The effect of visible and ultraviolet irradiation on cultured fungi

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Content THE EFFECT OF VISIBLE AND ULTRAVIOLET IRRADIATION ON CULTURED FUNGI A Thesis Presented to the Faculty of the Department of Botany University of Southern California a | K a | e 3 | e 3 | c ^ e a # e a | e a t e 3 | c 4 e 3 | c 3 | c In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy 4 c * 4 c * * * * 4 c 4 c * 4 c * by Samuel Joseph Pusateri Hay 26, 19?0 UMI Number: DP21715 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. I MI Dissertation Publishing UMI DP21715 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest' ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 This dissertation, written by L— Lf. ......... under the guidance of h. tF ac ul ty Committee on Studies, and approved by all its members, has been presented to and accepted by the Council on Graduate Study and Research, in partial fu l fillment of requirements for the degree of D O C T O R O F P H I L O S O P H Y Dean Date Committee on Studies ■ 4t-.LV- -X:VL<<}Ac&^ J Chairman ACKNOWLEDGMENTS Sincere thanks are expressed to the many persons who guided and advised the author during this research problem, I am indebted to Dr* Geo R. Johnstone for his helpful assistance in solving many of the problems encountered in this undertaking* Grateful acknowledgment is also extended to Dr. Thomas Fuller, Dr. Bartholomew and Dr. Milo D. Appleman for their comments and suggestions during the preparation of this manuscript. Further thanks are extended to Dr. John Backus, of the Department of Physics at the University of Southern California, and to Morton Roberts, from the Depart ment of Physics at Occidental College for their advice regard ing the measurements of radiant energy. Dr. R. B. King, of the Department of Physics at the California Institute of Technology contributed much helpful information regarding light measurements. The author is sincerely grateful to Marshall Odeen and M. E. Stickney of the National Technical Laboratories in Pasadena for their advice on technical prob lems and also for the use of the Beckman quartz Spectropho tometer. Final appreciation is expressed to Earl De Gowin who formerly used the visible irradiating equipment, and offered valuable information regarding the techniques used in his investigations. S. J. P. TABLE OF CONTENTS Page I. INTRODUCTION — REVIEW OF LITERATURE . . 1 Lethal effects of radiation ..... .......... 1 Action of ultraviolet on yeast ................ 3 Action of ultraviolet on molds ••••••••• 5 Stimulation by ultraviolet .••*••••••• 6 Effect of ultraviolet on fermentation ........ 7 Ultraviolet and pigmentation.................. 8 Ultraviolet and mutations ......... 9 II. THE PROBLEM........................................11 III. APPARATUS..........................................12 Fluorescent lamp ...... .................. 12 Carl Zeiss carbon arc ............. 12 Type C carbon ............. 12 Gelatin filters..................................1*+ Quartz filters......... lb IV. ORGANISMS - STRUCTURAL AND GROWTH CHARACTERISTICS . 16 Polyporus sulphureus............................16 Armillaria meiiea ..............................17 Agaricus campestris ................. . . . . . 18 Pleurotus ostreatus ................... 19 Penicillium expans urn............. 19 Penicillium digitatum......... 20 Aspergillus oryzae ............... 21 Aspergillus niger ......................... .. 21 Saccharomyces cerevisiae . .................... 22 Rhizopus nigricans ••••••••••••••• 2 Alternaria solani ..... .......... ..... 2 Cephalothecium roseum ....................2? Neurospora si tophi la ....................25 Monilia nigra ....................... 26 V. METHOD..............................................28 Preparation of cultures ..... .............. 28 Preparation of stock cultures .............. 28 Preparation of media............. 29 Agar slants and Petri plates............. 29 Water blanks ............. 30 Broth cultures ................... |0 Moist chamber cultures ............. 31 Page VI. PROCEDURE.......................................... 3** Method of Irradiation............................3^ Visible irradiation • •••••••••••• 3^ Ultraviolet irradiation • •••••••••. 36 Method of growth determination .•••••••• 38 Visual comparison . . . ......................38 Weighing method . ♦ . . ♦ ........ • • • • • 39 Use of Weston foot-candle meter • •••••• 39 Measurement of gas formation (Frost gasometer) h-0 Method of determining radiant energy ...... WO Carbon Arc Lamp • •• •• ....................**1 Calorimeter ............................ **1 Method of Determining Energy Ratios by means of the Calorimeter .................. VII. RESULTS OF INVESTIGATION............................ V8 Visible irradiation ••••••.. .......... . **8 Ultraviolet irradiation ........................ 58 VIII. DISCUSSION OF RESULTS.............................. 70 IX. SUMMARY............................................ 76 X. LITERATURE CITED.................................... 78 XI. SUPPLEMENTRAY DATA.................................. 83 LIST OF TABLES TABLE PAGE I. Data on the Radiant Energy Transmitted Through the Visible Spectrum Filters............... **2 II. Data on the Amount of Energy Received by the Various Organisms During Irradiation with the Carbon Arc .......................... *+3 III. Record of Growth of Pleurotus ostreatus After Exposure to Fluorescent Daylight Lamps.......... *+9 IV. Record of Growth of Polyporus sulphureus After Exposure to Fluorescent Daylight Lamps.......... 51 V. Record of Growth of Polyporus sulphureus after Exposure to Fluorescent Daylight Lamps . .... 52 VI. Record of Growth of Polyporus sulphureus After Exposure to Fluorescent Daylight Lamps . .... 53 VII. Record of Growth of Agaricus campestris After Exposure to Fluorescent Daylight tamps ..... 59 VIII. Record of Growth of Agaricus campestris After Exposure to Fluorescent Day 1 ight Lamps......... 60 IX. Record of Growth of Agaricus campestris After Exposure to Fluorescent Daylight Lamps ..... 61 X. Lethal Effeet of Ultraviolet Rays on Various Organisms.................................... 83 XI. Lethal Effect of Ultraviolet Rays on Various Organisms..................................... 8b XII. Lethal Effect of Ultraviolet Rays on Various Organisms ........ •••• 85 XIII. Lethal Effect of Ultraviolet Rays on Various Organisms .......... ..••• 86 XIV. Lethal Effect of Ultraviolet Rays on Various Organisms ........... 87 XV. Number of Colonies After Exposure to Ultraviolet Light Through Filter #791...................... 88 TABLE XVI. XVII. XVIII. XIX. XX. XXI. XXII. XXIII. XXIV. XXV. The Effect of Ultraviolet Light Through Filter #791 on Quartz Test Tube Agar Slant Cultures . . Growth Determination Using the Weight Method . . Growth Determination Using the Weight Method . . Growth Determination Of Saccharomyc es cerevislae Using the Weston Foot-Candle Meter . ...... Growth Determination of Monilia nigra Using the Weston Foot-Candle Meter ... ................ Data Summarizing the Results of the Lethal Effects of Various Wavelengths of Ultraviolet Light on Fungi After an Exposure 90 Minutes to the Carbon Are....................... ............ Data Summarizing the Results of the Lethal Effects of Various Wavelengths of Ultraviolet Light on Fungi After an Exposure 90 Minutes to the Carbon Arc .................................... Data Summarizing the Results of Pigment Formation in Fungi Irradiated with the Fluorescent Lamps . Data Summarizing the Results of Pigment Formation in Fungi Irradiated with the Fluorescent Lamps Data Summarizing the Results of the Effects of Various Wavelengths of Ultraviolet on Gas Forma tion in Saccharomvces cerevisiae .............. PAGE 89 90 91 92 93 9 * 1 * 95 96 97 98 LIST OF FIGURES FIGURE Page 1. Visible Irradiation chamber showing arrangement of exposure boxes in respect to the 60 watt Fluor escent Lamps .............. ........... 13 2. Carbon Arc lamp complete with cover and trans former ................................ 13 3. Arrangements of carbon in the Carbon Arc lamp used for irradiating the various organisms .... 13 *f. Graph showing radiant energy values of Carbon C at different wavelengths ........... 15 5. Percentage transmission curves of the Corning Glass Co., Quartz filters ............... 32 6. Van Tiegham cells used for irradiating organisms in liquid and solid media....................... 32 7. Corning Glass Co., Quartz filters used in conjunc tion with the Carbon Arc l a m p............... 32 8. Percentage Transmission Curves of Eastman Kodak Co. Daylight Filters #29, ^9, 61, 70, 71, 72, 75, 88, Xlf 73, and 7b . . . ............................. 35 9. The Calorimeter used in determining radiant energy transmitted through the various Daylight Filters from the Fluorescent Lamp .......... Ml- 10. Graph showing radiant energy values of the Fluor escent Lamp at various wavelengths............... **7 11. Formation of rhizomorphs in Armillaria mellea ex posed to daylight filters ........................ 55 12. Showing formation of chlamydospores in Polyporus sulphureus ........................... 57 13. Showing formation of sporophores in cultures of Agaricus campestris exposed to the blue end of the spectrum......... 57 l*f. Showing phototropic response in Pleurotus ostreatus 57 FIGURE Page 1?. Showing formation of sporophores in cultures of Agaricus campestris exposed to the blue end of the spectrum ............................... 57 16. Cultures showing lethal effect of ultraviolet rays on Saccharomyces cerevislae ................. 63 17. Cultures showing lethal effect of ultraviolet rays on Alternaria solan! . . '••••#... 63 18. Cultures of Saccharomyces cerevlsiae showing the effect of ultraviolet rays on gas formation . * . 63 19. Cultures showing lethal effect of ultraviolet rays on Saccharomyces cerevisiae ........ 63 20. Cultures showing lethal effect of ultraviolet rays on Penicillium expans urn................... 66 21. Cultures showing lethal effect of ultraviolet rays on Cephalothecium roseum ........... 66 22. Cultures showing lethal effect of ultraviolet rays on Penicillium expansum........................ 66 23. Cultures showing lethal effect of ultraviolet rays on Penicillium digitatum..................... 66 2*f. Cultures showing lethal effect of ultraviolet rays on Monllia nigra................. 68 25. Cultures showing lethal effect of ultraviolet rays on Neurospora si tophi l a ........... 68 26. Cultures showing lethal effect of ultraviolet rays on Monllia nigra................. ........... ••• 68 27. Cultures showing lethal effect of ultraviolet rays on Monilia nigra................................ 68 28. Data showing effect of ultraviolet rays transmitted through filter #586 on Neurospora sitophi la . . . 99 Lower - Data showing effect of ultraviolet rays transmitted through filter #986 on Neurospora sit- ophila............ * ................... 100 FIGURE Page 29. Upper - Data showing effect of ultraviolet rays transmitted through filter #791 on Monllia nigra . 100 Lower - Data showing effect of ultraviolet rays transmitted through filter #586 on Monllia nigra . 100 30* Upper - Data showing effect of ultraviolet rays transmitted through filter #77** on Monilla nigra . 101 Lower - Data showing effect of ultraviolet rays transmitted through filter #791 on Rhizopus nigri cans ................................... 101 31, Upper - Data showing effect of ultraviolet rays transmitted through filter #586 on Cephalothecium roseum.......................................... 102 Lower - Data showing effect of ultraviolet rays transmitted through filter #77** on Cephalothecium roseum . . ................... 102 32. Upper - Data showing effect of ultraviolet rays transmitted through filter #970 on Rhizopus nigri cans .................................. 103 Lower - Data showing effect of ultraviolet rays transmitted through filter #791 on Cephalothecium roseum.......................................... 103 33. Upper - Data showing effect of ultraviolet rays transmitted through filter #791 on Alternaria so- lani............................................ 10*f Lower - Data showing growth results of Polyporus sulphureus exposed to various daylight filters . . 10*f 3**. Data showing growth results of Polyporus sulphureus exposed to various daylight filters ............ 105 35. Data showing growth results of Armillarea mellea ex posed to various daylight filters .............. 106 36. Upper - Data showing effect of ultraviolet rays transmitted through filter #986 on Penicillium ex pansum .................................... 107 Lower - Data showing effect of ultraviolet rays transmitted through filter #791 on Penicillium ex pansum ............. 107 FIGURE Page 37. Upper - Data showing effect of ultraviolet rays transmitted through filter #791 on Penicillium digitatum • . . 108 Lower - Data showing effect of ultraviolet rays transmitted through filter #97Q on Saccharomyces cerevisiae . ................. 108 38. Upper - Data showing effect of ultraviolet rays transmitted through filter #986 on Saccharomyces cerevisiae ............... . . ........... 109 Lower - Data showing effect of ^ultraviolet rays transmitted through filter #586 on Saccharomyces cerevisiae . . . .............. • 109 39. Upper - Data showing effect of ultraviolet rays transmitted through filter #77** on Saccharomyces cerevisiae........... 110 Lower - Data showing effect of ultraviolet rays transmitted through filter #791 on Saccharomyces cerevisiae ........... ... 