MARINE BIOLOGICAL LABORATORY. Received ^^g^s-t* 1940 Accession No. 4^504 Givenby C. V« llosby Co. Place, St, Louis, Mo. *;^*I4o book OP pamphlet is to be pemoved fpom the Ijab- oratory tuithout the permission of the Trustees. MEDICAL MYCOLOGY p ii MEDICAL MYCOLOGY FUNGOUS DISEASES OF MEN AND OTHER MAMMALS BY CARROLL WILLIAM DODGE, Ph.D. MYCOLOGIST, MISSOITRI BOTANICAL GARDEN; PEOFESSOR, HENRY SHAW SCHOOL OF BOTANY, WASHINGTON UNIVERSITY ST. LOUIS ILLUSTRATED ST. LOUIS THE C. V. MOSBY COMPANY 1935 COPTEIGHT, 1935, BY THE C. V. MOSBT COMPANY (All rights reserved) Printed in U. S. A. Press of The C. Y. Mosby Company St. Louis TO MY WIFE WHOSE CONTINUED ENCOUEAGEMENT AND HELP FOE A DECADE HAS MADE THIS BOOK POSSIBLE PEEFACE This work was undertaken at the suggestion of the late Professor Roland Thaxter while the writer was a member of the faculty of Harvard University. After a preliminary survey of the literature, a course in Medical Mycology was offered in 1924 and the book has grown out of that course. The manu- script was completed in February, 1932, but unavoidable delays in publishing have prevented its earlier appearance, though it has been used by my classes and research students for several years and has frequently been revised. It contains a summary of all the literature in this field to the end of 1933 except for a few scattered references in relatively rare periodicals which are marked by asterisks in the bibliographies at the ends of the chapters. I have also included all of the references available to me up to July 1, 1934, either at the library of our own Medical School or in the Boston Medical Library, but probably important papers in this period have been missed, as many titles had not found their place in abstracting and indexing journals ; work in my own laboratory during this period has been incorporated, even though the papers resulting from the work have not yet been published. During the correction of proofs I do not intend to include any references unless new names or combinations proposed in them affect the nomenclature of species I have already recognized. For the first time in this field, a relatively complete and accurate bibli- ography of existing literature is presented. In such a large bibliography, the abbreviation of names of journals presents a serious problem. The code of the Leagaie of Nations* was received too late to be followed. If the abbreviation be too brief, it may cause ambiguity to one who is not well acquainted with the journals of a given country or of a given subject, especially when journals have similar names. For example, Arch. Derm, might refer to Archives of Derma- tology and Syphilology, Archiv fur Dermatologie und Syphilis or Archivio Italiano di Dermatologia e Sifilologia. When Ann. is confused with Arch., as is regularly done bj'' the writer of one text in this field, we have also to consider Annales de Dermatologie et de Syphiligraphie and the Anales Dermatologicas y Sifiliograficas. More rarely we have several journals with the same name, when it is desirable to add the name of the editor, especially if the journals are contemporary. For example, the abbreviation Jour. Bot. is almost mean- ingless. It might refer to the Journal fur die Botanik, Journal de Botanique (Desvaux), Journal de Botanique (Morot), Journal of Botany (Hooker) or Journal of Botany, British and Foreign (edited successively by Seemann, Triemen, Britten and Rendle, and sometimes cited as Seemann' s Jour., etc. In cases where the usual abbreviation would indicate a journal only by the •League of Nations International Institute of Intellectual Cooperation, 1930. Interna- tional Code of Abbreviations for Titles of Periodicals. 12 pp. and Supplement 18 pp. 1932. 8 PREFACE presence or absence of a diacritical mark, I have not abbreviated owing to ease with vt^hich diacritical marks are overlooked in proofreading. The num- ber of the series has been given in Roman numerals, the volume number in boldface, and where the pagination is not continuous, the number of the article or part is given in ordinary type between colons. Some care has been taken to ascertain the dates of publications where new species or new combinations are proposed, since this date is the effective one for deciding questions of priority. If it is desirable to record the first isolation of an organism as a matter of historical interest, it should not be connected with the scientific name in bibliographical citations. From bitter experience one learns to disregard the dates given by certain authors, or to add from one to ten years to the dates given. When an author indulges in this practice with reference to his own new species and uses the date of publica- tion or even misquotes the latter in the case of species proposed by others, one cannot help but suspect intentional dishonesty. Another objectionable practice of some authors is the copying of a bibli- ography without verifying the citation or reading the article quoted. For example, many authors quote Bohin 1847 when they really mean Bohin 1853. In Robin's doctoral thesis, Les vegetaux qui croissent sur les animaux vivants, viii, 120 pp., 3 pis., Paris, 1847, he uses only the name Achorion Schoenleini, although he summarizes the work of previous authors very carefully. "When the thesis was reissued in book form under the title Histoire naturelle des vegetaux parasites qui croissent sur I'homme et sur les animaux vivants, x, 704 pp., Paris, 1853, with an Atlas of 15 plates, the text was greatly expanded and the various organisms were given scientific names. Hence all names attributed to Robin should be cited 1853 not 1847. It follows that when an author cites Robin 1847 for a species name, he is copying without having read the very rare thesis of that date. Such carelessness tends to throw doubt upon an otherwise acceptable piece of work. It is recognized that errors occur in proofreading, but it is felt that by giving complete bibliographic data as to volume and year, there is little chance that both will show the same typographic errors. The author will be grateful for corrections of errors, for information of the location of refer- ences marked with an asterisk or for references to significant work which has been overlooked or to new literature, looking toward a revision. The chapter on microscopy and staining has been kindly contributed by Dr. Morris Moore and the section on hydrogen ions (pp. 34-38) by my wife. The text-figures have been redrawn from original sources, duly acknowledged in each, by Dr. Gladys Baker, Mr. Albert Heinz, Dr. Morris Moore and the late Mr. Thomas O'Brien, most of those except the yeasts by the latter. The drawings by Dr. Morris Moore are the result of his own research. While the author assumes full responsibility for the statements of this book, he is grateful to Dr. Margaret B. Church of Urbana University, to Dr. Morris Moore of the Barnard Free Skin and Cancer Hospital of St. Louis, and to Dr. Joseph Swartz of the Medical School of Harvard University for PREFACE 9 reading the text of the chapters covering their respective fields and for their constructive criticism; to the late Professor Thaxter of Harvard University and to Dr. Charles Thorn of the Bureau of Soils for the kind interest and advice they have so generously given; to Mr. James F. Ballard and Miss Lotta McCrea of the Boston Medical Library and to Miss Ella B. Lawrence of the Library of the Washington University School of Medicine for their sympathetic help in finding incorrectly cited references ; to many of my former students for helpful suggestions ; to the Chancellor of Washington University and to the Director of the Missouri Botanical Garden for a leave of absence to complete reading in the files of rarer periodicals in Boston before begin- ning my duties in their respective institutions ; and to my wife who has helped throughout in the dreary tasks of preparing the manuscript for the printer, correcting the proofs and preparing the index. — C. W. D. St. Louis. August 1, 1935. CONTENTS CHAPTER PAGE I. General Morphology of Fungi ______________ 37 " Vegetative structures, 17; Eeproductive structures, 19; Sexual organs and sexuality, 21; Classification, 24; Bibliography, 25. 11. Physiology of Fungi With Special Reference to Reproduction _ _ _ _ 31 Water, 31; Inorganic salts, 32; Carbon, 33; Nitrogen, 33; Hydrogen ion concentration, 34; Oxygen requirements, 38; Temperature require- ments, 39; Influence of light, 39; Tropisms, 40; Radium, 40; Bibliog- raphy, 40. III. Culture Media, Their Preparation and Sterilization _______ 44 Cleaning glassware, 44 ; Sterilization, chemical methods, 45 ; Physical agents, 46; Culture media, 48; Liquid media, 49; Solid media, 50; Bibliography, 54. IV. Isolation op Microorganisms _______________ 57 Transfer, 57; Isolation from skin, 57; Isolation from feces, tongue scrapings, etc., 58; Dilution, 58; Inliibitors, 58; Microcultures, 59; Giant cultures, 60; Single cell cultures, 61; Ascospore detection, 61; Fermentation, 62; Animal and human inoculations and recovery of organisms from lesions, 64; Bibliography, 65. V. Microscopy (by Morris Moore) ______________ 66 Mounting media, 66; Spore stains, 67; Stains for fungi in skin, i Stains for fungi in other tissues, 68 ; Stains for hair and scrapings, 69 ; Stains for sputum, 70; Vital staining of fungi, 70; Fixing agents, 71; Parafiin method, 71; Nitrocellulose method, 71; Bibliography, 73. VI. Botanical Nomenclature ________________ 75 Historical sketch, 75; International Rules of Botanical Nomenclature, 76. VII. Phycomycetes ___________________ 97 Mucorales ____________________ 97 Mucoraceae, morphology _______________ 98 Mucor Micheli __________________ 110 Absidia Tieghem _________________ 111 Bhisopus Ehrenberg ________________ 115 Mortierella Coemans ________________ 118 Bibliography ___________________ 119 Vin. Ascomycetes __-______-_--------- 121 IX. Endomycetales __________________ 126 Spermophthoraceae _________________ 127 Ashbyaceae ___________________ 128 Piedraia Fonseca & Area Leao; Piedra of hair ________ 131 Pichiaceae _________--__------- 136 Guilliermondella Nadson & Krassilnikov __________ 136 10 CONTENTS 11 CHAPTER PAGE Ascoideaceae ___________________ 137 Endomycetaceae __________________ 137 HanseTmla Sydovv _________________ 142 Eanseniospora Zikes ________________ 143 Dipadascaceae ___________________ 145 Actonia Dodge _________________ 145 Coccidioideaceae __________________ 147 Coccidioides Stiles; Coccidioidal granuloma _________ 147 Bhinosporidiii/ni Minchin & Famthani ___________ 151 Histoplasma Darling ________________ 152 Paracoccidioides Almeida ______________ 155 Neoffeotrichum O. Magalhaes _____________ 156 Protomycetaceae __________________ 157 Taphrinaceae _ __________________ 159 X. Eremascaceae __________________ 161 Zymonema Beurmann & Gougerot ____________ 165 Oleina Tieghem _________________ 179 Octomyces Froilano de Mello & Gonzaga Fernandez _______ 180 Bargellinia Borzi _________________ 182 Hemispoi-a Vuillemin ________________ 182 XI. Eremascaceae Imperfectae ______________ 186 Methods of study, 186; Morphology, 190; Classification, 194; Eespira- tory infections, 204; Thrush, 204; Sprue, 205; Skin infections, 205; Keys, 206. Proteomyces Moses & Vianna _____________ 208 Geotrichum Link _________________ 215 Mycoderma Persoon ________________ 223 Candida Berkhout ________________ 229 Schizohlastospoi-ion Ciferri ______________ 234 Pseudomycoderma Will _______________ 235 Parendomyces Queyrat & Laroche ________ ____ 238 Castellania Dodge _________________ 246 Parasaccharomyces Beurmann & Gougerot _________ 265 Monilia Bonorden _________________ 270 Syringospara Quinquaud _______________ 272 Blast odendrion Ota ________________ 282 Beduellia Ciferri _________________ 289 Mycotoruloides Langeron & Talice ____________ 290 Mycocandlda Langeron & Talice ____________ 293 Pseudomonilia Geiger _______________ 295 Doubtful position _________________ 297 XII. Saccharomycetaceae ________________ 300 Schizosaccharomyces Lindner _____________ 307 Debaryomyces Kloecker _______________ 308 Saccharomycopsis Schionning _____________ 316 Sacchnromyces Meyen _______________ 317 XIII. Saccharomycetaceae Imperfectae ____________ 325 Cryptococcus Kuetzing ; Torula meningitis _________ 326 Pseudosaccharomyces Laer ______________ 841 "/(.So^ 12 CONTENTS CHAPTER PAGE Atelosaccharomyces Beurmann & Gougerot _________ 342 Torulopsis Berlese _______ _________ 347 Eutorula Will _________-_____--- 354 Asporomyces Chaborski _______________ 356 Microilastosporion Ciferri ______________ 357 Trigonopsis Schachner _______________ 357 XIV. MALASSE.ZIA BaILLON; CLINICAL DISCUSSION OF SEBORRHEA AND ACNE _ 358 Bibliography of Endomycetales _____________ 371 XV. Plectascales _________-_____--- 425 Gymnoascaceae __________________ 425 Ctenomyces Eidam ________________ 429 Gymnoascihs Eidam ________________ 430 Ateleothylax Ota So Langeron _____________ 431 XVI. Trichophytoneae (Gymnoascaceae Imferfectae) _______ 425 Clinical discussion of lesions produced on epidermis, 435; Tinea im- bricata, 436; Tinea circinata, 436; Eczema marginatum, 438; Tinea unguium, 440; Lesions of hair and hair follicle, 440; Favus, 441; Tinea tonsurans, 444; Microsporum infections, 444; Trichophyton in- fections, Sabouraudia type, 445 ; Malmstenia type, 445 ; Neoendothrix type, 447; Sycosis, 448; Kerion Celsi, 448; Granuloma of Majocchi, 448; Colony characteristics, 449; Morphology of organisms, 449; Variations and mutants, 455; Pleomorphism, 456; Phylogeny, 457; Classification, 462; Geographic Distribution, 465; Physiology, 466; Therapeusis, 471; Immunology and related phenomena, 474. Pinoyella Castellani & Chalmers ____________ 476 Epidermophyton Sabouraud ______________ 477 Endodermophyto'n Perry _______________ 489 Ectotrichophyton Castellani & Chalmers __________ 493 Megatrichophyton Neveu-Lemairc ____________ 509 Favotrichophyton Neveu-Lemaire ____________ 512 Trichophyton Malmsten _______________ 527 Microsponom Gruby ________________ 537 Achorion Eemak _________________ 551 Bibliography _ __________________ 562 XVII. Aspergillaceae ___________------- 608 Aspergillus Micheli ________________ 621 Penicillvum Link _________________ 639 Paecilomyces Bainier ________________ 642 Scopulariopsis Bainier emend. Thom ___________ 643 Phaeoscopulariopsis Ota _______________ 651 Allescheria Saccardo & Sydow _____________ 652 Onygenaceae __________--------- 653 Bibliography __________--------- 653 XVIII. Fungi Imperfecti — Hyphomycetes _____________ 665 Mucedinales __________---------- 665 XIX. Toruleae. ____________------- 669 Coniosporiww. Link ___________..__-- 673 Pullularia Berkhout ____________---- 67 3 CONTENTS 13 CHAPTER PAGE Dcmatkim Persoon; Carate or pinta ___________ 676 Madurella Brumpt; Mycetoma _____________ 680 Indiella Brumpt _________________ 685 Bibliography ___________________ 687 XX. ACTINOMYCETEAE __________________ 694 Morphology, 694; Methods of study, 702; Classification, 703. ActinoTnyces Harz _________________ 705 Bibliography ___________________ 767 XXI. Sporotrichieae __________________ 786 Aleurisrna Link _________________ 786 Trichosporium Pries ________________ 791 Acremonium Link _________________ 795 Acremoniella Saccardo __________--^__ 798 Sparotrichum Link; Sporotrichosis ___________ 798 Bibliography ___________________ 811 XXII. Chalareae ___________________ 822 Chalara Corda __________________ 822 Cephalosporieae __________________ 823 Syalopus Corda _________________ 824 Cephalosporium Corda _______________ 826 Corethropsis Corda ________________ 831 Phialophoreae __________________ 833 Phialophora Thaxter ________________ 833 Gonatobotrytideae _________________ 834 Thomiella Dodge _________________ 834 Botrytideae ___________________ 835 Acladium Link _________________ 836 Monosporium Bonorden _______________ 837 Verticilleae ___________________ 841 Spicaria Harz __________________ 843 Haplographieae __________________ 843 Catenularia Grove ________________ 843 Eaplographium Berkeley & Broome ___________ 844 Eoi-7nodendron Bonorden _______________ 845 Periconieae _______________---- 850 Gomphmaria Preuss ________________ 850 Hyalodidymeae __________________ 851 DiplosporiMm Link ________________ 853 Phaeophragmieae ___________-__---- 853 Acrothecium Preuss ________________ 854 Spondylocladi'n/m Martins ______________ 855 Phaeodictyeae ____________------- 856 Altcrndria Nees ab Esenbeek _____________ 856 Stilbaceae _________-_--------- 857 Dendrostilbella Hohnel _______________ 858 Tilachlidmm Preuss __________------ 858 Tuberculariaceae _________--------- 858 Fusarvum Link __________--_---- 859 Doubtful position _______-_--------- 860 Bibliography ______------------- 861 ILLUSTRATIONS FIG. PAGE 1. Hypnospore formation ___.__________-__-- 18 2. Oidial formation ____________________ 20 3. Coremium _______________________ 21 4. Sporangia __________________----- 99 5. Showing the development of the sporangium of Sporodinia grandis ______ 100 6. Blakeslea trispora ____________________ 102 7. Syncephalastrum cinereum __________________ 103 8. Mortierella niveovelutina ______________---- 105 9. Zygospores ______________________ 106 10. Development of the zygospore of Sporodinia grandis __________ 107 11. Pyronema confluens ____________________ 122 12. Pyronema confluens ________________---- 123 13. Spore shapes in the Endomycetales __________--_-- 127 14. Spermophthora Gossypii __________________ 128 15. Piedraia Hortai _____________________ 129 16. Eremothecvwm Gossypii ____________--_---- 130 17. Nematospora Coryli _________ __________ 131 18. Endomyces Magnusii ___________________ 138 19. Endomyces decipiens ___________________ 139 20. Endomycopsis fibiiliger ___________________ 140 21. Endomycopsis Lindneri __________-___----- 141 22. Dipodascus albidn,s ____________________ 144 23. Coccidioides immitis (Pseudococcidioides Mazzai) ___________ 148 24. Coccidioides immitis _______________----- 150 25. Ehinosporidiiom Seeberi ___________________ 152 26. Mistopla-sma capsulatum ___________-__---- 153 27. Histoplasmu pyriforme ______________----- 154 28. Protomyces macrosporus ____________-__--- 158 29. Taphrina deformans _____________------- 159 30. Eremascus fertilis ______-____--------- 162 31. Eremascus albus ______________------- 162 32. Zymonema capsulatum _____-______-_----- 163 33. Zymonema dermatitidis ___________-------- 164 34. Oleina nodosa Tiegh _______________---- 165 35. Zymonema Harteri _________-_--------- 178 36. Hemispora stellata __________---------- 183 37. Hemispoi-a coremiformis ____________------ 184 38. Blastodendrion intermedvum _________________ 189 39. Types of blastospores ________----------- 192 40. Showing Shrewsbury 's cell types in Eansemda ____________ 193 41. Proteomyces infestans ________-__-------- 209 42. Proteomyces asteraides _______-____------- 211 43. Proteomyces cutaneus _______------------ 212 44. Proteomyces Balseri __________--------- 213 45. Geotrichum ________-------------- 216 46. Geotrichum versiforme ________----------- 222 47. Candida {Geotrichoides Langeron & Talice) ____________ 230 14 ILLUSTRATIONS 15 FIG. PAGE 48. Candida Krusei _____________________ 232 49. Monilia (Candida Langeron & Talice) ______________ 271 50. Syringospora {Mycotorula Langeron & Talice) ___________ 273 51. Syringospora clilamydospores _________________ 277 52. Blast odendrion intermedium _________________ 284 53. Blastodendrion Pinoyi ___________________ 286 54. Eedaellia elegans Cifeni __________________ 289 55. Mycotoriiloides _____________________ 290 56. Mycocandida ______________________ 294 57. Schizosaccharomyces octosporus ________________ 301 58. Zygosaccharomyces Chevalieri _________________ 301 59. Torulaspora Bosei ____________________ 302 60. Saccharomyces cerevisiae __________________ 302 61. Nadsonia fulvescens ____________________ 303 62. Debaryomyces Kloecheri __________________ 303 63. Saccharomycodes Ludwigii _________________ 305 64. Debaryomyces Hudeloi __________________ 310 65. Debaryomyces Leopoldi ___________________ 313 66. Saccharomycopsis guttulatus _________________ 316 67. Saccharomyces gramdatus __________________ 318 68. Saccharomyces