frequencies \vere so named because of the lack of a visible response of a certain species at the same time and in the same field where other species frequency range was given this \\-ere responding optimally. The “confusion” name because the organisms were obviously sensing the field, were disturbed, and sometimes spun around their centers in the manner of a pinwheel. In one case, however, at 100 megacycles Paramecium have been seen to spin with tremendous velocity about their long axes \vhile stopped or migrating in the north-south direction. An early observation in an an immobilized Paramecium showed that certain asymmetric cytoplasmic inclusions were oriented when the electromagnetic field was impressed and reverted at once to their original position when the field was released. This led us to assume that it might be possible to affect selectively, as a function of frequency and voltage, certain structures or molecular aggregates within the cell. These initial observations on Paramecium were subsequently verified with Amoeba proteus. Fig. 5a.-Polystyrene spheres (1.171 ,u diameter) in random distribution. Fig. 56.-Same preparation as Fig. 50, with applied field at a frequency Experimental
Cell Research 20
of 27 meg.
lYnicellular
organisms in an electromagnetic field
555
Experimental
Cell Research 20
556
A. A. Teixeira-Pinto, L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
Fig. 6a.-Euglenoidina with random orientation. Fig. 6b.-Same preparation as Fig. 64 with applied field of 10 meg. Experimental
Cell Research 20
Unicellular
organisms in an electromagnetic field
Fig. 7a.-Random polystyrene spheres of 1.171 ,u diameter in the interelectrode space. Fig. 7h.-Same preparation as in Fig. 7~2, with spheres mapping field at IO meg. Experimental
Cell Research
70
55s
A. A. Teixeira-Pinto,
L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
When an unattached amoeba with pseudopods extended so that it had a long axis was placed in a 5 megacycle field, it oriented east-west while asymmetric cytoplasmic inclusions oriented in the same direction. This required careful observation because of the cytoplasmic flow, particularly with extending pseudopods. However, when the frequency was rapidly changed to about 27 megacycles, an ,4moeba immediately pivoted 90” to point northsouth, but the cytoplasmic inclusions still oriented east-west vvithin the organism. If an amoeba had its pseudopods extended so that lines could be drawn through three pseudopods, the amoeba as a whole responded to the field according to the resultant of the vectors of the three lines. The above phenomena in Amoeba vvere obtained with moderate voltages of approximately 300 to 500 volts peak to peak across the electrodes with a 15 microsecond pulse duration and a repetition rate of 500 to 1000 pulses per second. As the voltage was increased still higher, Amoeba proteus, but not Amoebu limax, responded by ceasing its pseudopodic extensions and becoming spherical. As can be seen in Fig. 9, there was pronounced flattening of the sphere with a tendency to form an ellipsoid with a major axis parallel to the field. The moment that the field was removed, the spherical form returned. When the field was sufficiently povverful, the sphere ruptured, with extrusion of the cytoplasmic contents, followed by rapid orientation and chain formation of these contents. The observation that intracellular material could be manipulated suggested that genetic material might be affected by such fields. Garlic root tips in vvater and Drosophila in air were therefore exposed to various fields at dill’erent frequencies and voltages. The initial findings in this area of investigation have been presented [31 and further data on chromosomal aberrations and mutations will be presented elsewhere. \Vhen an electromagnetic field is distorted by an object having greater or extends for some less conductivity than the media, the field distortion distance on either side of the object. An example of this could be seen vv-hen observing the behavior of both Amoeba and Euglena together in a held. As the Euglena came into a position relatively close to the Amoeba, they were very rapidly drawn towards the Amoeba and, once they had come virtually into contact with the wall, they rotated with an enormously rapid spin. A similar phenomenon was observed when the amoeba in the field occasionally
Fig. So.-Euglenoidina
in between
Fig. Sb.-Euglenoidina 27 meg.
between
Experimental
Cell Research 20
electrodes.
electrodes
in a north-south
orientation
with
an applied
field at
Unicellular
organisms in an electromagnetic Jield
550
Experimental
Cell Research 20
560
A. A. Teixeira-Pinio,
L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
Fig. S.-Ellipsoidal inclusions.
