AN INVESTIGATION OF ULTRAVIOLET INDUCED MUTANTS OF FOUR TRICHOPHYTON SPECIES Thesis for fihe Degree 01‘ M. S. MICHIGAN STATE. UNIVERSITY DAVID RECHARD FLEMING 19-6 6 WEEK‘S L'ie‘fl R Y ' yawn" ; 2cm ‘o'xaidllb J. A}? IIVLLSTIG{TI-"’1‘: OF ELTH.’=.V'11.3LE.T I‘ZDUGLD YUTAHTS OF FOUR THICECPHYTOH S37CI‘S by David Richard Fleming The microconidic of four speciee of Erictophzggg, Trichofhyton mentegggpgzggg (Robin) Blanchard, 1896, TrichOphytgg gelling; (Hegnin) Silva and Benhmm, 1932, Erichophxton rubrum (Caetellcni) Saboureud, 1911, and Trlchophytog,megginii Blanchard, 1896 were exposed to a selected ultraviolet irradiation (25}? angstroms) for verioue periods or time (2-16 minutes). Colonies germinated from these eporce were subjected to 3 nor. phological and physiological comparison with parent controls and other known species of the some genus to determine whether the mutant: resembled established species. Only the moat variable colonies were selected, 35 in e11. Upon comparicon with other epeciee, the mutants of 2‘_mjntegrophxtee and‘g‘ gallinae were found to resemble 2‘_gchoenleinii; E‘bconeentricgp, and'z‘ toneurens. The mutants were examined microscoDicelly by the elide culture technique. A few lost the capacity to produce conidie but most had typical Trichoghzton etruc- turee euch es chlamydoeporee and variation in also: of microconidie. None produced.macroconidie on the media used. David Richard Fleming 2 All species and their mutants were innoculated onto media composed of one or more of the following: an organic or inorganic nitroEen source, thiamin, inositol or nico- tinic acid, and an amino acid. A few mutants showed vari- stions but 80% of the nutants had nutritional requirements similar or identical to the controls. Ultraviolet induced mutants were found to be similar to naturally occurring variants of a species: (1) they display s variety or macroscOpic cultural phenotypes, (2) microscOpically only typical ErichOphyton features are noticeable, and (3) nutritionally, most or the mutants have similar or identical requirements to that or the parent strain. All but two of the thirteen mutants of g; mentagro- Ehztes had the same nitrogen and vitamin requirements as the controls. The mutants of 2;.gallinae did not grow as well as the controls on media containing ammonium n1- trate, while all were stimulated by the addition of histi- dine witn the exception of one mutant. GAL-0801, which could utilize neither ammonium nitrate or histiuino. The mutants of 2;_megninii were not able to utilize ammoni- um nitrate unless histidine was added to the medium. an INVLSTLCATLUH or ‘LTRAVIuLET IRLUCLD HUTaMTS 0F FDUR TRICEQfihYTON SéhCIhS By David Ricnard Fleming k TAESI3 Submitted to the School of Graduate Studies of Michigan State University in partial fulfillment of requirements for the degree of MAQTAR OE SCI:HCE Department of Botany and le1t FatnoloEy 1956 To my wife Sally ii AC KNOW Liz-.L‘lGl-LiirgNTS The writer wishes to express his sincere appreci- ation to Dr. E. S. Beneke whose dedicated interest, direction and encouragement during the course of this work has made possible its completion. The writer also wishes to express his thanks to Drs. H. E. Wade and R. O. Balding for their willingness in giving helpful suggestions and constructive criticisms. iii TABLE OF CONTENTS Ir‘lTRODUC'IIION O O O O O O O O O O O O O O O O 0 LITERATURE REVIEW . . . . . . . . . . . . . . Ultraviolet Irradiation and Its Effects on Organic Matter Irradiation Procedures Physiological Requirements Affecting Colony Growth and Korphology MATERIAL AND METHODS . . . . . . . . . . . . . RESULTS . . . . . . . . . . . . . . . . . . . Irradiation Studies of Mutants Morphological Studies of Mutants Physiological Studies of Mutants DISCUSSION .4. . . . . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . . . . . BIBLI OGRAPIiY O O O I O O O O O I O O O O O O 0 iv Page 11 1h 2.3 29 29 33 60 71 78 81 Table I. II. III. IV. V. VI. VII. VIII. IX. X. XI. LIST OF TABLES Bacto-TrichOphyton agars for differenti- ating the dermatophytes. The lethal effects of ultraviolet irradi- stion. The number of observed mutations at each irradiation period. morphology and growth of Trichoph ton mentggroonztes mutants at the en of— SIIHEys. Morphology and growth of Trichorhitog gsllinae mutants at the end 0 eye. Morphology and growth of Trichoghzton rubrum mutants at the end of 21 days. Morphology and growth of TrichOphyton megninii mutants at the end of 21 days. Nutritional modifications or TrighOphyton mentagrgphites mutants on bacto-TrlchOphy- ton agars. Nutritional modifications or Trichophyton gallinse mutants on Bacto-TricnOphyton agars. Nutritional modifications of TrichOpnyton rubrum mutants on Bacto-TrichOphyton agars. Nutritional modifications or TrichOphyton megninii mutants on Baoto-Triohophyton agars. Page 28 31 32 3h h3 h9 S7 61 66 67 I Figure l. 2. 3. h. S. 6. 7. 8. 9. 10. 11. 12. 13. 111. 15. 16. 17". 18. 19. 20. 21. 22. 23.. LIST OF FIGURES Trichopnyton mentagroPQytes control. . . . . Mutant MEN-OZOu. . . . . . . . . . . . . . . Mutant MEN-OMOS. . . . . . . . . . . . . . . Mutant MEN-OhOé. . . . . . . . . . . . . . . Mutant MEN-0u07. . . . . . . . . . . . . . . Mutant MEN-0211. . . . . . . . . . . . . . . Mutant MEN-OHIZ. . . . . . . . . . . . . . . Mutant MEN-0813. . . . . . . . . . . . . . . Surface View of Trichophyton mentagroyhztes mutants O C O C O O O O O O O O O O O O O O O Reverse side view of Trichoehzton mentagro- Ehztea mutants . . . . . . . . . . . . . . Trichophyton gallinae control. . . . . . . . Mutant GAL-160M. . . . . . . . . . . . . . . Mutant GAL-1207. . . . . . . . . . . . . . . Mutant GAL-1008. . . . . . . . . . . . . . . Mutant GAL-0809. . . . . . . . . . . . . . . Mutant GAL-0805. . . . . . . . . . . . . . . Trichopnxton rubrum control. . . . . . . . Mutant RUB-0205. . . . . . . . . . . . . . . Mutant RUB-1201. . . . . . . . . . . . . . . Mutant RUB-100a. . . . . . . . . . . . . . . Mutant RUB-OMOZ. . . . . . . . . . . . . . . Mutant RUB-OhOB. . . . . . . . . . . . . . . Trichophyton magninii control. . . . . . . . vi Page 38 38 38 38 no no no no hl L11 116 1.16 116 as 118 as 53 53 53 53 5’4 Sh 59 6 f i v " b p | 1 I c L s D Q I o I r r ’ O D l A I D O t o 0 o ' I a c a V a o b ' . v - \ o - . I V - s .e O l ' Q . . . . . - e 0 I ~ ' 1 0 fl s ‘ I n o - o o - O - O . s - . . . e - o , v o I s . u o - . o n .- . g - 0 O n s o O A # . e Q ' n - - u v I I Q Q s o e v e A l, v p I a v - I ~ o I Q r I I D \ o v v <1 l V 9 I . . - a k 0 - I I o J I . - s s < - I o \ e O O a ‘ a a I s f 1 c r I O V A 4 Figure 2h. 25. 26. 27. 28. 29. 30. 31. 32. 33. 3h- MUtant MEG'OBOI. e o s e e e e e e e TrichOphiton mentagrOphytea control. Mutant MEN-0813. e e e s e s e e s e Matarlt I’LEN'OLLOYQ o o e e o e o o e e Mutant MEN'OZOLLO e o e e e o e e e o. Trich0pnxton gallinae control. . . . Mutant GAL-114,030 e o e o s e e o. e e Pintant GAL-0805 e e e e e o e o o s o MUtMt GAL-0809. o e e e e o e e e o Trichopnzton magninii control. . . . ”intent MEG-08030 e e e e e s e e e 0 vii Page 5 9 62 62 62 62 61+ 6h 6h 6h 68 68 INTRODUCTION The dermatOphytes are a group of fungi that are of particular interest to man. They are not only parasitic to the body of man but to his domestic animals as well. Their existence is well known by those who have been infected and by those who have worked with the treat- nent of the diseasea. These organisms can be found in the aioroflore of soil living as sapropnytes with a potential for parasitism. They are noted for their destruction of hair, nails, and the keratin-containing layers of the skin. The problem of correct identification of each species is of concern not only to the taxonomist but also those who must choose proper medical treatment on the basis of specific species. One of the first attempts at classical identifica- tion of the dermatophytee was that of Sabouraud (1910). He divided an. highly variable group into five genera and a multitude of species. Since this time the genera have been reduced to three by Emmons(193h): TrichOphIton, Microeporum, and Mpidermophyton. In the genus Irichophzton, Sabourmid recogniztd 31 species. It was not realized at the time how variable the organisms are. Since then.asny of these TrichOphytons have been found to be variants of better established species. Many investigators are looking for a characteristic or a set of characteristics that will distinguish each true species and its variants from all the others, while some investigators are trying to establish an explanation for their variable nature. Some have designated this variation as pleomorpnism, others have called it spon- taneous mutation, While one researcher claims that the variants are intermediate stages between the potential parasites living as saprOphyLes in the soil and the more highly developed parasitic forms. It is the purpose of this investigation to shed some light on the problem of identification by develop— ing mutants that resemble other species morpholOgioally, to check for any modifications of nutritional requirements, and to see if these might be similar to changes that have occurred in nature. LITERATURE MEVIEH Ultraviolet Irradiation ggg‘ltg Effect 2g Organic Matter In physics, irradiation is the process of transmit- ting energy through space. It is in the form of highly accelerated particles;or electromagnetic waves. The electromagnetic waves range in length from 0.05 to 5.0 angstroms for gamma rays and high energy x-rays to radio waves hundreds of miles in length. At 500 angstroms low energy x-rays merge into ultraviolet and at 3800 angstroms ultraviolet becomes visible light. The ex- tremes of the ultraviolet spectrum are 10 angstroms and 3800 angstroms. Below 1850 angstroms air is not pene- trated by ultraviolet. Thus, the portion of the spec- trum useful for irradiation is between 1850 and 3800 angstroms. The effects on matter of any form of irradiation depend greatly on two factors: (1) the amount of energy carried by the radiation and (2) the physicochenical makeoup of the matter. The amount of energy carried is greater for short wavelengths and the degree of pene- tration or absorption is highly influenced by the density of the target material. It is the penetration of matter and the effects upon the matter that are of vital signi- ficance in attempting to understand the changes caused by ultraviolet irradiation on organic materials. 