anginae __________________ 319 69. Saccharomyces annulatus __________________ 323 70. Malassesia ovalis ______ ______________ 368 71. Amuuroascus verrucosus __________________ 426 72. Gymnoascus setostis (Eidamella spinosa) _____________ 427 73. Gymnoascus gypseus and Ctenomyces serratus _____----___ 427 74. Ctenomyces serratus ___________________ 428 75. Section through hair from a case of favus, caused by Acharion Schoenleini _ _ _ 442 76. Section through hair from a case of tinea tonsurans microsporica, caused by a species of Microsporum ___________________ 444 77. Section through hair from case of tinea tonsurans __________ 447 78. Mycelium ______________________ 449 79. Nodular organs _____________________ 450 80. Arthrospores _____________________ 451 81. Pedicellate chlamydospores _________________ 452 82. Intercalary chlamydospores _________________ 452 83. Showing closterospores and their transition to chlamydospores ______ 453 84. Aleurospores ______________________ 454 85. Endodermophyton Boquettei _________________ 492 86. Favotrichophyton camerounensis ________________ 516 87. Microsponmi ferrugineiim _________________ 546 88. Aphanoascus cinnaiarimis _________________ 609 89. Monasous ruber _____________________ 610 90. Diagrammatic radial sections of colonies _____________ 611 91. Diagrammatic radial section of a colony of Paecilomyces Varioti ______ 612 92. Thielavia basicola ____________________ 613 93. Types of phialides ____________________ 614 94. Conidial stages _____________________ 615 95. Gliocladiitm roseum ___________________ 616 96. Penicillium Brefeldianum __________________ 616 97. Aspergillus nidulans ___________________ 617 98. Penicillium Wortmanni ___________________ 618 16 ILLUSTRATIONS PIG. PAGE 99. The ascospores of AspergilMs ________________ 620 100. Aspergillus Amstelodami __________________ 631 101. Aspergillus Jeanselmei __________________ 638 102. Penicillium Bertai ___________________ 640 103. Paecilomyces Varioti ___________________ 643 104. Scopulariopsis brevicaulis (Sacci.) Bainier ____________ 644 105. Alternaria sp. _____________________ 666 106. Hormiscium stilbosporum Corda and Eormiscium pinophilum Nees _____ 672 107. Pullularia Jeanselmei ___________________ 675 108. Demati/wm articulatum Pers. _________________ 676 109. DematiAim, hispidulum __________________ 677 110. Madurella mycetomi ___________________ 680 111. Indiella Mansoni ____________________ 686 112. Actinomyces II isolated from soil _______________ 696 113. Actinomyces XVIII ___________________ 698 114. Actinomyces alius (A. griseus Krainsky?) ____________ 699 115. Actinomyces XVIII ___________________ 700 116. Actinomyces OMreaus ___________________ 701 117. Actinomyces Lavendulae __________________ 702 118. Actinomyces scahies (Thaxter) Giissow _____________704 119. Acremoniwm alternatum Link ________________ 796 120. Acremonvum Potronii Vuillemin _______________ 797 121. Bhinocladium coprogenum Saccardo & Marclial ___________ 800 122. Sporotrichum, Schenki var. Beurmanni _____________ 807 123. Sporotrichum Lesnei Vuillemin ________________ 810 124. Thielaviopsis paradoxa __________________ 823 125. Chalara fusidioides Corda _________________ 823 126. Eyalopus muscorum Corda _________________ 825 127. Cephalosporium. Acremonvum Corda ______________ 827 128. Cwethropsis paradoxa Corda ________________ 832 129. Phialophora verrucosa Thaxter in Medlar ____________ 833 130. AcladiAim conspersum Link _________________ 836 131. Acladvum Castellanii ___________________ 836 132. Monosporium spinosum Bonorden _______________ 838 133. Monospwvum apiosperrmim Saccardo ______________ 839 134. Verticillvum agaricinum, (Link) Corda _____________ 842 135. Eaplographium chlorocephalum (Fresenius) Grove __________ 844 136. Hormodendron olivaceum Corda _______________ 846 137. Hormodendron Fontoynonti _________________ 847 138. Gomphinaria Pedrosoi __________________ 851 139. Arthrohotrys superia Corda _________________ 852 140. Trichothecium roseum Link _________________ 852 141. Diplosporinim vaginae __________________ 854 142. Acrothedum nigrwm ______---_-_---- _- 855 MEDICAL MYCOLOGY CHAPTER I GENERAL MORPHOLOGY OF FUNGI The Fungi form a large heterogeneous group of plants, including all those lacking chlorophyll, which are not closely nor obviously related to other groups. In a few primitive families, the vegetative body is naked and amoeboid. In the rest of the fungi, it is surrounded by cell walls and usually appears as septate filaments, called hyphae. The vegetative hyphae are collectively known as mycelium. Under certain conditions of nutrition, as in solutions of very high or of very low osmotic pressure, the hypha grows by sprouting, when a small protuberance enlarges, rounds off and is abjointed (cut off by a septum) from the mother cell. The daughter cell, called sprout cell or blastospore, continues to increase in size and eventually separates from the original group of cells. In certain groups, as in the yeasts, no other type of vegetative body is known. When conditions for growth are unfavorable, the protoplasm contracts, rounds up and secretes a special thick wall, forming resting cells, called hypnospores or chlamydospores. (Fig. 1.) With the re- turn of favorable environmental conditions, these hypnospores again develop normal vegetative mycelium. In a few groups of fungi, the hyphal wall gives the cellulose reaction, in most others that of chitin. In fructifications and resting cells, the hyphal wall first appears as a thin, hyaline membrane which becomes thicker and may be further differentiated by secretions and deposits of minerals or resins, or colored by pigments. A relation between the fundaments of the wall and mitosis has been demonstrated in only a few cases, as in ascospore formation. In general, the wall is gradually differentiated from the cytoplasm without nuclear aid. The septum often forms by furrowing (annular thickening of the walls, like an iris diaphragm in a camera) . For the maintenance of inter- cellular communication, the septa are frequently pierced by a few openings through which pass protoplasmic threads. During rapid growth, there may be a delay in the formation of septa, later compensated by simultaneous or successive development of septa. In the Phycomycetes, the septa are wholly suppressed ; the whole mycelium is then a single, branched, multinuclear mass of filaments, becoming septate during the formation of reproductive organs, or during conditions of poor nutrition or of senescence. The individual hyphae generally are intertwined in feltlike masses. Such a group of hyphae, called the mycelium, usually absorbs food at any point 17 18 MEDICAL MYCOLOGY over its whole surface. Small mycelial branches, which serve to attach the mycelium to the substrate and to absorb the nutrients, have become special- ized in form and function in several groups. When the absorbing organs are rootlike, filamentous, finely branched and tapering, they are called rhizoids (frequent in the Chytridiales). When they are intracellular, clavate, bluntly lobed, coarsely branched or coralloid organs of parasites which do not imme- diately injure or kill the host cell, they are referred to as haustoria (frequent Fig-. 1. — Hypnospore formation in 1 ; 2, Mucor racemosus; S, M. dimorphosporus ; i, Rhizopnis arrhisus. (After Lendner 1908.) in plant parasites). When the function seems to be purely one of attachment and the mycelial branches are flattened and disciform, bluntly lobed or branched, or even coarsely filamentous, they are called holdfasts (e.g., Bhizopus of the Mucorales). Appressoria are holdfasts which are superficial or non- penetrating, although they may be intercellular (frequent in the sooty molds). In many cases, the hyphae grow together in groups, intertwine, adhere, and form a thick tissue, called plectenchyma. If the single hyphal elements are GENERAL MORPHOLOGY OF FUNGI 19 still recognizable as such, they are referred to as prosench3rma ; if the hyphae have lost their individuality so that they lie adjacent, with the cells (in sec- tions of the tissue) appearing isodiametric and continuous, they are called pseudoparenchyma since they are formed by cell division in a single plane while the parenchyma of higher plants develops from cell division in three planes. Sclerotia are small hard masses of plectenchyma with a firmer pseudo- parenchjrmatic rind and a looser prosenchymatic core. These structures serve to carry the organism over unfavorable environmental conditions and, with the return of normal conditions, germinate to the usual mycelium or to a fructification. Sclerotia are formed on drying out of the culture in many species of Aspergillus. Bulbils are small sclerotia formed of a few layers of cells and are often present in large numbers. The grains in the lesions of mycetomas (Madura foot, etc.) belong in this general category. Reproductive Structures. — In most fungi, at a definite age and imder favorable conditions of nutrition, reproductive structures develop on the mycelium. The products of the reproductive processes are chiefly spores. Spores may be defined as characteristically formed cells or groups of cells which separate from the mother plant and may grow independently to new individuals. They serve either for propagation (multiplication and dispersal) or for resisting unfavorable conditions of the environment (prolonged desic- cation, overwintering, etc.). Spores specialized for the latter function are often called hypnospores (Fig. 1). In the simplest case, hyphal cells separate from the parent hyphae and develop into new hyphae. These individual cells are called arthrospores or thallospores and are homologous with the cells of a chain of blastospores, though the latter arise by sprouting rather than by the breaking apart of the cells of a hypha (Fig. 2). From arthrospores there is a gradual transition to more typical spores with characteristic color, form, and sculpturing of the wall. In many cases they are abjointed directly from the cells of an ordinaiy hypha ; in other cases they arise on specialized sporophores. AVhere these sporophores form the spores within specialized sporogenous cells, sporangia., they are called spor- angiophores and the spores, if they are enclosed and nonmotile, sporangia- spores (e.g., in the Mucorales). Sporophores which abjoint their spores ex- ogenously at their tips are referred to as conidiophores and their spores conidia (e.g., in the Fungi Imperfecti). A chlamydo spore is any thick-walled asexual spore without further regard to its morphologic significance. In the higher fungi, the mycelium surrounding a group of conidiophores (or sexual organs) is known as a fructification. When these groups are in fascicles, they are called coremia (Fig. 3) ; if they form widespread cushions, they are called sporodochia (Tuberculariaceae) or acervuli. The tissue from which they arise is then known as their stroma. When the conidiophores develop in cavities in the stroma, the finictifications are called pycnia, and the conidia are often called pycnospores or stylospores. These different spore 20 MEDICAL MYCOLOGY forms, blastospores, arthrospores, chlamydospores, conidia, etc., usually de- velop on the liaplont (thallus of the haploid stage of the life cycle). A single species which successively produces several spore forms is called polymorphic. The spores of a second type are connected with sexuality and develop after fertilization or meiosis in the spore mother cell. These changes are connected respectively with the beginning or the end of the diploid phase. The spore forms following fertilization are recognizable morphologically since they are encysted zyg-otes (the products of a sexual act) ; biologically they usually develop as hypnospores or resting spores (e.g., the zygospores of the Mucoraeeae) . The spore forms which follow meiosis are morphologically recognizable because they form tetracytes (as daughter cells of gonotoconts, the organs in Fig. 2. — Oidial formation in i; S, Mucor racemosus ; S, Mucor Prainii. de Botanique.) (After Chodat, Principes which meiosis occurs). Apparently, since they are products of meiosis, they have become constant in number, usually 8 (in the Ascomycetes) or 4 (in the Basidiomycetes) ; biologically they are also hypnospores in the higher fungi. When the sporogenous cells which serve as gonotoconts form their spores internally through free cell formation, they are called asci and their spores ascospores (whence the term Ascomycetes). When the spores are cut off externally from the gonotoconts, they are called basidiospores and the sporogenous cells basidia (whence the term Basidiomycetes, the saprophytic mushrooms, puffballs, etc., and the parasitic rusts and smuts). As the conidiophores of the haplont, these gonotoconts are usually grouped in a superficial layer of tissue, known as a h3nnenium. This tissue generally OENERAL MORPHOLOGY OF FUNGI 21 develops on a fructification whose structure is highly differentiated. In the higher groups, the fructifications containing the gonotoconts become visible to the naked eye and are called the perfect forms. The other spore forms are referred to as imperfect or secondary spore forms. Sexual Organs and Sexuality. — The sexual function in its simplest stage involves but two processes : (a) fertilization, a fusion of two nuclei, recurring periodically in the course of development, initiating specific stimuli for fur- ther development; and (b) meiosis, a return to the single chromosome num- ber. This rotation (haplont -^ fertilization — > diplont -^ meiosis — >) comprises the changes of nuclear condition in most organisms. A few fungi seem to exist without such a change; they seem to live an unlimited number of "gen- erations" without reconstruction of their nuclei and to propagate themselves Fig-. 3. — Coremium. Hemispora coremiformis. (After M. Moore 1935.) by imperfect stages only. Such fungi with incomplete, or incompletely known life cycles are called Fungi Imperfecti. The fungi with complete life cycles are divided into homothallic (her- maphroditic) and heterothallic forms. In contrast to the higher plants and animals, the division of sexes is here limited to the haplont, i.e., the mycelium. The homothallic form is often indicated by the symbol ±, the heterothallic forms as + or -. The + and - mycelia of the latter group may be distinguished from each other only physiologically, as indicated by their sexual tendencies, or morphologically as well (e.g., in growth form, sexual dimorphism). Fertilization in fungi, as in protozoa, may occur in many ways. The simplest normal type of fertilization, when two spatially separated, not closely related sexual cells fuse to form a new entity, is called amphimixis. If the sexual cells arise as daughter cells of a differentiated mother cell and are themselves characteristically formed, they are called gametes and 22 • MEDICAL MYCOLOGY the mother cells gametangia. The copulation of two gametes of this type is called merogamy. If the gametes appear wholly of the same size and shape, the copulation is isogamous; if the gametes are differentiated, their copulation is heterogamous (anisogamous). When the female gamete is a large and non- motile egg and the male gamete is a small and motile sperm, their copulation is oogamous, a condition confined to primitive fungi, although present in most higher animals and primitive plant phyla. The more advanced fungi show various stages in the degeneration of merogamy. At a comparatively low stage, the differentiation of gametes is suppressed and the contents of the gametangia remain polyenergid. Thus the original copulation of gametes disappears, being replaced by many sec- ondary processes which compensate for the loss of the original merogamy. All these secondary processes are classified as deuterogamy. The higher algae and flowering plants have also developed these processes, while the primitive merogamy has persisted through the highest vertebrates. In deuterogamy, the gametangia assume the function of their daughter cells, the gametes, and their coenocytic content fuses without further differ- entiation. The sexual act occurs between two sexual organs instead of between two sexual cells and sexual attraction passes from the latter to the former. This type of copulation is called gametangial. It assumes close contact be- tween two gametangia and has a biologically obvious consequence that one gametangium can fertilize only one other which must be located nearby ; it has the advantage that the fusion of gametes is no longer fortuitous, since the gametangia provide that their nuclei can come into contact and fuse. Thus gametangial copulation is an efficient type, since most of the nuclei of both gametangia succeed in their activity with an increase in the number of zygote nuclei. The fate of the gametangium hangs upon the occurrence or nonoc- currence of a single sexual act, from which results one single, very strong, coenocytic zygote instead of many smaller unicellular zygotes. Also a single mature gametangium no longer depends on a definite medium (e.g., water) for the copulation of its gametes, a condition which has facilitated the transi- tion from water to land habitats and to parasitism. Whether gametangia are borne on specialized branches or whether hyphal branches as a whole complete the act of fertilization, they are called copulation branches. When sexual dimorphism is present, the male is called an antheridium and the female an ascogonium. Among the higher Ascomycetes, the fundaments of the antheridium are gradually reduced, whereby cross-fertilization generally ceases and is replaced by self-fertilization, i.e., a new group of deuterogamous processes, between daughter cells of the same mother cell or between nuclei of the same cell, which are included in the term automixis. Automixis is represented in fungi by two forms : parthenogamy and autogamy. Parthenogamy is fertilization which occurs between two female cells, i.e., in fungi usually between two cells of the ascogonium. In some groups this parthenogamic fusion of two specialized cells is replaced by the GENERAL MORPHOLOGY OF FUNGI 23 pairing of nuclei within a single multinucleate cell of the ascogonium. This automictic fertilization within a cell is called autogamy. From these forms in which the sexual organs (or in any case the female organ) are apparently typical in form but no longer functional and serving only as a site of automictic processes, there is a series of intermediate stages to the other extreme where the sexual organs disappear entirely, the sexual processes occurring in the mycelium between any two sexually differentiated cells. The latter process is called pseudomixis. Since the copulating cells are not morphologically distinguishable from other vegetative cells and since only the release of specific developmental stimuli marks this anastomosis of two vegetative cells as a sexual process, pseudogamy is often difficult to dis- tinguish from the usual pseudosexual anastomoses which are brought about by food relations. Its true character is recognizable cytologically only in the pairing of nuclei. If pseudogamy occurs between two sprout cells, tliej^ are sometimes wrongly called gametes. In order to avoid misunderstanding, this term should be reserved for merogamous gametes. The ambiguous term pedogfamy, often employed in other senses, should be used to indicate pseu- dogamy between adult and young cells. The special case of pseudogamy between mother and daughter cell is called adelphogamy. Apomixis, the entire loss of fertilization, represents the last step in this series of reduction of sexuality where growth from reproductive cells occurs vegetatively without cell or nuclear fusion, or any external stimulus of de- velopment. If the new individual (in the absence of fertilization) arises from haploid sexual cells, the process is called parthenog'enesis ; if they arise (in the absence of meiosis) from diploid sexual cells, the process is called apogamy. In the study of fungi, there is the further difficulty that the original processes of fertilization are replaced by all sorts of substitutes. Among the lower fungi, there is simple fertilization when a fusion of the cytoplasm of two sexual cells (plasmogamy) is immediately followed by a fusion of both haploid nuclei into a diploid zygote nucleus, a syncaryon (caryog-amy). In most fungi, however, caryogamy is delayed and is only completed just before meiosis. Thus the sexual haploid nuclei, while remaining spatially separate, unite only to form a dicaryon, where the paired nuclei divide synchronously (conjugately) while retaining the same ability to activate somatic develop- ment as after complete caryog*amy. This phenomenon is analogous to that in Cyclops, in which the parent chromosomes remain distinct up to the time of egg formation (synapsis) although they are surrounded by the same nuclear membrane, whereas in the fungi they remain Avithin their original nuclear membranes. In this retardation of caryogamy, the binucleate "zygote" continues its growth without completing nuclear fusion, developing a new mycelium whose cells, morphologically virtually diploid, contain two sexually differentiated haploid nuclei. This new phase, intruded between plasmogamy and caryog- amy, is called the binucleate phase. In the Ascomycetes, this phase is mostly limited to the ascogenous hyphae and the hymenium of the fructification, but in the Basidiomycetes, it is usually the most conspicuous phase of the life cycle. 