Harris, Franklin I. The action of ultraviolet light on certain bacteria in relation to specfic absorption by amino acids "library -.. IRVINE^ \}f' •ilVERSlTY OF CALlFCRfllA LIBRARY. IRVlNt UNIVERSITY OF CALIFORNIA PUBLICATIONS IN PATHOLOGY Vol. 2, No. 21, pp. 245-250 March 25, 1919 THE ACTION OF ULTRAVIOLET LIGHT ON CERTAIN BACTERIA IN RELATION TO SPECIFIC ABSORPTION BY AMINO ACIDS BY FRANKLIN I. HARRIS and HUBBARD S. HOYT UNIVERSITY OF CALIFORNIA PRESS BERKELEY ■^.>f UNIVEESITY OF OAUTOENIA PTJBLIOATIONS Note. — The University of California Publications are offered in exchange for the pnbU- cations of learned societies and institutions, universities and libraries. Complete lists ol all the publications of the TJnlverBity will be sent upon request. For sample copies, lists of publications or other information, address the Manager of the University Press, Berkeley, Oalifomia, V. 8. A. All matter sent In exchange should be addressed to The Ezchang* Department, XJsiyeisity Library, Berkeley, California, V. S. A. PATHOLOGY. — ^Frederick P. Gay, Editor. Price per volume $2.50. Volume I (pp. 349) completed. Volume n in progress. Cited as Univ. Calif. Publ. Path. VoL 1. 1. On the Quantitative Separation of the Globulins of Hemolytic Senun, -with Special Beference to the Carbon Dioxide Group, by Olarence Quinan. Pp. 1-5. July 11, 1903 ..._ „ ,05 2. On the Hydrolysis of Protamine, with Especial Beference to the Action of Trypsin, by Aionzo Englebert Taylor. Pp. 7-31. February 17, 1904 — - M 3. On the Synthesis of Fat through the Eeversed Action of a Fat-spUtttng Enzyme, by Aionzo Engelbert Taylor. Pp. 33-42. March 29, 1904. 4. On the Occurrence of Amido-Aclds in Degenerated Tissues, by Aionzo Englebert Taylor. Pp. 43-47. March 30, 1904. Nos. 3 and 4 In one cover ~ .26 5. On the Autolysis of Protein, by Aionzo Englebert Taylor. Pp. 49-63. June 24, 1904. 6. On the Beversion of Tryptic Digestion, by Aionzo Englebert Taylor. Pp. 65-70. June 24, 1904. Nos. 6 and 6 in one cover _ — _ _ 26 7. Studies on an Ash-Free Diet, by Aionzo Englebert Taylor. Pp. 71-86. July 30, 1S04 ....- - 20 8. On Fermentation, by Aionzo Englebert Taylor. Pp. 87-341. February 16, 1907 - - $2.00 9. On the Synthesis of Protein through the Actl«n of Trypsin, by Alonso Englebert Taylor, Pp. 343-347. February 21, 1907 _ 06 No. 8, On Fermentation, is also published separately, in a bro- chure of 255 pages. Price $2. Vol. 2. 1. The Fixation Beaetion In Eolation to the Formation of Specific Serum Precipitates, by Frederick P. Gay. Pp. 1-22. August 22, 1911 .20 2. A Comparison between the Bordet-Gengou Fixation Beaetion and the Wassermann Beaetion, based on the Eelative Dosage of the Beacting Substances, by Frederick P. Gay. Pp. 23-28. August 22, 1911 05 8. On the Eolation between Eat and Human Leprosy, by L. S. Schmitt Pp. 29-37. September 2, 1911 - — .06 4. The Non-Antigenic Properties of Lipoids Extracted from Human Livers with a Method of Preparation of Antigen for Use in the Wasser- mann Eeaction, by J. G. Fitzgerald and J. B. Leathes. Pp. 3^-16. May 17, 1912 - W 5. Systemic Oidiomycosis, a Study of Two Oases Developing Terminal Oidiomycetic Meningitis, by Glanville Y. Busk. Pp. 47-58. June 21, 1912 1» 6. Studies on the Origin of Antibodies I. The Persistence of a Soluble Antigen In the Serum of Immunized Babbits, by Frederick P. Gay and G. Y. Busk. Pp. 69-72. June 21, 1912. 