3 h The literature indicates that very little is known about the direct changes caused by ultraviolet irradi~ ation on exposed matter. However, the work of Robert Platsman (1959) indicates that although the various forms of irradiation are distinct from one another they do have many similarities. Thus, it is possible to acquire knowledge of the characteristics of ultraviolet by studying the effects of other forms of irradiation. Ionizing radiation in its strict application means, ”the ability to penetrate matter and displace electrons from atoms and molecules" Platzman (1959). Further, the same author indicates that ionising radiation is restrict- ed tc those forms or radiation that are of gamma ray or higher magnitude of energy. Ultraviolet, being of lower magnitude than gamma rays, has been left out by mmey researchers as having an ionising effect. It is thought that ultraviolet irradiation has a low penetration ability and not enough energy to form ions by removing electrons from their planetary orbits about atoms and molecules. However, it is possible for waves of low energy levels to alter the motion of electrons and cause a state or molecular or atomic excitement. This excitement can lead to a reorganization of the electrons that hold the atoms together which can change the molecular complex. The change caused by molecular excitement may or may not be a permanent effect. In organic material. a morpho- 5 logical or physiological change may be noticed and then a return to normal. During the stage of physicochemicsl change the ex- cited.molccules spontaneously dissociate. After a free. tion of a second unis yields a combination or stable molecules and chemically unstable molecular fragments known as free radicals. The structure of the target substance is highly unstable as these fragments enter into reactions with one another and uith.nolecules not directly affected by the radiation. When equilibrium is restored, the chemical bonds are reestablished in new configuration: new molecules have been synthesised and new preperties anarted to the substance. Thus a chemical change has taken place. In organic compounds ultraviolet irradiation can induce direct carbon to carbon links between adjacent molecules. The number or cross links is directly pro- portional to the dose of radiation and the necessary dose is less as the concentration or the material is reduced. The amount of absorbed radiation is more meortant than the amount of radiation. As the number of spores is re- duced, more radiation is absorbed by each spore. At different concentrations the pattern of cross linking also changes due to the available active ends. If the concentration or chemicals is low, the free ends or a single molecule may Join causing short and called mole- cules, Charlesby (1959). In 1932 it was thought that ultraviolet irradiation had an all or nothing effect upon living organic chemicals. Thus prior to this time no sublethal effects were eXpected nor found. Since this time a few experiments with vari- ous wavelengths of ultraviolet have been tested. Hollaender and Romans (1939). (l9hl) experimented with the effectiveness of eight wavelengths between 2180-2967 angstroms. as to their ability to cause mor- phOIOgical and physiological changes in fungi. They found wavelengths of 2537 and 2650 angstroms to be the most effective in causing mutations. This coincides with the high absorption coefficient of nucleic acids. However. nucleic acids may not be the only call com- ponents responsible for the observed changes. Other chemicals within the cell soon as enzymes and those involved in the structure of the cell may be so changed as to prevent normal cell functions or create functions that are completely new to the cell as indicated by Hollaender and Stapleton (1959). When the conidia of fungi are irradiated, such factors as spore clumping, presence or nacroconidia. pigment. multinucleate spores, and the density of the medium containing the spores can influence the amount or radiation received by each spore. It is ideal to have spores of uniform size and uninuclsate. One or the characteristic changes after irradiation is the physiological delay in germination as compared to the controls. Schankel (1960) also noticed this in ultraviolet irradiation of Escherichia 321i. Emmons and Hollaender (1939) found that fungal colonies differing only in amount of colonial growth from the controls reverted back to normal upon transfer. True mutagenic characteristics verified by several sub- cultures under controlled conditions are colony form, growth rate, pigment production. microscOpic morphoIOgy, and nutritional requirements. They found the degree of mutation resulting in these modifications to depend pre- dominantly on the amount of radiation absorbed by the spores. The ultraviolet absorption spectra of different regions of the spores were determined by Cole (l9ul) by means of a zeiss ultraviolet microscope. The absorption spectra were of three types: (1) regions of high density, (2) regions of medium density, and (3) regions of low density, thus indicating that a single spore does not absorb an equal amount of irradiation throughout the entire cell. Duggard (1936) noted that all of the in- fluencing factors in regard to absorption can cause many different mutations at one wavelength and one irradiation interval. Such experiments are nearly im- possible to repeat due to the many factors that cannot be duplicated. Even the concentration of spore sus- pensions that are irradiated has an influence on the amount of radiation absorbed. Bofsten (l96h) found that the killing effect of ultraviolet on a single or- ganism was less in denser populations. Attempts have been made to discover what intracell- ular changes occur when cells are exposed to ultraviolet irradiation but the picture is still not clear. Klein (190h) states that it is not known whether the mutation occurs in the nucleus of the conidium, the nucleus of a hyphel cell. or whether it is nonnucloar. Nofsten (196h) in her work with cultures of Optics- ;ng wiannulatm correlated the change in growth mor- phology with the decrease in DEA content of irradiated cultures. At the and of a 60 second exposure period from.a Phillips TIV 15, low pressure, mercury vapor tube at an emission of 2537 angstroms, the DEA content of the spores was reduced to one half. In contrast, Samoilova and Ovchinnikova (1963) found that the concentration of DEA increased after paranccia were exposed to a lethal dose of ultraviolet. Shankel (1960) in his studies of Escherichia 221;,found.changes in the genetic material of the mutants obtained after irradiation but made no correla- tions of the changes. The knowledge that sublcthal doses of ultraviolet ir- radiation can induce mutation has been used by a few re- searchers to study and attempt to correlate the induced variations among the derratonhytes with the natural variation within a species or the dermatopnytes. At the time when many of the 'n‘ionopnyton species were classified. the ability of these organisms to undergo spontaneous mutation was not fully realized. In 19hS. Emmons and Hollaendsr used ultraviolet irradi- ation at a usvclsngto or 2650 angstroms to induce mu- tations in Tricnopnyton mentagrophzggg. They found that not of the conidis irradiated produced mutations. The induced mutants differed from.the original so widely that if the parent organism had not been known. many mutants could not have been identified witn the origi- nal. Some mutations resembled other named species so closely that s mutation origin for some known species is suggested. The only means available to distinguish between some of the induced mutations and a known species was to determine tnsir virulence for animals. Many of the mutations were similar to spontaneous mutations in old cultures. It is conceivable that ultraviolet merely accelerates the rats of mutation (Emmons sod Bollsendsr. 1939). walker (1958) irradiated a suspension of ErichOpgxggn gglggrggg spores at s wavelength of 2537 angstroms and produced five categories of mutants that differed from tne parent strain in colony morphology and color. Most of these mutants resembled other known species in one cate- gory or another. 