24 MEDICAL MYCOLOGY In spite of this removal and retardation, caryogamy always occurs in definite organs. The organs in which the fertilization processes are completed and the dicaryon ends are called zeugites. As caryogamy is delayed until the necessity for meiosis appears, the zeugites also frequently function as gonotoconts. These two processes, the transformation of the cells which com- plete the sexual act and the division of the sexual act itself into plasmogamy and caryogamy, separated in time and space, are both fundamental and very useful in the study of phylogeny and classification. In the present state of our knowledge, the various groups of fungi are apparently polyphyletic and unrelated. Some members seem more closely related to other groups of plants than to other groups of fungi; e.g., the Chlamydobacteriales and perhaps the Thiobacteriales seem more closely re- lated to the Myxophyceae than they are to other groups of bacteria or of fungi. The following list of classes of fungi illustrates the main subdivisions while not necessarily assuming that all the fungi of these larger groups are monophyletic. In time some of these groups will probably be further divided, e.g., the Phycomycetes seem quite heterogeneous. For our purposes, how- ever, only one order of these, the Mucorales, has been shown to have members attacking man and other mammals, and needs consideration here. Schizomycetes : bacteria in the larger sense. Myxomycetes: slime molds, having many resemblances to Protozoa in some stages of their life cycle. Phycomycetes: coenocytic fungi of varied origin and relationship. Ascomycetes : a large poljonorphic group with a common method of spore formation in asci. Basidiomycetes : a very large group with spores borne on a specialized gonotocont, the basidium, furnishing most of the conspicuous fungi. Fungi Imperfecti : a heterogeneous group whose life cycles are unknown in full, or which have degenerated until sexuality has been lost, provisionally grouped together until they are better known. Schizomycetes. — Bacteria. While this group is by far the most important from the standpoint of the physician and the surgeon, it is also the best known to the medical profession and will not be given further consideration here. The bacteria seem almost wholly unrelated to the other groups of fungi, and some of the higher forms are suggestive of Myxophyceae which have lost their chlorophyll. Myxomycetes. — The slime molds are a very interesting group with many stages suggestive of similar conditions found in the Protozoa, especially the Rhizopoda, while other stages are analogous to those in other groups of fungi. The stages commonly observed being plantlike, they have been studied mostly by botanists and only in the last decade has any long continued or thorough attempt been made to follow the life cycle in detail or to study the cytology. At present the group is known to cause plant diseases but has not been suggested in relation to animal disease. Any related organisms attacking man have undoubtedly been studied and classified as Protozoa. GENERAL MORPHOLOGY OP FUNGI 25 Phycomycetes. — This group in its more primitive members seems distantly related to the Flagellata of the Protozoa. Most of the members have a vegeta- tive body of mycelium and in all but one order reproduce by a flagellated body. Most of the group are saprophytes or parasites on plants except a few facultative parasites of fish or invertebrates. The only group at present known to produce human parasites is the Mucorales which will be treated later in the appropriate chapter. Ascomycetes. — The more primitive members show distant relationships to the higher Phycomycetes on the one hand and to the Ehodophyceae (Red Algae) on the other. After a half century of bitter controversy on this ques- tion, there is still little agreement. Here the vegetative body is a typically uninucleate, septate mycelium and sexual reproduction occurs by means of cells produced within an ascus, following meiosis in all forms where this has been carefully studied. Of the twenty orders into which this group is usually divided, members of only two, the most aberrant and primitive (or degener- ate), have been shown to cause human disease. The bulk of the group are saprophytes or parasites of the leaves and bark of plants, while the most highly specialized (Laboulbeniales) are at present known only as parasites of living insects. Basidiomycetes. — While the life cycle shows a certain parallelism to that of the higher Phycomycetes and the Ascomycetes, it is difficult to assign to these any very close relationship with other groups. The vegetative body again consists of uninucleate or binucleate mycelium, forming a conspicuous fructification on which the reproductive structures are borne. Reproduction occurs by means of basidiospores. So far as is known the group is saprophytic on decaying vegetable matter in the conspicuous species, such as the mush- rooms, punks, puffballs, etc., or parasitic, usually on leaves, in the Uredinales (rusts) and Ustilaginales (smuts). Fungi Imperfecti. — These are a large group, artificially classified together while we await more knowledge of their life history. The greater part, whose life history has been discovered, has been found to be Ascomycetes, but it is never safe to predict that several members of a given genus of Fungi Im- perfecti will necessarily belong to the same genus of Ascomycetes or even will be Ascomycetes; e.g., when Oedocephalum was carefully studied, one species was found to be a Phycomyeete, another an Ascomycete, and a third a Basidiomycete. Various subdivisions have been proposed, but none has proved altogether satisfactory. Further considerations may well be delayed to a subsequent chapter (p. 665). In the following chapters only those orders will be discussed in which mammalian pathogens have been found, and the reader is referred to Gau- mann and Dodge (1928) for information on other groups. BIBLIOGRAPHY The following bibliography includes references to early work in which it is difficult or impossible to be sure of the group of fungi involved, also gen- 26 MEDICAL MYCOLOGY eral review articles, textbooks, etc., covering most of the groups of human pathogenic fungi. These have not been repeated in the bibliographies of the individual groups, even though they often contain as much information about a single group as do the references given for that group. Asterisks (*) de- note that I have not read the reference in question, but it is quoted from a fairly reliable author or abstracting journal. I have not tried to quote any references from certain writers whose references are notoriously incorrect, often showing errors in over half the titles of a short bibliography. Agostini, Angela. 1934. Miceti della Somalia, Atti 1st. Bot. B. Univ. Pavia TV, 4: 191-201, 4 fig. Bail. 1867. Ueber kraukheiteneizeugende Pilze, Wiener Med. Woch. 17: col. 977-979; 993- 996. Baillon, H. 1889. Traite de botanique medicale cryptogainique, Paris, 376 pp. [see pp. 246- 249]. Baumgarten, Paul Clemens. 1884. Ueber patliogene pflanzliclie Mikroorganismen. I, Deutsch. Med. Zeitschr. 1884i: 149, 150; 163-165; 175-178; II. Die pathogenen Schizomyceten, Ibid. 18842: 169-173; 181-185; 193-197; 205-208. — . 1890. Lehrbuch der pathologischen Mykologie. Vorlesungen fiir Arzte und Studierende. Harald Brulm; Braunschweig, IX, 973 pp., 1 pi, 101 figs. Beurmann, Lucien de. 1912. Les nouvelles mycoses, exoascoses (ex-blastomy coses) oidio- mycoses, sporotrichoses, botrytimycose, oosporose, hemisporose, Paris, Masson & Cie. 165 pp. Beurmann, L. de & H. Gougorot. 1909. Les Exascoses, endomycoses, et parendomycoses (Mu- guet) saccharomycoses (mycose de Busse-Buschke) et parasaccliaromycoses. Zymone- matoses (Mycose de Gilchrist) Envision et demembrement de I'ancien groupe des Blastomycoses, Bull. Mem. Soc. Med. Hop. Paris III, 28: 222-246; 250-263. — . 1912. L'etat actuel de la question des mycoses, Biol. Med. 10: 133-166; 187-222, 18 figs. Bianchini, Giuseppe & G. Paolo Manfrini. 1924. La micologia del cadavere umano nei rispetti della cronologia della morte e delle trasformazioni tanatologiche, Atti B. Accad. Fisiocrit. Siena IX, 16: 335-351, S figs. Blanchard, Raphael. 1896. Parasites vegetaux a 1 'exclusion des bacteries, in Bouchard, Charles. Traite de pathologic generale 2: 811-926. — . 1899. Sur une affection causee par les spores d'un champignon parasite du roseau ou canne de Provence (Arundo donax L.), Arch, de Parasitol. 1: 503-512. Bodin, E. 1902. Les champignons parasites de I'homme, Paris, Masson et Cie, 208 pp. Brocq Eousseu. 1921. Les recherches mycologiques en medecine veterinaire, Bull. Soc. Myc. France 37: 99-103. Brooke, Gilbert E. 1908. Aids to tropical medicine. New York, William E. Wood, 163 pp. Brumpt, E. 1927. Precis de parasitologic. 4 ed. Paris, Masson & Cie, viii, 1452 pp., 5 pis. Castellani, Aldo. 1912. Note on the importance of Hyphomycetes and other fungi in tropical pathology, Brit. Med. Jour. 2: 1208-1212. — . 1917. Notes on tropical diseases met with in the Balcanic and Adriatic zones, Jov/r. Trop. Med. Hyg. 20: 157-164; 170-174; 181-186; 198-202; 209-214; 219-223. — . 1918. Alcune osservazioni sulla malaria e su altre malattie tropicali della zona Balcanico-Adriatico, Ann. Med. Nav. 24i: 169-213. — . 1920. Higher fungi and human pathology, Lancet, 198: 847-852, 895-901, 943-946 [also reprinted in Jo^tr. Trop. Med. Hyg. 23: 101-110, 117-125, 132-138]. — . 1923. Medical mycology, Brit. Med. Jour. 2: 937-1041 [also reprinted Jour. Trop. Med. Hyg. 27: 49-53, 61-65, 1924]. GENERAL MORPHOLOGY OF FUNGI 27 — . 1924. Tropical Dermatology, Int. Conf. Health Problems of Trop. Amer. [United Fruit Co.] 485-499. — . 1925. Observations on some diseases of Central America, Jour. Trop. Med. Hyg. 28: 1-14, 31 figs. — . 1926. Chronic bronchites with hemorrhagic sputum of nontubercular origin, N. Orleans Med. Surg. Jour. 79: 20-42. — . 1927. Notes on certain bronchomycoses which may simulate pulmonary tuberculosis, Am. Bev. T^tiberculosis 16: 541-574. — . 1927-1928. Fungi and fungous diseases. Arch. Derm. Syphilol. 16: 383-425; 571-604; 714-740, 1927; 17: 61-97; 194-220; 354-379; 1928 [reprinted Chicago, Amer. Med. Assn. 203 pp., 44 figs. 1928]. Castellani, Aldo & Albert J. Chalmers. 1910-1919. Manual of tropical medicine, New York, WilUam Wood & Co., XXV + 1243 pp.; ed. 2, XXXII -f 1747 pp., 16 pis., 909 figs., 1913; ed. 3, X -F 2436 pp., 16 pis., 909 figs., 1919. Castellani, Aldo, MacKenzie Douglas, & J. Thomson. 1923. Further observations on tonsil- lomycoses, Jou,r. Trop. Med. Hyg. 26: 19-24. Castellani, Aldo, & Henri Tejera. 1923. Notes on the etiology of ''cute," Jour. Trop. Med. Hyg. 26: 183, 184. Cavara, V. 1928. Le micosi oculari, Siena, 494 pp. Chiurco, Giorgio Alberto. 1922. Simbiosi tra ifomieeti patogeni e schizomiceti, Biv. Biol. 4: 684-688. * — . 1922. Simbiosi tra ifomieeti patogeni e schizomiceti, in rapporto al loro potere patogeno, 1st. Bot. B. Univ. Siena, 75 pp. — . 1923. Micosi chirurgiche sperimentali, Pathologica. 15: 487, 488. — . 1925. Esperience nelle cavita articolari tessuto muscolaree cutaneo dei ratti e delle cavie in rapporto alia simbiosi ifoschizomicetica, II, Atti B. Accad. Fisiocrit. Siena IX, 16: 171-207, Pis. 1, 2. — . 1927. Experimentell hervorgerufene mykotische Arthritiden und Myositiden, Derm. Woch. 84: 228-232. Coupin, Henri. 1909. Atlas des champignons pathogenes de I'homme et des animaux. 58 planches renfermant 1000 dessins reproduits d'apres les travaux originaux, Paris, Octave Doin et Fils. Dubreuilh, William. 1891. Des moisissures parasitaires de I'homrae et des animaux superieurs, Arch. Med. Exp. Anat. Path. 3: 428-447, 566-592. Durante, G. 1927. Les mycoses meconnues. Bull. Mem. Soc. Med. Hop. Paris [51] : 1513- 1516. Fairman, Ch. E. 1920. The ascomycetous fungi of human excreta, Lyndonville, N. Y., 11 pp., S figs, 1 table. Fleisher, M. S., So M. Wachowiak. 1924. The relation of fungi imperfecti to diarrhoeal con- ditions. Am. Jour. Med. Sci. 168: 371-380. Fonseca filho, Olympio Oliveira Eibeiro da. 1928. Algumas consideragoes de ordem geral sobre as dermatomycoses, Sciencia Med. 6: 565-569. — . 1929. As mycoses na clinica dermatologica e syphilographica da faculdade de Medicina do Eio de Janeiro, Bev. Med. Cirurg. Brasil. 37: 126-128. — . 1929. Notas sobre os exames de laboratorio na pesquiza e diagnostico das mycoses, Bev. Med. Cirurg. Brasil. 37: 169-179. Frescoln, L. D. 1916. Mycology as a part of practical dermatology, Internat. Clin. 2: 170- 172, 1 col. pi. Frey, Ludwig. 1888. II. Fungus articulat. talo-calcaneae. Injection con Jodoformather Heilung und voUkommene Wiederherstellung der Functionsfahigkeit, Wiener Med. Presse. 29: col. 1325, 1326. Furbringer, Paul. 1876. Beobachtungen iiber Lungenmykose beim Menschen, Arch. Path. Anat. Physiol. [Virchow] 66: 330-365, PI. 15. 28 MEDICAL MYCOLOGY Giaumann, Ernst A., & Carroll William Dodge. 1928. Comparative morphology of fungi, New York, McGraw Hill Book Co., 701 pp., 398 figs. Gautier, L. 1907. Eecherches biologiques sur quelques champignons parasites de I'homme el des animaux (diastases & toxines), Brest, P. Gadreau, 149 pp. Gedoelst, L. 1902. Les champignons parasites de I'homme et des animaux domestiques, Bruxelles, 199 pp., 1£4 figs. Grazia, Francesco de. 1903. I microorganismi dei polmoni dei Cardiaci, Bif. Med. 19: 709- 713. Greco, Nicolas V. 1916. Origine des tumeurs (etiologie du cancre) et observations de mycoses (blastomycoses, etc.) Argentines, La Semana Medica: Buenos Ayres. VI + 853 pp., 29 pis., 492 figs. Greenwood, Arthur M. & Joseph H. Swartz. 1927. [Fungous diseases of the skin] a review of the literature [of the last two years]. Arch. Derm. Syphilol. 15: 404-414. Greig, E. D. W. & G. C. Maitra. 1918. Observations on the occurrence of hyphomycetic elements m ulcers of the skin in 19 cases, Indian Jour. Med. Res. 5: 481-490, 1 fig. Grohe. 1870. [Experimente . , . iiber die Injection von Pilzsporen], Berlin. Klin. Woch. 7: 8, 9. Griitz, O. 1927. Die Hyphenpilze oder Eumyceten, Hand. Path. Mikroorg. [Kolle So Wasser- mann, ed. 3] 5: 133-320, 10 pis., 66 figs. Gueguen, Fernand. 1904. Les champignons parasites de I'homme et des animaux. Generalites, classification, biologie, teclinique. 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Precis de pathologie exotique (maladies des pays chauds et des pays froids), ed. 4, Paris, Octave Doin, v. 1, 942 pp.; v. 2, 1232 pp. *L6wenberg. 1880. Des champignons parasites de I'oreille humaine, Paris. GENERAL MORPHOLOGY OF FUNGI 29 Magalhaes, Octavio de So Aroeira Neves. 1926. Ensaios de mycologia. Contribuigao para o estudo dos cogumelos em Bello Horizonte, Janeiro 1912-Juiiho 1920, Mem. Inst. Oswaldo Cruz 19: 245-283, Pis. 56-91 [translated: Essays on mycology, contribution to the study of fungi in Bello Horizonte. Jan. 1912- June 1920, Ibid. 19: 285-322, 1926]. Maillard, J. P. 1933. Les mycoses du conduit auditif externe, These Fac. Med. Univ. Paris 537: 1-73. Marchoux, E. 1922. Mycose pulmonaire, BiM. Soc. Path. Exot. 15: 919-920. Mattlet, J. 1926. Mycoses dans I'Urundi, Ann. Soc. Beige Med. Trop. 6: 1-41, 18 figs. Mayer, A. C. 1815. Verchimmelung (Mucedo) in lebenden Korper, Deutsch. Arch. Physiol. [Meckel's] 1: 310-312. Mendelson, Ealph W. 1921. Tropical bronchopulmonary mycosis, Jour. Am. Med. Assn. 77: 110-112, 4 figs. — . 1925. Some interesting diseases observed in the clinic of the Bangkok Central Hospital, Jour. Trop. Med. Hyg. 28: 393-405, SI figs. Mohler, J. 1904. Mycotic stomatites of cattle, U. S. Dept. Agr. Burewu Animal Industry, Circ. 51: 1-6. Nannizzi, Arturo. 1934. Repertorio sistematico dei miceti dell'uomo e degli animali, Siena, S. A. Poligrafica Meini xii + 557 pp., 224 figs. [Received in 1935 after this book was iA galley proof, too late to be considered except for changes of nomenclature of species already included.] Neumann, L. G. 1892. Traite des maladies parasitaires non microbiennes des animaux domestiques, ed. 2, Paris, XVI + 768 pp., 364 figs. Neveu-Lemaire, Maurice. 1911. Parasitologie des animaux domestiques. Maladies para- sitaires non bacteriennes, Paris, J. Lamarre & Cie, 1257 pp. — . 1921. Precis de parasitologie humaine, ed. 5, Paris, J. Lamarre, VI -l- 466 pp. Niethe, Ulrich. 1926. Die Bedeutung der gewohnliche Schimmelpilze fiir die menschliehe Haut, Arch. Derm. Syphilis 152: 358-364. Ota, Masao. 1928. Champignons parasites de I'homme (Etudes morphologiques et sys- tematiques), Jap. Jour. Derm. Urol. 28: [202 pp. in Japanese, 7 pp. of French summary, 5 pp. of bibliography]. Perazzi, Piero. 1926. T miceti dimoranti nello- regione genitale della donna, Atti B. Accad. Fisiocrit. Siena 17: 361-410, Pis. 1-S. Perin, Arrigo. 1925. Le micosi pulmonari e generalita sui miceti patogeni. Siena, Libreria Editrice Senese, viii -|- 294 pp. Pinoy, E. 1903. Les champignons pathogenes; leur classification d'apres les caracteres botaniques. Bull. Inst. Pasteur. 1: 761-774. Plant, Hugo C. 1903, 1912. Die Hyphenpilze oder Eumyceten, Handb. path. Mihroorganis- men [Kolle & Wassermann] 1: 526-660; ed. 2, 5: 1-154, 7 pis. Eamsbottom, John, & Arthur Whitfield. 1931. Fungi pathogenic to man, Syst. Bact. in Relation to Medicine [Med. Res. Council] 8: 11-70. Raymond, Victor & Jacques Parisot. 1916. £tiologie, prophylaxie et therapeutique de I'afEection dite gelure des pieds, C. B. Acad. Sci. 162: 694-696. RedaeUi, P. 1931. Tecnica micologica medica, Bologna, L. Capelli. 227 pp., 58 figs. Robin, Charles. 1847. Les vegetaux qui croissent sur les animaux vivants, These Fac. Sci. Paris, VIII + 120 pp., 3 pis. — . 1853. Historie naturelle des vegetaux parasites qui croissent sur I'homme et sur les animaux vivants, Paris, x + 704 pp. Atlas 15 pis. Rockwell, George E. & John H. Highberger. 1927. The necessity of carbon dioxide for the growth of bacteria, yeasts, and moulds. Jour. Inf. Dis. 27: 438-446. Rockwood, Ethel M, 1930. A study of fungus infected nails. Arch. Derm. Syphilol. 22:. 395-400, 7 figs. Eoeckl, G. 1884. tJber Pneumonomykose. Deutsch. Zeitsrhr. Thiermed. Vergleich. Path. 10: 122-132, PI. 12. 30 MEDICAL MYCOLOGY Eouyer, E. & J. Pellissier. 1915. Contribution a 1 'etude de certaines mycoses de blessures de guerre, Ann. Inst. Pasteur 29: 551-555. Eudolph, Max. 1914. tJber die brasilianische Figueira, Arch. Schiffs-Tropenhyff. 18: 498. Sartory, A. 1920-1923. Champignons parasites de I'homme et des animaux, 14 pts. 1 suppl. 1-86, 1920; 91-484, 1921; 487-721, 1922; 727-895 and index 1-47, 1923; suppl. 1-78, 1923. Schmidt, Hans. 1921. ijber Beziehung zvvischen Tuberkelbazillen und Schimmelpilzen, Ztschr. Klin. Tu'berlculose. 46: 456-459. Schmidt, P. W. 1933. Dermatomykosen, Derm. Zeitschr. 66: 241-261. See, Pierre. 1922. Les mycoses. Gas. Sop. Civ. Milit. 