SMITHSONIAN MISCELLANEOUS COLLECTIONSVOLUME 87, NUMBER 10
LETHALACTION OF ULTRA-VIOLET LIGHTON A UNICELLULAR GREEN ALGA(With Two Plates)
'^A \
BYFLORENCE E. MEIER
AUG 17 1932
Division of Radiation and Organisms, Smithsonian Institution
(Publication 3173)
CITY OF WASHINGTONPUBLISHED BY THE SMITHSONIAN INSTITUTIONAUGUST 17, 1932

SMITHSONIAN MISCELLANEOUS COLLECTIONSVOLUME 87, NUMBER 10
LETHALACTION OF ULTRA-VIOLET LIGHTON A UNICELLULAR GREEN ALGA(With Two Plates)
BYFLORENCE E. MEIERDivision of Radiation and Organisms, Smitlisonian Institution
(Publication 3173]
CITY OF WASHINGTONPUBLISHED BY THE SMITHSONIAN INSTITUTIONAUGUST 17, 1932
Z^i £oti> (§aitimovi (PrcecBALTIMORE, MD., U. S. A.
LETHAL ACTION OF ULTRA-VIOLET LIGHT ONA UNICELLULAR GREEN ALGA 'By FLORENCE E. MEIERDivision of Radiation and Organisms, Smithsonian Institution(With Two Plates)INTRODUCTIONThe stimulative and lethal action of ultra-violet irradiation onhigher and lower plants and animals has been the subject of interestingresearch during the past 50 years. Unfortunately, the lack of sufficientphysical data makes a correlation of the various results difficult andoften inconclusive.An accurate determination of the action of ultra-violet light onplants and animals can be obtained only by the use of monochromaticlight and by measuring its actual intensity at the surface of theorganisms.For the work described here a quartz spectrograph was constructedfor the purpose of exposing algae under sterile conditions to mono-chromatic light and thereby observing the effectiveness of a wide rangeof wave lengths in a definite time. This spectrograph was designed byDr. F. S. Brackett and was constructed in the shop of the Division ofRadiation and Organisms of the Smithsonian Institution. A delicatethermocouple ' made possible the unselective determination of the rela-tive energy of the different wave lengths.I wish to express my gratitude to Dr. C. G. Abbot, Secretary of theSmithsonian Institution, and to Dr. F. S. Brackett, Chief of the Divi-sion of Radiation and Organisms of the Smithsonian Institution, fortheir aid and suggestions. The work was done with the cooperation ofDr. E. D. McAlister of the Division of Radiation and Organisms, whocarried out the spectroscopic manipulations and physical measure-
^ This paper reports investigations made under a grant from the NationalResearch Council to the author as National Research Fellow in the BiologicalSciences.
' The thermocouple of special design developed in the Division of Radiationand Organisms, similar to those described in the paper, The automatic record-ing of the infra-red at high resolution, by Brackett and McAlister, Rev. Sci.Instruments, vol. i, pp. 181-193, 1930, was constructed by Dr. E. D. McAlister.Smithsonian Miscellaneous Collections, Vol. 87, No. 10
2 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 87
ments. In a paper in process of publication Dr. F. S. Brackett andDr. E. D. McAlister will discuss more fully some of the physical prob-lems that arise in connection with this work.RESULTS OF OTHER INVESTIGATORSThe unicellular green algae, such as Chlorella, Pleiirococcus, Scene-destnus, and Chlamydomonas, because of the similarity of their cells insize, shape, and contents when in pure culture, can be grown fairlyhomogeneously on a plate for exposure in the spectrograph. They thusform excellent material for the study of the effect of ultra-violet light.As early as 1882 Engelmann placed green cells of Oedogouium,Cladophora, and other algae in the spectrum of a microspectroscope toobserve the movement and accumulation of oxygen-loving bacteria inthose regions most favorable to assimilation. He used a constant gaslight and an incandescent electric light as sources of illumination.Because of the great light intensities he was able to use a narrow slitand so obtain a very pure spectrum. Ingenious as this method is, hisvalues are only approximate. Pringsheim ( 1886) using the samegeneral method found quite different values.Ward ( 1893) exposed plates of agar uniformly covered with bac-teria to the spectrum and then observed the behavior of the illuminatedregions after incubation. He used the solar and " electric " spectra andfound that no detrimental action was perceptible in the infra-red, red,orange, and yellow regions, but that all the bacteria were destroyed inthe blue and violet regions and far into the ultra-violet.Hertel ( 1905) was the first worker who made quantitative measure-ments of the intensities of monochromatic light used for ultra-violetradiation. His monochromator with its quartz prism and lenses wassimilar to those now used in ultra-violet microscopy. He determinedthe relative intensities of four lines of the ultra-violet part of the spec-trum by means of a thermopile and he varied the intensity by regulat-ing the amperage of the metallic arc. He found the region 2,800 A.'to have a very destructive action on paramoecia and bacteria.In the past few years Cernovodeanu and Henri (1910), Browningand Russ (1917), Mashimo (1919), Bang (1905), Bie (1889, 1905),Bovie (1915), and a number of other workers have used the quartzspectrograph for the study of the bactericidal action of light. Raybaud(1909) made a spectrogram of three fungi. Hutchinson and Ashton(1929), and Weinstein (1930) have studied the effect of monochro-
^ A. ^ Angstrom units, i/u = i,(X!om/[i ^ 10,000 A. There are 100,000,000 A.to the centimeter.