10 It is evident from exocrimontal data (Charleeby, op. cit. and others) that ultraviolet irradiation by its electromagnetic waves, can cause a molecular excito- ment that results in two kinds of change on organic material: (1) a permanent change and, (2) a short dur- ation change. The indications are that these changes result from a1 alteration of the DNA molecule. This is supported by the known.cnengce in one concentrations after ultraviolet irradiation, and that the greatest amount and degree of mutation occurs when wevelengthe of 2537 and 2650 algctrome are used. These wavelengths are identical with the absorption coefficient of nucleic acids. All chaigee after irradiation may not be genetic and are therefore only temporary. The degree of chmnge may be attributed to the amount of irradiation absorbed by the spores. Absorption is influenced by both intra~ cellular and extracellular densities of matter. The changes that were induced by Emmone and Hollaender (IQuS). and Walker (1958), were definite genetic changes. The organisms remained changed after many euboulturee were made. 11 Irradiation Procedures Emnons end Hollsender (1939) irradiated spores of Triohophzggg’mentegrcgnltgg suspended in saline at s concentration of 70X106/ml. A water cooled. high pres- sure, quarts capillary. mercury vapor lamp was used as a source of ultraviolet irradiation. The wavelength used ranged from 2280 to 2950 angstroms. The spore sus- pension was rapidly and constantly stirred during expos- ure. At intervals, 0.1 cc. of the suspension was removed and plated out on corn noel agar and incubated at 30°C. for 5 to 6 days. The outgrowing colonies were transferred to slants containing modified Ssboursud's agar and check- ed for phenotypic changes. Single spore isolations were also made to insure pure cultures. Kihlberg and Fries (1957) irradiated conidis of g; nentagropnztgg that were shaken out of cultures on agar slants with 5 ml. sterile water and poured into flasks. The density of the spore suspension was deter- mined in a counting cell and diluted to SXloh/nl. Two milliliters of the suspension were transferred to each of the sterile weighing glasses. The glasses were placed under the ultraviolet tube, lids removed, st s distance of 25 on. and exposed for 15 seconds, 30 seconds, hS seconds. 1 minute. 2 minutes and 5 minutes respective- 1:. The weighing glasses were kept in constant rotary motion. Spore dilutions of SOOO/ml., 500/ml., and SO/ml. 12 were obtained and from these 1 ml. of each was trans- ferred to sterile petri dishes and suspended in melted Sabouraud's agar. The best eXposure period for mutation was one minute. Isolation procedures were the same as those described by Fries (19u8). All cultures were in- cubated at 30°C. ‘ Walker (1958) irradiated a spore suspension of Trichoghlggg eulfureum in sterile isotonic saline solu- tion that had been filtered through sterile cotton wool to remove fragments of mycelium. The filtrate had a spore density of 125,000/ml. A 3 ml. volume was placed in an eXposure cell and rapidly and constantly agitated. The suspension was eXposed to a wavelength of 2537 angstroms for periods varying from 60 to 320 seconds; after each eXposure 0.1 ml. was withdrawn. The 0.1 ml. volumes were diluted to 1:50 concentration with sterile isotonic solution and ten 0.1 m1. quantities of each were plated onto malt extract agar plates and incubated at 26°C. The controls were checked at the end of 7 days and the irradiated plates after 1h days. Hammer and Knight (1959) irradiated tne Spores of 1‘ mentsgernites. The spores were obtained from colonies growing on Sabouraud's dextrose agar maintain- ed in 300 ml. prescription bottles at 37°C. After 10 days incubation, 30 m1. of sterile distilled water con- taining glass beads was added to the slants and tilted 13 back cud forth so that the beads dislodged the spores. The spore suspension was standardized in a haemocyto- meter to about 2X107/m1. by dilution wit. attrila water. Seven milliliters of suspension were ylcced in a sterile petri dish and stirred while being irradiatcd wito ultra- violet light. The irradiation was for #5 seconds at a distance of 10 cm. from an 8 watt General Electric Ger- micidal lamp rated at 2537 angstroms. A one tenth milliliter portion of the irradiatcd Spore suspension was spread ovcr the surface of the basal synthetic medium and incubated at 30”C. for 3 days. The basal medium was that of Robbins and Na with Oeafi Lnarginine as the nitrogen source. The mutants were tranefarred to Sabouraud'a dextrose agar. nutritive requirements were determined by the addition of nutrients that would permit growth. All mutants infected guinea pigs with strikingly variable degrees of pathogenicity. 1h Physioloaicsl Esquiromcnts Affecting ColoniGrowthsnd Horphologl If one know the nutritional requirements of all the established species or s genus, one might be able to dis- tinguish species and newly occuning variants more readily. Robbins and Ho (l9u5) investigated 2; mantsgroghztss in regard to vitamin, nitrogen source, and amino acid growth requirements. They found that the organism was 'sutotrOpnic for all vitamins tested and unable to use ammonium nitrate as a nitrogen source. Growth was better on s mixture of amino acids than any single amino acid. ,This characteristic was attributed to its inabil- ity to use an inorganic nitrOgcn source for tne snabolism of needed amino acids for protoplasmic protein. The plsomorphic forms of g; montagronnytes were studied. They grow better than too non plsomorphic forms on both ammonium nitrsts and ssparsginc. It is suggested that plsomorphic forms had a bettsr mechanism for transforming ammonia or s single organic nitrOgen source into cell substance. It also was found that less pleomorphism occurred at lower temperatures of incubation. Hackinnon and Antsgsvogtis-Allende (l9h8) cultivated 5 strains or Trichoghyton discoides and 2 strains or ErichoPnztgg’ocnracsum on synthetic media witn pyridoxine, thiamine, and DL-inositol, clone and in all possible com- binations. From the erratic results they concluded that 15 vitamin requirements cannot be useful for identification or species because of the many variations of vitamin requirements of strains within a species. Benham (l9h8) realized the effects or media and variation in nutrients on the morphological deveIOpment of organisms. She reported that 50 strains or Tricho- phxton rubrum had good microscOpic characteristics de- veloped on corn meal agar and all but one strain pro- duced macroconidia on blood agar base. It is her con- tention that tryptoee is the causative agent of macro- conidia production in the blood agar base. McVeigh and Campbell (1950) thought there might be a differential basis among the variants of species in regard to nitrogen source. They tested six strains or ‘2; mentagroghzggg on asparagine, ammonium nitrate, casein hydrolysate, and a variety of amino acids singly and in ill possible combinations. The results verified those 0! Robbins and Ma (l9h5) that casein hydrolysate is the most favorable nitrogen source for all strains tested., The most extensive nutritional research with the dermatOphytes has been performed by L. K. Georg and her associates (l9h9). She related the influence of vitamins, amino acids, and other growth factors in natural products to good growth and the production or conidia in 1;_rubrum, 16 Trichophzton schoenleinii, Trichoghyton ravirormg, and Trichgphztgn,violaceum. The following year she investigated the growth requirements or 1; faviforme and found both thiamine and inositol were needed. She also found good mycelisl growth and conidial pro- duction on casein hydrolysate as a nitrcgen source (1950). In 1951, Georg found that‘g; violaceum proved to be deficient in thiamine synthesis but was capable of surviving on ammonium nitrate. her work with Trichophlton magninii and Trichophxtongallinae (1952) proved to be of value in distinguishing these two species. 2; megninii was found to be incapable of growing on ammonium nitrate and has a histidine re- quirement for growth. In the case of 2;_gallinae, the organism was found to be autotrophic for vitamins and grew well on ammonium nitrate agar. Later Swarts and Georg (1955) in determining the nutritional needs of Irishophlggg_tonsurans, round thiamine necessary and the best growth was on casein hydrolysate.‘ One of the typi- cal strains tested demonstrated a definite stimulation by the addition of thiamine. One of the most extensive checks on nutrition re- quirements was that performed by Georg and Camp (1957). It involved 100 strains or Trichophitgg’verrucosum, 50 strains of z; schoenleinii. 19 strains of Trichophxtgg concentricum. 60 strains obe; tonsurans, 69 strains 17 of 2;‘mentagroph tes, uh strains or g; rubrum, 13 strains or g; violaceug, 1h strains or g; ferrugineum, 13 strains of‘2:,mpgninii, 7 strains of g&>gallinae, and 13 strains of 2; eguinum. The outstanding nutritional requirements found were a complete requirement for thiamin by 2; verrucosum, a requirement of niootinio acid by 2; e uinum, a requirement or L-histidine for growth or g; megninii. and that both'2;,mentagrophztes and g; rubrum are com- pletely sutotrOphic for vitamins. With an emphasis on g; gellinae and g; magninii, Silva (1952) checked morphological variations on various established media. Good colony characteristics and pigmentation were found on Sabouraud’s dextrose and honey agsrs. For good macroconidia production a greater vari- ation in media was needed. TrichoyhytonAgentagropnytes requires wart agar, corn meal agar or heart infusion tryptose agar for good macroconidia production. Tricho- phzton rubrum will produce macroconidia on heart in- fusion tryptose agar. TrichoPniggg gallinae has good macroconidis production on casein hydrolysate medium and yeast extract agar. 0n potato dextrose agar and on corn meal infusion agar many or the species show submerged growth in the medium. TrichOphyton rutrum produced pigment only when dextrose was added to corn meal agar. The nitrogen, vitamin, and amino acid re- 18 quiremcnts were similar to the results reported on the same organisms by George and others. One of the most striking characteristics or the dermatophytes is pigment production. Some authorities have tried to distinguish between the species on the basis of pigmentation. When strains or TricnOphyton mentsgrophytos produce red pigment it is difficult to distinguish from Trichophyton rubrum. This problem led Silva and Benhsn (l95h) to study pigment production in in regard to pH changes, amino ecids, and pathosenicity. It was found that pigment production occurred at pH 7.0 to 8.2 end is influenced by different smino acids as well as increased smounts of the some amino acid. Pig- mented strains orig; mentegrOphytes were found to be more virulent than nonpigmented strains when injected into guinea pigs. It has been found that pigmentation is not the result or e single pigment but s complex of pigments as pointed out by Uirth, O'Brien, Schmitt, and Sohler in their work with 2; rubrum (1957). Stockdule { (1953) has reported that the hydrogen ion concentration or the medium has s tendency in some TrichOpnytons to influence the color. In ecid media the mycelium is yellow while in basic media the color is red. Yuki. Fujii. end Hossws (l96h) found the absorption spectre of pigment combinations for 2; rubrum, 3; tonsurans. 