Amoeba profeus, being exposed to a strong field
Fig. lO.-Spinning
pseudopodic
Experimental
Cell Research 20
fragment
(arrow)
with
oriented
intracellular
of Amoeba profeus exposed to a strong field.
Cnicellular
organisms in an electromagnefic field
Fig. Il.-Amoeba
proteus following
exposure
561
to field.
lost the tips of two of its pseudopods on opposite sides of the organism. The fragments became spherical, migrated down the length of the amoeba, and immediately began to spin very rapidly (Fig. 10). A drop in voltage could stop the spinning completely, and a reversion to the previous conditions could induce it again. One other phenomenon has been noted with Amoeba proteus. If the organism had been exposed to considerable voltage after having assumed a spherical shape and then the field was turned off, the Amoeba seemed no longer capable of normal purposeful pseudopodic movement. Instead, there are wavering cytoplasmic extensions together with an increase in the width of the hyalin layer (Fig. 11). In contrast to the above phenomenon, the ability to cause other microorganisms to orient either with or across the lines of force is a phenomenon which is reproducible without any apparent visible damage to the organisms, provided they are not subjected to any significant heating. Preparations have been placed in the field intermittently for as long as 4 to 5 days with no ostensible evidence of damage. The north-south orientation of living microorganisms was a phenomenon 37 - 60173253
Experimental
Cell Research 20
562
A. A. Teixeira-Pinto, L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
which was unique within the limits of the experimental materials and frequencies used. All other non-biological materials have always aligned along the lines of force or in an east-west direction. When Euglena were killed thermally by a few long pulses at a carrier frequency at which the protozoa moved north-south when alive, the organisms maintained their original orientation. The individual cells, however, no longer remained separated when dead but instead formed chains extending in an east-west direction with the long axis of each cell perpendicular to the chain. If additional heat was introduced while still maintaining the same frequency, the organisms pivoted and lined up end to end in a chain which also was parallel with the field. If, on the other hand, the initial frequency was such that the Euglena were going east-west and they were thermally killed, they immediately took up the end to end chain position. A north-south chain formation has never been observed. In contrast to the normal lining up to form chains of cells in mutual contact which was seen in almost all unicellular non-motile organisms, Chlorella seemed to be a notable exception. Chlorella formed a general east-west type of chain, although at low frequencies it looked more like a network with the predominant axis being east-west. In no case, however, was it possible to induce Chlorella to come within several diameters of its nearest neighbor. It has been noted that when a conductor is in a field, the lines of force dip into it. This frequently causes alignment of particles along the lines of force leading into the conductor. In a preparation with Heliozoa, a similar phenomenon was observed. As soon as the field was turned on, other protozoa immediately appeared to become stuck upon the tip of each spicule. In a similar way, Euglena aggregated with their posterior ends in contact with the tips of the pseudopods of Amoeba and nowhere else upon its surface.
DISCUSSION
As was previously stated on p. 548, the early work with electromagnetic fields at nonthermal levels dealt primarily with the formation of chains by colloidal particles. The explanation later evolved postulated the creation of induced electrical dipoles on each particle [6, 7, 81. Thus the formation of chains by dipole interaction was plausible when applied to the emulsified liquid used as a test system. Another theoretical approach can be evolved by considering the fact that in a given force field, any object which is more or less conductive for the Experimental
Cell Research 20
Unicellular
organisms in an electromagnetic field