NO. 10 ULTRA-VIOLET LIGHT ON GREEN ALGA MEIER 3
matic light on paramoecia. Hutchinson and Newton (1930) have con-tributed quantitative data on the effect of irradiation on yeast. Bucholtz(1931) found that the cells of higher plants are more resistant to thelethal action of ultra-violet light than bacteria and paramoecia. Wein-stein (1930), Bucholtz (1931), and many of the other authorshave made comprehensive reviews of the literature on ultra-violetirradiation.Of the recent investigators, Gates (1929. 1930) has most clearly
'demonstrated the value of the use of monochromatic light of differentintensities in the study of the lethal eft'ect of 10 lines of the mercury-vapor spectrum on bacteria. By the use of a specially constructedmonochromator and a thermopile he found the wave-length limits ofthe bactericidal action to be between 3,130 and 2,250 A., although thelower limit could not be positively ascertained.EXPERIMENTAL PROCEDUREChlorella vulgaris, the alga which was used in this experiment, is aunicellular green alga, the spherical cell containing a parietal chromato-phore and one easily visible pyrenoid. The diameter of the cell isusually 3-5/x, although some giant cells exceed iO;ti,. It multiplies byoval or elliptical spores, usually two to four in number. This alga hasbeen maintained in pure culture in my collection for two years.The nutritive solution in which the algae were grown is Detmer 1/3,a modified Knop solution, made up in the following proportions andthen diluted to one-third
:
Calcium nitrate . . . : i- gramPotassium chloride 0-25Magnesium sulfate 0.25Potassium acid phosphate 0.25Distilled water i- literFerric chloride ^ tracePetri dishes 9.5 mm in diameter containing the above solution plus2 per cent agar were sterilized in the autoclave at 15 pounds pressurefor 20 minutes. When the media, which was about 4 mm thick, hadsolidified, a suspension of green cells of Chlorella vulgaris that hadbeen growing in Detmer 1/3 solution in diffuse light was poured overthe agar in the petri dish. This suspension of green cells was allowedto remain on the media for 24 hours, then the excess was poured off.The covered culture was placed under a bell jar and grown in diffuselight from a north window during the month of July. After a
4 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 8/
month's time the surface of the agar plate was covered with a quiteuniform green growth of algal cells.The cover was removed, and the lower part of the petri dish wasimmediately covered with clear cellophane that had been soaked in99 per cent alcohol. The culture was then placed in position in thespectrograph. It is necessary that no absorbing medium shall be pres-ent between the measured incident energy and the exposed algae.However, Johnson ( 1931) found that the percentage transmission ofcellophane as compared to air is close to 100. Browning and Russ(1917) have demonstrated that no difference can be detected in thedensity of the growth of bacteria over the irradiated and non-irradi-ated portions of agar ; consequently, ultra-violet irradiation has noappreciable effect upon agar.After exposure of 21 minutes to the spectrum the cover of a sterilepetri dish was placed over the cellophane-covered lower dish and thepetri dish culture was returned to the bell jar in diffuse light.Xo change was observed in the growth of the algae on this first plateuntil one week after exposure. Then white lines resulting from thecomplete decolorization of the chlorophyll and death of the green cellscorresponded to the typical mercury lines for all wave lengths shorterthan 3,000 A. just as they would be seen on a photographic positive(see pi. i).A slightly different technique was developed for the preparation ofsubsequent plates for exposure in the spectrograph. The surface of aglass plate of dimensions 8 by 10 cm was ground so as to retain theagar poured on it. The plate was placed in a large petri dish 15 cmin diameter and covered with Detmer 1/3 agar 2 per cent, sterilized,and inoculated as described above with a suspension of green cells ofChlorella vulgaris. After a month's time the agar plate covered withgreen cells was cut out of the surrounding agar in the petri dish andplaced upright in a closed sterile brass container with a quartz window.A decker was arranged in front of the slit of the spectrograph to per-mit the exposure of different portions of the plate for different lengthsof time.The second plate was subjected to five irradiation periods of 6 and 20minutes, i, 3, and 18 hours. When the plate was removed from thespectrograph at the end of 222 hours the effect of the 18-hour exposurewas clearly visible. Three lethal regions of the 3-hour exposure werealso visible. Plate 2, Figure i is a photograph made of the plate assoon as it was removed from the spectrograph. The algal plate wasplaced in a sterile petri dish in a bell jar in diffuse light. Within twodays the results of all five exposures were evident.