19 25’mentagr09h3tes, and 2; megninii. The spectra were found to be different for each, thus these species can be differentiated on this basis. Silva (1953) attempted to show a difference in pigment production among the mrichophytons in regard to the influence or a nitrogen source. Five strains of 2; rubrum, two strains of g; mentagrOQh tee, six strains or g; megninii, and two strains of 2;_gallinae were used. The nitrogen sources tested were ammonium nitrate, casein hydrolysatc, and histidine. by compar- ing the results on all nitrOgen sources, the strains tested were distinguishable on the basis of pigment color. Baxter (1963) found two pigment characteristics that may help in identifying strains or g, rubrum in addition to other taxonomic criteria. Four strains of ‘2; rubrum and three strains of 2; nenteErOphxtgg were each grown on Saboursud's dextrose agar and Lab-lsmco beer extract dextrose agar. 0n the Lab-lemcc dextrose zglrubrum produced pigment earlier than on other stan- dard media and the shade of the pigment was unique as com- pared to E; mentagrOpnytes on the some media. In the study of both pleomorphic forms and irradi- ation induced mutations many specimens are lost when seeded on ordinary media. Benham (1953) in her work with a strain or Epidermophzton floccosug and a plea- 20 morpnic form of it, found that they would not grow unless supplemented with yeast extract, neOpeptone, or bactepeptone. The vitamins and amino acids found in yeast extract were added individually and in combina- tion to a basal medium. In no instance was growth ob- served. It is concluded that yeast extract contains some unknown factor that promotes growth. The problem or pleomorphism has lead many people to study its characteristics. wilhelm (19h?) believed pleomorphism to be a result or heterocaryosis and nu- clear separation rather than a genetic change result- ing in mutation. In contrast to this idea, McVeigh and Campbell (1950) contend that pleomorphic forms of g; gentagrOphztes are naturally occurring mutations. They based their opinions on the results of six pleo- morpnic strains. These pleomorphs indicated very stable characteristics in regard to growth rate, pa, morphology, and nutrient requirements. The strains were maintained without change for six months by transfer every 7 days. Pleomorphism can be retarded by incubating at low temper- atures of about 26°C. At higher temperatures it is possible to reduce pleonorphism by raising the pH to 3.0 (196i). 1 The question as to whether pleomorphisn is Just variation within the species or whether it is a genetic mutation has been studied by various investigators. 21 Evidence for the snsuer is presented by Hsitsman (l96h) in her work with gicrosporqg Eggssum. The rediscovery of sexual reproduotion in this organism hss led to ss- perimsntstion.uith cross mstings between pleomorpnio cultures and toe wild type. The results demonstrated thst pleomorphism is the result of one or more chrono- soasl gens nutsttons and is inherited in simple Nonde- lisn tsshion. It is suggested that the torn plsomoro phism be discarded in tsvor of the torn mutation. It is known that derustOphytss can be isolated from microrlors or soil, ss sspropnytes and the same species fron.mammals. se psrssites. Tho sspropnytio form is thought to be primitive although it has s oom- plets morphology ss campersd to the more highly evolved but morphologicslly simpler psrssitio form. There srs distinctive adaptations lesding from the ssproyhytio stage to the parasitic. This is not only morphologlv oslly true but physiologically true as well. Tbs ms~ Jority of adaptations occur in the parasite within the host. It is this host street that remains unclear. Hany adaptations that require time are involved in the change from s ssprophytio form in s soil environment. to s parssite inm‘waru.bloodod host. In so doing there develops s rsnge of forms from ssprophytss with pars- sitio potential to vary highly devologod psrssites with sun; intermediate forms in between. It is on 22 this basis that lesbsnoff suggests s new classifica- tion for the dermstOphytes (1965). It is evident that morphology slone is not suffi- cient for differentiation, and that nutritional studies have helped in the separation of some species. Howevar. new investigations in the future may help with further study in dittorentisting species and verisnts of the dormstoyhytes. MATERIAL AND METHODS Four TrienOpnyton species were used for ultraviolet induced mutants. These were: Tricnopnitgg‘gentsgrognltgg (Robin) Blanchard, 1896, obtained from Swiss white mouse skin scrapings at Michigan State University, Tricqugx- £22 rubrum (Castellsui) Seboursud, 1911, obtained from a chipped fingernail in Lansing, Michigan, grionopnxtgg negninii blsncnard, 1896, (uZuCLC) obtained from the Communicable Disease Center in Atlanta, Georgia, and Trichognxtgg_gsllinse (Moguls) Silva and Benhsm, 1952, obtained from the culture collection at Hicnigsn State University. The cultures were grown on Ssboureud's glucose agar in 8 inch test tubes and incubated at 25°C. At the end of 20 days a spore suspension was made by washing out the conidia with 10 ml. of sterile distill- ed water. A spore count was made with s nsemooytometer and the spore density was adjusted to ux106/m1. by addition of sterile water. Spore clumping was not noticeable but fragments of hydro could be seen. Those were not filtered off as it was assumed that too initial exposure period (2 win.) would be lethal to all viable hyphse. A witndrswal of 1 ml. of suspension was saved for the control. The remaining 9 ml. or suspension were placed in a sterile petri dish at a distance of 30 cm. from s 15 watt, General Electric, 23 2h Germioidal Lamp emitting its radiation at a wavelength of 253? angstroms. After intervals of 2, h, 8, 10, 12, 1h, and 16 minutes, 1 ml. of exposed spore suspension was pipetted into separate sterile dilution blanks. Additional dilutions were made before plating out the spores. During the entire esposure the suspension was constant- ly agitated. This was done for two reasons: (1) to reduce spore clumping and (2) to increase the chance for each spore to receive an equal amount of irradiation. Three dilutions of spores for each exposure period were made in order to select one of the dilutions where the colonies were not too crowded in the plates to facilitate picking out mutant colocdes. For each ex- posure period, 1 ml. of each dilution was pipetted into sterile plastic petri dishes and distributed into warn Sabouraud's dextrose agar. A 0.5 percent yeast extract was added to insure a more complete medium. All plates were incubated at 25°C. and checked every three days from the time of seeding. This served as s check for accelerated germination due to the effects of irradiation. However, none of the exposed spores germinated before the controls. Host of the seeded plates showed colony formation by the third and fourth week. 25 Hyphae from all colonies showing the slightest change from the normal were isolated as soon as possible to prevent then from being overgrown by other colonies. A transfer of each control colony was also maintained for comparison. All isolations were placed on Sabouraud's dextrose and yeast extract agar. Approximately 150 isolations were made. At the end of 15 days growth s single spore isolation was made from the stable variaits onto Sebouraud's dextrose and 0.5 percent yeast ex- tract agar, and incubated at 25°C. About 75 Percent of the variants isolated reverted back to the normal and were discarded. At the end of 21 days, the observable changes in the colonies were recorded including: growth rate, texture, margin, pigmentation, and pleo- morphism. The slide culture technique was used for observing microscopic characteristics. The medium used was 2 percent corn meal agar with 0.1 percent yeast extract, 1 percent dextrose, and 0.1 percent malt. About 15 ml. of this medium were poured into a sterile petri dish, allowed to solidify and cut into 1 on. square blocks. The culture apparatus consisted of a sterile petri dish, filter paper covering the bottom, a flamed wire stand, a glass slide, and a cover slip. The flamed glass slide was placed on.the wire stand in the petri dish. A 1 cm. square block of the prepared agar was placed in the 26 middle of the slide. The organism was transferred from the single spared cultures to the four exposed sides of the agar block. A sterile cover slip was then placed on the upper surface or the agar block. To prevent the agar from drying out, about 3 ml. of sterile distilled water was added to the petri dish. The slide cultures were incubated at 25°C. for 10 days and then checked daily for reproductive structures. At the end of 21 days all cultures were prepared for microscOpic obser- vation. The agar block and cover slip were removed and a drop of lacto-phenol cotton blue was used for the slide meant. MicroscOpic features examined were: chlamy- dospores, microconidia, macroconidia, and hyphal defor- mities. Modifications of nutritional requirements were checked on Bacto-TrichOphyton agars (Table I). The stock media consisted of casein agar and ammonium nitrate agar. Each medium was prepared according to the formulations by Georg and Camp (1957), based on the nutritional requirements of the various Trichophy- 333 species. Six nutritional factors were tested, three vitamins: inositol, thiamin, and nicotinic acid, two nitrogen sources: casein as an organic nitrOgen source and ammonium nitrate as an inorganic nitroEen source, and an amino acid; histidine. Ten ml. of each : ilk: , ENFEHIE’ :auw. .,.,. 