95: 469-476. Senator, H. 1891. [Pneumaturie und Diabetes], Intern. Beitr. Wiss. Med. 3: -319-332 [also cited Festschrift Kudolf Virchow]. Shear, Cornelius Lett. 1927. Mycology in relation to human pathology. Am. Nat. 61: 151-159. Siebenmann, Friedrich. 1889. Die Schimmelmykosen des mensclilichen Ohres. ed. 2, Wies- baden, 118 pp., 4 pis. Sorokin, N. 1882. Rastytel'nye parazity cheloveka i zhivotnykh kak prichina zaraznykh boleznei dlia naturalistov, vrachei, studentov i veterinarov. 4 vol. and atlas. S. Peterburg. Souls, Ferdinand Xavier Felix. 1891. Contribution a I'etude des otomycoses, These Fac. Med. Pharm. Univ. Bordeaux 9: 1-48, 2 -figs. Stein, E. O. 1914. Die Fadenpilzerkrankungen der Menschen. Miinchen, 1914, 99 pp., S2 pis. [also cited Lehmanns Med. Atlanten. 12: 1-99, 3^ pis., 1914]; ed. 2, 128 pp., 32 pis., 1930. Strong, Eichard P. 1930. The African republic of Liberia and the Belgian Congo. Based on observations made and material collected during the Harvard African expedition, 1926-1927. Cambridge [Mass.], Harvard University Press, v. 1, xxvi + 568 pp., 6 pis. Strong, Eichard P. & George C. Shattuck. 1930. Medical and pathological investigations in Liberia and the Belgian Congo, Contr. Dept. Trap. Med., Inst. Trop. Biol. & Med. 55: 210-461. Talice, Eodolfo V. 1930. Le concept actuel des mycoses medicales de I'appareil respiratoire, Bev. Stid-Amer. Med. Chir. 1: 181-188. Thibierge, G. 1925. Les enseignements dermatologiques de la guerre 1914-1918, Ann. Derm. Syphiligr. 6: 481-514; 641-661; 705-724. [III. Mycoses cutanees, pp. 641-653.] Uhthoff, W. 1883. Beitrage zur pathologischen Anatomic des Auges. III. Partielle Necrose der mensclilichen Hornhaut durch Einwanderung von Schimmelpilzen, Arch. Ophthal- mol. 29: 3: 178-181. Virchow, Eudolf. 1856. Beitrage zur Lehre von den beim Menschen Vorkommenden pflanz- lichen Parasiten, Arch. Path. Anat. Physiol. [Virchow 's] 9: 557-593. Vuillemin, Paul. 1931. Les champignons parasites et les mycoses de I'homme. Paris, Paul Lechevalier & Fils, 291 pp. Weidman, Fred D. 1929. Light from the botanic field on medical mycologic problems, Arch. Derm. Syphilol. 19: 867-877. Wright, James Homer. 1913. Non-bacterial fungus infections — the mycoses, in Osier, William & Thomas McCrae, Modern medicine in theory and practice — ed. 2, 1: 1033-1060. Xavier, A. A. 1926. Apontamentos sobre micologia (Notas de um curso), Sciencia Med. (Eio de Janeiro) 4: 60-68. Young, James. 1921. Organism cultivated from carcinomata, Brit. Med. Jour. 1: 933. Ziirn, Friedrich Anton & Hugo C. Plaut. 1887-1889. II. Die pflanzliche Parasiten in Ziirn, F. A., Die Schmarotzer auf und in dem Korper unserer Haussaugetiere sowie die durch erstere veranlassten Krankheiten, deren Behandlung und Verhiitung. Weimar, 837 pp., 4 pis. CHAPTER II PHYSIOLOGY OF FUNGI WITH SPECIAL REFERENCE TO REPRODUCTION The life of a fungus may be divided into three periods: (1) vegetative growth, (2) multiplication by asexual means, and (3) the development of sex- ual organs usually resulting in a spore able to resist unfavorable factors of the environment. Since studies of classification and evolution of the various groups of fungi are usually based on this last stage which presents the great- est diversity of phenomena, much of laboratory work in the last half century has been directed toward securing the production of sexual stages. After Bary, Brefeld, Hansen and Zopf had developed the technic of cultivation, Klebs in the years from 1896 to 1904 laid the foundations of much of our pres- ent knowledge by detailed studies of the conditions necessary for vegetative growth and reproduction in a few organisms. Subsequent work of C. H. Kauffman and his students has done much to extend Klebs' generalizations to further groups of fungi and to confirm his well-known dicta. These may be summarized as follows : 1. Among all organisms, growth and reproduction are life processes, Avhich depend upon different conditions : among lower organisms, probably environment determines whether growth or reproduction will occur. 2. As long as the environment is favorable for growth, reproduction will not occur. An environment favoring the latter is usually more or less un- favorable to further growth. 3. The limits of each factor of the environment are narrower for reproduc- tion than for growth. Therefore growth can still take place, although repro- duction is limited by a too weak or too strong influence of some factor. 4. Before reproduction can occur growth must have been sufficient to have stored the products necessary for the reproductive processes. As a consequence of these dicta, most specialized methods for securing sexual or even asexual reproduction in a given group consist essentially in growing the organism for a time as nearly at optimum conditions as possible, then suddenly changing one or more factors of the environment to a condition less favorable for growth but still within the limit for reproduction. Exam- ples of this will be given in the following analysis of some of the chief factors of the environment. Water. — The water requirement and transpiration have been little studied in fungi. While spores often have mechanisms for resisting desiccation over long periods and some very complex specialized structures are developed among the higher fungi for preventing the reproductive organs from drying out, in general the fungi both grow and reproduce in relatively high humidi- 31 32 MEDICAL MYCOLOGY ties. The optimum humidity seems very closely related to oxygen supply and respiration. Many of the lower fungi grow well, or even normally are found, immersed in water. Many others will form a thick hyphal mat over the sur- face of a liquid culture medium, although in this case much more oxygen is required for normal development than in purely aquatic fungi and the repro- ductive structures are usually produced in the aerial mycelium. Still other groups will grow well and reproduce only on solid substrata, and a few grow best in comparatively low humidities. Growth is usually much slower at low humidities.* Some species have a narrow range while others will tolerate a very wide range. Transpiration rates and humidity are often important in initiating repro- duction. Very frequently reproductive structures are produced only when vegetative growth is severely checked by the drying out of the media. This, however, is usually associated with partial exhaustion of nutrients and the accumulation of toxic products of metabolism (staling) so that it is often difficult to evaluate the influence of the various factors. (Klebs 1896, Coons 1916.) Nutrition. — The most emphasis has been placed upon this factor and a great body of literature on culture media and their effects has been produced. The complexity of the vast majority in use does not lend itself to an analysis of the separate factors involved. These may be considered under the head- ings of inorganic salt requirements, carbon and nitrogen supply, and relations to concentration of hydrogen ions, although these are closely interdependent and also related to the physical factors of the environment. Inorganic Salts. — The requirement of these seems much the same as for the higher plants, traces of potassium, magnesium, iron, and calcium being necessary to secure good growth and reproduction. Calcium, however, is much less important for fungi than for the flowering plants. Of the nonmetal- lic elements, phosphorus, sulphur, carbon, and nitrogen are important. Other elements are often present and may influence growth and reproduction, but they are relatively unimportant in comparison with the above (e.g., McHargue & Calfee 1931). Phosphorus is usually supplied as a phosphate, where it is often useful in regulating the concentration of hydrogen ions in the solution. In many media it is supplied as nucleoproteins, nucleic acid, and lecithin, and seems equally available in these forms. Sulphur. — This element is usually supplied as a sulphate, but is also utilized from sulphocyanates, thiosulphates and the sulphur-containing amino acids and their compounds. The literature on this subject was well sum- marized by Armstrong (1921) and will not be covered here. In various or- ganic media probably a large portion of the necessary sulphur is available from the proteins since practically all of them contain some cystine or cysteine. Where large amounts of sulphur are available, particles of sulphur may be ♦This fact may b© utilized in stock cultures by transferring to a 4-5% agar instead of l%-3% commonly employed. Since growth is slow and, water loss is slower from the harder agar, one safely can allow a much longer time to elapse between transfers. PHYSIOLOGY OF FUNGI 33 deposited in the cells or hydrogen sulphide may be produced, although these phenomena are rare under ordinary cultural conditions. Carbon. — The possible sources of carbon are almost infinite, since there are many possible combinations from which it may be utilized in dilute solu- tion. For practical purposes the carbohydrates and organic acids are em- ployed most. Of these, glucose has the widest use although the other hexoses, especially mannose and fructose, are easily utilized, and also sucrose (cane sugar) by organisms which secrete invertase. The other sugars and aldehydes, celluloses, starches, etc., are variable in effect, those with low molecular weights generallj^ being toxic. The lower alcohols are also generally toxic, although some of higher molecular weight, such as glycerol (glycerin) and mannitol (mannite), are very readily assimilated. In general, substances hav- ing a three- or six-membered carbon chain are the most assimilable. Among the organic acids, the amino acids and those of the aliphatic series having a low hydrogen ion concentration, are readily utilized. Acids of the cyclic (benzene) and heterocyclic series are rarely metabolized unless there is a side chain of at least three carbon atoms, as in the amino acids, phenylalanine, tyrosine, and tryptophane. There are also scattered observations on utiliza- tion of depsides, tannins, alkaloids, amines, etc. The proteins and their de- composition products often furnish a suitable carbon source, although they are primarily regarded as a nitrogen source. In many of the compounds assimilability depends upon the ability of the organism in question to secrete some enzyme which will hydrolyze or so break down the higher compound that one or more of the resulting products will be utilizable while none will be toxic, e.g., in depsides and tannins, usually the hexose is utilized and the galloyl compounds are not attacked. Many of the older authors attempted to arrange carbon compounds in the order of decreasing availability, but this is rather futile, as the exact order varies Avith the organism in question and with other environmental factors. (Pasteur 1860, 1862 ; Naegeli 1879, 1882 ; Reinke 1882 ; Duclaux 1885, 1889 ; Linossier & Roux 1890; Went 1901; Ekman 1911. For action of cyclic carbon compounds, see Waterman 1913, Bokorny 1920.) Nitrogen. — While from time to time atmospheric nitrogen has been indi- cated as a source of nitrogen for fungi, the inadequacy of methods of deter- mination of total nitrogen has tended to discredit this source for most fungi. Duggar and Davis (1916) have carefully summarized the literature and dis- cussed the methods employed. Klotz (1923) has adequately summarized the literature for other sources. Older authors attempted to group fungi as nitrate, ammonium, amino acid, peptone or protein organisms, but in view of the large number of variables involved and the difficulties of adopting a suit- able criterion for growth these groupings are quite inadequate. While some fungi are capable of utilizing nitrates or ammonium salts, in the groups in which we are most interested here, they are so few that they need not be con- sidered. Czapek (1902) made a very extensive study of substances as nitrogen 34 MEDICAL MYCOLOGY sources and concluded that amino acids and compounds nearly resembling them, are most fully and easily utilized, since the principal use of nitrogen in nutrition is in the production of proteins. Lutz (1905) extended and con- firmed these observations. Ritter (1909, 1912, 1914) studied the effect of the acid produced when ammonium salts were used. Kossowicz (1912, 1914) ex- tended his studies to purines and related compounds. Brenner (1911) extended his observations to include many compounds which proved toxic. While these and many other later papers have much interest for the prob- lem of the mechanism of protein synthesis in fungi, they have little direct bearing on the problems of cultivation. Few studies have been made on the vitamin requirements of fungi, and the evidence is not very conclusive at present. (See excellent review of litera- ture by Sergent 1928, Peskett 1933.) However, the possibility of such fac- tors should be borne in mind in nutritional studies (see Freedman & Funk 1922; Robertson & Davis 1923). The problem of nutrition of fungi is closely bound up with the varying degrees of parasitism. The division of fungi into saprophytes and parasites is rather artificial since there are so many intergrading forms. However, it is often convenient to speak of facultative parasites, species which normally are saprophytic, but under especially favorable conditions may develop in the tissues of other organisms, usually as secondary invaders. Other fungi may develop during part of their life cycle as parasites and reproduce sexually only on the dead tissues of the host, or, vice versa, with, a reproductive cycle in the living host and saprophytic vegetative existence outside, as in Cocci- dioides immitis. A few, such as the plant rusts and the Laboulbeniales on living insects, complete their whole life cycle on the living host, and have not been successfully cultivated apart from their hosts. Even more artificial and confusing are the terms employed for symbiosis and related phenomena to express varying degrees of the interaction of two organisms which may vary all the way from parasitism at one extreme to epiphytism at the other. Hydrogen Ion Concentration. — Although biologists have ceased attempt- ing to explain nearly all phenomena by hydrogen ion concentrations, yet these do play an important part in metabolism and should be considered. At this point it might be well to review briefly the underlying concepts and the methods of determining this concentration of hydrogen ions. In a given ionizing solvent, of which water is the only one which can concern us here, solutions of various substances are observed to conduct an electric current to a greater or lesser degree. Pure, distilled water is for aU practical purposes a nonconductor. Solu- tions of sugar for instance, are quite as unaffected by electric current as pure water. Acetic acid in solution conducts a given current slightly, while substances such as HCl, NaCl, and NaOH are strong conductors when dissolved. It is quite reasonable, then, to assume that in conducting solutions there are present conducting units and that the quantity of current conducted is in some way proportional to the numbers of these units. Now, since most of these substances that are good conductors PHYSIOLOGY OF FUNGI 35 when dissolved are nonconductors in the absence of moisture, we must postuUite some change in the solute. Without going any further into the proofs for electrolytic dissociation, we will state here very simply the long accepted fact that if a substance when dissolved in water is found to be a conductor of electric current, it dissociates from the uncharged form into ions capable of carrying current and yet balancing one another in electric charges. The graphic representation of this fact may be made as follows: XY :^ X+ + Y". Arrows are used to indicate that this is an equilibrium, not a completed reaction, that must adjust itself to whatever other equilibria other solutes may necessitate. Let us take, now, the hypothetical acid HA. It dissolves in water and dissociates accord- ing to the following representation: HA ^ H^ + A- At the instant of solution we may visualize the undissociated acid breaking up into H* and A- ions and the dissociated ions recombining to form HA at such rates that the equilibrium for the given acid will be established. Thereafter the rates of dissociation and reassociation must be equal. If, now, we add to the solution, some salt BA, then the concentration of the A" ions will be materially increased. If by any chance we have doubled the concentration of A" we have doubled the possibility of collisions between H* and A" and doubled tlie velocity of the reaction from right to left. If now, we add another acid so that the concentration of H* will be doubled, we have, obviously, quadrupled the possibility of collision. The velocity, then, in a given direction is proportional to the products of the concentrations of the reacting groups. Eepresenting concentrations by square brackets, we may express this as follows: Velocity from right to left kj [H+] [A~] where k^ is the proportionality factor. At the same time, however, we are having HA redissociating at a rate proportional, more or less, to the concentration. Velocity from left to right k^ [HA]. Since, as we have already stated, equilibrium is established, the velocity in one direction must be equal to the velocity in the other. Equating these two expressions, we get: k, [H^] [A-] = k, [HA] or [Hi [A-] ^^^;g- [HA] k, which will be a determinable quantity, characteristic of the solute for which it is determined and referred to as equilibrium or ionizing or dissociation constant. Similarly, a hypothetical base BOH, dissociating as follows: BOH ^ B* + OH- will have a characteristic ionization constant expressed by the equation: Kb = [B-] [0H-] [BOH] And the salt BA: BA ^ B^A- Kba = [Bi [A-] [BA] Assume, now, that we mix equal quantities of two solutions, one of the acid and one of the base, in equivalent concentrations. We will have then in solution the four ions: H+, A", B*, OH". These will adjust themselves so that the constants, Ka, Kb, Kba, 'wiU be satisfied and, in addition, another constant which will express the equilibrium between the H+ and OH" ions as follows : ^ _ [Hi [0H-] 36 MEDICAL MYCOLOGY Since HOH is water and since the quantity formed in a given solution by reassociation of ions is negligible in comparison with the sum total of molecules present, we may for all useful purposes write the equation: Kw ■= [H-] [0H-] The above is a relationship that holds in all aqueous solutions. However concentrated the hydrogen ions, there still must always be enough hydroxyl ions present to satisfy the constant ; however concentrated the hydroxyl, there must still be enough hydrogen ions. Thus, for instance, we might express the acidity of solutions in terms of the hydroxyl ion concentra- tion. As a matter of convention, but not necessity, we express acidity and alkalinity in terms of hydrogen ion concentration. Kw has been determined with considerable care to be practically or lO-i*. Thus 1014 [H+] [0H-] = 10-14 always holds, whatever the solute or solutes. In chemically pure, distilled water, which is of course entirely neutral, [H+] c= [0H-] = 10-7. A neutral solu- tion, then, is one in which this equation holds. For higher concentrations of hydrogen ions, those from [H+] = IQi up to [H+] = 10"^ (remember the exponent is negative) solutions give acid reactions. For lower concentrations, between [H+] = 10"^ and [H+] = lO'i*, solu- tions are alkaline. As a convenience in manipulation, the reciprocal of the hydrogen ion concentration is generally used. This obviates the use of the negative exponent. Thus, in alkaline solutions, varies between 10^ and IQi*, another way of expressing the facts expressed above, but somewhat simpler. It is still simpler, and quite permissible, to use the logarithm of the reciprocal _ _ = 10" logj, = x logjJO. Since tlie logarithm of ten is unity, [H+J H+ log = X. This X, the logarithm of the reciprocal of the hydrogen ion concentration, is H* what is referred to as the pH of a solution. It follows, then, that from pH 1 to pH 7 a solu- tion is acid and from 7 to 14, a solution is alkaline. There are two common methods