NO. 10 ULTRA-VIOLET LIGHT ON GREEN ALGA MEIER 5RESULTSDecolorized regions appeared where the plate was exposed to wavelengths 2,536, 2,652, 2,699, 2,753. 2,804, 2,894, 2,967 and 3,022 A.Those algae exposed to wave lengths 3,130, 3,341, 3,650 A. wereunharmed and the cells were filled with green chlorophyll. Further-more, it may be noted that wave lengths 3,130 and 3,650 A. are moreintense by actual thermocouple measurements, as shown in Table i.McAlister, by the use of the double monochromator and extremelysensitive thermocouples, has accurately measured the energy distribu-tion in the mercury arc in the ultra-violet region between 2,000 and4,000 A. In Plate i the first algal spectrogram obtained in this experi-ment is superimposed on McAlister's record of the mercury-arc spec-trum. The ordinates given here in centimeters of galvanometer deflec-tion are proportional to the intensities measured with the double mono-chromator. For quantitative comparison these intensities have beencorrected for the relatively lower transmission of the fused quartzsystem of the spectrograph used in this experiment.Table i gives the intensities of the lines used and the computationof the relative lethal sensitivity to each line. Duplicate natural-colorplates were made of the first algal spectrogram which was exposed for21 minutes over the entire length of the slit. Black and white copiesof these color plates were then made, and a densitometer record wasdetermined on a Moll recording microphotometer. The curves of thedensity of the silver in the photographic emulsion correspond here tothe algal density. A photometer record was also made of the compositesuperimposition of the two negatives of the color plates, the photo-graph of which is shown in Plate 2, Figure 2. The areas under thephotometer curves corresponding to the intensity of the lethal effectwere measured with a planimeter. In cases where the plates wereobviously so thin that the densitometer record appears as a truncatedpyramid, extrapolation to a normal curve has been made in order tocorrect as far as possible for this source of error. The probability isthat the stronger lines are still undercorrected. The average of theareas of these three densitometer records gives the best available datafor the first traverse of the color plate.A second traverse, that is, a densitometer record across anotherregion of the plate, was made in order to obtain a better representativedetermination and so minimize the inhomogeneity of this plate. Theuniformity in the second traverse is such that equal weight has beengiven to it with the average area determinations of the first traverse.Mean area (A) is the average of the areas from the first and secondtraverses. These areas should give a reasonably good measurement
SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 8/
o o c o6 6 6 6 6 6 I'^i o t^00 -IN "
o o o o
o o o o6 6 6 6
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NO. lO ULTRA-VIOLET LIGHT ON GREEN ALGA MEIER
of the lethal effect, for they should be proportional to the algae killed,within the limitations due to plate thickness and other possible causes.If these figures are divided by the relative intensity of the lines, anapproximate value is obtained of the relative lethal effect of a givenquantity of incident energy of different wave lengths. These valuesare given in the ratio — While these values have been griven to twofigures for the sake of uniformity, the significance of the quantitiesdiffers greatly for different wave lengths. Values for wave lengths
!