field than the surrounding media will cause field distortion. In such a situation, the particles will take up a position which minimizes field distortion. Both of these approaches could also apply to the chaining phenomenon organisms which align eastobserved with polystyrene spheres, nonmotile west, and motile organisms which are constrained to move in an east-west direction. One major objection to both theories, however, lies in the consideration of the dielectric properties. Due to the technical complexities of attempting to measure dielectric properties of various cells and their media into which they are constantly taking LIP and excreting metabolites, polystyrene spheres and water were used to evaluate theory. In both the field distortion and dipole theories, one would expect to find no frequency optima in terms of alignment within the range where the dielectric constant of polystyrene spheres and water remained unchanged. The fact that frequency optima were found within this range tends to make both theories deficient. Existing theory is also deficient in explaining the orientation of an Amoeba versus its cytoplasmic inclusions. At a certain frequency, the organism orients east-west with its intracellular contents forming chains extending in the same however, the organism assumes alignment direction. At higher frequencies, in the north-south direction with its cytoplasmic inclusions still forming chains in the east-west direction. The Amoeba’s orientation also illustrates that the effect of the field is not directly upon motile apparatus such as flagellae or cilia. Thus, there can bc an action of the field upon the cell as a whole as well as a definite interaction with cellular components. This can be seen best in the Amoeba but has also been observed in some other organisms. Thus, the north-south orientation of organisms and the ability to obtain frequency optima within a frequency range where the dielectric constants probably do not vary prevent the utilization of a simple field deformation or induced dipole theory. Electrotropism of cells need not be considered for the explanation of orientation because the glass-insulated electrodes preclude any d.c. component, as is evidenced by the fact that there is no preferential direction-either east or west-taken by organisms reacting to an “east-west” frequency. Furthermore, motile and nonmotile organisms respond similarly in their east-west orientation. Many other theoretical possibilities have been considered, including resonance, magnetic polarization, point heating, electrostriction, etc. No single explanation or group of explanations are completely adequate at present. Further exploration into the biophysical parameters will be presented elsewhere. Experimental
Cell Research 20
564
A. A. Teixeira-Pinto, L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller SUMMARY
1. Unicellular organisms reacted in a variety of ways to high frequency, high voltage electromagnetic fields. 2. Non-motile organisms aligned parallel to the lines of force at frequencies in the lower megacycle range and some of them at right angles to the lines of force at higher frequencies. 3. No non-viable matter has been noted to orient at right angles to the lines of force. They have always aligned parallel to the field and formed chains. 1. Motile microorganisms were constrained to travel only parallel to the field at lower frequencies and at right angles to the lines of force at higher frequencies. 5. Intracellular structures were affected by appropriate selective frequencies. 6. Other phenomena of the behavior of cells and groups of different organisms in electromagnetic fields are described. REFERENCES T., Personal communication. 1. GILLESPIE, J. H., Proc. Third Int. Symp. on the Reticuloendothelial System (in press). Ronald 2. HELLER, Press, New York. J. H. and TEXEIRA-PINTO, A. A., Research Bull. 4, 10 (1958). 3. HELLER, 4. ~ Nature 183, 905 (1959). J. F., Personal communication. 5. HERRICK, W., Arch. Phys. Therapy. 21, 362 (1940). 6. KRASNY-ERGEN, Hochfreq. techn. Elektroak. 48, 126 (1936). 7. . , ibid. h9,-195 (1937). 8. ~ P., Arch. Phys. Therapy 19, 736 (1939). 9. LIEBESNY, 10. MUTH, E., KoZZoid Z. 41, 97 (1927). Heat, p. 55. Elizabeth Licht, Publisher, New 11. SCHWAN, H. P., in Licht, Sidney: Therapeutic Haven, Connecticut, 1958. A., WAKIM, K. G., HERRICK, J. F. and KRUSEN, F. H., Arch. Phys. Med. 40, 12. WILDERVANCK, 45 (1959). H. H., MERRIT, L. L., Jr. and DEAN, J. A., Instrumental Methods of Analysis, 13. WILLARD, pp. 314-319. D. van Nostrand Company, New York, 1951. D. R., MONAHAS, J. P., NICHOLSON, W. J. and ALDRICH, J. J., IRE Transactions 14. WILLIAMS, on Medical Electronics, PGME-4, 17 (1956).
Experimental
Cell Research 20
548 THE
BEHAVIOR
OF UNICELLULAR ELECTROMAGNETIC
A. A. TEIXEIRA-PINTO,l J. L. CUTLER,’ New England
Institute
for
Medical
Cell Research 20, 548-564 (1960)
ORGANISMS
IN AN
FIELD
L. L. NEJELSKI,
JR.,
and J. H. HELLER Research, Ridgejield,
Conn., U.S.A.
