LETHAL AND QUASI-LETHAL EFFECTS PRODUCE BY MONOCHROMATIC ULTRA-VIOLET IRRADIATION

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1 474 LETHAL AND QUASI-LETHAL EFFECTS PRODUCE BY MONOCHROMATIC ULTRA-VIOLET IRRADIATION BY A. L. MCAULAY AND M. C. TAYLOR Physics Laboratory, University of Tasmania (Received 26 April 1939) (With Three Text-figures) SCOPE OF EXPERIMENTS THE following is an account of some experiments made on lethal and quasi-lethal effects over a range of biological material, using monochromatic ultra-violet radiation. Experiments of this type have been made in the past on bacteria, but even with this material the results obtained do not seem to be consistent. For example, Giese (1938) states "The 3660 A. is generally considered beyond the region of destructive action. On the other hand Coblentz and Fulton consider this region in very high doses bactericidal...." NATURE OF BIOLOGICAL MATERIAL The biological material used was spread over as large a range as practicable in our circumstances, in both animal and vegetable kingdoms. The materials used and the effects looked for were as follows: (i) Fungi. The spores of species of the genus. Chaetomium were irradiated and the total energy per sq. cm. that prevented 50 % of the spores from germinating was recorded. The total energy per sq. cm. will henceforth be described as the dose. (ii) Protozoa. Samples of Paramecium were irradiated, and the dose required to kill most of them was recorded (25 % survival was taken as a criterion). (iii) Arthropoda. Insect larvae (Drosophila melanogaster) were irradiated as soon as possible after hatching and the dose required to prevent 50 % developing into imagos was recorded. (iv) Bacteria. An innoculation of bacteria (B. coli) was irradiated and the dose recorded that just prevented colonies forming. (v) Angiospermae. Pollen. Maize pollen was irradiated and the smallest dose found which prevented pollen tubes from growing. It was of course recognized that many factors would affect the results observed, but as the effects looked for were of a large scale, sources of error of the order 20 %, if inconvenient to eliminate, were ignored. The principal precautions taken were

2 Monochromatic ultra-violet irradiation 475 aimed at making the conditions of a set of experiments uniform. The accuracy to be expected with the various materials can be estimated from the accounts of experiments given later. It is roughly in the order in which the material has been arranged above; Chaetomium experiments being the most accurate, pollen experiments the least. GENERAL NATURE OF RESULTS The range of wave-lengths investigated was from 254 to 365 m/x, and all the materials showed the same general behaviour. Fig. 1 records the doses required to produce the lethal effects described above. These doses in joule/cm. 2 are plotted Fig. 1. Lethal and quasi-lethal doses in joule/cm. 1 plotted against wave-lengths for the materials Chaetomium, Paramedum, Drosophila, B. coli and maize pollen. Curve E gives the intensity of solar radiation at sea-level, zenith distance o. as ordinates against wave-lengths as abscissae over the range m^x. A large ordinate thus represents a high resistance to radiation of the wave-length given by the abscissa. Three different scales are necessary, the ratios of the ordinates being as 100 : 10 : 1, on account of the greatly differing resistance of the materials used. It is worth noticing that the ratio of the maximum to the minimum doses recorded in the figure is as 20,000 : 1. The most striking feature of the series of experiments is seen to be the sudden rise of the resistance in all the material examined between 297 and 313 m/x, and the fact that in every case the lethal dose is approximately independent of wave-length from 254 to 297 m/z. At 313 m/x the lethal dose is from 100 to 500 times greater than it is at 280