7. Note on the Negative Effect of lodlpln on the Output of Artificial Hemolysins, by Frederick P. Gay and G. Y. Busk. Pp. 73-76. June 21, 1912. Nos. 6 and 7 In one cover - - •»» 8. An Improved Eapid Method of Producing Precipitins and Hemolysins, by Frederick P. Gay and J. G. Fitzgerald. Pp. 77-82. June 26, 1912 - - - - 0* 9. A Comparison of Chemical with Microchemlcal Methods for the Deter- mination of Varying Amounts of Glycogen in the Liver, by G. Y. Eusk. Pp. 83-89. October 31, 1912 06 10. A Eapid Method of Producing Bacterial Agglutinins, by Edna Locke. Pp. 91-96. October 31, 1912 06 UNIVERSITY OF CALIFORNIA PUBLICATIONS IN PATHOLOGY Vol. 2, No. 21, pp. 245-250 March 25, I9I9 THE ACTION OF ULTRAVIOLET LIGHT ON CERTAIN BACTERIA IN RELATION TO SPECIFIC ABSORPTION BY AMINO ACIDS BY FRANKLIN I. HAERIS and HUBBARD S. HOYT (From the Department of Pathology and Bacteriology, University of Caiifornia) Within recent years considerable interest has been shown in the study of the ultraviolet radiations in relation to their toxicity for living protoplasm. It was early found that these radiations exert a highly toxic influence on protoplasm exposed to them. Henri,'' - in conjunction with various co-workers, has done pioneer work in this field, and was the first worker to point out the possibility of a practical application of this subject, namely, in the sterilization of various substances and solutions. Further work on the germicidal effect of ultraviolet light has been done by Houghton and Davis,^ who found that the rays produced by the Cooper-Hewitt mercury arc exert a strong bacterial action on various species of bacteria, including spore forming organisms. The action of these radiations was recognized as a photo- chemical process, and was supposed to be due to the absorption of the rays by the bacterial protoplasm. In a previous report^- ^ we studied this phase of the problem and proceeded on the basis of the first law of photochemical action, that in a photochemical system, to be effective, the rays must be absorbed, usually by specific constituents. We h.nve further shown that the toxic 246 University of California Publk-atioiis in Pathologi) [Vol. 2 action of ultraviolet light obeys this law and is due to the absorp- tion of the rays by certain constituents of all living protoplasm, namely, the tyrosine and phenylalanin radicals of the protein molecules. These two acids are the specific absorbents in living protopla.sm for the rays of the mercury arc. The object of the present work was to confirm this work, using bacteria as a biological test, and further to study the rela- tive speed of absorption of the ultraviolet raj^s by the protoplasm of the various types of bacteria. jMethods Three organisms were selected as typifying thi-ee general groups of bacteria, which are classed on the basis of the posses- sion or lack of protective structures, spores and capsules. 1. A non-sporulating, non-capsulated organism, Staphylo- coccus aureus. 2. A sporulating. non-capsulated organism. Bacillus suh- tilis. 3. A capsulated, non-sporulating organism, B. niucosus capsulatus. For the exposure of these organisms various methods were tried. The organisms were grown on agar slants for twenty-four hours, and then washed off with sterile 0.85% NaCl. A pre- liminary exposure was made with a given amount of each saline suspension exposed directly to the rays. As no consistent results were obtained by this method, due undoubtedly to the absorption of the rays by the upper layers of the bacterial suspension, this method was abandoned. A number of plating methods were then tried, ^lelted agar was poured at 42° C and allowed to harden in ten centimeter jictri dishe-s. To prevent condensation it was found best to cover with sterile tile covers. After hardening three methods of inoculation ■were tried. a. Two separate streaks were made with a loop full of saline suspension, one on cithei- side of the diameter of the plate. Half of the plate was then exposed, the other half being protected by a glass cover, covered with black paper. The plate was then li'li'I Han-is-lloijl: Aclioii of I'llrariolcl Lif/lit on Barleria 247 ineubated twcjily-iuur lioiuvs, and tliu colonics were identified directly, or, in siispicioiLs eases, smears were made. The objection to this method was that there was no certainty tliat the control streak and the exposed streak were similarly inoevdated. /;. To overcome this objection a single large streak in the shai)e of the letter "Z" was