27 of the seven medic were pipetted into six inch test tubes and slanted. Each of the 35 mutations and the four normal strains werc inoculated on the slants. A uniformly email amount or the inoculum was used for each transfer. The slant cultures were incubated at 25°C. and compared to the controls at the and of 12 days growth. The characteristics compared were changes in rate of growth on cach medium and changes in pigmen- tactic“. 28 Anmoav aaco cad moooo ho vouccnchm a<¢ cw Ocmfl o c o o c 0 cm H00 0 c o c c 0 .w 0.0: 0 90H 0 o c o c 0 cm mcH O c c O O c wand coaadna as. ONOOmflocoocco cmHOOOOcoccc Om 0.0:. 0 o I c 0 0 cm 30H 0 o l c o c c .w m.N . nuCn0£< swam oaouopmam oucupooam daecwnb HouflnocH HouwaccH I . O. C O U .0 . cud: c c¢m< couhzaoncahascuoam o O O I c c o c O O haw‘I-OHO¢~N . . . . . . .ouahflam Esaaoawc: . . . . . . . . ouchuxcasouodm . . . .ouanaaonm naancauoaccox c c c c c 0 O .QUIHHHZ aggogflu‘ cud: a coma couhnoonoahaoouccm mafia H me< couhsaozoaussowocm spa: A hamq :oumaaozcahancacca nu“: H camd coumsaoncaaauoucam . . . . . . . . . . huw¢uouccm o o o o o c oQDGHHflW gfiflQflde c c c c o O O c Ono-NgnaaIOQOUnw . . . .ouanooonm asanumuoaocox . . ..au«o¢ cadacnco coax cuemua>touoan mononuauacoo H hdw< councconouna ocumnaonoaha cowhnacnudha couhcqoncahe couhzaonoaha coahnaoncdua cauhnaonoaha mace: ¢.noanmcucEpoc can wcwumaucoccuuao Lou comma coahnmccownaocuoum H mundk RESULTS Irradiation studies 25 mutants At the end of 12 days, plate counts of the number of germinating conidia were made for each organism at each irradiation interval. The percent kill was determined by finding the difference between.the calculated and actual number of viable conidia in each plate, then dividing the difference by the calculated number of spores and multiplying by one hundred to obtain percent. The results indicate an abrupt lethal increase between 8 and 10 minutes for all four species and a gradual in- crease in mortality from 10 to 16 minutes. These re- sults are shown in more detail in Table II. The data indicate that 2; mentagroghxtes and g; megninii were the first to show lethal effects of irradiation. 2; gallinae was effected the most at the end of 6 minutes by showing a to percent lethal increase. 2;.megninii indicated a greater sensitivity to the lethal effects of irradiation by showing a 13 percent increase at the end of h minutes exposure and a mortality of 99% at the end of 10 minutes exposure. The number of observed mutations at each irradiation period is given in Table III. The greatest number of mutants for gzlmentagrOphytes and 2;_gallinae occurred at the 16 minute exposure. TrichOphxtonwrubrum had its 29 30 highest observed mutation at the 2 minute exposure period and'ghrmggninii showed its greatest number of mutation at h minutes exposure. 31 .60.: 3333: e3 do ocoou on :33ch deduce»: choc 3503 ed .HI\QOHK: do venom-c NH no use one on 93139703 m.ee mmm nm.eo eo- oe.oo oped o~.eo mmmd access on 33 Ram m3... l chem 2.2. Son 3.8 nmmw o8; .: o.oo wee: m.mo enam a m.mo meme :.oo snap cease: ma o.eo man: A «.mo «we» o.mo name m.>ov mesa cease: on o.He “was” n.3e. npemd o.~m omens m.o: noon” oozes o o.mn moon o mmnm o as»: m.» soda can: a 0 ends 0 head o moan o nmmm as: w o mmm a new o can 0 den 4: o Walflflfleoodn «a 33598 33 a «saunas SQ 33.393 issuance o m Madmen mm Ste: «H ”4% Mi .mflflazuzel «M Luann“. 3 ecoausdoeanu negates»? Wm. H H "ma—gun. essence Hana.” can 32 .nuaOhw when NH no use one as penance one: condensate .60.: edohasnuv on» do snack on case: as Hl\ooaxd_nc oceans g “M .o «leaned 4M .o a”? 4w... .9 nadoamoucei 4H .d o a m : mmm ooaw one” «mna cease: on o a a o some eeem seau nmmm cease: an o a a o wees. onem name page cease: ma 0 a n 0 me»: ”was name mesa accede on n o n w «News mamas embed amass ooze: a a m o m .mwmn emnm «he: son: 03:: a o m o m onus heme moan mmmm an: m o o o o mmm wmw can gen 4: o v e at a b ;c mr s nausea: denounce «squeegee assau> taaaeaeoo seduce oeeaeeoo Mo «spasm no sense: we .cm sodasuoe9h% veueasoaeo ocanem douaeaoshhu nose mm.ndo«ususn vephoeno_mm. HHH muncfi Lennon 4“ oak 33 Morpholcgical studies gg'mutants Many of the mutants displayed a variety of colonial characteristics very different from the controls while a few varied only slightly. All cultures were grown on Sabouraud's dextrose and yeast extract agar. They were subcultured three times as single spore transfers to - check for stability of the mutant colony. Those mutants lacking or rarely producing conidia were trans- ferred by the three point inoculating method to insure a transfer of stable form of the organism (Fig. 12). The colonial characteristics of the 2; mentagro- phytes mutants are described in Table IV. One of the most interesting charges, in contrast to the control with a white, velvety smooth surface and a tan reverse side (Fig. 1) was a mutant colony with a gray, wrinkled and raised myoclial surface and a pale tan reverse side. This organism grew only a few centimeters less than the control. This mutant developed after 16 minutes of irradiation (Fig. 2). Another mutant of varied features displayed a myosliun that separated from the agar because of deep wrinkles and grooves. Its surface was gray, velvety. and the reverse golden brown. A pleomorphic form can also be seen in this mutant colony (Fig. 3). A very restricted slow growing sulfur yellow colony, mutant MEN-Ohoo, hads deeply grooved surface with an amber yellow reverse side (Fig. h). A slow growing 3h TABLE IV Morphology and growth 2; Trichophyton mentagroghytes “flu-eel.“ Colony MicroscOpic Colony Characteristicse Structures** Diameters Key: 2 3 Organism Control 1. Fast growing, with a white, velvet , smooth surface. Reverse is ten. (Fig. l . 2. Numerous chlamydospores and oval microconidia (1,121.). 3. 8h millimeters. MEN-1601 1. 2. 3. HEN-0802 1. 2. 3. MEN-1603 l. 2. 3. MER-OZOh 1. A white, cottony smooth surface with s gran- ular margin. Reverse is pale tan. The exposure was 16 minutes. Lacked conidia and chlamydospores. 63 millimeters. A white, velvety smooth surface with wrinkles at the margin. ReVerse is pale tan. The exposure time was 8 minutes. Lacked conidia and chlamydoapores. 35 millimeters. A white,smooth,velvety center with margins cottony. Reverse is white at the margin and tan near the center. The echsure was 16 minutes. Lscked conidia and chlamydospcres. h? millimeters. The colony is gray, velvety, raised, and fold- ed in the center. The margins are velvety white. The reverse is ten at the margins TAELE IV HBN-OZOh (cents) MEN-OQOS Mhfi-Ou06 MEfi-OhO? MEN-1608 35 (continued) 2. 3. 2. 3. 1. 2. 3. 1. 3. 1. 2. 3. varying to a dark brown in the center. The alposure was 2 minutes. (Fig. 2). Lacked conidia and chlamydospores. 50 millimeters. A grayish velvety surface with extreme folding. Folds at the margin have lifted from the agar. Shows a pleomorpnic form at the margin. Reverse is golden brown. The emposure was h minutes. (Fig. 3). Has small numbers of oval microconidia 3 to an in size. Ho chlamydospores. 27 millimeters. The surface is velvety white with deep radial furrows and an irregular margin. The reverse is tuber yellow. Slow grow- ing colony. The emposure was u minutes. (Fig. (4)0 Numerous chlamydospores. No conidia. 17 millimeters. A sulfur yellow, velvety colony witn an irregular margin. The center is wrinkled and raised. The reverse is bright yellow at the margin and golden yellow at the center. The exposure was a minutes. (Pigs 5)e Numerous chlamydospores and no conidia. 33 millimeters. The surface is velvety white with radial grooves and a raised center. The reverse is ten. The exgosure was 16 minutes. Has many elongate clavate shaped micro- conidie about 7u long. No chlamydospores. 28 millimeters. as TABLE IV MEN-Oh09 MEN-1510 MEN-0211 MEN-OhlZ MEh-Ool3 36 (continued) 1. 2. 2. 3. 1. 3. 1. 2. Hearly the same as MEN-1608, excepting rate of growth is more rapid. The exposure was (4, 31111“ tel e Numerous oval microconidia, about hu., and a few chlamydospores. ha millimeters. The colony growth is very similar to the control. The reverse is darker brown than the control and slower growing. The expos- ure was 16 minutes. Has many microconidia, elongate and elongate clevate shaped, about Tu. long. 30 millimeters. Colony white, velvety, with deep radial furrows and a raised center. Yellowish brown droplets are on the surface. The edges are irregular. The reverse is yelldwish-tan. The exposure was 2 minutes. (Figs 6). Few oval microconidia about hu. in size. No chlamydosporee. 26 millimeters. A raised smooth, white, velvety, colony with very compact, dense, growth. The margin is entire. The reverse is amber. The exposure was h minutes. (Fig. 7). Few oval microconidis about u to Su. in diameter. No chlamydospores. 