110 *+0» Data showing growth results of Saccharomyces cer evisiae exposed to various daylight filters when grown on malt, agar........... Ill REVIEW OP THE LITERATURE I. REVIEW OF THE LITERATURE The lethal effect of radiant energy upon microorganisms was first observed when surfaces exposed to sun radiations, became partially or wholly sterilized (Carnoy,*1870). It was concluded that the lethal effect was not caused by the action of the heat rays but by the ultraviolet of which the sun is a good source. Investigations employing artificial ultraviolet wave lengths for lethal action on microorganisms have yielded significant results. The carbon arc and mercury vapor lamps have made it possible to open this phase of technology. Lethal Effects of Radiation- The prolonged exposure to solar radiation has long been known to exert an inhibitory effect on the development of various fungi. One of the first men to discover this phenomenon was Fries (1821). Numerous workers since that time have supported this statement by irra diating fungi with radiation derived from artificial means. Carnoy (1870) and Regel and Vines (cf. Duggar 1936) have carried on qualitative studies to determine the inhibitory effect that sunlight has on the growth rate of fungi. By the use of various known filters, bands of solar energy could be isolated to determine what rays were responsible for this in hibitory effect. Regel and Vines, two of the earlier workers in this field, using liquid light filters, showed that the blue end of the spectrum retarded growth. Ashton and Johnson (1930) and Newton (1930), working with the ultraviolet spec trum, reported that the metabolism of fungi is inhibited more between 2600 8 and 36OO 8 than in the visible spectrum and that the shorter ultraviolet rays were more injurious than the longer ones. In the early researches, the quality of light intensity and wave lengths were not accurately measured. This, at times offered a very elastic interpretation of some of the results so that conclusive facts could not be established. Investigations by Ehrismann and Noethling (1932) and Os ter (193*0 considered the amount of energy necessary to kill Ehrismann and Noethling measured the energy necessary to kill 1 to 10# of the organisms irradiated, while Os ter measured the energies necessary to kill 50# of the cells exposed to the ultraviolet. Although the wave lengths used in the two investigations were different, the curves showed similar re sults with a minimum at 2650 8, when wave lengths are plotted against lethal quantities of energy. Oster1s results revealed another minimum below 2300 &. On the basis of 50# killing, he showed that b57 ergs per sq. mm. are necessary at 2652 fi, as compared with 23,?00 ergs per sq. mm. at 3022 8. He sug gests the above results may be due to the absorption of energy 3 by nucleoproteins which are found in yeast cells and many other organisms. Dillon-Weston and Hainan (1930) showed that increasing the intensity of the light increases inhibitory and lethal effects. Absolute measurements of intensity, with a thermo pile or photo-electric cell, have been made by Wyekoff and Luyet (1931)* The results obtained by Wyekoff, Luyet, and Schreiber (193*0 and Oster (193*0 can all be recorded in the form of typical S-shaped survival curves when the per-cent of survivors is plotted against the energy of light used. Action of Ultraviolet on Yeast, Duggar (193&) revealed that Lacassagne (1930) exposed Saccharomyces ellinsoides to radiations of wavelengths between 2800 § and 38OO 2 and found the cells to fall into three classes, according to their sus ceptibility to the rays. Some died immediately, some survived several minutes, and others exhibited only a retarded rate of cell division, but ultimately recovered their power of repro duction. Wyekoff and Luyet (1931) aade similar observations and noted the production of some giant cells. In the work of Oster (193*+) * earefully prepared suspen sions of Saccharomyces cerevisiae. containing practically no clumps of cells, were employed. Following irradiation, ob servation with a microscope revealed certain abnormalities in cell growth and reproduction. There was apparently a per iod of survival during which normal colonies could not be formed, after irradiation by each of the wave lengths b (222? - 3132 8) studied. There could be distinguished single cells of normal size and not visibly altered; single cells of giant size; two-celled groups usually from three to eight times the size of normal two-celled groups and more spherical in shape and often showing a long filament-like process con stricted at intervals; three to eight cell groups of giants, which at this stage either cease budding or go on to furnish buds of normal size; and finally, larger groups which, al though retarded in their ability to form normal-sized colonies, show little evidence of giantism. At all wave lengths, a ^ definite, measurable retardation in reproduction as based upon the criterion of culture formation, could be demonstrated. The size of the colonies increased at a rate roughly propor tional to the increase in incident energy. The relation be tween the percentage inhibition and the incident energy ap peared to be logarithmic over the inhibitory range. When the period of exposure was increased beyond this point, various degrees of injurT' occurred and finally all budding and growth stopped. As a criterion of lethal action, Oster adopted the inability of the yeast cells to form two or more daughter cells rather than merely the inhibition of macroscopic colonies. The survival-ratio is then the ratio of the number of exposed cells. For each of several wave lengths shorter than 2900 8, curves were plotted of the survival-ratio against the energy in ergs per sq. mm. In the case of the longer wave lengths, very much more Incident energy was required for lethal effects. 5 From the individual survival-ratio curves plotted against the incident energies applied, Oster and Arnold (cf. Duggar 1936) calculated the number of quantum hits necessary to kill at various wave lengths. The results indicated a "multiple- quantum hit-to-killf l relation, the minimum number of quantum hits varying from four to about six. The shape of the curve was believed to suggest that other factors than single quant um hits on several molecules are involved, as suggested by Rahn (1912), or multiple-quantum hits as a sensitive volume element, as suggested by Wyekoff and Luyet (1931). Action of Ultraviolet on Molds. Reports on molds are conflicting. Some workers state that molds are killed with out much difficulty by exposure to ultraviolet light. Others report that molds are killed by ordinary exposure only when the molds are present in very small amounts. Some molds have protection in the form of fatty or waxy secretions. The lat ter may shield the mold from some radiation. Houghton and Davis (191^) made comparative studies of molds and bacteria, and revealed that molds showed a more marked resistance to ultraviolet than did bacteria. Fulton and Coblentz (1929) exposed spores of 27 species of fungi for one minute to ultraviolet radiation from a mercury tungsten arc at a distance of six inches. There was complete killing of the spores of 16 species and a survival of less than 1% in *+ species. It was hoped that this process could be applied to the killing of molds and fungi on oranges. Such a dosage 6 caused no injury to fruit. The limitation in the method, however, lies in the fact that the rays do not penetrate sufficiently beneath the surface to afford complete disinfec tion of the fruit. Stimulation by Ultraviolet. There have been suggestions that sublethal doses of radiation in the lethal wave length range may produce Injuries to the cells, by virtue of which products capable of stimulating the growth of other cells are formed. Hollaender (1921) observed that cells from the 10# which survived a certain irradiation in isotonic salt suspen sion by the 2650 fi rays, increased in numbers when transferred to salt solutions, in which normal cells died. Loafbourow and Morgan (19*+0) claimed that substances which stimulated the respiration of other cells are produced in cells by ultra violet light. Subsequently, these observers devised a method for demonstrating the formation of growth-stimulating substances in the injured cells. The active substances were allowed to diffuse from the injured cells through an agar gel upon the surface of which a fresh colony was placed. Colonies grew more rapidly on agar above injured cells, than on agar above normal cells. Stevens (1928) believed that ultraviolet rays activate the germination of the teleutospores of Puccinia coronifera and P. triticina and to a lesser extent P. graminis. According to Bailey and Ramsey (1930), irradiation by a quartz mercury arc stimulates spore formation in cultures of Macro- sporium and Fusarium cepae. 7 In earlier experiments, Pulkhi (cf. Duggar 1936) had been unable to produce a stimulant for Bacillus mycoides by the addition of yeast cells. Abderhalden (cf. Duggar 1936) was un able to alter significantly the course of alcoholic fermenta tion due to yeast cells by the addition of alcoholic extracts of irradiated yeast, irradiated yeast macerated juice, or er- gosterol. Woodrow (1927), Bailey (1932), and Fuller (1932) believed irradiation of yeast culture media produces a non volatile toxic material derived from the sugar present, but Oster (193*+) was unable to detect any toxic substance liberated by the organism during its irradiation. Effects of Ultraviolet on Fermentation. Zeller (1926) (cf. Duggar 1936) found sunlight inhibited the output of car bon dioxide from yeast after a temporary, slight stimulation. Guerrini (cf. Duggar 1936) inoculated yeast into 5% glucose solutions and incubated the cultures in darkness, in daylight and in light filtered through red, green, yellow, and blue screens. He claimed to find the liberation of carbon dioxide to be greater in cultures exposed to light than in those in darkness. In general, the greatest quantities of COg were produced in red light, the next greatest in yellow, then in green and least in blue light. The last gave practically no co2 . Tanner (1923) found ultraviolet light to have only a de pressing action on fermentation, whether the yeast, the fer menting culture or the glucose broth was the subject of 8 irradiation. According to Reynolds and Wynd (cf. Duggar 1936), the inhibition of fermentation by radiations between 2f>00 and 3000 S is a specific effect independent of general injury to the cells. According to Keeset (cf. Duggar 1936) glucolysis of 1% glucose solution in the presence of yeast cells was accelerated by irradiation with red light for 30 minutes, but white, green or blue light had little effect. Pigmentation. The role of pigments in fungi has long been a subject for investigation. The fact that color pre dominates more in organisms grown in the light than those grown in the dark, has been validated by many researchers. This observation was made by Bonorden (1851) almost one hun dred years ago. Smith and Swingle (190*0 have also shown this to be a fact, as many of the forms they used produced no color when grown in the dark. Humbolt and Seyne (cf. Duggar 1936), however, showed that many fungi produce pigments irrespective of light conditions. Lieske (1921), working with various Actinomycetes, showed conclusively that formation of pigment in these organisms is Independent of light. It was revealed by Smith and Swingle (1938) that color formation in Fusarium oxvsnorum is correlated with light intensities; the higher the intensity the more pronounced the coloring.. The effect was not of a permanent nature, since after irradiation any new growth was white. It is generally agreed by Smith and Swingle (1938), Robinson (1926), and McCrea (1928) that the blue end of the spectrum is responsible for the formation of pigment in some species of fungi. MeCrea, working in the red end of the spectrum, found that it had no inhibiting effect on pigment formation. Dillon-Weston (1931)» working with different varieties of wheat rust, shows that white and orange varieties are more susceptible than red or gray to lethal rays Although no mention was made of the formation of these pig ments during irradiation, it is suspected that they offer pro tection against the rays. Mutations. Giese, A. C. and Lederberg, E. Z. (19^6) studied the effect of ultra-violet irradiation upon Neurospora and found that mutant reversions occurred after repeated ex posures. Strains which at one time lost their ability to utilize certain carbohydrates, after exposure, regained this characteristic after subsequent culturing. Hollaender, A. (19W), and Ford, J. M. (19**8) found that repeated exposure to ultra-violet light resulted in altered mutants which re mained constant with further sub-culturing. Oster, R. H. (193*0 was able to produce mutant forms of Saccharomyces cerevisiae by continued irradiation with ultra-violet rays. These new forms showed definite formation of mycelium. Bonner D. M. (19**6) working with various species of Penicillium found that exposure to short ultra-violet rays interrupted chemical reactions within the synthetic media due to the inability of the mutant to hydrolyze certain compounds. Introduction of the altered enzyme, by artificial means, causes the organism to continue the chemical process. Lindegren, C. C. and 10 Lindgren C. (191 *!) found this to he true with Neurospora sit- ophila. Beadle, G. W. and Tatum, E. L. (19^5) working with Neurospora showed alteration in nutritional requirements fol lowing exposure to ultra-violet light. The resulting mutants were judged by their inability to utilize chemical compounds as well as morphological changes. A recent paper by Pridham, T. G. (19^9) reviewed the literature of the effects of ultra-violet irradiation on micro organisms. He showed that the long ultra-violet (3500 8 - 3000 8) stimulated the formation of riboflavin in Ashbya gossvpii. This corroborates the finding of Bailey, A. A. (1930), Davidson, J. N. (19*K», and Giese, A. C. (19^2) who found this to be so in many other fungi. II. THE PROBLEM 11 The purpose of this investigation is to determine the effect of various wave lengths of light on a number of species of fungi. Since this field has a wide range, the investiga tion has been limited to the following specific problems: to determine (1) what wave lengths of light are most lethal to fungi, (2) the stimulating effect of shorter wave lengths on spores and vegetative tissue, (3) the effect of irradiation on gas production in Saccharomyces cerevisiae. (*0 the form ation and significance of pigments during irradiation. III. APPARATUS 12 Fluorescent lamps. A series of seven, daylight, Mazda, fluorescent lamps were used as a source of visible light to radiate the various fungi (fig. 1). These lamps were used in conjunction with Eastman gelatin filters so that desired bands of visible light could be isolated. They were operated on 60 watts with 110 volts AC and about 0.5 amperes of current. Carl Zeiss carbon arc lamp, (fig. 2). This was operated on 110 volts AC and about 30 amperes. Two carbons are used in the lamp, one being stationary while the other is movable. An even flow of light could be maintained by adjusting the dial on the lamp so that the two carbons were spaced at the right distance (fig. 3). Coming Gla.ss Co. quartz filters were used in conjunction with the carbon arc to Isolate de sired bands of ultraviolet light. The carbons used in irradiating the different organisms were of the C type. This is a polymetallic type of carbon in which several types of metals are used in the core. The me tals are iron, nickel, aluminum and silicon. The C carbons are designed to emit ultraviolet wave lengths with a minimum of the visible rays. This carbon produces antirachitic radi ations from 2500 8 to 3020 fi and is the strongest of all ther apeutic carbons. The principal interest in most sources of radiation used in light therapy centers between 2200 and 3200 8. The C carbon Fig. 1 Upper - * > Apparatus used for isolating various rays of the visible spectrum. Each box contains a gelatin filter which allows only certain bands of light to enter. The light sources below are seven fluorescent daylight lamps. 