made of one loopfnl of bacterial suspension, and half the plate exposed as before. (■. Another plating method experimented with was to flood the entire plate with a definite amount of saline suspension and then expose one side, as in the above. Plating methods were also abandoned finally because they did not yield uniform results, due undoubtedly to some oi-ganisms getting in under the agar and being protected by the protein material. The method finally employed was a cover slip method suggested by Professor Ivan C. Hall. Upon one .surface of a sterile cover .slip one loopful of saline suspension of a twenty-four hour agar growth was placed and allowed to dry in a sterili> peti'i dish. Assuming the saline suspension to be uniform each cover slip therefore had approximately the same number of organisms. When dry the cover slips were exposed directly to the rays by placing them in a petri di.sh 12 cm. below the arc of the Cooper- Hewitt machine. After the given exposure the cover slip was picked up with sterile forceps, dropped into a tube of broth, the broth was incubated for forty-eight hours, and the results observed. The growth of these three organisms in broth is quite characteristic, and no further examination was usually necessary. In doubtful eases agar plates were streaked from the broth and the organisms were identified by the usual methods. The exposures varied from five seconds to 200 seconds. Some- what over 100 exposures were made, and although there were slight discrepancies in the results, due to the objections mentioned to plating methods, consistent results were obtained by the cover slip method, so that we may definitely say that : Bacillus mucosus capsulatus was killed after 20 seconds. Staphylococcus " " " 90 " B. suhtilis " " "150 " 248 JJniversitij of Valifoviiia PuhUcaiionsin Paflioloyij [Vol.2 These figrires represent the relative resistance of these three organisms. The protective action of tlie amino acids was then studied. The cover slips were exposed as before, but between the cover slip and the arc a quartz beaker containing the given amino acid was interposed, so that the rays before striking the organisms passed through the amino acids. The results obtained confirm our previous work. With B. suhtilis, whose normal extermina- tion period is 150 seconds, we found exposure for forty minutes to ultraviolet light passed through 1% tyrosin solution exerted no toxic effect upon the bacilli, a good growth being obtained in forty-eight hours. Similarly, Staphylococcus aureus gave good growth after forty minutes; B. mucosus cwpsulafus, though not tested after longer exposure, gave satisfactory growth after ten minutes. A good growth was also obtained with amino-benzoic acid after exposure of these organisms for 3200 seconds to ultraviolet light detoxicated by passing through this substance. Phenyla- lanin could not be secured, but there is little doubt that similar results could be obtained with it. These re.sults confirm those of our previou.s report*''' and indicate that the aromatic amino-aeid radicals are the absorbing substances in bacteria as well as in protozoa. Kober's" work placed the absorption band for tyrosin at 248 to 297/i, or 2480- 2970 Angstrom units, of wave length, and for phenylalanin 236- 27 V, or 2360-2710 Angstrom units. Therefore ultraviolet light, of wave lengths 2360-2970 Ang- strom units, should contain practically all of the rays toxic for protopla.sm. Two recent papers have appeared, however, which report different results. Browning and Russ" found the toxic action of ultraviolet light falling off sharply at 2960 A. U.. which would apparently correspond with one edge of the tyrosine band. The\' report, however, an apparently constant toxicity from 2960-2100 A.U., and did not investigate below 2100 A.U. New- comer'*-" reported also a constant toxicity from 2100 A.U. up to a little less than 21)00 A.U., the toxicity falling off to