of measuring hydrogen ion concentration. The most direct method is by measuring the potential of the so-called hydro- gen electrode in a portion of the solution to be determined. It has been found that with this quantity measured, the concentration may be deduced very simply according to the following formula : Potential , 1 = log Numerical factor [H"^] With the factor once determined and the potential directly measured, there should be no further difficulty. The apparatus, however, is expensive and while apparently simple to manipulate is actually unreliable except in the hands of an expert who appreciates the various possible sources of error and is ever on the alert for them. There are many types of potentiometer on the market, but it is safe to say that none of them is fool-proof. The indirect method, the colorimetric, depends upon the fact that certain series of organic compounds exhibit great changes in color with varying pH. Phenolphthalein, for instance, is colorless in acid solution, but becomes ma- genta at pH 9. Litmus is violet in alkaline solutions, red in acid, changing at approximately pH 7. Suitable indicators may be found for almost any desired PHYSIOLOGY OF FUNGI 37 pPI or range of pH's, notable among which are a long series of sulphon- plithaleins and another of azo compounds. There is not space here to go into the theory of indicators or even to list them. Thorough and satisfactory discussions may be found elsewhere. While apparently crude, this method, in addition to the obvious advantages of being cheap and quick, may, in the hands of a normal, intelligent manipulator, be developed to quite a high degree of accuracy. It must be remembered that the color perception of some individuals is very imperfect so that no amount of training will give them satisfactory re- sults Avith a colorimetric method in biochemistry or bacteriology. The ex- tremely simple method of matching colors Avith a colored chart, such as fur- nished by Clark (1920, 1922), is only a very rough approximation and is practically useless Avitli even slightly clouded solutions. A more reliable method, in Avhich the unknown + indicator is compared Avith a standard + indicator as seen through an equal depth of solution Avithout indicator, is quite commonly used and is satisfactory if the unknoAvn solution is not highly colored. It is important that the vessel be of the same size, shape, material, color, and thickness, and that the light intensity be equal. Simple devices may be purchased quite cheaply. By carefully eliminating sources of error by use of a colorimeter of the Duboscq type and varying the light intensity, the Avriter (Dodge 1919, Duggar & Dodge 1919, Duggar 1919) Avas able to secure as great accuracy as is usually attained by the potentiometric method, and extend the range of indicators in both directions so that only half as many need be used as in the usual series proposed by Clark & Lubs (1917). It should also be kept in mind that the alkali sloAvly dissolves from glass, in- creasing the alkalinity of solutions so that standards sealed in glass should not be used indefinitely. The safest Avay is to prepare carefully one's OAvn stand- ards and store them in paraffin lined bottles, for use as occasion demands. These standards, usually mixtures of buffers, are solutions of Aveak acids and their salts, which can maintain their pH almost unaltered on the addition of considerable quantities of strong acids or bases. Any substance capable of remoAdng hydrogen or hydroxyl ions from the solution either physically or chemically Avill act as a buffer; e.g., Bovie (1915) has shoAvn that charcoal has a buffer action. We have, however, to deal only with chemical buffers, a simple explanation of Avhich might not be amiss. Acetic acid (we will indicate the acetate group, CH3COO-, here by the simplified Ac-) is a weak acid. That is, though in a normal solution there is one gram atom of ionizable hydrogen per liter, most of this remains as un-ionized HAc and the reaction of the solution is only slightly acid, the pH being only a little below 7. Sodium acetate, being a salt, is almost completely ionized. NaAc :;±: Na+ + Ac- The Ac- ion will immediately start to take up the H* ion of the water until the dissocia- tion constant for HAc is satisfied. This will liberate an excess of OH- ions which will remain virtually uncombined because NaOH is a very highly ionized base. Thus a normal solution of pure NaAc in pure water will give a definitely alkaline reaction. 38 MEDICAL MYCOLOGY Assume now that we have in a given solution a mixture of solutions of HAc and NaAc in such proportions that the resulting reaction of the mixture is acid. We have, then, present large quantities of Na*, 0H-, H* and Ac- ions and of un-ionized HAc, practically no un- ionized NaOH. Let us now visualize what would happen if to such a solution we add a solution of a strong acid, such as HCl, almost completely ionized to H+ and C1-. The reaction does not, as one might hastily assume, turn sharply acid. Before the pH can be materially lowered, the free H+ added must first adjust its equilibrium with the free Ac- and OH- in the solution. This will be accomplished by association into almost un-ionized HAc and HOH and until this is accomplished, addition of HCl will not materially affect the reaction of the solution. By suitably selecting the acid and its salt, we may secure satisfactory buffers for almost any region of the whole pH scale. Boric acid, H3BO3, and phosphoric acid, HjPO^, are among those most commonly used. These have the double advantage of being weak acids and of ionizing in three stages: e.g. H3PO, ^ H* + H„PO,- H,PO,- ^ H+ -1- HPO,- HPO,= ;^ H+ -f-PO/ The dissociation constants of these three stages are successively smaller, the possible use in buffering correspondingly greater. The actual composition of the buffer solutions we will not go into here, save to state briefly that boric acid is usually used in conjunction with borax, while, instead of using phosphoric acid itself, there is used a mixture of the two salts KH.PO, and Na.HPO, (or, rather, its hydrate). Clark (1922) gives a series of possible buffers, with directions for their preparation and tables showing their pH range. For a comprehensive discussion of the subject one should consult such works as those of Clark (1922) and Michaelis (1926). This matter of buffers is very important in the preparation of media and in interpreting the results of older authors, who measured the total acidity, usually using phenolphthalein as an indicator. It was customary to titrate a sample medium and add the calculated amount of acid or alkali necessary for a given total acidity. If the medium was made up of phosphates, etc., as some of the early media were, this added acid had little real effect, while if few buffers were present it might change the reaction very much. While generalizations are unwarranted, a large number of observations indicate that most bacteria grow best betwen pH 7 and 9 while most fungi grow best between pH 5 and 7 with some, especially members of the Asper- gillaeeae, able to grow slowly at much higher acidities. This fact is sometimes utilized in restraining the growth of bacteria in isolations by using media to which a small quantity of an organic acid, such as acetic or lactic acid, has been added (see p. 59). Klebs' dicta hold in this case as well as in other factors of the environment, since growth is possible at a much wider range than is reproduction. Talice (1930) records optima, maxima, and minima of about thirty common human pathogens on three usual media. (See also notes of Mallinckrodt-Haupt 1929, 1932; Kadisch 1929.) Oxygen Requirements. — Since the fundamental processes of respiration are the same for plants and animals and are already known as to conditions and end products, no attempt will be made to discuss aerobic and anaerobic PHYSIOLOGY OF FUNGI 39 respiration here. Such works as that of Kostyehev and Lyon (1927) and cur- rent textbooks on general physiology may be consulted for further informa- tion. However, one should clearly distinguish between oxidation and fermen- tation phenomena. Space will not permit of a full discussion of this problem here, but some practical suggestions will be given in connection with the methods for the study of fermentation (see p. 62). In general, little atten- tion need be given to a consideration of oxygen supply under the ordinary conditions of culture (Kadisch 1933). It sometimes happens, however, that in small, tightly plugged test tubes there may not be a sufficient amount of oxy- gen present to support sexual reproduction. In some cases a sudden change in oxygen pressure may stimulate reproduction, especially if applied along with other changes in the environment. Temperatiu-e Requirements. — These vary greatly with the species, some of which grow well only at body temperature while others will make good growth at all temperatures between room temperature (20° C.) and body tempera- ture (37.5° C). The earlier authors spent much time in search for an opti- mum temperature, little realizing that a temperature may be optimum for an organism under one set of conditions while far from optimum under another set of conditions (Blackman 1906). Later studies considered the effect of temperature on rate of growth and found that growth roughly follows van 't Hoff's law of doubling the rate for every ten degrees of tem- perature in the middle part of the temperature range, but with a rapid falling off of rate after a certain critical point is reached. The limits of growth are usually much wider than those of reproduction as Klebs postulated. In gen- eral, spores are adapted to withstand higher and lower temperatures than vegetative structures, and ordinarily, thick-walled spores are much more re- sistant than thin-walled spores, while spores resulting from the sexual act are more resistant than those of asexual origin. Influence of Light. — That light has strong morphogenic influence has long been recognized from observations in nature (well summarized by Elfving 1890). Since some organisms develop reproductive organs only in response to light stimuli, light may be of considerable importance in cultures. On the other hand, many fungi seem to develop as well in the dark as in the light. Elfving suggested that light acts as an inhibitor of organic synthesis and that the closer the food available is to the constituents of the protoplasm, the less action the light has. In most plants light, especially of shorter wave lengths, tends to restrict vegetative growth. Many subsequent investigators have ex- tended these early observations. Neidhart (1924) reports lethal action of x-rays and radium in Sporotrichum and Ectotrichophyton gypseum. Nadson & Phillippov (1925) report interesting effects of x-rays on sexuality in Muco- raceae. Dome & White (1931) report differential action of x-rays in different groups of fungi, while using the x-rays for therapeutic purposes. (See also Liebesny, Wertheim & Scholz 1933.) 40 MEDICAL MYCOLOGY Chemotropism. — The strongest chemotropic reaction of liyphae is usually negative ; the hyphae grow away from regions which have been staled by the products of their own metabolism (Clark 1902, Fulton 1906, Balls 1908, Graves 1916, Brown 1922, 1923, 1925, Pratt 1924). A simple example of this reaction is found in the circular growth of mycelia. In so far as a clear field is avail- able, the hyphae tend to grow equally in all directions from the point of in- fection. The same factor may account for the alternate dense and sparse zones which characterize many fungal colonies. Energetic growth results in the deposition of catabolic substances, and growth is accordingly reduced until a few hyphae pass beyond the inhibiting zone and give rise to a new ring", or frequently the germination of fresh spores outside this zone produces a similar effect. Ammonia and potassium bicar- bonate are often among the substances producing staling, as this phenomenon is called. Incubation at higher temperatures hastens staling. Hydrotropism may occur but is difficult to prove. Phototropism. — Many reproductive structures are very sensitive to light and by means of this reaction adjust themselves in a position favorable to the distribution of their spores, since the direction of light is usually that of the direction of open spaces (BuUer 1909-1931, Jolivette 1914, Parr 1918, and Blaauw 1914). It is probable, however, that there is little positive phototropism among the human pathogens. Radium. — Little work has been done on the effect of radium on patho- genic fungi. Sartory & Meyer (1926) report that in Aspergillus fumigatus on media containing salt, exposure to 3-7.2 millicuries, either discontinuous or continuous, produced an increase of conidiophores, with a tendency to de- crease the size of the head and approach conditions found in Penicillium. On media nearly free from salts, there was a tendency to form large-celled oidia rich in oils, or large, thick-walled spores, 3-8/a in diameter, singly or in pairs, and large pseudosporangia, 30/i, in diameter, with echinulate walls but no spores observed within them. It was noted at the same time that in dissociated media reducing power was lowered and pH was increased. (Sucrose 5 gm., gelatin 7.5 gm., NaCl 1 gm., carrot juice q. s. for 100 c.c.) In undissociated media, reducing power was increased and pH was decreased. "With higher dosage (10.2 millicuries per sq. cm.), hard, fusiform sclerotia were produced in submersed mycelium. These contained perithecia. BIBLIOGRAPHY Armstrong, G. M. 1921. Sulphur nutrition; the use of thiosulfate as influenced by hydrogen ion concentration, Ann. Missouri Bot. Gard. 8: 237-281. Balls, W. Lawrence. 1908. Temperature and growth, Ann. Bot. 22: 557-591, 11 figs. Blaauw, A. H. 1914. Licht und Wachstum I, Zeitschr. Bot. 6: 611-703. Blackman, F. P. 1906. Optima and limiting factors, Ann. Bot. 19: 281-295, ^ fiffs. Bokorny, Th. 1917. Benzolverbindungen als Nahrsubstanzen, Zentralbl. Physiol. 32: 55-63. Bovie, William T. 1915. A direct reading potentiometer for measuring and recording both the actual and the total reactions of solutions, Jour. Med. Bes. 33: 295-322, 14 figs. PHYSIOLOGY OF FUNGI 41 Brenner, W. 1911. Untersuchungen iiber die Stickstoffernalirung des Aspergillus niger und deren Verwertung, Ber. Deutsch. Bot. Ges. 29: 479-483. Brown, William. 1922. On the germination and growth of fungi at various temperatures and concentrations of oxygen and carbon dioxide, Ann. Bot. 36: 257-283, 4 figs. — . 1923. Experiments on the growth of fungi in culture media, Ann. Bot. 37: 105-129, 7 figs. — . 1925. Studies in the genus Fusarium. II. An analysis of factors which determine certain microscopic features of Fusarium strains, Ann. Bot. 39: 373-40S. BuUer, Arthur Henry Reginald. 1909-1934. Researches on Fungi, vols. 1-6. Longmans Green & Co. London. Cerutti, Pietro. 1933. Conccntrazione idrogenionica e sviluppo degli ifomiceti patogeni: ricerche sperimentale e cliniche, Patkologica 25: 32-37. Clark, Judson F. 1902. On the toxic properties of some copper compounds with special refer- ence to Bordeaux mixture, Bot. Gas. 33: 26-48, 7 figs. Clark, William Mansfield. 1920, 1922. The determination of hydrogen ions, Baltimore, Williams & Wilkins Co., 317 pp., 1920; ed. 2, 480 pp., 1922. Clark, William Mansfield So H. A. Lubs. 1917. The colorimetric determination of hydrogen ions and its applications in bacteriology, J. Bad. 2: 1-109. Coons, George H. 1916. Factors involved in the growth and the pycnidium formation of Plenodomus fuscomaculans. Jour. Agr. Bes. 5: 713-769. Czapek, Karl. 1902, 1903. Untersuchungen iiber die Stickstoffgewinnung und Eiweissbildung der Schimmelpilze, Beitr. Chem. Physiol, u. Path. 1: 538-560, 1902; 3: 47-66, 1903. Dodge, Carroll William. 1919. [The fate of] Tyrosine in fungi: chemistry & methods of studying the tyrosinase reaction, Ann. Missouri Bot. Gard. 6: 71-92. Dome, Maurice & Cleveland Wliite. 1931. Treatment of superficial infections with the long wave length Roentgen rays (Grenz rays), Arch. Derm. Syphilol. 24: 409-416. Duclaux, E. 1885. Sur la valeur alimentaire de diverges substances pour I'Aspergillus niger, C. B. Soc. Biol. 37: 91-94. — . 1889. Sur la nutrition intracellulaire, Ann. Inst. Pasteur. 3: 97-112, 413-428. Duggar, Benjamin Minge. 1919. The microcolorimeter in the indicator method of hydrogen ion determination, Ann. Missouri Bot. Gard. 6: 179-181. Duggar, Benjamin Minge & A. R. Davis. 1916. Nitrogen fixation, Ann. Missouri Bot. Gard. 3: 413-437. Duggar, Benjamin Minge & Carroll William Dodge. 1919. The use of the colorimeter in the indicator method of hydrogen ion determination, Ann. Missouri Bot. Gard. 6: 61-71. Ekman, Gunnar. 1911. Studien iiber den Nahrwert einiger KohlenstoffqueUen fiir Aspergillus niger van Tiegh, ofversight af FinsTca Vetensh Soc. Forh. 53A: 16: 1-43. Freedman, Louis & Casimir Funk: Nutritional factors in the growth of yeasts and bacteria, I. Vitamines, /. Metai. Res. 1: 457-468. II. Protein hydrolysates, IMd. 1: 469-480. Fulton, Harry R. 1906. Chemotropism of fungi, Bot. Gas. 41: 81-108. Graves, Arthur Harraount. 1916. Chemotropism in Rhizopus nigricans, Bot. Gaz. 62: 337- 369, 4 figs. Janke, Alexander & Stephan Kropacsy. 1929. Die Bestimmung des Wasserexponenten mittels des Stufenphotometers. I, Biochem. Zeitschr. 213: 154-169. Jolivette, Hally D. M. 1914. Studies in the reactions of Pilobolus to light stimuli, Bot. Gaz. 57: 89-121, 1£ figs. Kadisch, E. 1929. tjber die Bedeutung der Nahrbodenalkalitat in der Mykologie, Derm. Zeitschr. 55: 385-396. — . 1933. Der Einfluss von Temperatur und Sauerstoif auf die Lokalisation der Infektionen. Wiigende Untersuchungen an Fadenpilzen. Untersuchungen mit dem Pulfrich Photo- meter an Hefen. Modifikation des Pulfrich Photometers, Arch. Derm. Syphilis 168: 438-475, 10 figs. 42 MEDICAL MYCOLOGY Klebs, Georg. 1896. Die Bedingungen der Fortpflanzung bei einigen Algen und Pilzen, Jena, G. Fischer, 543 pp., Spls. Klotz, L. J. 1923. Some aspects of nitrogen metabolism in fungi, Ann. Missmiri Bot. Gard. 10: 299-368. Kossowicz, A. 1912. Die Zersetsung von Harnstoff, Harnsaure Hippursaure und Glykokoll durch ScMmmelpilze, Zeitsch. f. Garungs-Physiol. 1: 60-62; 2: 51-54. Nitritassimila- tion. Ibid. 2: 55-58. tJber das Verhalten einiger Schimmelpilze zu Kalkstickstoff, Ibid. 2: 124, 125, 1912. — . 1914. Zur Kenntnis der assimilation von Kolilenstoff- und Stickstoff-verbindungen durch Schimmelpilze, Bioch. Zeitschr. 67: 391-399. tJber das Verhalten von Hefen und Schimmelpilze zu Nitraten, Ibid. 67: 400-420. Kostychev, S. & C. J. Lyon. 1927. Plant respiration, Philadelphia, P. Blakiston's Son So Co., 163 pp. Liebesny, P., H. Wertheim & H. Scholz. 1933. tJber Beeinflussung des Wachstums von Mikro- organismen durch Kurzwellenbestrahlung I, Klin. Woch. 12: 141-145. Lutz, L. 1905. Sur 1 'assimilabilite comparee des sels ammoniacaux, des amines, des amides et des nitriles, C. B. Acad. Sci. Paris 140: 665-667. McHargue, J. S. & R. K. Calfee. 1931. Effect of manganese, copper, and zinc on growth and metabolism of Aspergillus flavus and Ehizopus nigricans, Bot. Gaz. 91: 182-193. Mallinckrodt-Haupt, Asta St. von. 1929. pH Messungen bei Pilzkulturen, Derm. Zeitschr. 55: 374-384. — . 1932. Der Wert der pH Messung bei Pilzkulturen, ZentralU. BaU. I. 125: 368-374. Meyer, Jacques. 1927. Contribution a 1 'etude de 1 'Aspergillus fumigatus Fresenius. ifitude experimentale de 1 'influence du radium et des milieux sur la production de phenomenes sexu^s, These Fac. Pharm. Univ. Strasbourg 31: 1-112, SO figs. Michaelis, L. 1926. Hydrogen ion concentration, its significance in the biological sciences and methods for its determinations. I. Principles of the theory. Authorized trans- lation by William A. Perlzweig, Baltimore, Maryland. Williams & Wilkins Company. Nadson, G. A. & G. S. Philippov. 1925. Influence des rayons X sur la sexualite et la forma- tion des mutantes chez les champignons inferieurs (Mucorinees), C. B. Soc. Biol. 93: 473-475. Naegeli, Carl. 1880. Ernahrung der niederen Pilze, Bot. Mitt. 3: 395-483. K. Ahad. Wiss. Milnchen, Math. Nat. CI., Sitsungsber. 10: 277-367. — . 