Received September 3, 1959
WHEN
radio frequency fields mere initiated as a device for applying thcrapeutic heat, questions were occasionally raised as to whether or not electromagnetic fields might have a biomedical efIect other than the simple production of thermal energy. The general consensus throughout the years has been that there is no effect other than that due to the production of heat. Since the advent of radar and microwave devices, pathological effects from exposure of individuals working near high po\ver transmitters have been noted [14]. Research in these fields again produced a consensus that thermal damage was the primary, if not the only efl’ect. To a great extent, investigators have tended to ignore some early observations of possible non-thermal effects. The first such observation was made by Xluth [lo] who described the formation of chains of emulsified fat particles in electromagnetic fields. Krasny-Ergen [6, 7, 81 analyzed these observations and explained the phenomena by the formation of electrical dipoles on each particle follo\ved by mutual electrostatic attraction. Additional analyses have been made by Liebesny [C3] and Schwan [ 111. More recently, this same phenomenon was reproduced using pulsed electromagnetic fields in place of continuous fields [12]. IJrom the standpoint the pulsed field is preferable to a continuous of biological investigations, field since the production of heat is minimized. Investigations into the possible significance of this type of alignment of particles as a nonthermal effect of high energy, high frequency fields in biological situations were reported in several preliminary papers from this laboratory [2, 3, 41. One of the phenomena noted was that motile bacteria were constrained in their motion in such electromagnetic fields. This observation led to the investiga1 Postdoctoral Fellow of the Gulbenkian Foundation (Portugal). de Histologia e Embriologia, Faculdade de Medicina, Universidade * Fellow of The National Foundation. Experimental
Cell Research 20
Permanent address: Instituto de Lisboa, Lisboa, Portugal.
Unicellular
organisms in an electromagnetic field
tion of the response of various series of interesting phenomena on these events.
higher unicellular organisms. As a result, a were observed. This presentation is a report
METHODS The experiments described herein utilized a breadboard assembly of electronic components constructed in this laboratory. It consisted of a Hartley circuit oscillating at from approximately 0.1 to 100 megacycles per second and suitably modified to deliver maximum field gradients of several thousand volts peak to peak per centimeter across the enclosure serving as the load. The oscillator was 100 per cent modulated by means of a General Radio Pulser No. 1217-A with several stages of amplification between the pulser and the oscillator. With lower field gradients, unmodulated carrier was also employed for studies principally involving non-viable material. Voltages were measured with a General Radio Vacuum Tube Voltmeter No. 1800-B.’ Wave form was monitored by a Tektronix oscilloscope No. 545.* The energy from the radio frequency source was coupled either directly or through a link from the tank circuit of the oscillator and brought into two electrodes which could be placed in a variety of configurations. One of the most commonly used assemblies was similar to one suggested by Herrick [5] and was constructed by taking a microscope slide and painting electrodes on it with silver printed circuit paint, leaving an air gap of from 2 to 5 mm between the electrodes (Fig. 1 A). The preparations to be examined were placed between two coverslips separated by a gasket of silicone grease (Fig. 1 R) and the sandwich was placed upon the electrodes. Mapping of the electromagnetic lines of force indicates that a rather homogeneous field between the electrodes existed in the horizontal plane (Fig. 7). An insulating plastic stage which could be placed on top of the metal stage of the microscope was made with a hole for optimal light transmission (Fig. 1 C). The various components in place can be seen in Fig. 2. It should be noted that there is no contact between the electrodes and the media or material exposed to the field. When larger enclosures were required to expose the root tips of plants or Drosophila, lucite chambers were constructed with metallic electrodes in their walls covered by 0.1 mm glass cemented to the electrodes. The glass-covered electrodes were separated by an air gap of from 4 mm to 1: cm (Fig. 3). Iron filings, starch particles, colloidal carbon particles of sizes ranging from 0.5 micron to 400 micra, homogenized milk, oil suspensions in water, and water suspensions in oil were studied at a variety of frequencies and field intensities. The substances studied were placed in various media, including air (in Drosophila experiments), water, oil, methyl cellulose solution, glycerol, acetone, sucrose and dextrose solutions, as well as various protozoan and bacterial media. A particularly useful substance for study was a series of excellent suspensions of colloidal polystyrene sphere9 where the particle size was quite precise with a very small standard deviation. Polystyrene spheres of 0.5, 0.8, and I.171 micra were used. Most colloids were in an aqueous 1 Kindly 2 Kindly 3 Kindly
donated by General Radio Corporation. donated by Tektronix, Inc. supplied by J. \I’. Vanderboff, Dow Chemical
Company. Experimental
Cell Research 20
550
A. A. Teixeira-Pinto, L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
Fig. I.-Microscope Fig. 2.-Microscope Experimental
cell assembly: (a) electrodes, (b) cell, and (c) plastic stage. cell assembly with all parts in operational position.