110 volts. Position of boxes above light is to equalize light intensity for each filter. Cultures to be irradiated are placed within the boxes. Pig. 2 Lower left - Carl Zeiss carbon arc lamp and trans former used in irradiating cultures of fungi. Pig. 3 Lower right - Arrangement of carbons within the lamp. This lamp operated at 30 amperes and ?0 volts across the arc. Organisms were irradiated at a distance of 20 cms. 13 lb is rich in this portion of the spectrum* Fig* b shows a double curve of ultraviolet emission; the upper curve repre senting wavelengths produced with 60 amperes AC current and 50 volts across the arc, while the second curve shows wave lengths produced with 30 amperes AC and 50 volts across the arc. Gelatin filters. Two sets of filters were used in this investigation for isolating desired bands of light* One set was obtained from the Eastman Kodak Company, Rochester, N* Y. and consisted of 11 gelatin filters mounted in glass* The wave lengths ranged from 7000 S . in the red end of the spectrum to 3500 X in the blue end of the spectrum* This included a small portion of the ultraviolet spectrum* These filters were used only with the visible radiation apparatus (fig. 1). For transmission data of these filters see fig. 8. Quartz filters. A set of 6 quartz filters (fig. 7) was obtained from the Coming Glass Works, Elmira, N* Y* These filters were used in conjunction with the mercury vapor lamp and the Carl Zeiss carbon arc lamp* Fig. 5 shows the per centage transmission values of these filters* Fig. b - Graph showing radiant energy value of Carhon C at various wave lengths. Each small square (1 cm.) represents 2?0 micro watts of radiant energy at a distance of 20 cm. from the carbon arc. The lower curve represents energy values when the carbon arc is operated at 30 amperes AC with ?0 volts across the arc. The small curves show the relative energy transmitted by the various quartz, ultraviolet filters used to irradiate the various organisms. Graph and data ob tained from the National Carbon Co. M icro u/att*9 Per 6 q . C m . Pe» »OOA E o lc J t v 4 m o \ l s q u a r e £ » cm.) r e p r e a e ^ ' V s HJSQ f'Ytcrovwortts raAvomV encvov^ A t - Ok. d »*>^snnce 0 ^ £.0 C*r\^. ^ r o w ArVve axe. 2500 I V; S^essaas: = = :aaB.III! ‘ ur1^1 ] > > ur rij, ‘ w M ‘ tii1 ■mi Ion ■ I mhmi kn — i ■ 'Ui"' am mm mm NATIONAL” C CARBON ilL Lower Curve, 30 Amperes, A. C. 30 W ilts across Arc IV. ORGANISMS 16 The organisms used were selected from the four classes of fungi, namely: Basldiomycetes, Ascomycetes, Phycomycetes and Deuteromycetes (Fungi Imperfecti). From the Basidiomy- cetes the following species were used: Polyporus sulnhureus (Bull.) Fr., Armillaria mellea (Vahl.) Fr., Agaricus campes- tris (I*.) Fr. and Pleurotus ostreatus (Jacq.) Fr. The Asco mycetes were represented by the species Penicillium expansum Link, P. digitatum Sacc., Aspergillus oryzae (Ahlb.) Cohn, A. niger Tiegh., Saccharomyces cerevislae Hansen, and Neurospora sitophila Dodge. The Phycomycetes were represented by the species Rhizopus nigricans Ehr. In the Deuteromycetes, cultures of Alternaria solani (E. & M.) Jones & Grout, and Cephalothe- cium roseum, Link, and Monilia nigra Cast. & Chaim, were used. Following is a description of the plant organisms which were used in this problem. These descriptions will help to explain the diverse results which occurred when they were exposed to ultraviolet and the visible spectra. Polyporus sulphureus (Bull.) Fr. This member of the Basidiomycetes can be found growing on Eucalyptus globulus Laibill. during the months of December and January. This fungus resembles the oyster mushroom in external appearance but contains pores in the underneath surface rather than radiating gills. One can detect this specimen at great dis tances because of its great size and brilliant yellow-orange color. The underneath surface of the sporophore reveals thousands of tiny pores which contain many basidia and basid- iospores. This organism can be easily cultured on artificial media. The mycelium grows very quickly and abundantly and at times may produce small sterile fruiting bodies. As the cultures mature, an abundance of chlamydospores are formed along the hyphae. This fact is of interest because these spores seldom form during natural growth. The color of the mycelium is pure white but takes on a yellow color during maturity. A great many septations can be seen in the hyphae and in the early stages, nuclei are visible within the cells. Vacuoles are completely missing in the early growth but form soon after. They are, at first, small and numerous but eventually fuse to form one large vacuole. At this stage, the hyphae appears void of any cytoplasm. The chlamydospores develop very readily and become quite large. These spores are hard to wet when suspended in water blanks. Armillarla mellea (Vahl.) Fr. This species was cultured in artificial media in order to study the effect of radiation on rhizomorphs. This fungus belongs in the Basldlomycetes and is classified as a parasite. It causes much destruction in fruit orchards in the form of root rot. The rhizomorphs are of interest because they are one of the means of dissem ination of the fungus. Sometimes these root-like structures can be followed for several feet underneath layers of bark or leaves. The fungus also reproduces by basldlospores. 18 The organism is hard to establish on artificial media but once started it grows very well and at times even produces small sterile fruiting bodies* The mycelium develops on the surface of the solid media but soon grows down into the media* The rhizomorphs are made up of several hyphal strands which have come together to function by absorption* These struc tures, as a rule, remain below the surface of the media and there absorb the nourishment for the mycelium and the pro spective sporophore. Agaricus camnestris (L*) Fr* One of the commonest spe cies of the Basidiomycetes is the edible mushroom, Agaricus camnestris* During the fall of the year, it can often be found in pasture land, particularly after rain* It can be identified in its early stages by its pink gills which later take on a brown color* The s tern is usually very short and contains a veil or web which is known as an annulus. The base of the stem, at times, may be bulbous but this species can be differentiated very easily from the poisonous forms* The cap or pileus is about two inches in diameter and light in color* In the later stages, it may start to flake, causing small brown pellicles to form* The fungus can be easily cultured in artificial media which results in an abundance of white mycelium. Under ideal conditions of temperature and moisture, fruiting bodies will form in the culture tubes and at times contain mature spores. The spores are brown in color and are easily miscible with water* 19 The mycelium is distinctly white in color and contains many cross walls. Clamp connections are abundant and at this stage the nuclei are borne in pairs. Due to the white color of the mycelium, formation of pigment was very easily studied, when it was exposed to ultraviolet radiations. Pleurotus ostreatus (Jacq.) Fr. This fungus is a wood- inhabiting form and appears very much like oyster shells. The organism is saprophytic and belongs in the Basidiomyeetes. It is readily cultured on artificial media and quite often produces many sporophores. Dhder some conditions, fertile spores are formed in these fruiting bodies. The fungus grows very rapidly and forms an abundance of pure white mycelium when grown in subdued light. No asexual spores, at any time, have been observed under artificial cul ture. The myeellal growth occurs on the surface of the media but at times, it tends to become slightly aerial. Very little subsurface growth occurs. Fruiting bodies appear as tiny pin points when first starting to form and in most cases form in areas where the surface mycelium has formed rhizomorphs. Penicillium expansum Link. This species of mold has a widespread distribution and is found very commonly on decayed fruits. It is a member of the Ascomycetes and reproduces both by asexual and sexual spores. The conidia are small round asexual spores borne in long chain-like structures on the end of the fruiting body. This fungus is present on decayed apples and forms characteristic structures called coremias, Coremias 20 are a result of several conidiophores forming a compact mass of asexual reproductive structures. The conidia germinate readily in artificial media forming a white mass of mycelium. During maturation, the mass takes on a bluish-green color due to the appearance of new conidia. The sexual stage of this form is rarely seen but at times may form in artificial media. Suspension of the conidia are dif ficult to make because of the waxy nature of the outside coat. Even after constant agitation, they will clump together and float on top of the liquid. Penicillium digitatum Sacc. This fungus is very simi lar to the above species but differs in some ways. It is found most commonly on decayed citrus fruits and forms char acteristic round green colonies. The colony is usually off set with a border of white, this being the young mycelium which has not formed conidia. As the conidia mature, they too, take on a green color. The name of this species of fungus is derived from the digitate form which the conidiophore assumes. The conidia are larger than those found in P. exoansum but do not occur in long chains as the ones in the above species. Coremias are never formed so that the colony appears smooth in contrast to the granular appearance of the colonies of those species form ing coremias. The waxy nature of the conidial walls makes them imper vious to water and also causes them to attract other conidia. 21 Clumping of conidia in this organism is not so extreme as in P. expansum. The size of the conidia may have something to do with this characteristic. Usually from 3 to 10 conidia may be seen grouped together, whereas in P. expansum, as many as 50 will form into one group. Aspergillus orvzae (Ahlb.) Cohn. This mold is a member of the Ascomyeetes. It forms many chain-like conidia on the sporophore so as to take on a globular appearance. The conidia are light in color when first formed but at maturity turn a brown color. Very old cultures appear very black and have a granular surface. The conidia, when suspended in liquids, cling to one another, but even suspensions can be formed by constant agitation. This organism grows vigorously in artificial media. The mycelium, when first formed is white but later becomes a yellowish-white. At a still later stage,* it becomes more pigmented. The color of the culture is due mostly to the conidia. When conidia start to form, they are yellow and later become a yellow-green. This color then gives way to dark brown. The mycelium forms both on the surface and under neath the solid media. Aerial mycelium is scarce. The hyphal strands are septated and contain very large vacuoles. Aspergillus niger Tiegh. This appears very much like the above species and at times can be confused with it. When both species of Aspergillus reach maturity, they can be differen tiated by the fact that A. niger forms black globular heads 22 of conidia, whereas the other species takes on a brown color. The conidia in the species A. niger are echinulated and us ually are larger in size. Other than this, the two species are very similar. Saccharomyces cerevislae Hansen. This yeast is a member of the Ascomyeetes. It is a saprophytic form and is found frequently on substrata such as fruits, nectaries of flowers and exudates from wounded plant tissues. Vegetative reproduction takes place by means of simple binary fission. This is a frequent type of reproduction and the process takes but a few minutes to be completed. After division, the daughter cells may adhere together so that eventually a large colony may result. The newly formed cells are very dense and do not contain vacuoles. The cell walls appear very thin but become heavier during maturity. Along with this procedure, the vacuoles start to form and soon will occupy the center of the cell. The greater part of reproduction in yeast takes place by budding. Small protuberances form on the cell wall and these soon enlarge and form a second yeast eell. Sometimes as many as a dozen cells can be seen clinging to the mother cell. Under ideal environmental conditions, asexual reproduc tion will continue. However, when conditions change the yeast cells will start to undergo sexual reproduction. The nucleus within the cells undergoes division until ^ to 8 small bodies result. Each of these in turn is surrounded by a small bit 23 of cytoplasm and soon a thin wall forms ahout the individual mass. These b to 8 spores thus formed are known as ascospores and are very resistant to adverse conditions. Yeast cells are readily miscible with water and form a homogeneous suspension with slight agitation. If this suspen sion is allowed to set for a few hours, the cells will even tually settle to the bottom. The yeast cells do not contain any pigment and are somewhat translucent during the early stages. As they mature, the cell wall becomes thicker and vacuoles start to form which causes the organism to become opaque. Rhlzopus nigricans Ehr. This fungus belongs to the Phy comycetes and is found in widespread areas. The mycelium of R. nigricans is multi-nucleated and nonseptate. It can be segregated into three groups of hyphaes rhizoids that pene trate the substratum, stolons which grow on top of the sub stratum, and sporangiophores which are aerial in habitat and produce the dark spores. The hypha is usually a light color and in the young stage shows protoplasmic movement. The older hyphae are largely vacuolated and streaming is seldom seen. There are definite transitional steps in the formation of the sporangiophores. The tips of the hyphae becomes gorged with cytoplasm and start to enlarge. At this stage, they do not differ greatly from ordinary vegetative hyphae. In a short time, nuclear divi sions occur and spores are formed which in turn develop hard 2b resistant walls. The spore wall color is gray. The outer wall of the hyphae thickens and forms the heavy sporangial covering. This is thicker and darker in color than the spore wall. Both the walls of the sporangium and the spore are very impervious to water. This characteristic is due to the pres ence of certain wax-like substances which are secreted by the cells. When spores come in contact with water they clump together noticeably. As many as 50 spores or more may group together. It is very rare to find isolated spores even after liquid cultures have been agitated. Alternaria solanl (E. & M.) Jones & Grout. This organism belongs to the Deuteromycetes and can be found on many fruits and vegetables. It causes blight on potatoes which is char acterized by brownish-black lesions on the leaves. These lesions, at times, show a series of concentric ridges appear ing very much like a t t target board11. This characteristic can be noticed when the organism is cultured on artificial media. The mycelium is an olive color, becoming very dark on reaching maturity. In cultures, it grows beneath the sub stratum as well as on top. The spores are very large, some measuring 296 microns x 10 microns. They are septate and as many as 5 or 10 cross-walls may form. The spore wall Is very thick and black in color. Alternaria solani is a very resis tant form and can withstand adverse conditions such as drought and heat. It grows very rapidly in culture and remains viable 25 for many months even though it is not subcultured* Its hard iness makes it a good organism with which to work* Cephalothecium rose urn Link* This organism is a member of the Deuteromycetes and is definitely proven to be a para sitic form. It is found frequently as a contaminant in lab oratory cultures and also on decayed and fresh fruit. In one case, a luxuriant culture of