1882. Ernahrung der niederen Pilze durch Kolilenstoff- und Stickstoffverbindungen. Un- tersuchungen uber niederen Pilze aus dem, Pflanzenphysiologischen Institut in Miinchen. 1-75. Neidhart, Leo. 1924. Beitrag zur Strahlenempfindlichkeit pathogener Hautpilze (Sporo- trichum Beurmanni und Trichophyton gypseum), Inaug. Diss. Med. FaTc. Univ. Zurich, 29 pp. Parr, Rosalie. 1918. The response of Pilobolus to light, Ann. Bot. 32: 177-205. Pasteur, Louis. 1860. Memoire sur la fermentation alcoolique, Ann. Chim. et Phys. Ill 58: 323-426. — . 1862. Memoire sur les corpuscules organises qui existent dans 1 'atmosphere, examen de la doctrine des generations spontanees, Ann. Chim. et Phys. Ill 64: 1-110. [IX. Sur le mode de nutrition des ferments proprement dits, des mucedinees et des vibrioniens, pp. 100-110.] Peskett, Geoffrey Lewis. 1933. Growth factors of lower organisms, Biol. Rev. 8: 1-45. Pratt, Clara A. 1924. The staling of fungal cultures. II. The alkaline metabolic products and their effect on the growth of fungal spores, Ann. Bot. 38: 599-615, 1 fig. *Eeinke, J. 1883. Unters. Bot. Lab. Gottingen 3: 13. Hitter, G. E. 1909, 1912. Ammoniak und Nitrate als Stickst off quelle fiir Schimmelpilze, Ber. Deutsch. Bot. Ges. 27: 582-588, 1909; 29: 570-577, 1912. PHYSIOLOGY OF FUNGI 43 — . 3914. Ammonnitrat und freie Saltpetersiiure als Stickstoff quelle fiir Schimmelpilze, Biochem. Zeitsch. 60: 370-377. Eobertson, R. C. & D. J. Davis. 1923. Food accessory factors (vitamins) in bacterial growth, observations on the ultimate source of accessory growth substances for yeast, Jour. Infect. Bis. 32: 153-158. Sartory, Antoine, E. Sartory, & Jacques Meyer. 1926. Etude de Paction du radium sur 1 'Aspergillus fumicatus Tresenius en culture sur milieux dissocies et non dissocies, C. B. Acad. Sci. 183: 77-79. — . 1926. La formation des peritheces chez 1 'Aspergillus fumigatus Fresenius sous 1 'influence du radium, C. B. Acad. Sci. 183: 1360-1362. Sergent, A. L. 1928. Les facteurs de croissance des microbes sur milieux artificiels, These Doct. Med. Paris, 182 pp. Talice, Eodolfo V. 1930. Le facteur pH en mycologie. Son influence sur la culture de certaines especes de champignons parasites de I'homme, Ann. Parasitol. Hum. Cornp. 8: 183-188, 2 talles. Waterman, H. J. 1913. Over eenige factoren, die de ontwikkeling van Penicillium glaucum beinvloeden Proefschr. Delft. [tJber einige Faktoren, welche die Entwicklung von Penicillium glaucum beeinflussen. Beitrag zur Kenntnis der Antiseptica und der Narkose. Centralbl. Baht. II 42: 639-688.] Went, F. A. F. C. 1901. Uber den Einfluss der Nahrung auf die Enzymbildung durch Monilia sitophila (Mont.) Sacc, Jahrb. Wiss. Bot. [Pringsheim] 36: 611-664. CHAPTER III CULTURE MEDIA, THEIR PREPARATION AND STERILIZATION Probably the oldest methods of cultivating fungi were developed in grow- ing the common mushroom of commerce {Psalliota campestris and related species), but these methods had little, if any, influence on the scientific study and cultivation of fungi. Undoubtedly many mycologists of the nineteenth century brought into their laboratories young fructifications attached to their substrate, and watched their development, also making many important ob- servations on material of early stages collected in the field. It remained, how- ever, for Pasteur and Koch in the decade 1873-1883 to develop methods of sterilization, isolation, and pure culture of bacteria to pave the way for our present technique. Earlier authors in several instances had anticipated these methods more or less completely, but their accounts had either been forgotten, buried under the debris of the theory of spontaneous generation, or lost in a little-known periodical of very limited circulation ; e.g., the work of Bizio on Serratia marcescens (Bacillus prodigiosus) which was published in 1823 and was only generally known among scientists after its translation and publication in 1924. For a more complete account of the historj^ of bacteriologic methods the reader is referred to the excellent short account in Conn & Conn's Bac- teriology and, for formulae of the media used to Desgardes (1921) and to Levine and Schoenlein (1930). In all work with pure cultures it is essential that everything used should be clean and sterile. This statement seems so axiomatic to the well-trained medical man that its emphasis here may appear out of place, but in this gen- eration when so much routine work is left to technicians and even humbler laboratory folk, it may not be out of place. Also in my classes I frequently find students with so little previous training in bacteriology that in the fol- lowing pages I shall not assume any previous experience in handling micro- organisms in pure culture. The cleaning of glassware, while one of the drudgeries of the laboratory, is very important.* Needless to say the glassware should be washed with soap and water until clean tap water will drain freely from it, and not hang in drops over the sides of test tubes, etc. This stage may be called physically clean. We must then be sure that it is chemically clean, i.e., that it does not contain any substances, minute traces of which may inhibit or cause abnormal growth of the organism to be cultivated. ♦If the glassware has been used for cultures, it should be sterilized by autoclaving (see pp. 47, 48) before proceeding' to wash it. Pautrier and Rietmann (1924) report infection of a laboratory worker with Trichophyton granulosum (ordinarily confined to horses) while clean- ing tubes containing year-old cultures ! 44 CULTURE MEDIA 45 Some new glassware when first received into the laboratory may still contain enough alkali to change considerably the hydrogen ion concentration of the medium to be employed. It may be necessary to heat this glassware with strong acids before using it for careful work, although it may still be used for many of the more tolerant organisms without further treatment. In many laboratories all glassware is treated with a strong oxidizing agent, such as an acid solution of sodium or potassium bichromate, commonly called cleaning solution. This tends to neutralize any free alkali in the glass as well as to destroy any life or organic material. This is especially necessary if dry sterilization of the glassware is expected, as failure to remove all organic material will result in a deposit of carbon which reduces visibility and is almost impossible to remove later, although it probably does not interfere with the growth of the organism. After treatment with cleaning solution (if necessary), the glassware should be thoroughly rinsed with distilled water. If dry sterilization takes place after hard tap water has been used for rinsing, a deposit of salt may be baked in, causing decreased visibility and, rarely, toxicity. The alternative method of rinsing with the solution to be used is not recommended in ordi- nary practice, a^ some liquid may adhere to the top of the tube of the neck of a flask and cause trouble in later procedures. Sterilization. — Many methods have been developed in the last half cen- tury, but none is equally good for everything, consequently the principles underlying each should be thoroughly understood and the proper method selected for the material in hand, with a clear understanding of its limitations. Chemical Methods (Disinfectants, Antiseptics). — These methods as com- monly applied in the laboratory consist in treating the material with a toxic chemical substance for a sufficient time to destroy all life, then removing the chemical substance and preserving the material from further contact with microorganisms until it is used. The methods are difficult and little used when other methods will serve. The chemical nature of the disinfectant must al- ways be considered, as well as its fungicidal power. Disinfectants may be grouped as halogens, salts of heavy metals, and organic compounds. Halogfens. — Fluorine and bromine are rarely used under present condi- tions. All of the halogens corrode metals and are difficult to handle. They may prove toxic to living tissue, although they are very valuable in some pro- cedures. Chlorine usually is applied as a solution of a hypochlorite which slowly gives off the chlorine in intimate contact with the material to be dis- infected. Improvements of this technic have been found useful as antiseptic dressings, disinfection of seeds in laboratory practice, etc. Calcium hypo- chlorite (bleaching powder, chloride of lime, sodium or potassium hypochlorite) is also used as a deodorant and disinfectant in outhouses and for sterilizing white clothing where other methods are not easily available. Iodine is usually used in alcoholic or potassium iodide solution or in the organic compound iodoform. This is one of the common disinfectants for 46 MEDICAL MYCOLOGY superficial lesions but causes unpleasant discolorations and in some individuals severe poisoning. It is rarely used about the laboratory as a disinfectant. The alcoholic solution is occasionally used as a counterirritant. Salts of Heavy Metals. — In general, salts of heavy metals are toxic to or- ganisms roughly in the order of the magnitude of their atomic weights, al- though the salt radical has some less clearly defined influence on toxicity. Copper compounds are frequently used in agricultural practice as a fungicide but rarely about the laboratory. Compounds of mercury have had great vogue, especially in some laboratories, but they have some severe drawbacks and probably many may be well replaced by other disinfectants. The oldest and most widely used is mercuric chloride (bichloride of mercury, corrosive sublimate). Toxic in extreme dilution, the solution is apt to dry, leaving tiny crystals which blow about the laboratory and occasionally prevent growth when all other conditions are favorable. Glassware which has been used for mercuric chloride solutions should never be used for cultures again, for if growth takes place at all, it is apt to be abnormal and stunted. In some per- sons it also causes severe dermatoses extending over several years. The recently developed mercurochrome has eliminated several of these objections. Silver nitrate (lunar caustic) has long been in use and recently some of the organic silver salts, especially nucleinates (argyrol and similar com- pounds) have been widely used about the mouth, ej'es, and genitalia. Organic Compounds. — Of the innumerable organic compounds only the lower aliphatic alcohols and aldehydes and the simpler compounds of the aromatic series, such as phenols and salicylic compounds, have found wide use. Methyl alcohol is quite efficient, but is rarely employed on account of the injury of the optic nerve by the vapors. Ethyl alcohol is perhaps the most widely used of the group. Absolute ethyl alcohol has little value, but the intermediate dilutions with water are good, especially for sterilizing cutting instruments which cannot be subjected to heat or to the stronger, but more corrosive, disinfectants. The higher alcohols are little used. Formaldehyde had a great vogue at one time as a gaseous disinfectant, but now is seldom used about the laboratory, except in aqueous solution (formalin) in seed disinfection and as a cheap preservative for class material. Phenol (often knoAvn in its 4% aqueous solution as carbolic acid) has stood the test of half a century, although its popularity has varied. For many years it was taken as a standard for comparison of the efficiency of antiseptics. Salicylic compounds belong rather to medicine, although salicylic acid itself is used externally in dermatology as a keratolytic agent. Volatile oils have been found useful with pathogens of the skin (Kingery et al. 1928, 1929). Physical Ag-ents. — Heat and light are the only sterilizing agents in gen- eral use at present, although it is probable that radiations of still shorter wave lengths would be effective if not so expensive. Light, especially the ultra- CUIiTURE MEDIA 47 violet end of the spectrum, at present belongs rather to the realm of thera- peutics than to laboratory practice and need not be considered here, although it may play a great part in hygiene. Hot dry air is one of the oldest methods employed and is still used for glassware, such as Petri dishes. The clean material, usually heat resistant glass, is put into a gas oven (rarely electric) heated to 150°-180° C. for from a half hour to an hour or more, depending upon the material to be sterilized and the length of time necessary to kill spore-forming organisms which may be found in a given laboratory. Passing material through a flame for a varying period is also a very old practice, now mostly used for sterilizing inoculating tools, such as needles, platinum spatulas, etc. The metal is ordinarily heated to redness in the flame, then allowed to cool to room temperature without touching any object which might contaminate it until used. The former surgical practice of cautery with red hot iron probably owed its success in part to this method of sterilization. It is obvious that this method is not available for tempered tools, such as scalpels, etc. A variant of this practice is to dip in alcohol and ignite, prob- ably less efficient but adequate in many cases. Steam. — None of the above-mentioned methods are useful for most cul- ture media, and it was only with the use of steam that culture media in the modern sense began to develop rapidly. Steam may be applied at atmospheric pressure in which case the medium reaches 100° C. and is held at that tem- perature for a period. This is usually carried out in an Arnold steam steri- lizer, a simple apparatus for generating steam with a minimum loss of water during the process. When spores were discovered in bacteria, Tyndall ap- plied this knowledge by proposing discontinuous sterilization, by heating the medium for a definite period, sufficiently long to kill all the organisms in the active vegetative state, then incubating sufficiently long to allow the spores to develop the vegetative stage, and heating again. This process must be repeated until the medium remains sterile on incubation. Under ordinary conditions the usual procedure is to heat in steam at 100° C. for an hour each day for three successive days. If the period between sterilizations is too long, the spores may have germinated and formed new spores, and if too short they may not all have germinated. The long time necessary to prepare media by this method, as well as several more theoretical objections, has tended to eliminate this method from the laboratory. However, some biologic products used as media are profoundly altered by higher temperatures, and it is neces- sarj^ to employ this method for these substances. Superheated Steam. — In this method the steam is confined in an autoclave up to any pressure desired, instead of being allowed to escape as in the ordi- nary steam sterilizer. A good steam pressure gauge on the autoclave is requisite, and a thermometer is not only desirable but also an additional safe- guard. The temperature ordinarily employed is 115°-125° C. or about 10-20 pounds pressure per square inch. An exposure to about 120° C. (or 15 lb.) for 15-20 minutes will sterilize most media, unless some very heat resistant 48 MEDICAL MYCOLOGY organism is present. The period of sterilization must of course be measured from the time the desired temperature is attained and it may require 10-15 minutes, even with a strong heating unit, to reach this temperature. These temperatures, especially if they are prolonged beyond the minimum necessary to secure complete sterilization, may injure or transform the more labile organic compounds, especially if the acidity or alkalinity of the medium is such as to favor hydrolysis of the higher carbohydrates to hexoses, etc. The autoclave may be heated by electricity or gas or connected with a steam supply pipe if there is sufficient pressure maintained. Care should be taken to see that sufficient water is present and that the lid is wiped free from dust and dirt before each sterilization. After the autoclave is full of steam and the thermometer registers 100° C, the vent is closed. It is not advisable to leave the autoclave without observation, although if the safety valve is properly set, steam will escape after the desired pressure is reached. As soon as this occurs one may safely cut down on the heat supply, since a rapid escape of steam soon exhausts the small supply of water, and often dislocates the cotton plugs or causes the medium to boil up and wet the plugs. When the necessary time for sterilization has elapsed, the gas is turned off, and the autoclave is allowed to cool until the temperature reaches 100° C. before open- ing. An experienced operator may open the cock and allow the steam to escape slowly before the pressure is wholly down, but this procedure is not advised for a beginner. Under field conditions, I have found one of the various aluminium pres- sure cookers now on the market very useful, although only a comparatively small amount of media may be sterilized at one time. Filtration. — The passage of the medium through a filter with pores smaller than bacteria is a possible though tedious process which may have to be used in cases where biologic products are so altered by heat that it is impossible otherwise to retain them in condition to use as media. Asepsis. — The careful excision of bits of plant tissue, under condition of asepsis approaching that obtaining in the operating room of a hospital, with thoroughly sterilized instruments, usually in a special room (culture chamber) where the air is kept relatively free from dust or microorganisms, is sometimes practiced. Such tissues should be incubated for a sufficient time to insure that they have not been accidentally contaminated during their preparation. This method in its simpler form is one of the oldest ways of securing culture media, but is not much used today. CULTURE MEDIA Media may be roughly classified as liquid and solid. The former were much used by the earlier workers, but at present are little used except in certain physiologic studies where solids interfere with chemical procedures and in a few other special cases. They are still employed in studies of spore srermination. CULTURE MEDIA 49 Liquid Media. — The earlier media were mostly naturally occurring liquids, such as tap water, milk, urine, etc., often with one or more other substances added. Physiologic studies then developed a series of solutions of known chemi- cal composition usually consisting of a basal solution containing all the metallic elements needed for growth and one or more organic compounds containing the requisite sources of carbon and nitrogen. Perhaps the solution known as Czapek's or Dox's solution is more frequently used by mycologists. Czapek's basal solution: Distilled water 1000. c.c. K,HPO, 1. gm KCl 0.5 gm MgS0,.7H,0 0.5 gm FeSO, 0.01 gm For other useful formulae see Levine and Schoenlein (1930). Besides a host of formulae of liquid media of definite chemical composition which have been developed in connection with physiologic studies on com- paratively narrow ranges of organisms, there are many, both solid and liquid, in common use, in which one or more of the principal constituents are aqueous extracts. These extracts may be classified as infusions and decoctions. Infu- sions are prepared by allowing the material to be extracted, more or less finely divided, to remain in contact with water, either cold or lukewarm, for a con- siderable length of time. Hay infusion was one of the very early media of this class but has practically disappeared. Meat infusions are still greatly used in the cultivation of bacteria, although much less important in the cul- tivation of most groups of fungi. The following directions may be taken as a sample of this type : IMacerate 1 part finely chopped lean meat with 2 parts distilled water in an ice box for 18 hours, stirring occasionally. Strain while cold through a fine cloth. Add 1.0% peptone and 0.5% NaCl to the filtrate. Heat until solu- tion is complete. Add NaOH until the reaction is slightly alkaline (practically neutral to phenolphthalein). Heat on a water-bath for 30 minutes and boil for 5 minutes over a free flame. Filter while hot through paper or cotton and cloth. Add 1.0% of desired nutrients. Adjust reaction as necessary (see p. 38). For the many variants of this method, consult Levine and Schoenlein. Most of the commonly employed media of this type may now be obtained in dehydrated form. In the preparation of these media the directions furnished by the manufacturer should be followed. Decoctions are usually employed with vegetable substances, as the process is more rapid and rarely are there suflScient proteins to cause trouble. Duggar (1909) proposes that for every 1000 c.c. of water in the decoction the equiva- lent of 50 gm. dry weight should be used. The plant product is washed, peeled if necessary, thinly sliced, and the necessary water added. It is boiled in a steam sterilizer for 2 hours or placed in the autoclave at 115° C. for 20 min- utes, or may be boiled over a free flame for a corresponding period, care being 50 MEDICAL MYCOLOGY taken that the vegetable does not cook on the bottom of the container and that water lost by evaporation is replaced. The decoction is then strained to remove the larger particles of vegetable and may be filtered through paper. Potato decoctions are very difficult to filter, as further precipitation may follow sterilization. Solid Media.