3 476 A. L. MCAULAY AND M. C. TAYLOR This is a striking result in view of the fact that the effects observed are very different with different types of material. In Paramecium the effect appears to be of the nature of osmotic disturbance; the animal swells and finally bursts, extruding protoplasm generally before it ceases to move. In Chaetonrium the effect is undetectable in the spore, which is dry and irradiated on glass. The dose that prevents a Drosophila larva forming an imago is less than a tenth of that required to kill an animal while irradiation is in progress. It would appear highly probable that the effect of irradiation on different materials is quite different, but has the common property of a rise of some hundreds of times between 313 and 297 m^, and is relatively independent of wave-length from about m/x. Correlation between lethal effects and radiation present in sunlight. A correlation indicated in Fig. 1 suggests an explanation of this result. A curve is included showing the intensity of the shortest ultra-violet in solar radiation at sea-level, zenith distance 0 (Abbot, 1937, p. 77). It will be noticed that the rapid rise in the resistance to radiation occurs at the wave-length of the shortest waves in daylight at sea-level. EXPERIMENTAL A. Irradiation, measurement of intensity, etc. The source of radiation was a mercury vapour arc lamp, and two quartz monochromators were available for selecting radiation of the desired wave-length. The first is a large aperture monochromator similar in design to one constructed for photo-chemical work (Bowden & Snow, 1934). The optical parts are of fused quartz. This was used for most of the experiments as it gives a spectrum of high intensity. The main disadvantage of this monochromator is the necessity for guarding against the comparatively high percentage of scattered light. The other, a Hilger instrument (D33), gives a much lower intensity but greater purity of spectrum. Intensity measurements were made with a Hilger thermopile (F86) calibrated by a Leslie cube, in front of which a small shutter in a large diaphragm' was opened and shut. The temperature of the surface of the Leslie cube when the shutter was open was measured with a small thermocouple for temperatures between 70 and ioo C. It is not claimed that the absolute intensities are accurate, but checks made with a furnace between 800 and C, using an optical pyrometer to measure temperature, show that they are of the right order of magnitude. They are probably not in error by a factor of more than 1-5. Purity of spectrum. It is most necessary to prevent contamination of the long wave-lengths by the short ones owing to the much greater effect of the latter, and when using the large monochromator, niters had to be inserted to eliminate scattered radiation. For the purpose of these experiments glass niters of thicknesses ranging from that of a microscope cover slip to plate glass were sufficient and convenient. Difficulties arose with the lines in the critical region, namely, 297 and 302 m^x, and as only a broad survey is intended in the present investigation, refinements were not attempted. It is hoped to make a detailed investigation of this

4 Monochromatic ultra-violet irradiation 477 region later. Two sources of trouble arise, contamination of 302 by 297 my, will make 302 m^, appear more lethal than it is, and contamination of 297 by 302 m^. will make the measured intensity of 297 m/x too great, and make it appear less lethal than it is. B. Methods used in irradiating material (i) Chaetomium. Spores of the fungus C. globosum were spread with a camel hair brush on one side of a diamond scratch on uranium glass, and placed in the focused spectrum of the required spectral line. The width of the band of spores was less than that of the slit image. The spores after exposure were transferred to malt agar in a Petri dish, and the number germinating was counted. This was done o be "40 a \ 254rna m/x Doses in joules per sq. cm Fig. 2. Percentage survival of Chaetomium spores plotted against dose for three different wave-lengths. for a range of exposures with each spectral line and a curve plotted giving the number which germinated for each dose. In some cases germination was greatly delayed by irradiation, the effect being most marked for a given percentage germination in the 313 my region. Fig. 2 shows representative curves. The two points shown as circles in the "365 my" curve were obtained using C. elatum instead of C. globosum. Both fungi were obtained from the Centraal-bureau voor Schimmelcultures, Baarn, Holland. (ii) Paramecium. A strain of Paramecium was obtained locally and kept in a mixture of pond water and straw infusion. Irradiations were made with both monochromators on this material. The difficulties met with were greater using the Hilger instrument and description of the technique will be confined to this instrument. The irradiations were made in the focused spectrum and the animals were confined in a channel of water about 1 cm. long by \ mm. deep by \ mm.