Cephalothecium roseum was found on used coffee grounds* The pink coloration is one of the identifying characteristics of this organism* When in culture, it forms concentric circles which are due to alternation of vegetative mycelium and spore-producing mycelium* Under the microscope, it may be readily identified by its cluster of three or four-celled conidia, formed at the ends of short conidiophores. The conidia are irregular in shape, usually taking on the shape of a pear* The apiculated nature of the conidia can be seen readily when it is detached from the stalk* When in artificial culture, C. roseum grows very rapidly and acquires a very brilliant color* This color fades during maturity and the culture takes on a granular appearance* When liquid cultures are made, even suspension occurs and clumping is unusual* Neurosuora sitophila Dodge. This is a frequent labor atory pest and causes a great deal of contamination in culture tubes. The spores are very light and are easily blown about by drafts* This organism formerly belonged in the Deuteromycetes 26 but at times will form ascospores in artificial media thus making it a member of the Ascomycetes. The organism is yeast-like in character but differs from yeast in that Neurospora produces an abundance of mycelium. The growth of mycelium is quite characteristic and can be picked out very easily from other forms. It is pink in color and forms a loose mesh of thread-like structures to the top of the culture tube. The conidia are borne at the very tips of the mycelium and form a striking mass of salmon colored powder. At times the growth will continue on through the cotton plug and sporulate on the outer surface. The conidia form by budding from pre-existing conidia and do not develop directly from the conidiophore. Under the mi croscope, the conidia appear translucent and colorless. The walls are very thin but highly impervious to water. When liquid suspensions are made, the conidia clump very directly and float on the surface. Even under extreme agitation, homo geneous suspensions can not be formed. Once the spores are wetted, they are easily killed by temperatures of 70°C. Monilia nigra Cast. & Chaim. This yeast-like organism belongs in the Deuteromycetes. When grown in artificial media, it forms dark pasty colonies. When the culture is young, it is yellow in color. This later turns to gray and eventually to a dark greenish-black. The black color is due to the for mation of the black conidia. 27 The conidia do not form at the tip of the hyphae, but occur as segmented portions along the hyphae. These conidia appear to form much the same as chlamydospores do in other species of fungi. After the conidia have formed, they immedi ately start to bud. The buds appear very much like yeast cells and at times the culture looks contaminated. As many as b or 5 buds can be seen on each conidium. The buds can develop into mycelium which in turn produce more conidia. The conidia are easily misclble in liquids so as to form a homogeneous suspension in broth cultures or water blanks. At the same time the budding yeast-like bodies are also dis lodged and separated. When in broth cultures, the buds usually germinate earlier than the conidia. V. METHOD 28 Preparation of stock cultures. The cultures used in this research were obtained from many sources. In the case of Alternaria solani and Cephalothecium roseum. they were isolated from potatoes and plums. They were removed from the substrata by the aid of inoculating needles and placed in Petri dishes containing malt agar. After a series of sub cultures, pure cultures were obtained. The other cultures were prepared from pre-existing stock cultures. Saccharomvces cerevislae was isolated from a Fleischman*s yeast cake. This again required numerous subinoculations on malt agar* The pH of this media was kept at 5.5# The method of Mounce (1929) vas used in preparing cultures of the members of the Basidiomycetes. In each case, fresh sporophores had to be used to make the initial culture. The cap of the fruiting body was first dissected with a sterile scalpel and a small amount of the tramal tissue was removed with a stiff platinum loop that had been previously flamed. This small piece of tissue was inserted into a sterile malt Agar culture and the tube replugged. After a few days, when growth could be discerned in the agar, a small bit of this mycelium was removed and re inocula ted into another sterile tube. This procedure was continued about three or four times. The cultures were labeled with the necessary data and small pieces of cellophane were wrapped 29 around the plug and the mouth of the tube in order to delay drying. Stock cultures were revived by subculturing at least every two weeks and in some cases, every week. Stock cultures were stored in a cold chamber until ready for use. Preparation of media. Cultures of the organisms used in this investigation were grown on agar slants, broth cultures and Petri plates. The media commonly used was malt agar which was purchased in prepared form from the Difco Co., Detroit, Michigan. The Difco medium was further prepared as follows: 55 grams of the powdered malt agar were weighed out and mixed with 1000 ml. of distilled water. This solution was then slowly heated and stirred until a homogeneous mixture occurred. In order to clarify the medium, it was filtered through coarse filter paper. After this procedure, the medium was titrated to determine its pH. After titration, the correct pH of 5.? was brought about by the addition of 1/10 normal HC1 or NaOH. Agar slants and Petri plates. After the medium was pre pared, it was poured into test tubes that had previously been sterilized. Two types of tubes were prepared; one set of tubes was filled with 5 Hil. of medium while a second set was filled with 10 ml. of medium. These in turn were plugged and placed in an autoclave and sterilized for 30 minutes under 15 lbs. pressure. After allowing the autoclave to cool, the tubes were removed and those containing 10 ml. of media were stored in a cold chamber until needed. These were for use in Petri plates. The remaining tubes were slanted when still in 30 a liquid condition so as to allow more growing surface for the organism. When plate cultures were necessary,' the media in the large test tubes were melted in a double boiler and then poured into sterile Petri plates. All precautions against contaminations were taken in this procedure. These plates were then stored in an incubator to expose contaminated plates. Because ordinary test tube glass transmits only a small amount of ultraviolet, quartz test tubes were used, which allowed these rays to pass through. These quartz test tubes were filled with 5 ml. of malt agar and plugged. These were then sterilized in the autoclave under standard temperature, and pressure. After inoculation these test tubes were exposed directly to the ultraviolet lamps for the desired length of time. Water blanks. Much irradiation was conducted in liquid suspensions, entailing the preparation of standard liquid suspensions. Water blanks were made by pouring 5 ml. of dis tilled water into sterile test tubes and these, in turn, ster ilized in the autoclave. These test tubes were not used for growth, but only for irradiation of the organism. Broth cultures. These were used to determine mold growth and formation of gas by some of the organisms. The broth consisted of beef broth base plus a 5% malt extract. Five ml. of this malt broth was poured into each tube and sterilized. For the study of gas formation, a 2% solution of glucose broth was madd from the preceding mixture. The malt-glucose broth 31 was placed in Smith fermentation tubes until the open arm was nearly filled. These were then plugged with cotton and all the tubes were sterilized under 15 lbs. pressure for 30 minutes. On removing the fermentation tubes from the steri lizer, the closed arms of the tubes were filled completely by tilting slightly. Both types of cultures were then stored in the cold chamber until needed. Moist chamber cultures- Small cultures and irradiation chambers were made by using the quartz filters, glass rings and thin glass covers (van Tiegham cells) (fig. 6). These chambers worked out very satisfactorily because observation by microscope could be made on the growing organism. The chambers (van Tiegham cells) were made as follows; the var ious quartz filters were used as bases and on top of these were attached 3 or ^ glass rings of 20 mm. diameter. These were fastened on to the filter by means of paraffin. A cover glass containing a drop of. media was then inverted over the glass ring and held in place by a small amount of sterile vase line. A drop of distilled sterile water was placed in the bottom of the chamber to keep the media from drying out. In oculation of the organism was made directly on the media be fore the cover glass was inverted into position over the glass ring. This apparatus was then placed over the ultraviolet lamp so that the rays passed through the filter and onto the organism. This was an ideal situation as the filters of vary ing wave lengths could be used as the bases of the chambers. Fig. 5 Upper - Chart showing percentage transmission of ultraviolet rays by various quartz filters. Fig. 6 Lower left - Irradiating chambers (van Tiegham cells) used in the research. These chambers were made up of glass cylin ders and the quartz filters as bases. For irradiating organisms on solid media, cover glasses containing small amounts of agar were inverted over the glass rings. The upper irradiation chamber was used for water sus pensions. Fig. 7 Lower right - Quartz ultraviolet transmitting filters used in Irradiating the various organisms. 32 M ID D LE U L T R A V IO L E T E R Y T H E M A L N E A R U L T R A V IO L E T V IS IB L E V IO L E T 100 90 8 0 70 6 0 5 0 \\ 4 0 V \ \ ' 3 0 20 3 4 35 36 37 38 3 9 40 41 42 33 As many as b different organisms could be Irradiated at the same time. These chambers were also used for irradiating liquid suspensions of the organisms. The van Tiegham cells were filled half-way with distilled water previously prepared in a sterile water blank. These in turn were covered with glass covers and the whole chamber placed in the Irradiating lamp. Contamination during this procedure was very low and if such did occur, the contaminated cultures were easily detected. During the preparation of these chambers, all precautions were taken. The chambers themselves were sterilized in 70% alco hol. The cover glasses were stored in alcohol and flamed just before use. The vaseline and paraffin used in sealing the cover glasses and rings were also kept in a sterile condition. 3^ VI. PROCEDURE Method of Irradiation — Visible spectrum. Before cul tures were placed in the irradiating chamber, the positions of the boxes above the fluorescent lights were arranged at measured distances from the lights. By adjusting the height of the frame upon which the boxes were fastened, (fig. 1), equal light intensity could be obtained for each culture to be irradiated. The standard light intensity which each cul ture received throughout irradiation was 5 microamperes. The amount of light delivered to each culture was kept constant by means of equipment and method of De Gowin 19*+0, who used a photometer and a microammeter. This amount of light intensity was used throughout the entire visible irradiation procedure. Because of transmission variation In the gelatin filters (see fig. 8), each had to be adjusted to a distance from the light source which maintained this standard of light intensity (table 1). Cultures to be irradiated were prepared 2b hours in ad vance so that growth might be ascertained. Twelve cultures were then chosen which showed vigorous growth. Care was Ex ercised to pick out cultures which showed like amounts of growth. These cultures were labeled with the name of the organism, date, and filter through which they were to be ir radiated. The culture tubes were then placed into their re spective boxes making sure that the inoculated surfaces faced Fig. 8 - Showing the percentage curves of visible light from filters used with the daylight fluorescent lamp. Filters on left reading top to bottoms #299 #**9* #61, #70. Filters on right reading top to bottom: #71* #72, #75, #88, #X1, #73, #7*. Photos copied from Wratten Filter Manual, Eastman Kodak Co. * - 0 0 1 P‘ M» A \ A\ H J " J « L I 1 * p O J I I J J U ] ss °\ 36 toward the light. The box lids were replaced carefully so that no extraneous light could enter, and the filters were adjusted so that the light openings were completely covered. One irradiating chamber of the series contained no filter and was totally darkened. The culture used in this chamber served as a control. A second box contained a glass filter which allowed all rays of the visible spectrum to enter. The 7 fluorescent lights were turned on and irradiation continued for a period of 6 to 8 days. The length of exposure was determined by the rate of growth of the different organisms, as some species of fungi grew more rapidly than others. After the desired period of exposure, the cultures were removed and the amount of growth recorded. Microscopic examination of the organism was also made at this time. Method of Irradiation — Ultraviolet spectrum. Cultures exposed to the ultraviolet irradiations were treated in the following ways: some organisms were inoculated directly on malt agar Petri plates and these in turn exposed to the ultra violet rays. Other cultures were prepared in quartz test tubes and the inoculated surface placed in the path of the ultraviolet rays. In the majority of experiments, the organ isms were irradiated in water blank suspensions and then re inoculated into a favorable growing medium, either malt agar or malt broth. The greater number of fungus organisms were irradiated in the moist chamber apparatus. These van Tiegham cells 37 (fig. 6), consisted of glass rings mounted on the quartz filters. The procedure in irradiating with this apparatus was as follows: the organisms were first suspended in a 5 ml. sterile water blank. These standard suspensions were prepared a few hours before being exposed to the ultraviolet lamp. The test tubes were agitated at various times in order to Insure an even suspension. Approximately 1 ml. of this suspension was then poured into one of the van Tiegham cells until it was half full. When all four cells were filled with the different suspensions, they were covered with a sterile watch glass and placed in position on the ultraviolet lamp. When irradiation was carried on with the Carl Zeiss carbon arc lamp, the chamber was placed over the opening on the stage (fig. 2). The dis tance of the suspensions from the flame of the carbon arc was approximately 8 inches. Prior to irradiating the organisms, the lamps were turned on for a period of 25 minutes in order to establish an even flow of ultraviolet light. The suspensions were then exposed to the ultraviolet rays for a period of 90 minutes. After each 10 minute period, Irradiation was discontinued for approx imately 1 minute, the watch glass was removed and a loopful of the liquid from each glass cylinder was used to inoculate a sterile malt agar slant. This slant was labeled with the name of the organism, filter