25 millimeters. Colony rose pigmented, dense, cottony surface smooth. The reverse is yellow at the margin changing to a red-wine color in the center. The exposure was 8 minutes. (Fig. 8). Few minute, oval microconidia about 1 to Zn. No chlamydospores. 37 TABLE IV (continued) MEN-0813 3. 70 millimeters. (oont.) #hoth colony characteristics and colony diameter were observed on Sabouraud's dextrose agar witn a“ yeast ex- tract. v #finicroscOpic structures were fOund on 2.0% corn meal agar (0.1% yeast eeract, 1% dextrose, 0.1» malt). ‘ . was-sW'fifr 38 Fig. 1. 'Trishophzton mentagrOphytes, control snowing s white, velvety smooth surface. Reverse side tan. The colony is fast growing. I 0.h. Fig. 2. Mutant MEN-OZOh showing a folded, raised, velvety surface varying from white to gray in color. Colony is moderately fast growing. Irradiation interval, 2 minutes. X 1. Fig. 3. Mutant MBN-OhOS showing colony edges raised above surface of agar and a very irregular surface. Pleomorphic at the upper edge. Irradiation interval, a minutes. I 1. Fig. h. hutunt M38-0h06 shows a deeply grooved and highly restricted slow growing colony. Re- verse side amber yellow. Irradiation interval, u minutes. x 1. Plant.“ 1s 1'1 ‘ gure 2. Q Figure (4s 39 co1ony with a sulfur yellow pigment was produced by mutant figs-Oh07, with wrinkled and raised surface. The reverse was varying shades of bright yellow to golden brown yellow at the center. This organism had been exyosed for four minutes (Fig. 5). The colonial features of mutant fiLE-Ole were noticeably different in two ways: (1) the surface was raised, white, with deep radiating furrows in the center and, (2) the surface had nunorous golden brown droplets of liquid (Pig. é). Another mutant, white on the surface, dis- played very restricted growth patterns as compared to the rapidly growing control (Fig. 7). Another was dis- tinguished by the production of a rose pigmented myoc- 1ium.(Fig. 8), The differences between the mutants and the control are very ayparent when they are placed next to each other and compared (Figs. 9 and 10). The microscopic morphology for‘2"mentar oohvtes mutants is also given in Table IV. lour of the mutants lost the ability to produce oonidis under cultural con- ditions as a result of the irradiation. One was nanosed 2 minutes, another 8 minutes, and the other two 16 minutes. T.s other mutants, r 3-1608 and ELL-1610, exp posed to 16 minutes of irradiation were the only mutants of this species to have long elevate shaped micron nidis. Eutsnt Lhfi-OSIB had very small microoonidis about 1 to 2 u. in diameter. The control nicrocanidia Fig. 5. F180 be Fig. 7. Figs 80 no Mutant NEH-0&0? showing a wrinkled, raised, velvety surface with a sulfur yellow pigment. Reverse yellow at the margin and golden yellow at the center. Irradiation interval, h minutes. X 1. Mutant HEN-0211 showing a wrinkled, raised surface with numerous drOplets of yellow-brown liquid. Reverse yellowish tan. Irradiation interval, 2 minutes. X l. Mutant HER-Ohlz showing a restricted, compact, white, velvety, colony. Margin entire. Reverse amber. Irradiation interval, h minutes. X 1. Hutant HEN-0813 showing a rose, velvety, smooth surface, similar to the rose colored naturally formed variant. Reverse yellow at the margin, red-wine in the center. Irradiation interval, 8 minutes. X O.h. wn Figure 5. Figure e.. Figure 7. Figure 8. hl Surface view of Tricnognxton mentagropnxtes mutants. From left to right: TOP HOW nas-ozou See Fig. 2 BOTTOM ROW Control See Fig. 1 nun-oulz MEN-OhOS nan-once See Fig. 7 See Fig. 3 See Fig. h MEN-0813 HEN-0&0? ERR-0211 See Fig. 8 See Pig. 5 See Fig. 6 Reverse side View of Trichophzton mentagrOphxtes mutants. From left to right: TOP ROW MEN-020k Pigment yellow to dark brown. BOTTOM ROW Control Pigment tan. MEN-OhlZ Pigment amber. HEN-0613 Pigment red- WING. MER-OhOS Pigment dark golden brown. Pleomorph tan. MEN-0&0? Pigment yellow to golden yellow. MEN-ouOG Pigment tan. MEN-0211 Pigment yellowish tan Figure 10. h2 were h to Su. in diameter. The other mutants that oc- curred had been exposed to 2 and h minute periods. Three spore conditions were noticed anon; then, normal nicroconidia and no chlamydospores, chlanwdospores and no nicroconidia, and both normal.nicreconidia end chlamydospores. Ho nacroconidia were seen in any of the cultures. There were no observed changes in the. hyphae of the mutants. The cultural characteristics of the Trichoghzton gallinae mutants are described in Table V. The control had typical characteristics of the species. The red pigment was diffused into the medium and the growth had a few wrinkles on the pink, velvety surface (Fig. 11}. A highly contrasting slow growing mutant is that of OAL-lbou. It is a raised and very irregularly folded colony. It has scream colored glabrous or waxy surface with a tendency to crumble when being transferred. The reverse is yellowish-orange and there is no diffusing pigment. The exposure was 16 minutes (Fig. 12). Mutant GAL-1207 has a curious characteristic of a thin layer of subnerged hyphae extending outward around the edge of the colony, with a crater shaped center and radial folds. It was exposed to 12 ninutes (Fig. 13). Mutant GAL-0809 was like the velvety surface and radial folds of the con. trol. Bowever,it lacked conidial preduction and had no red diffusing pigment. The surface was white and the RB TABLE V Morphology and growth of Trichoghzton gallinae mutants 22 th. and 2!: £2; d§.e Colony MicroscOpio Colony Characteristicsv Structuresfifi Diameter. Key: 1 2 3 Organism Control 1. Colon! pink, velvety on the surface with radi wrinkles and a regular margin. The red pigment is diffused in the medium. The reverse side has a red-wine color (Figs 11). 2. A few oval nicroconidia, 3 to nu. in size. No ohlamydospores. 3. 65 millimeters. GAL-0801 1. Surface velvety, light pink in color, radial wrinkles, and an irregular margin. The diffused pigment is not as intense as the control. The underside is red. The exposure was 8 minutes. 2. A few chlamydospores, no conidia. 3. 58 miliineters. GAL-1602 1. The appearance sinilar to GAL~OBOS, raised, wrinkled, and brown in the center with a white margin. See Fig. 16. Slow growing colony. Exposure was 16 minutes. 2. No conidial formation. 3. 32 millimeters. GAL-lac) l. The surface velvety white with few radial wrinkles, and an irregular margin. Has no diffused pigment. The reverse side is yellowish-tan. The exposure was in minutes. 2. Few chlamydospores, no conidia. 30 S7 M111Mtdl‘le _— TABLE V (continued) GAL-460).; 1 e 2. 3. 2. 3. 2. 3. GAL-1207 1. 2. The colony is creamy glabrous, raised, and folded on the surface. The margins are very irregular. The underside is yellowish-orange. The exposure was 16 minutes (Fig. 12). A few chlamydospores, no conidia. 16 millimeters. The surface is velvety, white, and entire at the margin. The center is heaped and has glebrous texture with radial furrows. The reVerse is creamy yellow. The expos- urO a” 8 Ethan's (Flee 16)e Few chlamydospores, no conidia. 20 millimeters. The surface is white and velvety near the center and cottony near the margin with radial folds. Pigment is not diffused. The reVerse is yellowish-tan. The expos~ ure was 10 minutes. numerous chlamydospores, no conidia. 62 miliimetcrs. Velvety wnite surface that is crateri- form with radial folds. The surface growth extends as a submerged layer of hyphae around the margin into the agar as a band about 10 mm. wide. The under- side is creamy yellow. There is no diffused pigment. The exposure was 12 minutes (Fig. 13). Hicroconidia are of two sises, one about 9u. long and elongate elevate shaped, the other about bu. and ownl. A few chlamydospores present. The exposure was 12 minutes. 16 millimeters. hS TABLE V (continued) GAL-1008 l. Colony is velvety, white with raised surface in.the center showing shallow radial grooves. No diffusible pigment. The reverse is yellowish-tan. The ex- pcsure was 10 minutes. (Fig. 1h). 2. Few ohlamydospores, no conidia. 3. 26 millimeters. GAL-0809 l. Colony has a white velvety surface with radial wrinkles and a regular margin. No diffusible pigment. The reverse side is pale yellow. The exposure was 8 minutes. (Fig. 15). 2. No spores produced. 3. 6h millimeters. GAL-1010 1. The surface has a white margin, powdery to velvety toward the center. No diffu- eible pigment. The underside is tan. The exposure was 10 minutes. 2. Chlamydospores present, no conidia. 3. 15 millimeters. fiBoth colony characteristics and colony diameter were ob- served on Sabouraud's dextrose agar with}! yeast extract. *flMioroecOpic structures were fbrned on 2.Q% corn meal agar (0.1% yeast extract, 1% dextrose, 0.1% salt). Piss 11s Figs 12s F13. 13s piss me ho Tridhoghlton gallinag, control showing a pink, velvety surface with radial fold . Diffused red pigment in nediun. Reverse red~wine. Mutant GAL-loch showing a highly folded, raised, creaa pigmented, glabrous surface. Reverse side yellowishnorange. Irradiation interval, 16 minutes. Mutant GAL-1207 showing a eraterifora and folded surface with thin.nycelial layer of subserged growth around colony edges. Re- verse side creamy yellow. Irradiation inter- V‘lg 12 3111qu a Mutant GAL-1008 showing a velvety, white, slow growing, folded, raised aycelius with- out pignant. Underside yellowish-tan. Irradiation interval, 10 minutes. Fi ure 12. Figure 11. 