8 SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 8/badly overexposed when adequate exposure is made for the weakerlines. Exposure 2 lasted 18 hours and shows perceptible lethal effecteven in line 3,022 A., which was scarcely noticeable in the color plate.The values of relative lethal effect for the color plate and each ofthe two exposures on the black plate have been plotted and are shownin Figure i. Those points connected by dotted lines are to be givenrelatively smaller weight. Of course, it should be emphasized thatthese measurements are only approximate in that different periods ofincubation and different times of exposure may modify the relativeeffects of different wave lengths. As the lines differ so greatly inintensity it is hoped that further investigation can be undertaken sothat the effects of intensity and time exposure may be studied.DISCUSSIONThe ultra-violet component of solar radiation at the earth's surfaceis from the limit of the visible spectrum, 4,000 A. to about 2,950 A.In nature, plants are exposed to invisible radiations in this region.The amount of ultra-violet light which the plant receives variesaccording to the altitude, atmosphere, and season of the year. Life asit is on the earth is possible only because of the ozone formed in theupper layers of the atmosphere by the action of the short wave lengthsof the ultra-violet of sunlight on oxygen. This ozone serves as alight filter and thus protects the life on the surface of the earth fromthe shorter destructive rays.Throughout the ages living organisms have probably become adaptedto solar radiation as it is received on the earth's surface and very pos-sibly with the same spectrum limit due to ozone. It is, therefore, notsurprising that radiation of wave lengths shorter than the solar limitproduce unusual effects. While large amounts of ultra-violet of cer-tain wave lengths are lethal, it is possible that very small amountsof the same wave lengths may be not lethal but, on the contrary, stimu-lating to the growth of green algae. With further experimentationwe hope to obtain more definite information in regard to the possibilityof this stimulative effect. SUMMARYIt is extremely interesting to note that in the regions where theultra-violet waves beyond 3,022 A., the approximate limit of ultra-violet irradiation in nature, were directed on the culture, the greenalgal cells were killed. These lethal regions appear as decolorized cellsin the green algal plate at the wave lengths 3,022, 2,967, 2,894 2,804,2,753, 2,699, 2,652, and 2,536 A. Wave lengths longer than 3,022 A.,
I
XO. 10 ULTRA-VIOLET LIGHT ON GREEN ALGA—MEIER 9
that is the wave lengths 3,130, 3,341, and 3,650 A., had no appreciablelethal effect upon the algae. Yet by the thermocouple measurementsa greater intensity of light was directed on the cultures at wave lengths3,130 and 3,650 A. LITERATURE CITEDAbbot, C. G.191 1. The sun's energy-spectrum and temperature. Astrophys. Journ., vol.34, pp. 197-208.Bang, Sophus.1904. Om Fordelingen af bakteriedraebende Straaler i Kulbuelysets Spek-trum. Meddelelser fra Finsens Med. Lysinstitut, vol. 9, pp. 123-135.Bayne-Jones, S., and Van der Lingen, J. S.1923. The bactericidal action of ultra-violet light. Johns Hopkins Hosp.Bull., vol. 34, pp. 11-15.BiE, Valdemar.1899. Unders^gelser om Virkningen af Spektrets forskellige Afdelinger paaBakteriers Udvikhng. Meddelelser fra Finsens Med. Lysinstitut,vol. I, pp. 33-74-BoviE, W. T.1915. Action of light on protoplasm. Amer. Journ. Trop. Diseases andPrev. Med., vol. 8, pp. 506-517.1916. The action of Schumann rays on living organisms. Bot. Gaz., vol. 61,pp. 1-29.1923. Ultra-violet cytolysis of protoplasm. Journ. Morph., vol. 38, pp. 295-300.Brackett, F. S. and McAlister, E. D.1930. The automatic recording of the infra-red at high resolution. Rev.Sci. Instruments, vol. i, pp. 181-193, 1930.1932. A spectro-photometric development for biological and photo-chemicalinvestigations. (To be published shortly by the SmithsonianInstitution.)Browning, C. H., and Russ, Sidney.1917. The germicidal action of ultra-violet radiation, and its correlationwith selective absorption. Proc. Roy. Soc, ser. B, vol. 90, pp. 33-3^-BucHOLTZ, Alex. F.1931. The eflfect of monochromatic ultra-violet light of measured intensitieson behaviour of plant cells. Ann. Missouri Bot. Garden, vol. 18,pp. 489-508.BUSCK, GUNNI.1904. Lysbiologi.—En Fremstilling af Lysets Virkning paa de levendeOrganismer. Meddelelser fra Finsens Med. Lysinstitut, vol. 8,pp. 7-III-Cernovodeanu, p., and Henri, Victor.1910. £tude de Taction des rayons ultraviolets sur les microbes. Acad, desSci. Comptes Rendus, vol. 150, pp. 52-54-1910. Comparaison des actions photo-chimiques et abiotiques des rayonsultraviolets. Idem, vol. 150, pp. 549-551-1910. Action des rayons ultraviolets sur les microorganismes et sur dif-ferentes cellules. Etude microchimique. Idem, vol. 150, pp. 729-731-