5 478 A. L. MCAULAY AND M. C. TAYLOR across. It was necessary to make the dimensions small as the spectrum was in a vertical plane and the water had to be maintained in position by surface tension; also the width of the spectral lines was made small so that the spectrum should be as pure as possible. With such a small amount of water available, evaporation was a serious problem. To confine it in a cell saturated with water vapour is effective only if all parts of the cell are kept closely at the same temperature. The water was kept in the desired place by outlining a strip on uranium glass with de Khotinsky cement. The difference of surface tension between the water-glass and watercement surfaces was found sufficient to enable the channel of water to be manipulated conveniently. In the Hilger monochromator only four or five animals could be used, but in the larger monochromator numbers of the order of thirty could be handled conveniently. Control strips of water were placed as near as possible to the experimental strips but outside the spectral lines and no difficulty was found in keeping the animals in them alive and healthy. It was evident that in the case of Paramecium many factors were operating which influenced the dose required to kill. The age and condition of the culture was one factor, the intensity of radiation another. The effect of intensity of radiation is the most important factor, as this probably influences the form of the curve as well as the absolute values of the lethal doses. The lethal effect appears to increase with increase in intensity and as 313 m^. is considerably more intense than the shorter wave-lengths, the sudden rise in lethality from 313 to 297 m/i will actually be greater than recorded. As a record of experimental results the figures should be read in conjunction with the intensities at which the irradiation was made. Curves plotting number surviving against dose drop sharply at the end, and an estimate of the dose required to kill three-quarters of the animals was taken as a reasonable measure of the average lethal dose. (iii) Drosopfala melanogaster. A strain of D. melanogaster kindly supplied by Dr W. L. Waterhouse, of the Department of Agriculture, University of Sydney, was kept on a food mixture of molasses, agar and corn meal, innoculated with a yeast culture. To obtain large numbers of larvae at one time, the flies were mated when between 4 and 6 days old. After about 4 hr., they were transferred to a bottle containing fresh food which was spread on a microscope slide for easy manipulation under a low-power microscope. The larvae began to appear after about 24 hr. and soon large numbers were available. These were picked off with a needle point and transferred to a channel of water confined by wax on a piece of uranium glass. The water surface was about 15x1 mm. and it could be placed in the focused spectrum or in the diverging beam below a slit to select a spectral line whichever was more convenient. A slit was used for all wave-lengths shorter than 313 m/x. The larvae were irradiate.d in batches of fifty and a range of doses was given with a ratio of about 10:1 between largest and smallest dose. The lethal dose was estimated by plotting the percentage of larvae which reached the adult stage as ordinate against dose as abscissa. The estimated dose which allowed 50 % of the

6 Monochromatic ultra-violet irradiation 479 larvae to reach this stage was recorded as. the lethal dose for the wave-length under investigation. Difficulties were met with in the variation of the condition of the food for the larvae. The controls showed a variation over a range of from 45 to 96 % survival and the average survival was slightly more than 70 %. Precautions were taken to ensure that the larvae used as controls were given exactly the same treatment as the experimental animals. Both were washed from a channel of water on a glass surface with a few drops into a 4 x i in. specimen jar which contained food to a depth of about i in. Precautions were taken to prevent contamination of the food by bacteria and fungus, but such contamination could not be completely eliminated. (iv) Bacterium coli. A culture of B. coli was kept on a nutrient medium; but as it grew well on the malt agar used for fungus culture, the latter was used in the experiments. Experiments were of two general types. In one the bacteria were irradiated on the medium, in the other in sterile water on a glass surface. For the first method a strip of the medium was spread with a suspension of bacteria in sterile water and placed in the focused spectrum of the large monochromator. A series of experiments was made with different strips covering a range of exposures for each line. In some, after incubating for a day at 28 0 C, the line showed clear and the bacteria had grown on either side of it. This was counted as "killed". In others no sign of where the line had crossed the medium was visible. This was counted as "not killed". When the position of the line was just visibly different from its surroundings, the dose was recorded as "just killing". The second method was used to eliminate the effect of irradiation on the medium. A fairly large drop of a suspension of bacteria in sterile water was confined by de Khotinsky cement on the upper surface of a flat piece of uranium glass. The shape of the channel allowed the drop to be placed in a spectral line or in the' diverging beam below a selecting slit. In order to prevent excessive evaporation the glass was held in a cell with a corex top. After an exposure a platinum loop was used to inoculate the malt-agar medium in four places. One Petri dish of medium was sufficient to take the inoculations for the range of exposures at one wavelength as well as controls. Although there was some delay in the appearance of colonies for the longer exposures, the dose which "just killed" was easily found. About 24 hr. was sufficient for all colonies to appear. The second method was used only at the wave-lengths 254, 265, 280, 313 m/x. For each wave-length it was found that the lethal dose was about four times that given by the first method. With both methods, the doses should be considered as possibly in error by a factor of 2. The two methods of exposure probably represent two distinct lethal effects. In the first the medium is involved, in the second it is not. The results tabulated and plotted were obtained by the first method. (v) Maize pollen. Fresh pollen was shaken from a tassel and dusted in an even layer along a narrow strip on uranium glass. This was arranged so that the pollen lay in the focused spectrum where a range of exposures was given for each wave-