6 Pi*ure 1h. Figure 13e b h? reverse was pale yellow. The exposure was 8 minutes (Fig. 15). Other examples of restricted growth are mutants GAL-1008 and GAL-0805 (Figs. la and 16). The microsOOpic changes induced in g; gallinae are given in Table V. No mutants were recovered at 2 and h minute exposures. All those exposed for 8 minutes were either non spore producers or had very few spores. Mutant GAL-1207 was exposed for 12 minutes. It had few chlamydosporcs and nicroconidia of two sites. One kind was elongate elevate, about 7-9u. long and the other was oval, about h-Su. in diameter. The mutants rc- covered at 1h and 16 minutes exposure were similar to those exposed for 8 minutes. One nutant, GAL-1602, had no spore formation and the others had a few chlamy- dospores and no nicroconidia. Ho changes were noticed in the structure of hyphae as compared to the control. No macroconidia were produced but elongate clavate microconidia of mutant GAL-1207 appeared.to resenble nacroconidia. They were distinguished by being smaller than most nacroconidia and not nulticellular. The culture characteristics of I; rubrun mutants are described in Table VI. This is the only species out of the four used that had mutants that produced colonies with larger diameters than the control. The control is a moderately fast growing organism with a pink-purple, velvety surface. It has a red reverse ha Fig. 15. Mutant GAL-0809 showing a colony similar to the control but completely white. Ho diffus- ible pignmnt. Reverse side yellowish tan. Irradiation interval, 8 minutes. Fig. 16. Mutant GAL-OCOS showing a white slow growing colony with a raised center of dark brown, glabroue folds. Irradiation interval, 8 minutes. P1811" 15 e h9 TABLE VI Morphology and growthqgg Triohopnlton rubrum mutants g; the and g; g; days Colony MicroscOpic Colony Characteristicsv Structuresee Diameters Key: 1 2 3 Ogganism Control 1. Colony has a velvety pink-purple surface with a regular margin. The reverse side is red (Fig. 17). 2. Small numbers of ohlamydospores. Numerous microconidia of two kinds: oval (3u.) on conidiophores; others elevate (6-7u.). 3. R3 millimeters. RUB-1201 l. The surface is yellow with a white margin. The center is velvety becoming cottony at the margin. The reverse side is bright yellow. The growth is nearly double the rate of the control. The exposure was 12 minutes. (Fig. 19). 2. ho spore formation. 3. 71 millimeters. RUB-Ohoz l. Colony has a cottony rose colored surface with a regular margin. The reverse side has a dark red center with a reddish- orange margin. The exposure was h minutes. (Figs 21). 2. Small numbers of mieroconidia, are elevate, about 7u. Ho ohlamydospores. 3. 50 millimeters. RUB-Ohm 1. Surface velvet , withapurple margin and a pinkish-purp e center. There are tiny droplets of liquid on the surface. The underside is blackish-purple. The ex- posure was h minutes (Fig. 22). 50 TABLE VI (continued) RUB-0&0} (cont.) RUB-100k RUB-0205 RUB-0206 RUB-1607 RUB-1h08 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. 2. 3. 1. Good production of elongate clavate micro- conidia. about 7n. Ho chlamydospores. 31 millimeters. Colony has a velvety white surface. with a raised smooth center. The reverse side is yellow with a light red center. The exposure was 10 minutes (Fig. 20). The hyphae were very irregular (some coil- ed). no conidia. Several ohlanydoapores. h9 millimeters. Surface is white and velvety at the center with a granular margin. The reverse side is a light, creamy yellow. The exposure was 2 minutes. (Fig. 18). Numerous chlamydoepores and swollen hyphae with a few oval microoonidia about bu. h3 millimeters. Has a velvety pink-purple surface with a few folds. The margin is regular. The underside is dark red. The exposure was 2 Minutfi. e Many chlamydoapores. Microconidia scarce and small (lu.), oval shaped. 28 millimeters. Surface is pink and velvety. The margin is regular. The center is raised. The reverse side is red. The exposure was 16 minutes. Many chlamydospores, no conidia. 35 millimeters. Colony has a velvety white surface with a regular margin, and radial wrinkles. The underside has pink margins with a red center. The exposure was 1h minutes. 51 TABLE VI (continued) RUB-lhOB 2. Sons oval nicroconidia, larger than control (cont.) (Su.). Many chlamydospores. 3. bl millimeters. 980th colony characteristics and colony diameter were ou-rved on Sabouraud's dextrose agar with.ii yeast extract. *flhicrosoopic structures were formed on 2.Q% corn meal agar (0.1% yeast extract, Ii dextrose, 0.1% halt). _: ‘i 7‘ . 52 side but no diffusing pignent (Fig. 17). In contrast to this, mutant RUB-0205 has deve10ped a white, thin, velvety, almost granular surface with an irregular margin. The reverse side is yellow (Fig. 18). One of the mutants developed a yellow surface and had a colony diameter almost twice the sise of the control (Fig. 19). Another mutant displayed a white velvety, slightly raised surface. Its growth was dense and very compact (Fig. 20). Two mutants distinguished them- selves by their color contrasts. One had a light rose colored surface with a reverse side of orange at the margin to red in the center (Fig. 21). The other had a surface alternating shades of purple pigment with a reverse side of blackish-purple (Fig. 22), and was moderately slow in colony growth. The microscopic changes noticed in g; rubrun are given in Table VI. Bo mutants were recovered at the 8 ninutes exposure period. At the 2 minute exposure period mutants had many chlanydoapores and a small nun- ber of microconidia. In.nutant RUB-0205 swollen hyphae were observed. Two mutants RUB-OhOZ and RUB-Oh03 were recovered at the h minute exposure period that had no chlamydoapores but did display elongated clavate micro- conidia. One mutant. RUB-lOOh, was recovered at the end of 10 minutes exposure, that had very irregular hyphae Figs 17s Pigs 18s Fig. 19s Figs 20 e 53 Trichopgyton rubrua, control showing a velvety pink-purple surface. Reverse red. Mutant RUB-0205 showing a white granular to thin velvety surface. Reverse side creas- yellow. Irradiation interval, 2 minutes. Mutant RUB-1201 showingzarapidly growing yellow colony, center velvety, margin cottony. Reverse side bright yellow. Irradiation interval, 12 minutes. Mutant RUB-icon showing a velvety, compact white colony, center raised. Underside yellow with red center. Irradiation interval, 10 Mi 11113.. e 3 .\I\.\ ; ? \ ._ 9 '; ’f _, Figure 17. Figure 18. "a. \ ' a a“ ‘ . Figure 1?. Figure 20. Fig. 21. F180 22 e 5h Mutant RUB-Ohoa with a roae colony like the control except growth ie less dense and more rapid. Surface ecttony. Reverse side orange at the margin with red center. Irradiation interval, h minutes. Mutant RUB-OhO} showing a dense, velvety, noderately alcw growing colony with alter- nating shades of purple at the margin and fine droplets of liquid near its center. Reveree aide blackieb purple. Irradiation interval, h minutes. Figure 21. Figure 22. 55 (some coiled) and many ohlamyaospores; no conidia. Mutant RUB-1201, recovered at the end of 12 minutes, lacked spores. Oval microconidia and many chlamydo- spores were observed in mutant RUB-thB, formed after 1h minutes irradiation. The only mutant recovered at the 16 minute level of irradiation had no conidia but several chlamydospores. The cultural characteristics of 2; megninii mutants are presented in Table VII. Very few variations were noticed in observing several hundred germinating conidia of this organism. Ho mutants were found after 8 minutes exposure. The most distinctive mutant was slower growing and had a yellow colored reverse side (Fig. 2h) as compared to the more rapidly growing control with a red-wine colored reverse side (Fig. 23). None of tne mutants nor the control displayed a diffus- ible pigment as is usual for the control,12;ygallinae. However, if natural mutation can result in nonpigmented forms similar to the induced mutations, the criteria of diffused pigment would only be of little value for species differentiation. The microscOpic variations of g;_megninii are given in Table VII. Only one mutant was recovered after h minutes exposure. It had oval microoonidia and a few chlamydoeporce. Following 8 minutes exposure three mutants were reco"cred, two had elongate clavate .5?" L— . <_‘ a u - .6 u‘.‘ 56 microccnidia about 7n. long and one had oval microccnidia. No changes in.the hyphae and no macroeonidia were ob- served. 57 TABLE VII Morphology and growth 2g Trichophyton megninii mutants 22 the end 2; 3; days. Colony Microscopic Colony Characteristics“ Structuresve Dianeterv Key 1 2 3 Organism Control 1. Surface is velvety with radial grooves. The center is pink with a rose margin. The reverse side is red-wine. The pigment is nondiffusible (Fig. 23). 2. Numerous oval microconidia. No chlamydo- spores. 3. 60 millimeters. MEG-0801 1. Surface that is velvety to cottony with radial grooves. The center is pink with a white margin. The reverse side is ‘ yellow with a dark yellow center. The pigment is not diffused. The exposure was 8 minutes (Fig. 2a). 2. Has many elongate elevate shaped micro- conidia about 6 to 7u. long. No chlamy- dospores. 3. no millimeters. MEG-0602 l. The colony has a surface that is velvety with radial grooves. The center is very light pink, almost units. The margin is white and entire. The reverse side is yellowish to tan. Pigment not diffused. The exposure was 8 minutes. 2. A few chlamgdospores and microconidia, oval, about hu. in diameter. 3. ’49 millime tera . 58 TABLE VII (continued) MEG-0603 l. 2. 3. MEG-Chou l. 3. The colony has the same characteristics as MEG-0802, except the rate of growth is slightly faster. Elongate clavate shaped microconidia scarce, about 6 to 7u. long; some chlamydospores. 53 millimeters. Surface is velvety with radial grooves, white with a pink margin. The underside is light pink near the margin and yellow- ish-tan near the center. The pigment is nondiffusible. The exposure was h minutes. Many oval microconidia and very few chlamy- dospores. 56 millimeters. #Both colony characteristics and colony diameter were observed on Sabouraud's dextrose agar with K yeast extract. vehicrcscOpic structures were formed on 2.0% corn meal agar (0.1% yeast extract, 1% dextrose, 0.xi malt). 