used, period of irradiation and the date. 38 These chambers were also used for irradiating organisms on solid media* A sterile cover slide containing a small drop of malt agar was inoculated with the organism to be ir radiated* The inoculum was obtained from a previously pre pared water suspension* A standard loopful of this material was spread over the thin layer of malt agar* After a drop of distilled water was placed on the bottom of the glass cylinder, the cover glass was Inverted and sealed on top of the ring by means of sterile vaseline* The other glass rings on the fil ter were prepared in the same manner* The chamber was then exposed to the ultraviolet rays* This procedure differed from the liquid suspension method in that the cultures were exposed to only one period of irra diation* A whole new chamber had to be prepared each time the organisms were submitted to a different irradiation period. This procedure was useful to determine the influence of the various irradiations on microscopic morphology. Method of Growth Determination. One of the difficulties encountered in this work was the inability to accurately mea sure the growth of some of these organisms* Due to the vari ation in growth characteristics, different methods were used in measuring the growth that occurred after the organisms were irradiated* The following methods were used throughout this research* Visual comparison. The first method was by visual com parison* This involved the comparison of culture growths with 39 the unirradiated control or determination of the number of colonies present. In cases where the colonies formed one large mass, such as in Polyporus sulphurous. the diameter in inches was a useful criterion. The weight of mold-pad method. The second method of growth measurement was by weighing with the analytical balance. This procedure worked very well for Penicillium cultures. When growing the molds in a liquid subculture medium, the pad which formed at the top was removed as a whole by means of a platinum loop. This, in turn, was placed on a piece of filter paper and allowed to dry for 2b hours at room temperature. After this period, it was weighed on the analytical balance and the weight recorded. Use of Weston foot-candle meter. The third method of growth determination was by the use of the foot-candle meter. This method worked very well for cultures that formed even suspensions in liquids. The apparatus consisted of a 10-watt light source enclosed in a light-proof box. At one end of the box was a small compartment large enough to house one test tube. Outside of this box and in the direct path of the light source, was placed a Weston foot-candle meter. The light com ing from the light souree would pass through the solution in the test tube and be recorded on the photometer in foot can dles. The working principle of the apparatus is that the clearer the solution, the greater the meter reading, and the more turbulent the solution, the less the meter reading. A bo control culture of the liquid itself was read before any growth cultures were measured. Before reading each culture, the tube was slightly agitated to form a uniform suspension. This method was fairly accurate as a small amount of turbidity in the liquid could be detected by a change in foot candle readings. Measurement of gas formation, . A Frost Gasometer was used in order to determine the amount of gas formed on Irradiated cultures of Saccharomyees cerevisiae. This is a very simple gauge made of cardboard on which percentages graduated from 0 to 100 are marked off. The gauge is so arranged as to ac comodate fermentation tubes of varying sizes. After the gas forms in the tube, the measurement is made in the following manner: the fermentation tube is lined up with the column of the chart that corresponds to the length of the closed arm. The top of this arm is raised until it coincides with the 0% mark. The percentage is read at the lowest point in the tube where gas Is present. Method of Determining Radiant Energy, The measuring of absolute radiant energy falling on an exposed surface is a problem in itself and one which requires advance training in optical physics and the necessary equipment. The energy val ues shown on tables I-II are calculated from data obtained from the General Electric Co. and the American National Carbon Co. hi Carbon Arc, The following method was used in determining the amount of radiant energy passing through the various fil ters from the carbon arc sources Percentage transmission cur ves were determined for each filter by means of the Beckman Quartz Spectrophotometer. These results checked exactly with the data shown in the Corning Glass Co. catalogue (fig. 5). The transmission curves of each filter was then plotted care fully on a graph showing the total radiant energy of Carbon C in watts per square centimeter. Fig. shows percentage transmission curves of the ultraviolet filters plotted against the 30 ampere carbon arc curve. Bach cm. square represents 250 microwatts of radiant energy. The area covered by Filter 586 is 10 squares of 2500 microwatts, (250 microwatts per square centimeter). Table II shows energy values for the quartz filters calculated by this method. Calorimeter, The use of a calorimeter for determining relative energy was suggested by Dr. R. E. Vollrath during the work of DeGowin (19kO). It was found that this apparatus was very sensitive to small amounts of light energy. The apparatus (fig. 9) consists of two flattened glass bulbs, coated with lampblack, 6 cm. in diameter and 8 mm. thick. These were connected by a capillary tube, with a 1.16 mm. bore, mounted on a millimeter scale. The bulbs were en closed within a box and separated by means of baffle plates. The one bulb was exposed to the exterior by means of an open ing ^ cm. in diameter. The other bulb was completely enclosed b2 TABLE I DATA ON THE RADIANT ENERGY TRANSMITTED BY THE VARIOUS VISIBLE SPECTRUM FILTERS EXPOSED TO THE FLUORESCENT LAMP WHEN THE INTENSITY OF LIGHT WAS EQUAL TO 5 MICROAMPERES„ Distance Calorimeter Microwatts Ratio of Filter Wavelength From Light Reading of Radiant Radiant Number in S Source in mm# Energy/cm2 Energy 29 (6800-7000) *17 0.7 5.20 1:2.3 *♦9 (3500-5100) 7 0.3 1>7 1:5.3 61 (1*850-6100) 12 0.3 1M 1:5.3 70 (5500-7000) 3 1.1 8.50 1:1.‘ f 71 (5800-7000) 3 0.8 6.00 1:2 72 (5000-7000) 1 0.2 1.50 1:8 73 (6800-7000) 1 0.3 l.b? 1:5.3 7k (5100-5750) 2 0.2 1.50 1:8 75 (*f750-5**00) (6900-7000) b 0.2 1.50 1:8 76 (6820-7000) (3300-^800) 8 0.1 0.75 1:16 88 (6800-7000) 1 0.2 1.50 1:8 ght introl (3500-7000) 2b 1.6 12.00 1:1 * Determined by a Weston photoelectric cell and ammeter according to method by DeGowin **3 TABLE II DATA ON THE AMOUNT OF ENERGY RECEIVED BY THE VARIOUS ORGANISMS DURING IRRADIATION. CARBON ARC LAMP Filter Number Distance from Lamp Total Energy in Microwatts Microwatts of Radiant Energy/cm2 Approx. Energy Ratio 585 20 cms. 3,000 120.0 *1:11.8 586 20 cms. 2,500 100.0 1: l.b 77b 20 cms. 22,200 888.0 1: 1.5 791 20 cms. 29,500 1180.0 1: 1.2 970 20 cms. 25,750 1030.0 1: l.b 986 20 cms. 11,500 b6o.0 1* 3 Carbon Arc 20 cms. 35,500 * Compared to l*f20.0 Carbon Arc 1: 1 Fig. 9 - Calorimeter consisting of two flat tened glass bulbs coated with lamp black and connected by a 1.16 mm. bore capillary tube. Energy absorbed by the exposed bulb will cause a deflection of the indicator within the tube. The greater the source of energy, the greater the deflection on the mm. scale. Figure shows the front, top and rear view of the calorimeter. and protected from temperature changes occurring in the ex posed bulb by means of baffle plates. De Gowin’s (19^) or iginal method of sealing the openings to the flattened bulbs with sealing wax was modified by the addition of two ground- glass stopcocks, which facilitated opening and closing the system* Method of Determining Energy Ratios by Means of the Cal orimeter. A drop of alcohol stained with acetocarmine was introduced into the capillary tube by means of a slender pi pette* Three drops of ether were then placed in each flat bulb and the stopcocks closed. The alcohol indicator was then brought to the 0 reading on the millimeter scale by opening one or the other stopcock and thus releasing pressure. The exposed flat bulb of the calorimeter was placed at the var ious irradiating distances, shown in table I, from the fluores cent lamp and each corresponding filter was inserted over the opening of the calorimeter. An exposure of one minute was made for each filter and the amount of deflection of the al cohol indicator noted. This procedure was repeated for each filter as well as for the fluorescent lamp (light control). The radiant energy falling on the calorimeter from the fluo rescent lamp at a distance of 2b inches was calculated to be 12.0 microwatts per square centimeter. This figure was de rived from data made available by the General Electric Co., Lamp Department (fig. 7). The deflection of the indicator in the capillary tube of the calorimeter is proportional to C-6 the amount of energy falling on the black bulb. Absolute figures of radiant energy were determined by comparing the ratio of deflection of the indicator through the various fil ters with the fluorescent lamp operating at a distance of Sc inches (Table I)* Pig. XO - Graph showing radiant energy values of the 60 watt fluorescent lamp at various wave lengths. The area of the Fluorescent Lamp curve represents a total radiant energy 11.5 or 0.3 watts per square centimetef. The graph and data were obtained from the General Electric Co. Lamp Bulletin. O M4 Oq$9 O009 oo&s 0006 o oS fr ooey o o t f n k ro s + v » r A . sft t i - v > 4 t < > o vnu W n } y www|J bo Z 1 - n 91 / L + \ 0 . 3 Watt** P a r 6^. C m . • I U V a . \ « N e O u tp u t « t V m * . 5 0 A if8 VII. RESULTS OP INVESTIGATION Visible irradiation- The growth resulting after visible irradiation proved to be very interesting. It has always been considered that fungi are plants that thrive only in habitats with restricted light. To a certain extent this may be true, but it is definitely known that some fungi are phototropic (Buller, 1929). Pleurotus ostreatus. although found in shaded wooded areas, responds very decidedly toward light (fig. 1*0. Results in this research showed variation in growth of the organism when exposed to the various wave lengths of the vis ible spectrum. Cultures of Pleurotus ostreatus grew almost as well in the light control culture as it did in complete darkness (TABLE III). It was noticed, howevert that the my celial growth in the darkened culture was very white, whereas the mycelium in the culture exposed to white light tended to ward a yellow color. Growth in all the cultures exposed from the red end of the spectrum to the violet showed some differ ences. The most vigorous growth occurred in the red end of the spectrum. The formation of a yellow-brown color was de tected in the culture exposed to the blue end of the spectrum. It was found that Pleurotus ostreatus formed fruiting bodies very rapidly under culture, and that the greater percentage of sporophore formation occurred in the green and blue-violet (*f800 fi to *fOOO fi) end of the spectrum. This was not true in all cases but the majority of the sporophores occurred in this region. *+9 TABLE III RECORD OP GRCWTH IN INCHES OF MEAN DIAMETER OP PLEUROTUS OSTREATUS AFTER EXPOSURE TO FLUORESCENT DAYLIGHT LAMP£> THROUGH VARIOUS EASTMAN GELATIN FILTERS. Gelatin filter Set 1 Set 2 Set 3 Set * » • # 29 red 0.500 in. 1.000 in. 1.750 in. 2.500 in # *+9 blue 0.125 0.500 1.125 2.000 # 6ln blue 0.250 0.750 1.125 2.125 # 70 red 0.250 1.000 1.500 2.125 # 71a red 0.500 1.500 2.125 2.750 # 72 amber 0.500 1.500 2.000 2.500 # 73 amber 0.250 0.750 1.500 2.125 # 7*+ green 0.125 0.500 1.000 1.500 # 75 blue 0.125 0.250 1.000 1.750 # X-. green 0.125 0.500 1.000 1.125 # 88a red 0.250 0.750 1.500 2.750 White light 0.062 0.500 1.500 2.125 Total darkness 0.500 1.125 2.000 2.500 5o Polyporus suluhureus simulated the above species very closely in growth characteristics. This fungus did not pro duce any fruiting bodies under culture, but did produce numer ous chlamydospores (fig. 12). These too were observed to be more numerous at the shorter end of the visible spectrum (1*800 8 to 3500 8). Microscopic observation revealed that the spores occurred more often along the mycelium and consequently gave the entire culture a yellow-brown color. Since this fungus produces one continuous round colony on Petri dishes, the rate of growth could be determined by measuring the diameter of the colony every two days. The most rapid growth occurred in the red end of the spectrum with a general decline of growth toward the green and blue-violet end of the spectrum (TABLES IV-VI, figs. 33-3*0. The growth between these two ranges, at times, was confusing, but in almost all cases a decided inhibiting effect could be seen in the blue-violet (**000 8 to 3500 8) end of the spectrum; whereas the growth was more pronounced and luxuriant in the red end (7500 8 to 6?00 8). Growth in the tubes kept in white light, showed the same results as those exposed to the blue-violet end; whereas the control cultures kept in the dark, showed, as a rule, vigorous growth comparable to that found in the tubes exposed to the red end of the spectrum. The effect of visible irradiation on Saccharomvces cere- visiae revealed very little change in growth variation. All cultures from the light and dark controls, and those exposed 51 TAB IE IV RECORD OP GROWTH IN INCHES OP MEAN DIAMETER OP POLYPOKDS SPLPHOBEPS AFTER EXPOSPRE TO FLUORESCENT DAYLIGHT LAMPS THROPGH VARIOPS EASTMAN GELATIN FILTERS. Gelatin filter Set #1 Set #2 Set #3 Set #f # 29 red 0.250 in. 0.750 in. 2.000 in. 2.750 in # **9 blue 0.250 1.000 1.750 2.250 # 6In blue 0.125 0.750 1.500 2.500 # 70 red 0.500 1.125 1.750 2.500 # 71a red 0.500 1.250 2.000 2.750 # 72 amber 0.500 1.250 2.000 3.000 # 73 amber 0.500 1.250 2.000 2.750 # 7** green 0.250 0.750 1.500 2.500 # 75 bine 0.129 o. 500 1.250 2.000 # X, green 0.125 0.750 1.500 1.750 White light 0.125 0.500 1.125 1.750 Total darkness 0.500 1.125 2.000 2.500 52 TABLE V RECORD OP GROWTH IN INCHES OP MEAN DIAMETER OP POLYPORUS SULPHUREUS AFTER EXPOSURE TO FLUORESCENT DAYLIGHT LAMPS THROUGH VARIOUS EASTMAN GELATIN FILTERS. Gelatin filter Set #1 Set #2 Set #3 Set # 29 red 0.250 in. 1.062 in. 1.750 in. 2.125 in # *f9 blue 0.125 0.250 0.500 0.750 # 6ln blue 0.062 0.500 1.000 1.750 # 70 red 0.062 0.750 1.500 2.125 # 71a red 0.062 0.500 1.000 1.500 # 72 amber 0.125 0.500 1.000 2.500 # 73 amber 0.125 0.750 1.500 1.750 # 7^ green 0.250 0.500 1.000 1.500 # 75 blue 0.250 0.500 1.000 1.500 # X, green 0.500 0.750 1.000 1.250 # 88a red 0.031 0.750 1.500 2.000 White light 0.031 0.125 0.500 0.750 Total darkness 0.750 1.000 2.000 2.250 53 TABLE VI RECORD OF GROWTH IN INCHES OF MEAN DIAMETER OF POLYPORUS SULPHUREUS AFTER EXPOSURE TO FLUORESCENT DAYLIGHT LAMPS THROUGH VARIOUS EASTMAN GELATIN FILTERS. Gelatin filter Set #1 Set #2 Set #3 Set # 29 red 0.500 in. 1.500 in. 2.750 in. 3.000 in # *f9 blue 0.062 0.500 0.750 1.125 # 6ln blue 0.125 0.750 1.750 2.125 # 70 red 0.250 1.125 2.000 2.500 # 71a red 0.125 0.750 1.750 2.125 # 72 amber 0.125 1.000 1.500 2.750 # 73 amber 0.125 0.750 1.500 2.500 # 7*+ green 0.062 0.500 1.750 2.125 # 75 blue 0.062 0.500 1.000 1.750 # green 0.125 0.750 1.125 1.500 # 88a red 0.125 0.500 1.125 1.500 White light 0.125 0.250 0.750 1.125 Total darkness 0.250 0.750 1.500 2.750 51 * - to the various wave lengths from the red to the blue-violet end of the spectrum, showed about the same amount of growth (fig. hO). Microscopic examination revealed that yeast cells taken from cultures exposed to the red end (7500 8 to 6500 8) of the spectrum showed the presence of a great many buds. The cells on the whole were larger and the vacuoles were about the same size as the ones found in the cells of the control culture. Cultures Irradiated in the green and blue-violet end of the spectrum (*+500 8 to 3?00 2) showed the presence of smaller yeast cells containing large vacuoles. The budding process was continuing but not so many buds were visible as in the control culture. The cell forms, besides being smaller, also appeared elongated. Microscopic examination of smears, stained to show spores, seemed to indicate that the blue- violet end (MXX) 8 to 3500 2) of the spectrum stimulated spore production. Armillarla mellea. the root rot fungus, showed some very interesting results after being exposed to the various wave lengths. This fungus produced very little surface growth but produces numerous rhizomorphs (figs. 10, 11). In nature, these rhizomorphs are usually hidden under the ground or be neath bark. They take on the appearance of roots and function as such. Under culture these structures, in most of the cases, grow under the surface of the agar. Cultures of this organism, when exposed to green and blue-violet wave lengths (5000 8 Fig# 11 Upper - Cultures of Armillaria mellea starting from left hand, cultures were exposed to the following filters: #29 red, #70 red. #73 amber, #72 amber, #7** green, #7? blue, oln blue. A decided decrease in growth can he seen in the cultures exposed to the short wave lengths of the visible spectrum. Lower - Cultures of Armillaria mellea showing the formation of rhizomorphs. These cultures were exposed to the red end of the visible spectrum through filter #29. 