practically zero at 2970 A.U. The region in which tyrosine and phenylalanin 1919] Harris-lloi/l ■ Ad ion tif UUraviolet Light on Bacteria 2V.) are both absorbed, i.e. :i48U to 2710 A. II., should be tlie most toxic for protoplasm, whereas the region containing wave lengths shorter than 2300 A. U. should be relatively non-toxic. Our former experiments eonelusively demoiistrated that a solution of tyrosine will absorb practically all of the toxic rays, those getting through not being sufficiently toxic to kill paramecia after exposure for forty minutes, whereas if the rays absorbed by tyrosine were allowed to act the paramecia were killed in 100 seconds. Conclusions 1. The aromatic amino acid radicals are among the sub- stances in bacteria affected by the action of ultraviolet light, as was shown for paramecium in a previous report. 2. The ultraviolet radiations produced by the mercury arc of wave lengths not aljsorbed by tyrosine and phenylalanin are relatively non-toxic. Therefore, using Kober's determinations for the wave lengths corresponding to these two absorption bands, the ultraviolet radiations which are toxic for protopla.sm are of wave lengths from 2480-2710 A.U. 3. In the three types of bacteria studied capsulated organisms were found to be most susceptible and sporulating organisms most resistant to the action of ultraviolet light. The work sug- gests strongly that the protoplasm of Bacillus mucosus capsulatus contains greater amounts of the above mentioned substances than the non-capsulated staphylococcus and the sporulating hay bacillus. Transmitted Fehruanj 6, 1919. 250 Unii-crsifi/ of Califuniia rublicationsin Pathology [Vol. 2 LITERATURE CITED 1 Henri, Victor, Helbrouuer, Audre, and de Eecldinghausen, Max. Sterilisation de grandes quantites d 'eau par les ravons ultraviolets. Compt. rend, de I'Aead. d. Sci., T. 150, 1910, pp. 932-934. 2 Bielecki, Jean, and Henri, Victor. Etude quantitative de rabsorption des ravons ultraviolets par les eetones, les dieetones et les acides ceton- iques. ' Compt. rend, de I'Aead. d. Sci., T. 156, 1913, pp. 1322-1324. 3 Houghton, E. M., and Davis, Lewis. A Study of the Germicidal Action of the Ultraviolet Rays. Am. Jour, of Public Health, vol. iv, 1914, pp. 224-240, 3 text figs. *. 5 Harris, F. I., and Hoyt, H. S. The Possible Origin of the Toxicity of Ultra-violet Light. Science, n.s., vol. xlvi, 1917, pp. 318-320. Review in The Jour, of the Am. Med. Assoc, vol. Ixix, 1917, pp. 1433-1434. 6 Kober, P. A. Spectrographic Study of Amino Acids and Polypeptides. Jour. Biol. Chem., vol. xxii, 1915, pp. 483-441 . " Browning, C. H., and Russ, Sidney. The Germicidal Action of Ultra- A^iolet Radiation, and its Correlation with Selective Absorption. Proc. of the Roy. Soc, Ser. B., vol. 90, 1917, pp. 33-38, pi. 3. 8 Newcomer, H. S. Bactericidal Fluorescence Excited by X-rays. Jour. Exp. Med., vol. xxvi, 1917, pp. 657-668. Abiotic Action of Ultra-violet Light. Jour. Exp. Med., vol. xxvi, pp. 841-848, pi. 66. TTNlVEESITY OF OALITOENIA PUBLICATIONS— (Oontlnnod) 11. A Stable Medium for the Production of Potent Tetanus Toxin, by Ivan C. Hall. Pp. 97-102. August 30, 1913 05 12. The Impossibility of Differentiation Between Monkey Blood and Human Blood by Means of Antisera Derived from Monkeys, by Hugh K. Berkeley. Pp. 105-110. September, 1913 _... .05 13. Studies on Conglutination, by Ivan 0. Hall. Pp. 111-125. September, 1913 - - 15 14. Preliminary Note on a Skin Eeaction Indicating Protection Against Typhoid Fever, by F. P. Gay and J. N. Force. Pp. 127-130. Novem- ber, 1913 - .05 15. Forensic Value of the Precipitin Test in the Enforcement of the Game Laws in California, by Frank C. Clarke. Pp. 131-138. April, 1914 05 16. Studies on the Locus of Antibody Formation II. The Effect of Benzol Intoxication and Consequent Leucopenia on the Formation of Arti- ficial Hei-iolysins and Precipitins, by G. Y. Rusk. 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