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Delf, E. Marion.1928. The influence of ultra-violet light on plants. Biol. Rev. and Biol.Proc. Cambridge Phil. Soc, vol. 3, pp. 260-269.Delf, E. Marion, Ritson, K., and Westbrook, A.1927-8. The effect on plants of radiations from a quartz mercury vapourlamp. British Journ. Exp. Biol., vol. 5, pp. 138-153-Ellis, Carleton, and Wells, Alfred A.1925. The chemical action of ultra-violet rays. New York.Eltinge, E.1928. The effects of ultra-violet radiation upon higher plants. Ann. MissouriBot. Garden, vol. 15, pp. 169-240.Engelmann, Th. W.1882. Ueber Sauerstoffausscheidung von Pflanzenzellen im Mikrospectrum.Bot. Zeit., vol. 40, pp. 419-426.1887. Zur Abwehr. Gegen N. Pringsheim und C. Timiriazeff. Idem, vol.45, pp. lOO-IIO.1888. Die Purpurbacterien und ihre Beziehungen zum Lichte. Idem, vol.46, pp. 677-689, 693-701, 709-720.Gates, F. L.1929. A study of the bactericidal action of ultra-violet light. I. The re-action to monochromatic radiation. Journ. Gen. Phys., vol. 13,pp. 231-248.1929. A study of the bactericidal action of ultra-violet light. II. Theeffect of various environmental factors and conditions. Idem, vol.13, PP- 249-260.1930. A study of the bactericidal action of ultra-violet light. III. The ab-sorption of ultra-violet light by bacteria. Idem, vol. 14, pp. 32-4^.Hertei, E.1905. Ueber physiologische Wirkung von Strahlen verschiedener Wellen-liinge. Zeit. f. Allgem. Phys., vol. 5, pp. 95-122.Hutchinson, A. H., and Ashton, Miriam R.1929. The specific effects of monochromatic light on the growth of para-moecium. Can. Journ. Res., vol. 4, pp. 293-304.Hutchinson, A. H., and Newton, Dorothy.1930. The specific effects of monochromatic light on the growth of yeast.Idem, vol. 2, pp. 249-263.Johnson, Frank H.1931. Cellophane covers for petri dishes for keeping out contaminationsand studying the effects of ultra-violet light. Science, vol. 73,pp. 679-680.Mashimo, Toshikazu.1919. A method of investigating the action of ultra-violet rays on bac-teria. Kyoto Imp. Univ. Col. Sci. Mem., vol. 4, pp. i-ii.McAlister, E. D.1929. The spectrum of the neutral mercury atom in the wave-length rangefrom I-2/J.. Phys. Rev., vol. 34. pp. 1142-1147.Meier, Florence E.1929. Recherches experimentales sur la formation de la carotine chez lesAlgues vertes unicellulaires et sur la production de la gelee chezun Stichococcus (S. mesenteroides) . Bull. Soc. Bot. de Geneve,vol. 21 (i), pp. 161-197. i
NO. 10 ULTRA-VIOLET LIGHT ON GREEN ALGA MEIER IINewcomer, H. S.1917. The abiotic action of ultra-violet light. Journ. Exp. Med., vol. 26,pp. 841-848.Pringsheim, N.1886. Ueber die Sauerstoflfabgabe der Pflanzen im Mikrospectrum. Jahrb.f. Wissen. Bot., vol. 17, pp. 162-206.Raybaud, Laurent.1909. De I'influence des rayons ultra-violets sur le developpement desmoisissures. Acad, des Sci. Comptes Rendus, vol. 149, PP- 634-636.Report of the Secretary of the Smithsonian Institution, 1931, p. 129.SCHULZE, J.1909. t)ber die Einwirkung der Lichtstrahlen von 280 mm Wellenlange aufPflanzenzellen. Beih. Bot. Zentralbl., vol. 25, pp. 30-80.Ward, H. Marshall.1893. The action of light on bacteria. Proc. Roy. Soc. London, vol. 54,pp. 472-475-Weinstein, Israel.1930. Quantitative biological effects of monochromatic ultra-violet light.Journ. Opt. Soc. Amer., vol. 20, pp. 432-456.

JSMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 87, NO. 10, PL. 1
ALGAL Spectrogram Superimposed on Mercury Arc SpectrumThe ordinates are proportional to the intensity given in terms of galvanometer deflections in;ntimeters. The abscissae are wave-lengths in Angstrom units.
SMITHSONIAN MISCELLANEOUS COLLECTIONS VOL. 87, NO. 10, PL. 2
Z600 Z800 3000 3Z00
I i t
I. P>lack plate showins' results of exposures of eighteen hours' and threehours' duration.
Z600 ZSOO 3000 izooJ I I I I I I
2. Composite <.if color plates showinu results of exposure of twenty-oneminutes' flm'atimi.ALGAL Spectrograms