55 56 to 3500 2), very seldom shoved rhizomorphs that eame to the surface* In the greater number of cases, they vere wholly embedded in the agar. The cultures exposed to the other wave lengths did reveal many of the root-like tips emerging from the agar. The culture in complete darkness showed this char acteristic very definitely. It was noticed that when tips of rhizomorphs emerged from the agar, mycelial strands making up the structures would tend to separate and start to grow on the agar surface. This fact was decidedly so in the control culture and in the cultures exposed to the red and orange part of the spectrum (7500 2 to 6000 2). This was not true, however, in cultures exposed to the blue-violet end of the spectrum. At times the tips of the rhizomorphs would emerge from the agar but no mycelial growth developed. As a rule, a branching would appear just below the agar surface and growth would continue in this fa shion. Agaricus camnestris grows very rapidly on artificial media, but rarely produces sporophores. Of the numerous cul tures prepared, only five produced fruiting bodies. Two of these cultures (figs. 13, 15) had been exposed to white light while the other three had been exposed to the violet end of the spectrum. The mycelium of this organism is very white in its natural growing state but when exposed to the short end of the spectrum, some of the vegetative growth turned a light brown color. Fig. 12 Upper left - Photomicrograph x M+O showing spore formation in hyphae of Poly- porus sulphurous. Note various stages of spore and vacuole formation. Fig. 13 Upper right - Cultures of Agarieus campestris exposed to the blue end of the visible spectrum. Note the presence of spor- ophores in two of the cultures. Fig. lb Lower left - Culture of Pleurotus ostreatus showing response to light. Sporo- phores showed a definite tendency to form and turn toward the light. Fig. 15 Lower right - Petri plate culture of Agarieus campestris grown from spores showing immature sporophores. Culture was exposed to the blue end of the visible spectrum for 8 days. 58 In measuring the growth of this organism, it was re vealed that the blue-violet end of the spectrum had an inhib iting effect on growth. The growth results in this case fol lowed those found in Polyporus sulphureus. The cultures kept in the dark as controls, and those irradiated in the red end of the spectrum showed the best growth (TABLES VII*IX). Ultraviolet irradiation, . The results of irradiation of organisms definitely showed that the most lethal rays existed between 2600 £ and 2800 £. Since wave lengths shorter than 2600 £ were not used, no statement can be made concerning this matter. The lethal effect of ultraviolet rays decreased as they approached the visible spectrum. When Saccharomvces cerevisiae was irradiated in water suspensions, by wave lengths between 2800 £ and 2600 £ (figs. 16, 19)9 complete lethal effect was accomplished in minutes. When it was irradiated on solid media, 60 minutes were required to kill it. The longer wave lengths showed de finite inhibiting effects but in no case were the cells com pletely killed even after exposures of 90 minutes. Yeast cells, irradiated in water suspensions, were more susceptible to the ultraviolet rays. It required periods of 10 to 1? minutes more to kill cells in solid media. The use of fermentation tubes showed the effect on gas formation by the yeast cells when exposed to the short ultra violet wave lengths. The organisms were irradiated in liquid suspensions and then transplanted in the fermentation tubes. 59 TABLE VII RECORD OF GROWTH ITT INCHES OF MEAN DIAMETER OF AGARICOS CAMPESTRIS AFTER EXPOSURE TO FLUORESCENT DAYLIGHT LAMPS THROUGH VARIOUS EASTMAN GELATIN FILTERS. Gelatin filter Set #1 Set #2 Set #3 Set #b # 29 red 0.2?0 in. 0.500 in. 1.125 in. 1.750 in # **9 bine 0.250 0.500 1.125 1.500 # 6ln bine 0.125 0.250 1.125 1.500 # 70 red 0.250 0.750 1.125 1.500 # 71a red 0.125 0.500 1.125 1.750 # 72 amber 0.250 0.750 1.000 1.500 # 73 amber 0.125 0.500 0.750 1.250 # 7b green 0.062 0.250 0.750 1.125 # 75 bine 0.062 0.500 0.750 1.000 # Xi green 0.125 0.500 0.750 1.125 White light 0.062 0.250 0.750 1.000 Total darkness 0.500 0.750 1.250 1.750 60 TABLE VIII HECOBD OF GROWTH IN INCHES OF MEAN DIAMETER OF AGARICBS CAMPESTRIS AFTER EXPOSURE TO FLUORESCENT DAYLIGHT LAMPS THROUGH VARIOUS EASTMAN GELATIN FILTERS. Gelatin Filter Set j»l . Set #2 Set #3 Set # 29 red 0.250 in. 0.750 in. 1.125 in. 1.500 in # ^9 blue 0.125 0.500 1.000 1.500 # 6In blue 0.125 0.500 0.750 1.125 # 70 red 0.250 0.750 1.000 1.500 # 71 red 0.125 0.500 1.125 1.750 # 72 anber 0.500 1.000 1.125 1.500 # 73 aaber 0.125 0.500 0.750 1.125 # 7k green 0.062 0.250 0.500 1.000 # 75 blue 0.125 0.500 1.000 1.500 # Xx green 0.062 0.025 0.750 1.000 White light 0.125 0.500 1.125 1.500 Total darkness 0.500 0.750 1.250 1.750 6 1 TABLE IX RECORD OF GROWTH IN INCHES OF M3 AN DIAMETER OF AGARICUS CAMPESTRIS AFTER EXPOSURE TO FLUORESCENT DAXLIGHT LAMPS fSffOUGH VARIOUS EASTMAN GELATIN FILTERS. Gelatin filter Set #1 Set #2 Set #3 Set #* # 29 red 0.250 in. 0.750 in. 1.500 in. 2.000 in # 1*9 bine 0.125 0.500 1.125 1.500 # 6ln bine 0.125 0.500 1.000 1.125 # 70 red 0.125 0.750 1.000 i.5oo # 71a red 0.250 0.500 1.125 1.750 # 72 amber 0.250 0.500 1.250 1.750 # 73 amber 0.250 0.500 1.250 1.750 # 7*f green 0.062 0.250 0.750 1.125 # 75 bine 0.062 0.250 0.750 1.000 # green 0.125 0.250 0.750 1.500 White light 0.062 0.250 0.500 1.125 Total darkness 0.500 1.000 i.5oo 2.125 62 In almost all cases, gas did not form in tubes that were ir radiated over 60 minutes (fig* 18). Numerous fermentation cultures were set up and in almost all cases results were identical (TABLE XXV). Microscopic examination of yeast cells submitted to the ultraviolet rays showed a decrease in the budding process* Buds that did form were, on the whole, very much smaller than in unirradiated yeast cells. Vacuoles were more predominate in the irradiated cultures and the cytoplasm appeared more granular than in normal cultures. Yeast cultures that had been exposed to the ultraviolet rays and then transferred to other agar slants showed a definite change in structure. This was more pronounced if irradiation was continued on these yeast cells. The cells appeared to elongate and the remaining round or oval cells appeared definitely smaller. The time required to completely kill Ceohalotheclum roseum was a little longer than that required to kill the yeast cells. On an average, 60 minutes of exposure to wave lengths of 2800 8 to 2600 8 were sufficient for complete lethal effect (fig. 21). Exposure to longer wave lengths eneouraged the formation of mycelium beneath the agar surface, and still longer wave lengths, such as found near the violet end of the visible spectrum, caused stimulation in spore formation. As a rule one or two spores are found on the end of the fruiting body but in some cases, after exposure to longer wave lengths, as many as four spores could be seen on the same stalk, when Fig. 16 Upper left - Cultures of Sacch. cerevisiae exposed to ultraviolet through u. 1r. sitica filter #791. No growth occur red in cultures irradiated over 50 min. One colony can be seen on culture irradiated h5 min. Fig. 17 Upper right - Cultures of Alter- narla solani irradiated by ultraviolet through U. V. filter #791. Note the inhibiting effect on cultures irradiated over 50 min. Fig. 18 Lower left - Fermentation tubes show ing the effect of ultraviolet, action on gas formation. Filter used was the U. V. silica #791. Reading from left to rights (1) control culture, (2) culture irradiated 10 min*. (3) 20 min., (*f) 30 min*, (5) bo min.. (6) 50 min. No gas formed in cultures irradiated mO and 50 min. Fig. 19 Lower right - Cultures of Saccharomv- ces cerevisiae exposed to ultraviolet irradia- tion through U. V. silica filter #791. Read ing from left to right : control, 15 min. irradiation, 30 min., b5 min., 60 min., 90 min. 63 I S MirnAt^ mutes M+nMtes M.npte* 6 h observed under the microscope. The culture took on a granu lar appearance due to the abundance of spores. Under normal conditions of growth this organism is a light pink color. Repeated exposures of the mycelium growing on Petri plates, to ultraviolet showed an intensification of color. The effect of pigmentation was not permanent as any new growth formed after irradiation reverted to its natural color. Rhlzopus nigricans was one of the most difficult organ isms to kill by exposure to ultraviolet light. At no time was complete lethal effect achieved, even after exposures of 90 minutes to wave lengths of 2800 fi to 2600 fi (TABLE XXI, fig* 30). A retarding effect could be observed when the or ganism was irradiated in quartz tubes; but after a few days, the organism would recover and produce as much growth as a normal culture. Since R. nigricans normally forms a great deal of subsurface growth, no increase in this type of growth could be discerned. Young cultures of this organism, when exposed to the longer wave lengths of ultraviolet, showed earlier maturation of sporangiophores and spores. Irradiated cultures showed the dark bodies appearing 12 to 29 hours in advance of normal cultures. When quartz test tubes cultures of Alternaria solani were exposed to ultraviolet, a definite retardation could be seen in the mycelial growth (fig. 17). The action of ultraviolet, no doubt, caused damage to the tips of the young growth, but 6? after a period of 2b to M3 hours, following exposure, growth was continued* A normal culture would produce conidia in about six to seven days* The difference in time was due to the period of recovery which the organism had to undergo after exposure to ultraviolet rays. No noticeable change in the structure of the mycelium or conidia could be discerned after irradiation. Observation of numerous control cultures revealed that subsurface growth is uncommon* Many of the irradiated cultures, however, showed definite subsurface growth. The mycelium formed under the surface of the agar showed less pigment than surface mycelium* Variation of color on the surface growth was not noticeable even after long exposures to ultraviolet. The conidia of both Penicillium expansum and P. dieltatum were very difficult to kill when in a dry condition. When water suspensions of the conidia were made, complete lethal effect was achieved in about 80 or 90 minutes (figs. 20, 22, 23, 36). Since these conidia are hard to wet, suspensions had to be prepared several hours before irradiation and agitated several times in order to insure an even suspension. It re quired more time to kill P. expansum completely than it did P. digltatum when exposed to ultraviolet under similar condi tions (TABLE XXI). Microscopic examination did not reveal amy morphological changes in either of these two species. The Aspergilli were difficult organisms to kill by ultra violet irradiation. The species Aspergillus niger was more Fig. 20 Upper left - Cultures of Peniclllium digitatum irradiated by ultraviolet through U. V. silica filter #791. Growth results shown here were typical for this organism. Some growth can be seen in tube irradiated 90 min. but in most cases complete lethal effect was achieved between 80-90 min. of irradiation. Fig. 21 Upper right - Cultures of Cephalothe- cium roseum exposed to ultraviolet through U, V. silica filter #791. No growth occurred in cultures irradiated over 30 minutes. Conidia were kept in water suspensions for 12 hours before exposure to the ultraviolet rays. Fig. 22 Lower left - Cultures of Peniclllium expansum irradiated by ultraviolet through U. 7. silica filter #791. Tubes reading from left to right: control, 15 min. irradiation, 30 min.. **5 min., 60 min., 90 min. This re action is not typical for this organism as most results showed growth occurring up to 80 min. of irradiation. Fig. 23 Lower right - Cultures of Peniclllium digitatum Irradiated by ultraviolet through tr. V. silica filter #791. Cultures reading from left to right: control. 15 min. irradia tion, 30 min. , *+5 min., 60 min., 90 min. Slight growth can be seen in tube irradiated 60 mih. No growth in last tube. 66 W M // C L o 67 difficult to kill than A. oryzae (TABLES X-XIV, XXI). The inhibiting effects of the ultraviolet rays could be detected in cultures radiated about 50 to 60 minutes (TABLES X-XIV), but exposures of 90 minutes or more were required for complete lethal action. Lethal action of ultraviolet on young mycelium was no ticed in just a few minutes time when young cultures on Petri dishes were exposed. The hyphal tips of the young mycelium were killed in about 10 to 15 minutes. After a period of recovery, which lasted about 2 to 3 days, the culture showed signs of activity. No lethal action could be detected on these two cultures when exposed;; to Ultraviolet wave lengths above 3500 8. Neurospora sitonhila is a strict aerobe producing an abundance of aerial mycelium and at times even grows through the cotton plugs of the test tubes. The action of ultraviolet on this mycelium was very interesting. The conidia were dif ficult to kill but the aerial mycelium showed the result of lethal effects of the ultraviolet rays In a very short time. Petri-plate cultures with vigorous mycelial growth revealed a definite retardation when exposed to the shorter wave of the ultraviolet (3000 8 to 2600 8) (fig. 26). After exposures of 60 minutes to these wave lengths, the aerial mycelium would appear collapsed. The culture, however, would recover after a period of 2b hours. 