59 Fig. 23. Trichcphxton megninii, control is a velvety, pink-rose colony with radial grooves. lo diffusible pigment. Reverse side red-wine. Fig. 2h. hutant 330-0801 showing a slow growing colony with white margin and pink center. Surface has radial folds. Reverse side yellow with a dark-yellow center. Irradi- ation interval, 6 minutes. Figure 23. Figure 21;. 60 Physiological studies 3; mutants All four species of Trichophlton were checked for nutritional requirements on the seven Bacto-trichOphyton agars. The amount of colony growth was recorded at the end of 12 days; incubation was at 25°C. The majority of mutants did not show much variation from the nutritional requirements of the control. The nutritional requirements for the 2; mentagrcphy- ‘tgg mutants are shown in Table VIII. The control (Fig. 25) and 13 mutants tested were autotrophio for the vitamins. The mutants grow well on the organic nitrOgen source, casein, but were not able to utilise the inorganic nitrogen source, ammonium nitrate, or the amino acid, hiatidine, as readily. Examples showing this are in Fig. 26, 27, and 28. None grew better with thiamine. Hutant MEN-0h07, and especially mutant HEN-Ohlz showed the least amount of mycelial growth with ammonium nitrate or with histidine in combination for the nitrogen source. The nutritional requirement for 2; gsllinse mutants are given in Table xx. The control (Fig. 29) and 10 mutants were all autotrophic for the vitamins. All mutants displayed good to maximum growth on casein agar. The control had good growth on ammonium nitrate. However, none of the mutants did as well. Mutants GAL-0801 and GAL-loch could not utilise ammonium nitrate. — u s 303050 on + I 5320 03h? .om .mam cements +H a morons hood .nm .wma cement +~ u nuance than .om .mmm sense +n a enacts coco .mm .wam some +: a nuaoto annexe: "ham +m +N +3 +3 +3 lb“ +3 connmamoczmx + + +3 +3 +3 +3 +3 magnum: +~ +~ 3 3 3 3 3 33.3: +N +N +3 +3 +3 +3 +3 0313....sz +~ +N 3 L 3 3 3 83°on +N +N +3 +3 +3 +3 +3 mooauzmz +N +N +3 +n +n +3 +3 ancho3003mx l +N +N +~ +3 +3 +3 +3 oo3ocmmm 6 +N +N +3 +3 +3 +N +3 mo3olzmz +N +N +3 +3 +3 +3 +3 ceaaomonmmx +N +N +3 +3 +3 +3 +3 needing... +N +N +3 +3 +3 +3 +3 NOQOIZMZ +N +N +3 l4“ +3 +3 +3 Hondlzm: +N +N +3 +3 +3 +3 +3 mechanoo annuaunmm one oneneaz once sausage .eeaamza “conned“ chosen auanemmo custom: ananonem 85360324 cadaudcmm 8 acaueocm h asm4 3 down m amm< 3 newm m Lewd N amw< a haw< .uaeme dcuwnnowwolaahuouoem mam monsoon HHH> mam<9 eeuunmonmeucea couummonoaaaawm.mmdaueouumooa Anecduaaenm Fise Fig. Fig. Figs Key: 25. 26. 27. 28. 62 Trichophyton menta ro h tea, control on Bacte- Trichophytcn agsra showing autctrcphism for vitamins and good mycelial growth on ammonium nitrate and in conbination.with histidine. Mutant HEN-0813 showing a similarity to the control by being autotrophie for vitamins, but unable to utilise ammonium nitrate or histidine as readily. Has a rose pigment on the casein agars. See Fig. 8. Mutant HEB-OhOY showing similar vitamin re- quirements to the control. The colony growth is much slower, and the rate of growth was less on ammonium nitrate and in combination with histidine. See Fig. 5. Hutsnt hEN-OZOh showing nutrition require- ments similar to that of the control. A change in colony characteristics can be seen between the casein agara and the nitrate agars. See Fig. 2. From left to right Tube 1. Vitamin free casein Tube 2. Inoaitol Tube 3. Inositol and thiamin Tube h. Thiamin Tube 5. Ricctinic Acid Tube 6. Vitamin free ammonium nitrate Tube 7. Ammonium nitrate plus histidine .. " I A.“ '3 t - . 0' .; . 2“ w a k ‘ Lb . . l Figure 25.(Be1ow) . Figure 26.(Above) w j". ”I ! Figure 27.(Above) Figure 28.(Below) _ 63 +~ ed +~ em .N +~ .H +n +n +1 Oadudaedm Qua . “mafia‘ an: anacoafld and ease . h heme swam eomavcs mm .wr— .53.. on .mhm 8m: ow .ur. 3? +A + 9“ +~ +H ow +N +N +m enough: o asmd IIIMI.IIIHn oem.mm nuance» a name .eas e no «has 3 oaoacnqw .: -han 3 33%;.“ .m somhun¢u n .23 a. 0440 en nQONWwwIQ<0 Hm eenoaauqco 37.20 em Noweldam .u 393:8 6 leuoeuao choose A head on aurbho a n hereto comm“ + “I flaszuhndh .a u nuzosc than +~ .- $3.5 3m: .m a 533 5.13" e a +4 , e: +n +3 +3 +m +n +3 em +n +4 +: +m +3 on +3 +3 +m em ed +n +n +~ +: +n +3 +n +3 +4 +# +M +3 +3 +n em e: en t: +M +m +: an e no #3 + «as onsewwwm Hana m< can. i a “can ~ a. camwmwoam : hom< n hand w: m as . endeaal KM mandfl 6h Fig. 29. Trichophyton gallinae control on Baoto- Triohophyton agars showing autotrophism tor vitamins and poorer mycelial growth on ammo- nium nitrate. The addition of histidine stnnulates colony growth. Fig. 30. Mutant GAL-lh03 demonstrates poorer mycelial growth on media.containing casein, niootinic acid, ammonium nitrate, or in combination with histidine. The organism is stimulated by thiamin. Fig. 31. Hutant GAL-0805 demonstrates a slow growing organism with a slight increase in growth in presence or thiamin. Poorer colony growth occurred with ammonium nitrateaor in combina- tion with histidine. See Fig. 16. Fig. 32. Mutant GAL~0809 demonstrates similar growth requirements to the control except for a trace of growth on.the ammonium nitrate I'diune 8.. Figs 15s Key: From left to right Tube 1. Vitamin tree casein Tube 2. Inositol Tube 3. Inositol and thiamin “b. h e Th1 find-n Tube 5. licotinic Acid Tube 6. Vitamin free ammonium nitrate Tube 7. Ammonium nitrate plus histidine # Figure 30.(Above} um I . l I l m. M.” --‘---v-'-""""' ”— Figure 31.(Above) Figure 32.(Below) _ 65 Mutant GAL-0809 (Fig. 32) showed only a trace of growth on ammonium nitrate. The control and all mutants except GAL-0801 increased in growth with the addition or histi- dine to ammonium nitrate. Mutant GAL-loch would not grow on ammonium nitrate but showed some mycelial growth when histidine was added. The nutritional requirements for g; rubrum mutants are given in Table x. The control and 8 mutants were autotrOphic for vitamins and all grow well on casein vitamin tree agar. The control and all mutants had poor mycelial growth on ammonium nitrate. There were no growth increases when histidine was added to the ni- trate agar. The only variations noticed were in mutants RUB-1201 and RUB-OhO}. Hutant RUB-1201 apparently need- ed thiamin and was retarded by inositol. It also grew' better after the addition of histidinc to ammonium nitrate agar. Mutant RUB-0h03 did not show an increase in growth when either inositol or thiamin was added, but when both were in the medium the mutant had maximum growth. The organism grew somewhat better with nicotinio acid as a single vitamin source than with either inoeitol or thiwmun. The nutritional requirements for 2; magninii mutants are given in Table II. The control (Fig. 33) and mutants MEG-0302 and KEG-0803 (Fig. 3h) were autotrOphic for vitamins but both mutants were inhibited by the addition or niootinic acid in the medium. Hutant 0801 grew well 66 .aneue nomwnnonodnaucaommemm.«oceans season +H +a +3 +« +a +3 +~ +H +3 +a +a +3 +H +H +3 +a +a +m +a +H .3 +~ +H +3 +3 +a +3 edaoauedx one evened: owed eusnuwa Idanosa< anaconfl< owedaooaz h hem< o num< m Lewd i I nuSOhO on + a corona conga +~ u hexane doom he a 5.8.5 as +m “I n3§6h0.6900 +3 a. na29§uaaid§£a +3 +3 +3 +3 +3 +3 +3 +3 +3 +3 +3 +3 +3 +3 +3 +N +3 +N +3 +3 +3 +3 +3 +N +3 +3 +3 casaana oneness a Hounaoan 3 ndw< m Lewd N nem< N flamdfi noaaeonu naeeso «new a mama mo:a-mam hooa-mmm eomoumam mowo-mam zooaamam moao-mam mo:o-m:m Homaumam Hetuooo widdemho mmwnmmmnonta mm.maoaeuandaeoa «accountaaz 67 mm .waa use. a N +H 0 MN +H 0 +H +M I +m + - +H . odo< .rr eased“: cauooaz oaaeaaeam ewua< saaaoae< om esw< chateau new“ e name 5 he a. +H +N +M. nuIOho 0. a sedan nwnnomo noon nnlbhu “dim nuIOho 6000 +: a 5.6.8 .555: dunedna duaeuna 3 new< +H +n +n +m +m +m m +3 + Hounuoan oaaaonn nsw< m eaw< m when :o:o-ou= Hm canomo-umx NOQOIwflt +M HOQOIONI +3 tHOhuflOO .0. ho naeeeo Inqddu H hswd fl g h 8 HM mam . e u w . . - - 8h Shankel, D. H. 1960. Studies on mutations induced by noncidal ultraviolet light. Dissertation Abate. 20(12):hh9l. Silva, H. 1953. Nutritional studies of the dermato- phytes-factora affecting pigment production. Trans. New York Acad. Sci. 15:106-110. Silva, H. 1953. The effect of amino acids on the growth and sporulation of Trichogglten rubrun: Possible application to diagnos s and therapy. Trans. New York Acad. Sci. 15:102-105. Silva, M. and R. H. Bonhan. 1952. nutritional studies of the dermatOphytes with special reference to Tricho h ton me'nini (Blanchard 1896) and Iricho- Egzton EaIIInae (Kegnin 1861). J. Invest. ermatol. 3 " ' s 31.1". He and Re We Bonhme 1951‘s ”atritLOMI .tUdio. of the dormatOphytes with special reference to the red-pigment producing varieties of Tricho ton nentagrophztes. J. Invest. Dermatol. 2?:533-gfi7. Stabbins. nary E. and William J. Robbins. 19h9. Mineral oil and preservation of fungous cultures. Mycologia. h13632-636. Stockdale, Phyllis H. 1953. Nutritional requirements of the dermatophytes. Biol. Rev. Cambridge Philos. 28 88h-10h. Swarts and L. K. Georg. 1955. The nutrition of Trichophlton tonsurans. Mycologia. h7(h):h75~h93. Walker, Jacqueline. 1958. Effect of ultraviolet irradiation on the spores of Trichoghzton sulfureun. AeHeAe Arche Donat°1e 73:1 " e Heitsman, Irene. 196h. Variation in Micros orun seun. I A genetic study of pIeonorpHIsn. gagouraudia. 3(3):l95-203. Wilhelm, Stephen. 19in. The dual phenonenon in the dermatophytes. Mycologia. 39:716-72h. Hirth, John 0.. Paul J. O'Brien. P. Louie Schnitt. and A. Sohler. 1957. The isolation in crystalline form of sons of the pigments of Trichopgxton rubrun. J. Invest. Dermatol. 29(1):u7-5'3'7~ '4. II | - _ m