7 480 A. L. MCAULAY AND M. C. TAYLOR length. As no medium could be found that would give a large percentage of pollen tubes with control pollen, the pollen was grown on the maize silks themselves. The silks, which had been protected from stray pollen, were cut off in the morning and kept in a warm moist atmosphere. Three silk ends about an inch long were placed on a microscope slide and the pollen transferred to the hairs on the silks with a needle point. The slide was then kept in a saturated atmosphere at 28 0 C. for 6 hr. or more. The silks were inspected under a low power microscope to find the number of pollen tubes growing. Owing to variations in the viability of the pollen, percentages were not recorded for the purpose of drawing curves to show dependence of lethal effect on dose. The experiments were used to find the smallest doses at the different wave-lengths which would just prevent the growth of pollen tubes. Pollen treated with half this dose would show a good percentage of tubes. For the wave-lengths investigated, 254, 265, 280, 313 m^., the lethal dose appears to be very close to that found for Chaetomium (Table I). The long exposures necessary for large doses introduce more serious difficulties with pollen than with the spores of Chaetomium. It is necessary to keep exposures for pollen down to a few hours. For this reason at 313 m/n contamination by 302 m/x was considerably worse than was the case with the other materials. This should be remembered when reading the lethal dose at 313 m/x from the curve for pollen in Fig. 1. Wave-length m/t S Chaetomium A 2-O i * 2000 Table I. Lethal doses in joule/cm. 2 Paramecium B I Drosophila o-oi B. colt D Joule/hr o-5-s 02-3 o-i Subsequent experiments have shown that thia dose is probably considerably too large, and that the corresponding curve should approach in form more nearly to that obtained for Paramecium. The doubtful figure is retained, however, as the check experiments were not made under the same conditions as the rest of the series. (See the section headed "Purity of spectrum".) RESULTS Fig. 3 shows as ordinate the logarithm of the lethal or quasi-lethal dose, arrived at as explained above for the various materials, plotted against wave-length in m/x as abscissa. This is the most convenient way of exhibiting the results of the whole investigation in one figure. Table I gives the figures from which these curves are plotted. It will be noticed that the ratio of the largest dose recorded to the smallest is a million to one, and that the dose for Chaetomium at 365 m/ii is the extremely large one of 2000 joule/cm. 2 A comparison curve E giving the intensity of solar radiation at sea-level, zenith distance o (Abbot, 1937, p. 77), is added to show the correlation that was found to exist. The last column in the table is included to give an indication of the intensity under the conditions of the experiment.

8 Monochromatic ultra-violet irradiation SUMMARY Lethal and quasi-lethal effects produced by monochromatic ultra-violet irradiation of biological material representing the phyla Bacteria, Fungi, Angiospermae, Protozoa and Arthropoda have been observed and recorded over a range of wavelengths from 254 to 365 mfm. Although the materials represent high and low forms in both vegetable and animal kingdoms, there is a striking similarity in their behaviour. There is a sudden increase in the lethal effect between 313 and 297 in every case m/x Fig. 3. Logarithms of lethal and quasi-lethal doses plotted against wave-lengths for the materials Chaetomium, Paramecium, Drotopkila, B. colt. Curve E gives the intensity for solar radiation at sea-level, zenith distance 0. Although the curves connecting lethal dose with wave-length are closely similar in all cases, the absolute magnitudes of doses are widely different. The ratio of lethal doses for different material may be as high as two hundred to one at corresponding wave-lengths. The ratio of the greatest to the least dose recorded is of the order of a million to one. Tables and curves are given recording lethal and quasi-lethal doses for the different materials at different wave-lengths, and brief accounts of the conditions in which the experiments were conducted are given in the text. An interesting correlation is exhibited between the form of the lethal dose curves and the ultra-violet radiation in daylight.

9 482 A. L. MCAULAY AND M. C. TAYLOR Our best thanks are due to the Trustees of the Science and Industry Endowment Fund for grants for apparatus, and to the Electrolytic Zinc Company (A'asia) Pty., Ltd., for their gift of the Hilger Monochromator. REFERENCES ABBOT, C. G. (1937). The sun as a source of continuous radiation. Measurement of Radiant Energy, edited by W. E. Forsythe, pp BOWDEN, F. P. & SNOW, C. P. (1934). Proc. Roy. Soc. B, 115, GIESK (1938). Biol. Bull. Wood's Hole, October.