68 Conidia kept in liquid suspensions for a longer period of time, succumbed to ultraviolet irradiation more readily than those kept in suspension for only a few minutes. Young growth of Monilia nigra could be killed by the shortest ultraviolet in periods of 60 to 70 minutes (figs. 2b, 26, 27). The older cultures would stand up against this ir radiation but the young hyphal tips would show the effect of lethal action. The mature culture of Monilia nigra appears very black and viscid. Cultures irradiated under ultraviolet irradiation between 3300 8 and 3000 8 showed that the pigments occurred much earlier than in normal cultures. Microscopic examination of irradiated cultures revealed that budding of conidia seemed to decrease under the shorter ultraviolet. As a rule, normal cultures reveal an abundance of buds adhering to the mature black conidia. Fig. 2b Upper left - Cultures of Monilia nigra irradiated by ultraviolet through Filter #586. An inhibiting effect can be seen in the last two cultures. Note compar ison of lethal action with this filter to that shown in Fig. 23, when the U. V. silica filter #791 was used. Fig. 25 Upper right - Cultures of Neuro- spora sitophlla irradiated by ultraviolet through U. V. silica filter #791. Reading from left to right: control, 15 min. irradi ation, 30 min., b5 min., 60 min., 90 min. No growth is present in last test tube. This organism showed extreme variability to ultra violet exposure. On the average, growth did not occur after irradiation from 60-80 min. Fig. 26 Lower left - Cultures of Monilia nigra exposed to ultraviolet through U. V. silica filter #791. Tubes reading from left to right were exposed: (1) Control tube, (2) 15 min., (3) 30 min., (*f) *f5 min., (5) 60 min., (6) 90 min. Fig. 27 Lower right - Cultures of Monilia tra exposed to ultraviolet through Filter 91. 69 VIII. DISCUSSION OF RESULTS 70 It is generally known that under adverse conditions or ganisms will deviate from the normal by developing protective devices or by hastening the completion of their life cycles (Kleba, 1928). When abnormal conditions exist In the envi ronment, plant organisms will at times produce reproductive structures in a shorter span of time than usual. This was ob served In cases of Polyporus sulphurous and Pleurotus ostrea- tus. Along with the above fact, organisms may react in other ways in order to protect themselves. When human skin is ex posed to the erythemal rays of the sun, it lays down a brown pigment which acts as a protective coat. This seems to occur in fungi. Many of the cultures that appear white under nor mal conditions, showed definite color change in the shorter end of the spectrum. In the case of Agarieus campestris. a brown color formed. This also happened in cultures of Poly- porus sulphureus (TABLE XXIII-XXIV). The formation of subsurface growth can, in many instances, be attributed to unfavorable conditions. The organism will react by growing into the media where more food is available. Although rhizomorphs are In the greater part subsurface fea tures, they do expose themselves at times. In the growth of Armillarla mellea. these structures came to the surface in control cultures and those exposed to the red end of the spec trum (fig. 11). This was not so in the cultures exposed to 71 the blue-violet end of the spectrum. Rhizomorphs, finding their way to the surface in some cultures, showed definite signs of inhibition since further surface growth did not occur. The formation of a lateral branch which continued to grow below the surface upholds this observation. The diverse characteristics of the organisms used in this research accounted for the varied results obtained after ir radiation with visible and ultraviolet light. These charac teristics were, firsts the morphological structure of the organism, second: the presence or absence of pigments, and lastly: the ability of the reproductive structures to resist liquids because of waxy impervious cell walls. Cultures that produced large fruiting bodies containing numerous spores or conidia were better adapted to protect themselves against the lethal effects of ultraviolet exposures than those whose re productive structures consisted of single spores or conidia. One of the hardest organisms to kill was Rhizopus nigricans. In reviewing the structures of this organism, it is noted that the asexual reproductive structure is made up of a heavy outer layer known as a sporangium. Within this structure are con tained numerous spores, each having a thick dark wall of its own. The ultraviolet rays would have to penetrate these two coats in order to produce any lethal action on the spores. There are a great many spores within a sporangium and there is no doubt that the outer spores serve as a protection for 72 the inner spores. For this reason, Rhlzopus nigricans was very difficult to kill (TABLES X-XIV). The mycelium of this organism develops substratum struc tures known as rhizoids. These rhizoids are protected from the ultraviolet by means of the agar. Even though ultraviolet rays are strong enough to kill surface growth, these protected rhizoids can, in a short period of time, rejuvenate the cul ture. Structure played an important part in the results found when the two species of Peniclllium were exposed to ultra violet irradiation. Penielllium expansum and P. digitatum appear very much alike except that the conidlophore of the former is more compact and contains many more and smaller conidia. P. expansum has the habit of forming many fruiting bodies which later grow together to incorporate themselves as one unit. These are spoken of as coremias. P. digitatum. on the other hand, grows very loosely. As results show, P. expansum requires longer periods of exposure than P. digitatum for complete lethal action. There is no doubt that the coremias offered protection to the Inner conidia and that most of the rays had been absorbed before reaching them. In water suspensions, due to their small size, the conidia of P. expansum clustered together so that again the inner conidia were not exposed to the direct rays of the ultraviolet. This clumping is also noticeable in P. digitatum but not to such a great extent. The conidia of these species of fungi contain 73 wax-like material on the cell wall that makes them almost impervious to water. It is this characteristic coating that causes them to clump so definitely. The difference in their clumping habits may be another reason why P. digitatum is killed more quickly than P. expansum (TABLES X-XIV). Alternaria solanl was able to resist ultraviolet action better than other fungi for two reasons. The first was be cause of the presence of dark pigment and the second was be cause of the maiiy-eelled arrangement of the conidia. There is no doubt that the pigment served as a protection as many of the ultraviolet rays probably could not penetrate the con- idium wall. The rays that did penetrate would kill only the outermost conidia leaving the inner ones to germihate when favorable conditions were present. This many-eelled condition of the conidia afforded the same protection as occurred in the Penicillium organisms when clumping of the conidia occur red. Monilia nigra possessed the dark pigment much the same as in Alternaria solani but lacked protection in that the conidia are single-celled. Along with this fact, the conidia expended some of their energy to form yeast-like buds which were readily killed by ultraviolet action. The cultures of this organism are very viscid and wet, and in water suspen sions even distribution can be obtained, thus exposing each cell to the full power of the ultraviolet. This organism showed characteristics similar to yeast cells but was harder 7 h to kill due to the protection of the black pigment (TABLES X- XIV). Investigation of Neurospora sitophila shows that the important protective features in this organism are its ability to resist water due to its waxy outer wa.ll and the presence of an orange pigment. The first feature accounts for the decisive clumping that occurs when the organism is suspended in a liquid. The conidial wall of this fungus is very smooth and translucent which, no doubt, refracts a great deal of the light waves hittings its surface. This fact, plus the pro tection afforded by the pigment, makes this organism very difficult to kill (TABLES X-XIV). Saceharomyces cerevisiae. in most eases, was completely killed by the short ultraviolet rays within 30 to *f5 minutes (TABLES X-XIV). This organism offered no protection in the way of pigments and consequently was fully exposed to the rays of ultraviolet light. Since this organism formed uniform suspensions in liquids, each cell was more prone to exposure to lethal waves. As was mentioned in the results, the organ isms appeared very much smaller than normal when exposed to some of the longer ultraviolet waves. According to many re searchers, organisms tend to shorten the period necessary to complete their life cycle when exposed to unfavorable envi ronment. At the same time these organisms can go through a stage of cell division before complete maturation, consequently, resulting in smaller cells. Spore formation was very prolific 7 5 near the longer end of the ultraviolet spectrum. There is no doubt that the ultraviolet stimulation of the yeast cells in some way caused this reaction. The two species of Aspergilli used in this investigation showed structural adaptations that afforded protection from the ultraviolet rays. The conidiophores are globular in form and bear hundreds of conidia which surround the club-shaped columella. The conidia which are located innermost are pro tected by the outer ones. This, however, does not explain their resistance to ultraviolet rays when in liquid suspen sion. In such cases, the dark pigment found in both forms must be the protective feature along with the characteristic clumping which may occur. IX. SUMMARY 76 This investigation was carried out to determine what effects visible and ultraviolet light has on fungi. The fungi selected, represent the four classes of Eumycophyta. They were selected on the basis of structural differentiation and ability to grow on artificial media. Data revealed that, under like conditions of intensity, the ultraviolet rays between 3000 2 and 2600 2, as transmitted by Wratten filter #7919 are the most lethal. Ultraviolet rays, longer than 3000 2, show only inhibiting effects on the growth of the organism (TABLES XXI-XXII). Cultures of Agarieus campestris and Polyporus sulohureus show the formation of pigment in the mycelium when exposed to light rays between **500 2 and 3500 2 (TABLES XXIII-XXIV). The action of these rays is not strong enough to show perceptible differences in mycelial growth. It is concluded that pigments act as a protective agent. Sporophores formed more readily in Agarieus campestris an(* Pleurotus ostreatus when exposed to the short end of the visible spectrum, (*+500 2 to 3500 2) than in the longer wave lengths of the spectrum. This was also true of asci and ascospore formation in Saccharomyces eerevlsiae. It Is ascer tained that ultraviolet above 3200 2 has a stimulating effect on this organism. Spores of Penicillium digitatum. Neurospora sitophila. Cephalothecium rosQum and cells of Saccharomyces eerevisiae. 77 when suspended in water, were more susceptible to the lethal effect of ultraviolet rays than when irradiated on dry media. Observations of various cultures which had been irradiat ed by ultraviolet between 2800 8 and 2600 8 shewed that veg etative tissue was killed more readily than sporogenous tis sue. This fact is concluded from results obtained from Saccharomvees cerevisiae and Monilia nigra. Irradiated cultures of Neurospora sitophila revealed that aerial mycelium was more susceptible to ultraviolet rays than surface mycelium. Hyphal tips, in the case of Alternaria solani, showed the lethal effect of ultraviolet radiation (2800 8 to 2600 8) more readily than older hyphal strands. LITERATURE CITED LITERATURE CITED 78 Bailey, A. A., 1932. Effects of ultraviolet radiation upon representative species of Fusarium. Bot. Gaz. 9^:225-271. Bailey, A. A., & Ramsey, W. L., 1930. Effects of ultraviolet upon sporulation in Macrosporium and Fusarium. Bot. Gaz. 89:113. Beadle, G. W. 19^5. Genetics and metabolism in Neurospora, Physiol. Rev. 25* 6^3-663. Beadle, G. W., & Tatum, E. L., 19^5. Neurospora II. Methods of producing and detecting mutations concerned with nu tritional requirements. Amer. Jour* of Bot. 32:678-791. Bonner, D. M.. 19**6. Production of bichemical mutations in Penieillium. Amer. Jour, of Bot. 33*788-791. Buller, A. H. R., 1929. 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Dillon-Weston, W. A. R.. 1931. Effects of ultraviolet on ure- dospores of Puccinia graminis tritici. Sci. Agron. 12:81-87. 79 Dillon-Weston. W. A. R., 1930. The fungicidal action of ul traviolet radiation. Phytopath. 20:959-965. Duggar, B. M., 1936. Biological effects of radiation, Vol II McGraW| Hill* Duggar, B# M. & A., 19*+0. Effects of monochromatic ultravio let radiation of fungous spores surviving irradiation. Am. J. Bot. 27:(10) 906-91^. t » Ehrismann, 0. & W. Noethling, 1932. Uber die bakterieide Wirkung monochromatischen Lichtes. Zeitsch Hyg. 113 s 597-628. Elliot, J. A.. 1917. Taxonomic characters of the genera Alternaria and Macrosporium. Am. J. Bot. *f:**39. Ferguson, M. C., 1902. A preliminary study of the germination of the spores of Agaricus campestris and other Basidio- mycetous fungi. If. S. Dept. Aerlc. Bur. Plant Ind. Bull. Feuer, B. & F. W. Tanner, 1920. The action of ultraviolet on the yeast-like fungi. J. Indus. Eng. Cfaem. 12s7^0-7l*'l. Ford, J. M., 19^8. Lethal mutation produced by ultraviolet and X ray irradiation of fungal spores. Aust. Jour, exp Biol. med. Sci. 26:2^5-251. Fuller, H. J., 1932. Some effects of radiation from a mercury vapor arc in quartz upon enzymes. Ann. Missouri Bot. Gard. 19:505-531. Fulton, H. R. < 5 c W. W. Coblentz, 1929. The fungicidal action Of ultraviolet radiation. £. Agric. Rea. 33:159-168. Giese, A. C., 19**2. Stimulation of yeast respiration by ultra violet radiations. Jour. Cell, Comp. Physiol., 20:35-l *6. Giese, A. C., 19^7. Radiations and cell divisions. Quart. Rev. Biol., 22:253-282. Giese, A. C. & Lederberg, E. Z., 19*+8. Induced reversions of biochemical mutants in Neurospora. Amer. Jour, of Botany, 35:150-157. Guilliermond, A., 1920. The yeasts, trans. by F. W. Tanner John Wiley, N. Y. Harvey, E. N., 19*+2. Stimulation of cells by intense flashes of ultraviolet light. Jour. Gen. Physiol. 25:^31-^3^. 80 Heyf G. L. & J. E. Carter, 1926. The effect of ultraviolet radiation on the vegetative growth of wheat seedlings and their infection by Erysiphe graminis. Phytopath. 