Cell Research 20
Unicellular
organisms in an electromagnetic field
Fig. 5.-Lucite
sample enclosure.
suspension, but other materials were substituted for the suspending phase where this was feasible. The biological substances used included mammalian erythrocytes, macrophages, E. coli, Cl. botulinum, E. typhi, B. sutilis, the root tips of common garlic (Allium sativum), Drosophila melanogaster (wild type), various species of Paramecia, Amoeba proteus, Amoeba limax, Euglena gracillis, and other Euglenoidina, Actinophrys sol, T’olvox, Pandorina, Eudorina, and Chlamydomonas.
EXPERIMENTAL One of the first phenomena observed when particulates were placeci in the electromagnetic field described above was the orientation of asymmetric with their long axes along the lines of force followed by chain formaparticles tion (Fig. 4). Symmetric spheres can show no preferential orientation since there is no long axis, and, hence, only chain formation can be seen in this situation (Fig. 5). Early observations indicated that the orientation and chain formation phenomena were frequency dependent as well as voltage dependExperimentul
Cell Research 20
552
A. A. Teixeira-Pinto,
L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
ent. Thus, if one takes a suspension of polystyrene and explores the effect of a variety of frequencies, there is one frequency band where optimal chain formation occurs with a minimum of voltage between the electrodes. As one departs from this optimum frequency band to either lower or higher frequencies, the amount of voltage required for chain formation, as judged by chain length and time required for their formation, increases by a considerable factor. In several cases these frequency optima \vere found to be within a range where the dielectric constant for both particles and suspending media do not vary. For instance, with polystyrene and water there is no significant change in dielectric constant between 1 and 100 megacycles [l ], yet optimum frequencies for chain formation are found within this range. As biological materials were studied, the frequency and voltage dependency became even more obvious. In one of our previous publications [a] it has been pointed out that motile microorganisms, when placed in the field, were constrained to travel along the electromagnetic lines of force instead of in random directions (Fig. 6). These lines were portrayed by taking microphotographs of the alignment of inert particles in the field (Fig. 7). Such lines of force also occurred around a single electrode with the other electrode at a virtually infinite distance. As various frequencies were explored, it was found that most motile organisms traveled “east-\vest” 1 along the lines of force at frequencies in the lower megacycle range for as long as the field was maintained. As the frequency was increased, however, the organisms made a pivot of 90” and moved “north and south” across the lines of force (Fig. 8). Different organisms took an east-west or north-south orientation at different frequencies. Thus, in a preparation of mixed Colpidium, Rhabdomonas incurva, and Astasia Klebsi at a frequency of 8.5 megacycles, an inter-electrode peak to peak voltage of 309 volts per centimeter was required to have all of the organisms traveling in an east-west direction. =\t 11.5 megacycles it was necessary to use a voltage of 1016 per centimeter to have Rhabdomonas traveling east-west and Astasia north-south, while Colpidium was almost random. At 27 megacycles, only 582 volts per centimeter were required to have all of the organisms travelling in a north-south direction. Hence, it was possible to choose the frequency 1 “East-west” denotes an axis along the lines of force, as portrayed in Fig. 7. “North-south” is the axis at right angles to the lines of force and parallel to the edges of the electrodes.
Fig. 4a.-Potato Fig. 4b.-Same Experimental
starch grains in random preparation
distribution.
as Fig. 4~7, with
Cell Research 20
applied
field at a frequency
of 10 meg.