^Some of the first media of this class were slices or plugs of vegetables, either raw or cooked (during the sterilization process). Some of these substances, such as string beans, prunes, squash, potato, and root crops, are still used in the study of plant pathogens and similar vegetable products have been used occasionally by isolated workers in the tropics with- out easy access to the more usual types of media. These are frequently very good for producing abundant vegetative growth but less satisfactory for secur- ing reproductive organs. If the vegetable is to be used raw, it must be carefully washed, the out- side thoroughly sterilized by alcohol which is allowed to evaporate or by repeated washing with sterile water in an atmosphere relatively free from dust or spores. Cylinders are then cut out with a sterile cork borer, slightly smaller than the diameter of the test tube to be used, and sliced diagonally. These pieces are then placed either in a special sterile test tube or in a test tube containing one or two glass beads and a small amount of water. If there is no objection to having the vegetable slant cooked, it may be prepared under clean but not necessarily sterile conditions and the whole sterilized together. Glycerol is sometimes added instead of, or in addition to, water in order to insure the surface of the slant remaining moist. Of course the glycerol should also be considered as a possible additional source of carbon. Rarely bits of meat or fish have been sterilized and used directly as media (Sawyer 1930, Rewb ridge. Dodge and Ayers 1929). The other solid media are all colloidal gels, either silicates, proteins, or carbohydrates. Silicates are somewhat difficult to prepare and are at present principally used where it is essential to know definitely the chemical con- stituents of the medium in physiological studies or in the rare cases in which the organism is capable of attacking and digesting the medium. Egg albumen and gelatin are the principal protein media used, mostly in the study of bacteria. Historically, gelatin has been used for a very long time and many of the important early methods and results were obtained with this medium. The directions of Dalmau (1929, 1930) for its preparation are useful, especially for workers in tropical countries. To 900 c.c. nutrient broth add 200 gm. of gelatin and heat in a water-bath until dissolved. The Bacto Gelatin and Pfanstiehl's brand are highly acid, about pH 3 or 4. Adjust reaction to about 6.5 or any desirable pH, let cool to 50° C, add whites of 2 eggs shaken in 100 c.c. nutrient broth, and bring it rapidly to a boil at 100° C. for 10 minutes in a double boiler with saturated salt solution in the lower compartment. The coagulum should clear the solu- tion. Filter through cotton, or cotton and gauze if necessary. Distribute in CULTURE MEDIA 51 tubes and sterilize at 100° C. for 15 minutes on each of three successive days. Cool rapidly after withdrawal from the sterilizer to obtain a hard gelatin, which will not liquefy at tropical room temperatures (28° C). At present it is little used except in studies of the ability of organisms to attack and digest protein. Sawyer (1930) has recently used egg yolk with very good results in his work on the Entomophthoraceae, fungous parasites of insects. The carbohydrate media are chiefly starches and agar. Commercial starches are used, either corn, potato (laundry) or inulin. Ten per cent starch is com- monly used and a colloidal solution obtained by short boiling. The medium is tubed and sterilized with or without the addition of salts or other nutrients. The hydrogen ion concentration of the added material should be carefully considered, as any considerable acidity or alkalinity will hydrolyze the starch, defeating the purpose of the medium by preventing solidification and provid- ing sugar. These media have not been widely used on this account. A variant of this medium, in which com meal mush is prepared, has been employed, al- though perhaps less frequently than the related corn meal agar. The container is partially filled with corn meal which is then thoroughly moistened with hot water (it is difficult to wet it thoroughly with cold water) and sterilized in the usual manner. This medium is never clear as the starch media may be, but it is easy to prepare, has enough of the inorganic compounds and; sources of nitrogen to support growth without further addition, and provides a medium rich in starch. The agars and similar compounds are complex substances which in part hydrolyze to galactose. They are produced during the metabolism of the marine algae, especially the Rhodophyceae (red algae), and comparatively little is known of their chemical structures. The agar of commerce is largely the product of various species of the Gelidiaceae found on the coast of Japan. It solidifies at a comparatively low temperature (about 40° C.) and melts at a very high one (about 95° C). The modern sources of supply have improved the quality very much, so that it seldom contains undesirable salts or nitrog- enous substances. Naturally, for very careful physiologic work, this point would be checked up before using it. Agar may usually be had in shreds, chopped shreds, or in powdered form. The variant methods of preparing agar found in various laboratories give about equally satisfactory final products. Since the medium carries practically no available nutrient, this is added in the form of a solution, infusion or decoction, or a combination of these. The formulae for these are almost infinite, Levine and Schoenlein recording 803 formulae, not counting the numerous variants in proportions and procedures. Of these, one of the simplest and perhaps the most widely used in culti- vating human pathogens is Sabouraud's conservation agar: Water 1000 c.c. Peptone 10 gm. Agar 18 gm. 52 MEDICAL MYCOLOGY Growth is slow on this medinm since the peptone furnishes both carbon and nitrogen, but pleomorphism of the dermatopliytes is greatly delayed. Per- sonally, following a suggestion of Thaxter who had long experience with other groups of fungi in culture, I prefer to add 40 gm. of agar instead of 18 gm. to media to be used for stock cultures. Growth is extremely slow and the cultures do not dry out so rapidly, both of which conditions are advanta- geous with routine stock cultures, as the labor of preparation of media and of transfer is greatly reduced. For isolation and study of the organisms, Sabouraud's test agar (1908), commonly called Sabouraud agar, contains 40 gm. of crude maltose. Sabour- aud has been severely criticized for using the crude sugar, as samples vary greatly, one sample used by Sabouraud on analysis yielding mostly glucose (Hodges 1928). For most organisms glucose gives equally good growth, in fact pure maltose is generally poorer. There seems little difference whether 10 or 40 gm. per liter are added. The firm from which Sal)ouraud secured his maltose ceased business during the World War (1914-1918) and since then, much research has been expended to secure a substitute product which will produce the same giant colonies as those figured by Sabouraud (1910). Sab- ouraud himself (1925) advocates the use of 8% honey, but again introduces a source of error, since honey varies very much according to the species of bee producing it and the flowers from which it is made; e.g., Berde (1926) failed to secure characteristic colonies on honey made from flowers of Rohinm or Stachys annua. Weidman «& Macmillan (1921) and Weidman (1928) in very extensive studies found that crude glucose gave equally good results, Fairchild's peptone being used instead of Chassaing. This medium is often referred to as the Pennsylvania medium. Goldschmidt (1924) proposed the following for English laboratories: Glucose 40 gm. Agar 20 gm. Peptone (Fairchild) 10 gm. Lemco (ordinary not laboratory) 5 gm. NaCl 5 gm. Tap water 1000 c.c. Lemco is an acid meat extract. Ingredients digested in a steamer for 1 hour, adjusted by "soda" to pH 6, sterilized 20 minutes each on 3 successive days. Gruetz (1923) proposed the following for German laboratories: Peptone (Knoll) 5 gm. Nervina Malz from Christiansen, Flensburg 80 gm. Agar IS gm. Water 1000 c.c. Pollacci (1922) proposed the following for Italian laboratories: 500 gm. ground beef in 1000 c.c. water; cook and filter; add 100 gm. Witte peptone, 5 gm, sodium chloride ; heat, filter, warm, and neutralize ; heat for half an CULTURE MEDIA 53 hour, filter, and add 70 gm. glucose and agar. Bruhns (1928) finds this medium superior to that of Gruetz. The medium proposed by Macleod (1928) for the cultivation of Malassezia furfur is quite similar: Agar 1.5 gm. Peptone (Cliassaing) 2 gm. Glycerol 2 c.c. Glacial acetic acid 1 m. Distilled water 100 c.c. Grigorakis (1931) obtained interesting results with 40 gm. of glycerol added to Sabouraud's conservation agar. Benedek advocates 8% crude glucose and peptone (both Merck products). Farley (1920) adds 3-5% human blood heated to 55° C. for 30 minutes to Sabouraud's test agar when cultivating Epider- mophyton. Gentian violet 1 :500,000 is a useful addition to restrain bacterial growth during the isolation of these organisms. I have found the prepared medium of the Digestive Ferments Company satisfactory as Avell as media prepared from ingredients furnished by them. Their peptone is too near neutral to reproduce exactly the giant colonies figured by Sabouraud (1910). On the other hand, there is the advantage of greater uniformity of different batches, something greatly to be desired when comparing results secured at different times. Recently Langeron & ]\Iilochevitch (1930) and others have returned to the cereal and dung media, of variable composition, but have secured inter- esting morphology not produced on the classic Sabouraud medium and its numerous variants. Nannizzi (1926), on the other hand, turned to equally variable animal products, such as bits of skin, nail clippings, horn, feathers, hair, and bone. In this direction the work of Karrenberg (1933) seems to be much the most promising. Using the brain medium of Hibler which was originally developed for anaerobic bacteria, he has maintained stock cultures over very long periods without pleomorphism or loss of virulence. While I have had no personal experience with this medium, it seems so promising that the details of its preparation may be given: Brains of recently slaughtered animals (within 24 hours) are freed from the pia mater, ground in a meat chopper and weighed. Two parts water to one of brains are added, rubbed through a sieve, and cooked for two hours in a steam sterilizer. The next day the medium is tubed and sterilized in the autoclave. Hach and Karrenberg both suggest 0.85% sodium chloride solution instead of tap water and Karrenberg found the medium .satisfactory without rubbing through a sieve. Independently Grigorakis (1933) has proposed a similar medium based on calf spleen. Pulp of calf spleen 500 gin. Peptone 10 gm. Agar 18 gm. Water 1000 c.c. 54 MEDICAL MYCOLOGY Many workers have reported excellent results with various fungi on car- rot agar. The following formula of Falchi may be considered typical: Carrots 500 gm. Water 1000 c.c. Agar 20 gm. Peptone (Rostock) 10 gm. Among the formulae for synthetic media, those of Currie (1917) and Fulmer & Grimes (1923) are widely used. Currie : Ammonium nitrate 2.5 gm. Potassium dihydrogen phosphate 1.0 gm. Magnesium sulphate 0.25 gm. Carbohydrate 10.0 gm. Water 1000.0 c.c. Agar q.s. Fulmer & Grimes: Ammonium chloride 1.88 gm. Dipotassium hydrogen phosphate 1.0 gm. Calcium chloride 1.0 gm. Sucrose 50.0 gm. Agar 15.0 gm. Water 1000.0 c.c. Both formulae have been used in studies of yeasts and fermentation. Acton & McGuire (1931) report very good results with Actinomyces from the medium described by Norris (1929). It consists of: Soluble starch Dipotassium hydrogen phosphate Calcium chloride Ferric chloride Sodium nitrate Asparagin Agar Water The medium is adjusted to pH 7.4. BIBLIOGRAPHY Biltris, R. 1929. Sur la variabilite des caracteres de I'espece chez les dermatophytes, Ann. Inst. Pasteur. 43: 281-358, 15 figs. *Bizio, Bartolomeo. 1823. Lettera di Bartolomeo Bizio al chiarissimo canonico Angelo Bellani sopra il fenomeno della polenta porporina, BiMioteca Ital. o sia Giornale di Letteratura Scienze e Arti Appendice 30: 275-295 [anno 8] [translated by C. P. Merlino, Jour. Bad. 9: 527-543, 1924]. Bruhns, C. 1928. Einige Bemerkungen iiber verschiedene Pilzarten und Pilznahrboden (Griitz agar, PoUacci agar), Dermatol. Zeitsch. 53: 104-112, 4 figs. Castellani, Aide. 1933. The advisability of using in laboratory work sugars tested by micro- biological methods. Jour. Trap. Med. Hyg. 36: 185, 186. 2.0 gm, 0.2 gm. 0.05 gm. 0.01 gm, 0.06 gm. 0.05 gm. 20.0 gm. )00.0 c.c. CULTURE MEDIA 55 Coim, H. W. & Harold J. Conn. 1923. Bacteriology, a study of microorganisma and their relation to human welfare, discussing the history of bacteriology, the nature of microorganisms, and their significance in connection with pathology, hygiene, agricul- ture and the industries, Williams & Wilkins Company, Baltimore, 441 pp. Currie, James N. 1917. The citric acid fermentation of Aspergillus niger. Jour. Biol. Chem. 31: 15-37, Pis. 1, 2. Dalmau, Luz Maria. 1929, 1930. Observations on mycologic technique with particular refer- ence to pathogenic fungi, Porto Rico Jour. Public Health Trop. Med. 5: 302-311, 1930 [tr. Maurice Langeron, Eemarques sur la technique mycologique, Ann. Parasitol. Hum. Comp. 7: 536-545, 1929]. Descouraux, J. M. C. M. 1926. Contribution a 1 'etude de nouveaux milieux de culture pour les dermatophytes, These. Fac. Pharm.. Univ. Strasbourg 24: 72 pp., 2 pis. Desgardes, DeribSre. 1921. Formulaire des milieux de culture en microbiologie, Paris, Le Francois, 98 pp. Dessy, G. 1933. La chimiotherapie des mycoses. III. Mucormycose 1, Experiences in vitro, Soc. Internaz. Microbiol. Boll. Sez. Ital. 5: 95-107; 2, Experiences in vivo. Ibid. 5: 201-206. Duggar, Benjamin Minge. 1909. Fungous diseases of plants, with chapters on physiology, culture methods and technique, Boston, Ginn & Company, 508 pp. Egyedi, Henrik. 1922. Zur Eeinkultivierung der pathogenen Schimmelpilze, Centralbl. BaTct. I, 87: 562-564. Farley, David If. 1920. The use of gentian violet as a restrainer in the isolation of patho- genic molds. Arch. Derm. Syphilol. 2: 459-465, £ figs. Fulmer, E. I. & M. Grimes. 1923. The growth of yeasts on synthetic agar media, Jour. Bad. 8: 585-588. Goldschmidt, W. N. 1924. A new medium for the growth and differentiation of the dermato- phytes, Brit. Jour. Derm. 36: 204-206. Grigorakis, L. 1931. L 'action des milieux glycerines sur le mycelium degrade par le pl6o- morphisme, C. R. Soc. Biol. 108: 94-96. — . 1933. Sur un nouveau milieu de conservation des dermatophytes (plfiomorphisme, car- actfire acquis, specificite tissulaire, C. R. Acad. Sci. 196: 60-62. GriitE, O. 1923. Beitrage zur Kultur der Dermatophyten und ihrer Artunterscheidung mittels deutscher Pilznahrboden, Derm. Wochenschr. 76: 568-573. Hedges, JB. S. , 1928. Cultures of ringworm fungi on Sabouraud's proof mediums and on mediums prepared with American peptones and sugars, Arch. Derm,. Syphilol. 18: 852-856. Karrenberg, C. It. 1933. Untersuchungen iiber Wachstum und Konservierung pathogener Hautpilze auf Hirnbrei nach v. Hibler, Arch. Derm. Syphilis 168: 438-475, 10 figs. Kingery, Lyle B. 1929. Thymol and cinnamon oil in the treatment of ringworm of the scalp, Arch. Derm. Syphilol. 20: 797-805. Kingery, Lyle B. & Alva Adkisson. 1928. Certain volatile oils and stearoptens as fungicides, Arch. Dermatol. 17: 499-511, 9 figs. Langeron, Maurice & S. Milochevitch. 1930. Morphologie des dermatophytes sur les milieu naturels et milieux a base de polysaccharides (Note preliminaire), Ann. Parasitol. Hum. Comp. 8: 422-436. Levine, Max & H. W. Schoenlein. 1930. A compilation of culture media for the cultivation of microorganisms, Monographs on Systematic Bacteriology 2: xvi + 969 pp. Macleod, J. M. H. 1928. An experimental study of the Pityrosporon of Malassez: its morphology, cultivation and pathogenicity, Brit. Jour. Derm. Syphilis 40: 139-148. Nannizzi, Arturo. 1926. Eicerche sui rapporti morfologici e biologici tra gvTiinoascacee e dermatomiceti, Ann. Myc. 24: 85-129, 6 pis., 12 figs. 56 MEDICAL MYCOLOGY Pautrier, L. M. & B. Eietmann. 1924. Trichopliytie cutanee a forme d 'epidermophytie dues au Trichophyton granulosum, et provoquee par une infection de laboratoire, Bull. Soc. Franc. Derm. Syphiligr. 31: Eeunion Strasbourg, 29-31. Pollacci, Gino &) Arturo Nannizzi. 1922. I niiceti patogeni dell 'uomo e degli animali descritti, delineati e preparati per I'osservazione al luicroscopio con notizie sopra i rimedi per combatterli. 1: prefazione. Siena. Eewbridge, Allan G., Carroll William Dodge & Theodore T. Ayres. 1929. A case of menin- gitis due to Endomyces capsulatus (new species), Am. Jour. Path. 5: 349-364, Pis. 71-73. Sabouraud, Kaimond. 1908. Milieux de culture des champignons dermatophytes (Technique de fabrication des geloses sucrees dites: Milieux d'epreuve), Ann. Derm. Syphiligr. IV, 9: 99-101. — . 1925. Note sur le remplacement de tout sucre dans les milieux de culture des dermato- phytes par le miel d'abeilles, Ann. Derm. Syphiligr. "VI, 6: 515-517. Sawyer, William H. 1929. Observations on some entomogenous members of the Entomo- phthoraceae in artificial culture, Amer. Jour. Bot. 16: 87-120, 4 pis. Weidman, Fred D. 1928. Comparison of ringworm culture ingredients. II. The nitrogen factor, Arch. Derm. Syphilol. 18: 829-837. Weidman, Fred D. & Thomas M. Macmillan. 1921. A comparison of ingredients of ring- worm culture mediums with special reference to American and French crude maltose, Arch. Derm. Syphilol. 4: 451-468, 16 figs. Weidman, Fred D. & Dorothy Spring. 1928. Comparison of ringworm culture ingredients. m, Griitz, Goldschmitt, Sabouraud (honey modification), Sabouraud 's glycerin and certain synthetic mediums, Arch. Derm. Syphilol. 