21: 695-699. Hollaender, A., 1921. Hereditary changes in plant species caused by ultraviolet rays. Science Supp. 93s (2*+12) 6 Hollaender, A., 19*+6. Effects of ultraviolet irradiation. Am. Rev. Physiol., 8:1-16. Hollaender, A., & Zimmer, E., 19*+5. The effect of ultra-violet radiation and X rays on mutations production in Penicil- lium notatum.. Genetics 30:8 (bst.) Hutchinson, A. H. & D. Newton, 1930. The specific effects of monochromatic light on the growth of yeast. Canadian J. Res. 2:2^+9-263. Hutchinson, A. H. & M. R. Ashton, 1930. The effect of radiant energy on growth and sporulation in Colletotrichum pho- moides. Canadian J. Res. 3s187-198. Klebs, Georg, 1928. Die Bedingungen der Fortpflanzung bei einigen Algen und Pilzen. Zweite, unveranderte Auflage, Verlag von Gustav von Fischer, Jena, Germany. Lacassagne, A., 1930. Difference de lfaction biologique pro- voquee dans les leveres par diverses radiations. Compt. Rand. Acad. Sci. Paris. 190:52*+-526. Lindegren, C. C., & Lindegren, G., 19*+1. X rays and ultra violet induced mutations in Neurospora., Jour. Heredity, 32:M) 5-^12. Loafbourow, L. A., & Morgan, N. T., 19*+0. Investigation of growth promoting factors and growth inhibiting factors by ultraviolet irradiation. J. Bact. 39s*+37-*+53. Luyet, B. J., 1932. The effect of ultraviolet, X rays and cathode rays on the spores of Mucoraceae. Radiology. 18:1019-1022. McCrea, A., 1928. A special reaction to light by the mycelium or Clavigeps purpurea. Papers Mich. Acad, Sci. 9s2*+5-252. Mounce, Irene, 1929* Studies of forest pathology. II The biology of Fomes p ini cola (S. W.) Cooke. Domin. Canada Dept. Agric. Bull. ^111 New Series. 81 National Carbon Co., 19**0. Radiation Characteristics« . National. Industrial and Therapeutic carbons. Cleveland, Ohio. Oster, R. H., 193**. Results of irradiating Saccharomyces with monochromatic ultraviolet. J. Gen. Physiol. lo:2^3-250. Oster, R. H. & W. A. Arnold, 1935. Results of irradiating Saccharomyces with monochromatic ultraviolet light. J. Gen. Physiol. 18:351-355. Pridham, T. G., 19**9. The effect of ultra-violet radiation on microorganisms with special emphasis on Ashbya gossypii and its synthesis of riboflavin. Unpublished disserta tion, Univ. of 111. Porter, C. L. & H. W. Bochstahler, 1928. Concerning the re action of certain fungi to various wave lengths of light. Proc. Indiana Acad. Sci. 38:133-135. Raper, K. B., Coghill, R. D./ & Hollaender, A., 19**5. The production and characterization of ultraviolet induced mutations in Aspergillus terreus II. Cultural and mor phological characteristics of the mutations. Amer. Jour, of Botany, 32:165-176. Smith, E. C.. 1935. Effects of ultraviolet radiation and temperature on Fusarium. Bull. Torrey Bot. Club. 62:^5-58. Stevens, F. L., 1928. Effect of ultraviolet radiation on various fungi. Bot. Gaz. 86*210. Stevens, F. L., 1928. The sexual stages of fungi induced by ultraviolet. Science N. S.. 67*511 *-515. Stevens, F. L., 1930. The effect of ultraviolet on various Ascomycetes, Sphaeropsidales and Hyphomycetes. Centrabl. Bakt. 2 Abt. *2:161. Stevens, F. L., 1930. Ultraviolet irradiation on Glomerella cingulata. Amer. J. Bot. 17:870-881. Stevens, F. L., 1931. Ultraviolet irradiation on Colletotri- chum lagenarlum. Mvcologia. 23:13*+-139. Tanner, F. W. & E. Ryder, 1923. Action of ultraviolet on yeast-like fungi. Bot. Gaz. 75*309-317. Thom, C., 1930. The Penlclllia. Williams & Wilkens Co. Baltimore. 82 Thom, C. & M. Church, 1926. The Asuergilli. Williams & Wilkins Co. Baltimore. Weitz, C. E., 19^6. General Electric Lamps. General Electric Lamp Department, Nela Park Engineering Division. Welch, H., 1930. The effect of ultraviolet light on molds, toxins, filtrates. J. Prevent. Med. 295-330. Woodrow, J. W., 1927. Effect of ultraviolet radiation upon yeast culture media. Plant Phv. 2:171-176. Wratten Light Filters, 1935. 15th ed. revised. Eastman Kodak Co. Rochester, W. Y. SUPPLEMENTARY DATA 83 TABLE X THE EFFECT OF ULTRAVIOLET LIGHT THROUGH FILTER #586 USING THE CARBON ARC LAMP AT A DISTANCE OF 20 CM. ON WATER BLANK SUSPENSION. LETHAL EFFECT OF ULTRAVIOLET RAYS ON VARIOUS ORGANISMS Organism Time in minutes Control 10 20 ^0 kO 50 60 70 80 90 120 CeDhalothecium roseum X X X X X X X X X X e Alternaria solanl X X X X X X X X X X X PeniciIlium exuansum X X X X X X X X X X X Penicillium dicitatum X X X X X X X X X X X NeurosDora sitODhila X X X X X X X X X X X Monilia nigra X X X X X X X X X X e Sacchr. eerevislae X X X X X X X X X e e Asuercillus nicer X X X X X X X X X X X Asnercillus oryzae X X X X X X X X X X X Rhizonus nigricans X X X X X X X X X X X x indicates growth o indicates lethal action e indicates doubtful growth 8 1 4 - TABLE XI the effect of ultraviolet light through filter #77*+ USING THE CARBON ARC LAMP AT A DISTANCE OF 20 CM. ON WATER BLANK SUSPENSIONS. LETHAL EFFECT OF ULTRAVIOLET RAYS ON VARIOUS ORGANISMS Organism_______________________________ Time in Minutes Control 10 20 Y> bo *>0 60 70 80 90 120 Cenhalothecium roseum X X X X X X X X X o o Alternaria solani X X X X X X X X X X e Penicillium ext>ansum X X X X X X X X X X o Penicillium digitatum X X X X X X X X X X e Neurosuora sitonhlla X X X X X X X X X e o Monllia nigra X X X X X X X X e o o Sacchr. cerevisiae X X X X X X X e o o o Asnergillus niger X X X X X X X X X X e Asuergillus oryzae X X X X X X X X X e o RhizoDUS nigricans X X X X X X X X X X X x indicates growth o indicates lethal action e indicates doubtful growth 8 5 TABLE XII THE EFFECT OF ULTRAVIOLET LIGHT THROUGH FILTER #791 USING THE CARBON ARC LAMP AT A DISTANCE OF 20 CM. ON WATER BLANK SUSPENSION. LETHAL EFFECT OF ULTRAVIOLET RAYS ON VARIOUS ORGANISMS Organism Time in Minutes Control 10 20 ^0 bQ 50 60 70 80 90 120 Cenhalothecium roseum X X X X X X X o o o o Alternaria solan! X X X X X X X X o o o Penicillium exnansum X X X X X X X X X X o Penicillium digitatum X X X X X X X X e o o Neurosuora sitoohila X X X X X X X X e o o Monilia nigra X X X X X X o o 0 o 0 Saechr. eerevisiae X X X X o o o o o o o Asnergillus niger X X X X X X X X o o o Asoergillus oryzae X X X X X X e o o 0 o RhizoDUS nizrleans X X X X X X X X X X e x indicates growth o Indicates lethal action e indicates doubtful growth 8 6 TABLE XIII THE EFFECT OF ULTRAVIOLET LIGHT THROUGH FILTER #970 USING THE CARBON ARC LAMP AT A DISTANCE OF 20 CM. ON WATER BLANK SUSPENSIONS. LETHAL EFFECT OF ULTRAVIOLET RAYS ON VARIOUS ORGANISMS Organism Time in Minutes Control 10 20 ^0 *fO ?0 60 70 80 90 120 CeDhalothecium roseum X X X X X X X e o o o Alternaria solanl X X X X X X X X X e o Penicillium exoansum X X X X X X X X X o o Penicillium digitatum X X X X X X X e o 0 o Neurosoora sitoohila X X X X X X X X X X o Manilla nigra X X X X X X X X X e o Sacchr. cerevisiae X X X X X X e o o o o Asoergillus niger X X X X X X X X X e o AsDergillus oryzae X X X X X X X X o o o Rhlzoous nigricans X X X X X X X X X x X x indicates growth o indicates lethal action e indicates doubtful growth 87 TABLE XIV THE EFFECT OF ULTRAVIOLET LIGHT THROUGH FILTERS #58? & #896 USING THE CARBON ARC LAMP AT A DISTANCE OF 20 CM. ON MATER BLANK SUSPENSION. LETHAL EFFECT OF ULTRAVIOLET RAYS ON VARIOUS ORGANISMS Organism____________ Time in Minutes Control 10 20 10 bO 90 60 70 80 90 120 Ceohalothecium roseum X X X X X X X X X X X Alternaria solani X X X X X X X X X X X Penicillium exoansum X X X X X X X X X X X Penicillium digitatum X X X X X X X X X X X Neurosoora sitoohila X X X X X X X X X X X Monilia nigra X X X X X X X X X X X Sacchr. eerevisiae X X X X X X X X X e e Asoergillus nicer X X X X X X X X X X X Asoergillus oryzae X X X X X X X X X X X Hhizoous nigricans X X X X X X X X X X X x Indicates growth o indicates lethal action e indicates donbtful grcwth 88 TABLE XV NUMBER OP COLONIES AFTER EXPOSURE TO ULTRAVIOLET LIGHT WITH THE CARL ZEISS CARBON ARC LAMP AT A DISTANCE OF 20 CMS. THROUGH FILTER #791. Organism Exposure in Minutes Control 15 30 b5 60 75 Sacchr. cerevisiae 12 8 3 - - - Monilia niera 3° 26 10 5 - - Penicillium dieitatum 10 12 9 8 6 b Asoergillus niger 8 7 7 5 b 3 Alternaria solan;L 20 22 2b 12 8 6 89 TABLE XVI THE EFFECT OF ULTRAVIOLET LIGHT THROUGH FILTER #791 USING THE CARBON ARC LAMP AT A DISTANCE OF 20 CM. ON QUARTZ TEST TUBE AGAR SLANT CULTURES Organism____________________ Time in Minutes Set #1 Control 15 30 * f r 5 60 75 90 Sacchr. cerevisiae x x x x x o o Cephalothecium roseum x x x x x x e Monilia nigra x x x x x e e Set #2 Sacchr. cerevisiae x Cephalothecium roseum x Monilia nigra x x x x o o o X X X X X X x x x e o o Set #3 Sacchr. cerevisiae x x x x e o o Cephalothecium roseum x x x x x x x Monilia nigra x x x x . x x e x indicates growth o indicates lethal action e indicates doubtful growth 90 TABLE XVII GROWTH DETERMINATION USING THE WEIGHT JffiTHOD WITH THE CULTURES 20 CMS. FROM THE CARL ZEISS CARBON ARC LAMP Penicillium digitatum Filter # 791 Exposure Weight in mg. after b days growth Set #1 Set #2 Set #3 Set # Control 0.30 0.25 0.32 0.35 10 min. Irradiation 0.30 0.25 0.35 0.37 20 " I f 0.32 0.26 0.30 0.35 30 " f t 0.30 0.20 0.32 0.30 ifO " I f 0.35 0.21 0.28 0.2 7 50 " I f 0.25 0.18 0.28 0.28 60 " t f 0.25 0.15 0.15 0.25 90 " I f 0.20 0.13 0.15 0.23 91 TABLE XVIII GROWTH DETERMINATION USING THE WEIGHT METHOD WITH THE CULTURES 20 CMS. PROM THE CARL ZEISS CARBON ARC LAMP Penicillium expansum Filter # 791 Exposure Weight in mg. after * + days growth Set #1 Set #2 Set #3 Set #f Control 0 .5 1 * - 0.50 0 > 2 0 .3 5 10 mln. irradiation 0 .5 1 * - 0.52 0.35 0.30 20 n tt 0.50 0.50 0.30 0.30 30 it ti 0.52 o.k9 0.28 0.28 bo it tt o M o.ko 0.26 0 .2 5 50 t » ft o M 0.20 0.17 0.20 60 t « ft 0 .3 5 0.22 0 .1 5 0.18 90 ft ft 0 .2 5 0.20 0.10 0 .1 5 92 TABLE XIX GROWTH DETERMINATION USING WESTON FOOT CANDLE METER WITH THE CULTURES 20 CMS. FROM THE CARL ZEISS CARBON ARC LAMP Saccharomyces cerevisiae Filter # 791 Exposure Light in Foot Candles Set #1 Set #2 Set #3 Set #*+ Control broth (clear) 15.2 8.9 15.2 15.0 Control broth culture 2.5 3.8 5.3 7.0 10 min. Irradiation 3.1 6.5 6.0 6.5 20 w » 6.5 8.0 7.5 8.0 30 " n 12.3 8.5 10.3 9.5 bO « n 12.8 8.7 11.5 12.0 o 3 " (no growth) 15.2 8.9 1^.5 12.5 60 " ” (no growth) 15.2 8.9 15.0 1^.0 93 - TABLE XX GROWTH DETERMINATION USING WESTON FOOT CANDLE METER WITH THE CULTURES 20 CMS. FROM THE CARL ZEISS CARBON ARC LAMP Monilia nigra Filter # 791 Exposure Light in Foot Candles Set #1 Set #2 Set #3 Set # Control broth (clear) 8.9 8.9 8.9 8.9 Control broth culture 2.8 3.0 1.5 3.0 10 min. irradiation 3.8 ‘ t.O 3.0 3.2 20 " n V .o if.if if.O >*.5 30 # ■ ti b.O if.1*’ h.5 5.5 bO ” n *+.3 5.0 6.0 7.0 50 « t » 5.5 7.5 6.5 8.5 60 " ft 6.0 8.0 7.5 8.5 70 M it 6.0 8.9 8.0 8.9 00 o 3 M (no growth) 8.9 8.9 7.0 8.9 90 1 1 “ (no growth) 8.9 8.9 7.5 8.9 TABLE XXI DATA SUMMARIZING THE RESULTS OF THE LETHAL EFFECTS OF VARIOUS WAVELENGTHS OF ULTRAVIOLET LIGHT ON FUNGI AFTER AN EXPOSURE OF 90 MINUTES TO THE CARBON ARC LAMP Number of Filter Filter Filter Filter Filter Filter cultures 586 58? 77*f 791 970 986 irradiated No, Results No, Results No. Results No. Results No, Results No. Results Saccharomyces — X 18 — X 10 X 3 . . . X 6 . . . X 5 . . . X cerevisiae 20 b — 0 2 — 0 3 0 3 . . . 0 7 . . . 0 5 . . . 0 1 — e 7 e lb mmm e 7 0 10 . . . 0 Penicillium 10 — X 10 — X 10 . . . X 7 mmm X 9 . . . X 8 . . . X expansum 10 1 mmm 0 1 . . . 0 2 . . . 0 2 mmm e Penicillium 10 ---X 10 — X 10 . . . X 5 mmm X 8 . . . X 9 . . . X digitatum 10 1 mmm 0 1 — 0 1 0 i f mmm e 1 . . . 0 Aspergillus 10 -X 10 — X 10 . . . X 3 mmm X 6 X 7 . . . X oryzae 10 5 m m m 0 2 — 0 3 — 0 2 mmm e 2 0 Aspergillus 10 -X 10- X 10 . . . X 6 mmm X 9 . . . X 10 X niger 10 3 mmm 0 1 . . . 0 1 . . . 0 x indicates growth o indicates lethal action 0 indicates doubtful grcwth TABLE XXII DATA SUMMARIZING THE RESULTS OF THE LETHAL EFFECTS OF VARIOUS WAVELENGTHS OF ULTRAVIOLET LIGHT ON FUNGI AFTER AN EXPOSURE OF 90 MINUTES TO THE CARBON ARC LAMP Number of Filter Filter Filter Filter Filter Filter cultures 586 585 77^ 791 970 986 irradiated No. Results No, Results No. Results No. Results No. Results No. Results Alternaria 10 . . . . . . X 10 — x 10 . . . . . . X 8 . . . . . . X 8 . . . . . . X 10 X solanl 10 1 — m 0 2 — — — 0 1 - - e Neurospora 8 — — — X 10 — x 7 — — X k mm-m. X 6 — — — X 7 X sltophlla 10 2 — — — 0 3 •mmm — 0 2 -- 0 h — — — 0 2 — 0 k --- e 1 --- 9 Monilia 8 — — — X 10 • * —« * x 6 — — — X 2 — X 6 — — X 5 X nigra 10 1 — 0 3 — 0 5 -- 0 2 — 0 b — — — 0 1 --- 0 1 9 3 9 2 — 9 1 - — — 9 Cephalothecium 9 . . . . . . X 10 — x 8 — X 5 X 6 — X ? — — — X roseum 10 1 — 0 2 -- 0 k 0 k 0 2 — 0 1 9 3 — 9 Rhizopus 10 •mrnmm. X 10 — x 10 X 8 X 10 — — — X 10 — X nigricans 10 2 -- 0 x indicates growth 0 indicates lethal action e indicates doubtful growth TABLE XXIII DATA SUMMARIZING THE RESULTS OP PIGMENT FORMATION IN FUNGI IRRADIATED WITH THE Filter Wave Number of Number lengths cultures irradiated 8 units P. sulphureus 10 29 6800-7000 A. campestris 10 P. ostreatus 10 P. sulphureus 10 1*9 3500-5100 A. campestris 10 P. ostreatus 10 P. sulphureus 10 61 1+850-6100 A. campestris 10 P. ostreatus 10 P. sulphurous 10 70 5500-7000 A. campestris 10 P. ostreatus 10 P. sulphurous 10 71 5800-7000 A. campestris 10 P. ostreatus 10 P. sulphurous 10 72 5000-7000 A. campestris 10 P. ostreatus 10 FLUORESCENT LAMPS. Number of % of cultures cultures showing pigment showing pigment 1 10.0 0 00.0 0 00.0 8 80.0 7 70.0 5 50.0 6 60.0 5 50.0 5 50.0 2 20.0 1 10.0 1 10.0 0 00.0 0 00.0 0 00.0 2 20.0 1 10.0 1 10.0 vO On TABLE XXIV DATA SUMMARIZING THE RESULTS OF PIGMENT FORMATION IN FUNGI IRRADIATED WITH THE FLUORESCENT LAMPS. Filter Number Wave Number of cultures irradiated Number of cultures showing pigment % of cultures showing pigment P. sulphurous 10 0 00.0 73 6800-7000 A. campestris 10 0 00.0 P. ostreatus 10 0 00.0 P. sulphureus 10 3 30.0 7k 5100-5750 A. campestris 10 2 20.0 P. ostreatus 10 2 20.0 P. sulphureus 10 6 60.0 75 k750-9*00 A. campestris 10 k ko.o P. ostreatus 10 k ko.o P. sulphureus 10 0 00.0 76 6800-7000 A. campestris 10 0 00.0 P. ostreatus 10 0 00.0 P. sulphureus 10 0 00.0 88 6800-7000 A. campestris 10 0 00.0 P. ostreatus 10 0 00.0 P. sulphureus 10 7 70.0 light - A. campestris 10 5 50.0 control P. ostreatus 10 5 50.0 P. sulphureus 10 0 00.0 dark - A. campestris 10 0 00.0 control P. ostreatus 10 0 00.0 98 TABLE XXV DATA SUMMARIZING RESULTS OP THE EFFECTS OF VARIOUS WAVE LENGTHS OF ULTRAVIOLET LIGHT ON GAS FORMATION IN SACCHAROMYCES CEREVISIAE AFTER AN EXPOSURE OF 90 MINUTES TO life CARBON ARC LAMP Number of Filter Filter Filter Filter Filter Filter culture 586 585 77*+ 791 970 986 Set #1 * 00 o 90# bO% no gas 20# 23# Set #2 100# 85# 70# IS* 1+0# 35# Set #3 100# 70# 35# no gas 10# 8# Set #+ 9?# 80# 50# trace 5# 10# Set #5 80# 60# 20# no gas trace 3# Set #6 87# 55# 10# no gas trace 2# * Gas measurements made with a Frost gasometer* HJ.MO90 JO J.unouy HJ.M099 JO NEUROSPORA. 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Asset Metadata

Creator Pusateri, Samuel Joseph (author)

Core Title The effect of visible and ultraviolet irradiation on cultured fungi

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Botany

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, botany,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-13426

Unique identifier UC11347701

Identifier DP21715.pdf (filename),usctheses-c17-13426 (legacy record id)

Legacy Identifier DP21715.pdf

Dmrecord 13426

Document Type Dissertation

Rights Pusateri, Samuel Joseph

Type texts

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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