Unicellular
organisms in an electromagnetic field
Experimental
Cell Research 20
554
A. A. Teixeira-Pinto,
L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
with a sample of mixed organisms where one type is going east-west and another type is simultaneously going north-south. Efforts to define specific east-west and north-south frequencies for different As an example, some Euglena were placed organisms were unsuccessful. betlveen two microscope corerslips sealed with silicone grease. At 5 megacycles the Euglena all went east-west, and at somewhat under 6 megacycles they made a 90” pivot and went north-south. Twelve hours later the organisms still responded by an east-west orientation at 5 megacycles, but the frequency now had to be raised to 18 megacycles to make the cells pivot and orient north-south. The only possible explanation for this response \\-as the fact that the metabolites, either in the cells or media, had been produced and destroyed in the intervening hours. Obviously, this was an extremely small change which caused major diiferenccs in terms of frequency response. This type of varying optima for orientation has been seen in virtually all living cells, depending upon difl’erences in the age of the preparation or in the composition of the media. The difrerence in frequency response was probably due to the change in dielectric constant of ions in the solution as a function of frequency [13]. In view of such sensitivity to very minute amounts of ions, the orientation frequencies could be determined only for a specific sample. In a given situation one can observe, in addition to east-west and northsouth frequencies, “dead” and “confusion” frequencies. The “dead” frequencies \vere so named because of the lack of a visible response of a certain species at the same time and in the same field where other species frequency range was given this \\-ere responding optimally. The “confusion” name because the organisms were obviously sensing the field, were disturbed, and sometimes spun around their centers in the manner of a pinwheel. In one case, however, at 100 megacycles Paramecium have been seen to spin with tremendous velocity about their long axes \vhile stopped or migrating in the north-south direction. An early observation in an an immobilized Paramecium showed that certain asymmetric cytoplasmic inclusions were oriented when the electromagnetic field was impressed and reverted at once to their original position when the field was released. This led us to assume that it might be possible to affect selectively, as a function of frequency and voltage, certain structures or molecular aggregates within the cell. These initial observations on Paramecium were subsequently verified with Amoeba proteus. Fig. 5a.-Polystyrene spheres (1.171 ,u diameter) in random distribution. Fig. 56.-Same preparation as Fig. 50, with applied field at a frequency Experimental
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of 27 meg.
lYnicellular
organisms in an electromagnetic field
555
Experimental
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556
A. A. Teixeira-Pinto, L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
Fig. 6a.-Euglenoidina with random orientation. Fig. 6b.-Same preparation as Fig. 64 with applied field of 10 meg. Experimental
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Unicellular
organisms in an electromagnetic field
Fig. 7a.-Random polystyrene spheres of 1.171 ,u diameter in the interelectrode space. Fig. 7h.-Same preparation as in Fig. 7~2, with spheres mapping field at IO meg. Experimental
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55s
A. A. Teixeira-Pinto,
L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
When an unattached amoeba with pseudopods extended so that it had a long axis was placed in a 5 megacycle field, it oriented east-west while asymmetric cytoplasmic inclusions oriented in the same direction. This required careful observation because of the cytoplasmic flow, particularly with extending pseudopods. However, when the frequency was rapidly changed to about 27 megacycles, an ,4moeba immediately pivoted 90” to point northsouth, but the cytoplasmic inclusions still oriented east-west vvithin the organism. If an amoeba had its pseudopods extended so that lines could be drawn through three pseudopods, the amoeba as a whole responded to the field according to the resultant of the vectors of the three lines. The above phenomena in Amoeba vvere obtained with moderate voltages of approximately 300 to 500 volts peak to peak across the electrodes with a 15 microsecond pulse duration and a repetition rate of 500 to 1000 pulses per second. As the voltage was increased still higher, Amoeba proteus, but not Amoebu limax, responded by ceasing its pseudopodic extensions and becoming spherical. As can be seen in Fig. 9, there was pronounced flattening of the sphere with a tendency to form an ellipsoid with a major axis parallel to the field. The moment that the field was removed, the spherical form returned. When the field was sufficiently povverful, the sphere ruptured, with extrusion of the cytoplasmic contents, followed by rapid orientation and chain formation of these contents. The observation that intracellular material could be manipulated suggested that genetic material might be affected by such fields. Garlic root tips in vvater and Drosophila in air were therefore exposed to various fields at dill’erent frequencies and voltages. The initial findings in this area of investigation have been presented [31 and further data on chromosomal aberrations and mutations will be presented elsewhere. \Vhen an electromagnetic field is distorted by an object having greater or extends for some less conductivity than the media, the field distortion distance on either side of the object. An example of this could be seen vv-hen observing the behavior of both Amoeba and Euglena together in a held. As the Euglena came into a position relatively close to the Amoeba, they were very rapidly drawn towards the Amoeba and, once they had come virtually into contact with the wall, they rotated with an enormously rapid spin. A similar phenomenon was observed when the amoeba in the field occasionally
Fig. So.-Euglenoidina
in between
Fig. Sb.-Euglenoidina 27 meg.
between
Experimental
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electrodes.
electrodes
in a north-south
orientation
with
an applied
field at
Unicellular
organisms in an electromagnetic Jield
550
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560
A. A. Teixeira-Pinio,
L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
Fig. S.-Ellipsoidal inclusions.
Amoeba profeus, being exposed to a strong field
Fig. lO.-Spinning
pseudopodic
Experimental
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fragment
(arrow)
with
oriented
intracellular
of Amoeba profeus exposed to a strong field.
Cnicellular
organisms in an electromagnefic field
Fig. Il.-Amoeba
proteus following
exposure
561
to field.
lost the tips of two of its pseudopods on opposite sides of the organism. The fragments became spherical, migrated down the length of the amoeba, and immediately began to spin very rapidly (Fig. 10). A drop in voltage could stop the spinning completely, and a reversion to the previous conditions could induce it again. One other phenomenon has been noted with Amoeba proteus. If the organism had been exposed to considerable voltage after having assumed a spherical shape and then the field was turned off, the Amoeba seemed no longer capable of normal purposeful pseudopodic movement. Instead, there are wavering cytoplasmic extensions together with an increase in the width of the hyalin layer (Fig. 11). In contrast to the above phenomenon, the ability to cause other microorganisms to orient either with or across the lines of force is a phenomenon which is reproducible without any apparent visible damage to the organisms, provided they are not subjected to any significant heating. Preparations have been placed in the field intermittently for as long as 4 to 5 days with no ostensible evidence of damage. The north-south orientation of living microorganisms was a phenomenon 37 - 60173253
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562
A. A. Teixeira-Pinto, L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller
which was unique within the limits of the experimental materials and frequencies used. All other non-biological materials have always aligned along the lines of force or in an east-west direction. When Euglena were killed thermally by a few long pulses at a carrier frequency at which the protozoa moved north-south when alive, the organisms maintained their original orientation. The individual cells, however, no longer remained separated when dead but instead formed chains extending in an east-west direction with the long axis of each cell perpendicular to the chain. If additional heat was introduced while still maintaining the same frequency, the organisms pivoted and lined up end to end in a chain which also was parallel with the field. If, on the other hand, the initial frequency was such that the Euglena were going east-west and they were thermally killed, they immediately took up the end to end chain position. A north-south chain formation has never been observed. In contrast to the normal lining up to form chains of cells in mutual contact which was seen in almost all unicellular non-motile organisms, Chlorella seemed to be a notable exception. Chlorella formed a general east-west type of chain, although at low frequencies it looked more like a network with the predominant axis being east-west. In no case, however, was it possible to induce Chlorella to come within several diameters of its nearest neighbor. It has been noted that when a conductor is in a field, the lines of force dip into it. This frequently causes alignment of particles along the lines of force leading into the conductor. In a preparation with Heliozoa, a similar phenomenon was observed. As soon as the field was turned on, other protozoa immediately appeared to become stuck upon the tip of each spicule. In a similar way, Euglena aggregated with their posterior ends in contact with the tips of the pseudopods of Amoeba and nowhere else upon its surface.
DISCUSSION
As was previously stated on p. 548, the early work with electromagnetic fields at nonthermal levels dealt primarily with the formation of chains by colloidal particles. The explanation later evolved postulated the creation of induced electrical dipoles on each particle [6, 7, 81. Thus the formation of chains by dipole interaction was plausible when applied to the emulsified liquid used as a test system. Another theoretical approach can be evolved by considering the fact that in a given force field, any object which is more or less conductive for the Experimental
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Unicellular
organisms in an electromagnetic field
field than the surrounding media will cause field distortion. In such a situation, the particles will take up a position which minimizes field distortion. Both of these approaches could also apply to the chaining phenomenon organisms which align eastobserved with polystyrene spheres, nonmotile west, and motile organisms which are constrained to move in an east-west direction. One major objection to both theories, however, lies in the consideration of the dielectric properties. Due to the technical complexities of attempting to measure dielectric properties of various cells and their media into which they are constantly taking LIP and excreting metabolites, polystyrene spheres and water were used to evaluate theory. In both the field distortion and dipole theories, one would expect to find no frequency optima in terms of alignment within the range where the dielectric constant of polystyrene spheres and water remained unchanged. The fact that frequency optima were found within this range tends to make both theories deficient. Existing theory is also deficient in explaining the orientation of an Amoeba versus its cytoplasmic inclusions. At a certain frequency, the organism orients east-west with its intracellular contents forming chains extending in the same however, the organism assumes alignment direction. At higher frequencies, in the north-south direction with its cytoplasmic inclusions still forming chains in the east-west direction. The Amoeba’s orientation also illustrates that the effect of the field is not directly upon motile apparatus such as flagellae or cilia. Thus, there can bc an action of the field upon the cell as a whole as well as a definite interaction with cellular components. This can be seen best in the Amoeba but has also been observed in some other organisms. Thus, the north-south orientation of organisms and the ability to obtain frequency optima within a frequency range where the dielectric constants probably do not vary prevent the utilization of a simple field deformation or induced dipole theory. Electrotropism of cells need not be considered for the explanation of orientation because the glass-insulated electrodes preclude any d.c. component, as is evidenced by the fact that there is no preferential direction-either east or west-taken by organisms reacting to an “east-west” frequency. Furthermore, motile and nonmotile organisms respond similarly in their east-west orientation. Many other theoretical possibilities have been considered, including resonance, magnetic polarization, point heating, electrostriction, etc. No single explanation or group of explanations are completely adequate at present. Further exploration into the biophysical parameters will be presented elsewhere. Experimental
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564
A. A. Teixeira-Pinto, L. L. Nejelski, Jr., J. L. Cutler and J. H. Heller SUMMARY
1. Unicellular organisms reacted in a variety of ways to high frequency, high voltage electromagnetic fields. 2. Non-motile organisms aligned parallel to the lines of force at frequencies in the lower megacycle range and some of them at right angles to the lines of force at higher frequencies. 3. No non-viable matter has been noted to orient at right angles to the lines of force. They have always aligned parallel to the field and formed chains. 1. Motile microorganisms were constrained to travel only parallel to the field at lower frequencies and at right angles to the lines of force at higher frequencies. 5. Intracellular structures were affected by appropriate selective frequencies. 6. Other phenomena of the behavior of cells and groups of different organisms in electromagnetic fields are described. REFERENCES T., Personal communication. 1. GILLESPIE, J. H., Proc. Third Int. Symp. on the Reticuloendothelial System (in press). Ronald 2. HELLER, Press, New York. J. H. and TEXEIRA-PINTO, A. A., Research Bull. 4, 10 (1958). 3. HELLER, 4. ~ Nature 183, 905 (1959). J. F., Personal communication. 5. HERRICK, W., Arch. Phys. Therapy. 21, 362 (1940). 6. KRASNY-ERGEN, Hochfreq. techn. Elektroak. 48, 126 (1936). 7. . , ibid. h9,-195 (1937). 8. ~ P., Arch. Phys. Therapy 19, 736 (1939). 9. LIEBESNY, 10. MUTH, E., KoZZoid Z. 41, 97 (1927). Heat, p. 55. Elizabeth Licht, Publisher, New 11. SCHWAN, H. P., in Licht, Sidney: Therapeutic Haven, Connecticut, 1958. A., WAKIM, K. G., HERRICK, J. F. and KRUSEN, F. H., Arch. Phys. Med. 40, 12. WILDERVANCK, 45 (1959). H. H., MERRIT, L. L., Jr. and DEAN, J. A., Instrumental Methods of Analysis, 13. WILLARD, pp. 314-319. D. van Nostrand Company, New York, 1951. D. R., MONAHAS, J. P., NICHOLSON, W. J. and ALDRICH, J. J., IRE Transactions 14. WILLIAMS, on Medical Electronics, PGME-4, 17 (1956).
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