18: 837-850. CHAPTER IV ISOLATION OF MICROORGANISMS Transfer. — This is perhaps the simplest process connected with the hand- ling- of organisms in culture and should be practiced by the beginner until the operations can be performed quickly and entirely without contaminations before more complicated procedures are attempted. Essentially the process consists in taking a small amount of mycelium or spores of fungi or a few bacterial cells and moving them from one receptacle of medium to another. The test tube containing the culture of the organism to be transferred and a tube of medium are held about half an inch apart between the thumb and fingers of the left hand with the thumb above. The plugs of both tubes are loosened in turn Avith a rotary motion, but not withdrawn. The transfer needle, loop, or platinum spatula, as the case may be, is grasped between the thumb and first finger of the right hand, the platinum is heated to redness in a blue flame and allowed to cool while still in the hand and without the heated por- tion touching anything. The cotton plug of the tube containing the organism to be transferred is grasped between the second and third finger of the right hand and gently withdrawn, care being taken to create as few air currents as possible. A needle or loop is inserted without touching the walls of the tube and touched to the spores or to the vegetative material if it is slimy, or a platinum spatula is used to cut away a little mycelium which should adhere to it. The needle is then gently withdrawn, the plug replaced and the other plug withdrawn, and the needle stroked along the surface of the agar (or the mycelium dislodged from the spatula). The needle is then gently withdrawn, the second plug replaced, and the needle (or spatula) flamed before laying it down, to destroy any organisms still adhering to it. The plugs may then be pushed in more firmly if necessary. In some laboratories it is customary to flame and quickly extinguish the cotton plugs. Isolation. — The simplest procedure is similar to simple transfer in which the needle or spatula is touched to the infective material and then transferred to the surface of agar in a test tube or Petri dish. This method is only occa- sionally successful, usuallj^ in those cases where the infective material is small and uncontaminated by other organisms than the one which it is desired to study. If contamination is only slight, a carefully executed transfer from the colony Avliich is desired may result in securing a pure culture. Otherwise, resort must be had to dilution and plating out. Isolations From Skin Lesions. — After cleansing the skin with 95% alcohol followed by ether, the lesion is scraped with a sharp, full-bellied, sterile scalpel or shaved with a sterile safety razor to the point where bloody serum just begins to ooze. A sterile Petri dish is placed beneath to catch the scrapings, 57 58 MEDICAL MYCOLOGY or, if they do not fall away readily, they are scraped off the blade into the Petri dish by another blade. On reaching the laboratory, a few scales are removed for microscopic examination after maceration in 40% KOH or some other similar treatment. Then a very thin film of Sabouraud maltose or glu- cose agar is poured into the plate containing the scales and detritus. Thus, the latter are caught by the nutrient medium and held. To reduce chances of bacterial contamination the scales may be moistened with alcohol for a short time, but this does not prevent the growth of some of the hardy saprophytes and may inhibit the growth of the more delicate pathogen. As soon as the colonies are visible to the naked eye, the suspicious ones are marked with a glass-writing pencil, each being given a serial number. The marked colonies are then transferred to Sabouraud 's sugar medium on slants and to the conservation medium (without sugar). This transfer is made early to prevent overrunning by the rapidly growing contaminants, such as Aspergillus and Penicillium, which may be present. Isolations From Feces, Tongue Scrapings, etc, — Poured plates are allowed to harden and then 25 points of contact are made in each with a platinum loop repeatedly soiled with the infective material. After about four days, when the colonies have developed, these are fished. In this way the percentage of points of contact of material with the medium which contains similar colonies gives a rough idea of the abundance of colonization in the material. (Dalmau 1930.) In connection with their studies with sprue, Weiss & Landron (1928) sug- gest as follows: Emulsify a small quantity of feces in a test tube of sterile distilled water and also in another tube containing whole ox bile in which has been incorporated 20% concentration of glycerol. By means of a glass rod bent at an angle of 30° a drop of each fecal suspension is spread over the surfaces of the plates of Sabouraud agar (pH 6.3) containing 4% glucose and 20% glycerol ; incubate at 35° C. Dilution. — About three tubes of agar (or other liquefiable medium) are heated gently on a water-bath (porcelain or glass beaker) until the agar melts. It is then allowed to cool until the end of the tube containing the agar can be held against the back of the hand without causing pain, i.e., until the solidifying point is almost reached. The tube is inoculated by the method suggested in simple transfer. After laying down the needle, the tube is rapidly rolled between the palms, while it is in a vertical position, to mix the contents thoroughly and scatter the inoculum. The loop is used to transfer a tiny drop of the molten medium to the second tube two or three times. This in turn is rotated, etc. The contents of the three tubes are then poured successively into three Petri dishes lying on a level surface. If the agar fails to wet the surface of the dish completely, the dish is tipped slightly to allow the agar to flow over the whole surface of the bottom. It is then allowed to solidify and is incubated bottom side up until growth is evident. Then individual colonies are transferred to slants. Sometimes it is necessary to repeat this process. In the above-mentioned processes it is desirable to work gently in ISOLATION OF MICROORGANISMS 59 order to set up as few air currents as possible, never to lift the cover of the Petri dish higher than necessary to insert the test tube for pouring, and to keep the test tubes plugged as much as possible. As soon as the agar is poured from the tubes, they should be lowered into a dish of water and boiled as soon as possible, both to kill the organism which may have adhered to the agar still in the test tube and to clean them before the agar has a chance to dry on. Inhibitors. — To keep back the rapidly growing organisms and allow the slower growing ones to develop, various substances may be added to the medium first used in isolation. Advantage is often taken of the fact that some groups of fungi grow at different hydrogen ion concentration from others. In these methods, varying small amounts of lactic or other organic acids are added to the medium to inhibit the growth of bacteria. Sometimes dyes are also used for this purpose, e.g., "gentian violet" (probably methyl violet) 1:500,000 (Farley 1920) or to indicate the presence of a small colony before it has developed sufficiently to be seen otherwise in order that it may be fished before it has been overgrown by a more rapidly growing organism. Indicators are very useful in this way if the organism one desires to isolate produces acid or alkali in the medium. Usually these special methods have been developed in connection Avith an intensive study of a single organism and are rarely useful unless the presence of a given organism is strongly suspected. Slight amounts of organic acids are useful, however, in keeping down rapid bacterial growth while waiting for the more slowly growing fungi to develop. Similarly, the choice of suitable media may do much to favor selectively the development of one organism while retarding another. No general rules can be given for these choices since they are largely the result of wide experi- ence and a shrewd guess as to the probable organism to be isolated. A thor- ough knowledge of the physiology of the various groups of fungi will be helpful, but in the present state of our knowledge generalization is very difficult. Microcultures. — In the study of the life cycle of many organisms it be- comes desirable to have a given spore or bit of mycelium under more or less continuous observation with the microscope. One of the early methods which has yielded much useful information is the hanging drop culture. A.n early and inexpensive form, usually referred to as a Van Tieghem cell, consists of a glass ring cemented to a microscopic slide with wax (made by melting to- gether pure beeswax and vaseline). The top of the ring is coated with vase- line. A drop of the culture medium or water is placed in the bottom of the cell thus formed. Another smaller drop is placed upon a clean cover slip of sufficient diameter to cover the ring. The inoculum is then placed in the center of this drop, and the whole seized by forceps and quickly inverted, care being taken that the drop does not spread too near the edge of the cover dur- ing the process. The cover (with the drop of medium or water hanging from it) is then lowered to the glass ring and pressed down gently until the soft vaseline (petrolatum) seals it to the ring. Thus we have a small moist chamber with the organism suspended in a drop from the cover. The drop placed in 60 MEDICAL MYCOLOGY the bottom of the cell prevents drymg out, since its vapor pressure is prac- tically the same as that of the hanging drop. The cell may be placed on the stage of a microscope and studied from time to time until the nutrient material in the drop is exhausted. One should be careful not to have the drop too large, since it will be difficult to focus to the bottom of it if the spore under observation should not be thoroughly wetted and lies in the surface layer of the drop, or is so heavy that it falls to the bottom of the drop. To avoid this, sometimes a thin film of agar is used instead of a liquid drop, in which case distilled water is usually placed in the bottom of the cell to prevent drying out. Sometimes a very young colony with a small amount of the surround- ing agar is cut out and placed on the cover glass, making a hanging block culture described by various authors including Dalmau (1929-1930). Studies made by these methods are especially valuable in following the early stages in the development of a spore or in the evolution of the life cycle. Various types of hollow-ground slides, etc., have been developed, but for convenience and general use, they have little advantage over an ordinary Van Tieghem cell. Giant Cultures. — At the other extreme from the microculture, we may use giant cultures. These colonies which are allowed to develop over a long period on an abundant supply of substrate, are very useful in giving gross morphology on various media and often are strikingly characteristic in ap- pearance for different species. They have had little favor in most labora- tories, as they must be allowed to grow from one month to two or three before this character can be ascertained. However, they have been utilized with very excellent results by Sabouraud and others in the dermatophytes and by Lindner in the yeasts. Since, under ordinary conditions a Petri dish is too shallow to hold sufficient medium, and allows it to dry out too readily and also to be subject to contamination, various specialized culture dishes have been devised. Perhaps the Roux flask, of which there are several types on the market, is the best known, although somewhat expensive. Ordinary cultures in Erlenmeyer flasks are satisfactory for most purposes, but they do not admit of either microscopic examination or photography without breaking the flask, which is frequently difficult to do without injury to the colony. To obviate this for photographic purposes, Shrewsbury (1931) suggests growing them in green glass medicine bottles of 10-12-ounce capacity (with flat sides if possible). The bottle may be evenly broken by application of a red hot nail along the sides. These bottles are also useful for growing cultures under diminished pressure since the glass is strong enough to resist almost complete exhaustion. He also suggests their use in exposing the inverted colony to fumes of various antiseptics, such as ethyl iodide. Karrenberg has suggested two ingenious devices which permit giant colonies to be photographed or studied with the low power of the microscope with a minimum chance for contamination. In 1926 he suggested an inner test tube carrying an agar slant from which the side had been removed. This was stored in a somewhat larger test tube plugged with cotton. When it is desired to photograph or to examine the colony, the inner tube may be easily ISOLATION OF MICROORGANISMS 61 removed and if care is taken to work in a relatively dust-free atmosphere, often several examinations may be made before the colony becomes contami- nated. The size of the colony is rather limited in this device. In 1927 he proposed a more elaborate flask. It is essentially a flask of the Erlenmeyer type, made in two pieces, a lower portion to hold the medium and a cover fitting over it rather more closely than the usual Petri dish cover. The pieces are held tog-ether by a metal plate underneath and a ring around the neck of the flask connected by three springs which hook into the upper ring to provide sufficient compression to prevent contamination. The flask is manipulated as an ordinary flask, the neck being plugged with cotton. After the colony is grown, the springs are unliooked from the ring and the top is lifted off. If care is taken in the examination, little contamination results. So far as I am aware, this type of flask has not been placed on the market. Sing^le Cell (Spore) Cultures.— Finally, there comes a time in the study of many fungi when the results of a study of cultures made by the above- mentioned methods are ambiguous, and it becomes desirable to work with mycelium and spores produced by a single spore in order that problems of sexuality or homothallism and heterothallism may be investigated, or that the relationships of apparent stages in a life cycle may be studied and verified. The problem has been variously met by different investigators, depending somewhat on the size and nature of the spore to be isolated and the instru- ments available for the work. If the spores are large and the hand is very skillful, it may be possible to pick up a single spore on a needle under the low power of the microscope and transfer it to a sterile tube or a hanging drop. Various mechanical devices have been developed to aid in this work, e.g., the old Barber spore picker or pipette or some of the modern micro- manipulators used in microdissection studies. In these devices, motion is secured by means of micrometer screws, and the spore is usually sucked into the end of a tiny pipette made by drawing out a piece of glass tubing to an inside diameter only slightly larger than the spore or cell to be isolated. This is then discharged into a hanging drop or a sterile culture tube. For detailed directions, those accompanying the instruments should be consulted. Ascospore Detection. — Since classification is based primarily upon the spore forms resulting from the sexual act (caryogamy), every effort to secure the sexual or perfect stage should be made before relegating an organism to the large heterogeneous group known as the Fungi Imperfecta There is no single method which is equally successful for all organisms, nor even for mem- bers of a single group. Perhaps patience is the first requisite. Frequently one finds evidence of sexuality by a diligent search of a colony 3-4 months old, after the agar has begun to dr5\ This seems very useful in the filamentous yeasts whose im- perfect stage is usually placed in the genus Monilia. Some media seem better than others, but usually a careful search will show traces of sexuality on most media upon which the organism has made a good growth. This method is very tedious, especially among pathogens when the physician wishes a prompt 62 MEDICAL MYCOLOGY diagnosis, at least to a genus name, and seems puzzled if the mycologist promptly reports that he has a species of Monilia and 2 or 3 months later changes his report to Zymonema. Among the true yeasts, several methods are in vogue, none of which is uniformly successful, but all of which may sometimes produce results. All should be tried before placing an unknown organism in the large and poorly defined genus Cryptococcus. Stelling-Dekker (1931) has summarized these methods and in most cases traced the method to the original author. The oldest method is that of Engel (1872), popularized by Hansen (1883), where the cells from an actively growing colony are scraped off and placed on a sterile block of plaster of Paris (gypsum) which is kept moist by sterile water, or malt extract (Klocker 1924) or by mannitol-phosphate solution (18 c.c. 2% mannitol + 2 c.c. 5% dipotassium phosphate) (Saito 1923). Gorodkova (1908) reported the use of an agar rich in nitrogen and poor in carbohydrate which usually bears her name. Distilled water 1000 c.c, agar 10 gm.,* peptone 10 gm.,* beef extract 10 gm., sodium chloride 5 gm., and glucose 2.5 gm. Various French authors, notably Guilliermond, have advocated the use of slices of carrot or potato. Beijerinck(1898) advocated the use of plain agar to which no nutrient had been added and which had been thoroughly washed to remove impurities which might possibly be a source of food. Wagner (1928) has made a more thorough study of conditions initiating ascospore formation. He emphasizes the importance of the sugars previously used in cultivating the organism and the hydrogen ion concentration of the medium. Kufferath (1928) attributes the success of his medium to its alka- linity. He prepares it as follows : Malt meal is hydrolyzed with sulphuric acid, the acid neutralized with calcium carbonate, and the agar added. It is then brought to the desired alkalinity with sodium hydroxide. In 1930 he studied the matter further. He found that in general the usual concentration of gelatin (15%) is as successful as higher concentrations. He studied the effect of alkalinity and found it rather more successful than acidity in producing ascospores, but occasionally the reverse is true. Before one can be certain that spores are not formed, one should try all the methods. Fennentation. — Another character to which some authors have attached much importance in some groups is ability to ferment or to utilize certain sugars. The term "fermentation" is used very loosely by various writers. Some, as Stelling-Dekker, would practically restrict it to the production of alcohol and carbon dioxide from a hexose, while Castellani would include all cases in which acid or gas appears in a carbohydrate-containing medium on which an organism has developed. It is probable that this different use of the term has occasioned much difference of opinion in regard to the fermenta- tive ability of a species. A further source of error is almost inherent in the methods in ordinary use, each of which indicates an equilibrium of several possible reactions, hence it is important to state clearly what method was used •Maneval (1924) recommends the omission of peptone, addition of 15 or 20 gm. agar, and reduces Liebig's meat extract to 3 gm. ISOLATION OF MICROORGANISMS 63 in determining fermentation if this character is to have meaning in the separa- tion of species. In the following discussion of methods, an attempt will be made to point out some of the sources of error and objections raised to each method. As in many other organic reactions, details of method often pro- foundly influence the point of equilibrium. Lindner's Microfermentation Method. — A hollow-ground microscopic slide is flamed and filled with sterile water. A relatively large number of yeast cells is suspended in the water and a pinch of the sugar to be tested is added. A cover glass is carefully lowered so as to exclude any air bubbles and sealed in place with lanolin or vaseline. This is placed in an incubator for 24 hours at the optimum temperature and the presence of bubbles (supposedly of car- bon dioxide) noted. In this method it is assumed that there will be com- paratively little growth and consequent production of CO2, due to absence of oxygen and the lack of nutrients. The amount of gas should be roughly proportional to the amount of the inoculum. If the organism ferments very slowly and a larger quantity of inoculum is used, the amount of glycogen trans- ferred with the yeast ceUs and that diffusing out of the dead cells may be sufficient to give gas production. Since the sugar is not sterilized, there is always a possibility of introducing some fermenting bacterium. Also there is a possibility that the seal is not tight and that subsequent evaporation may give the appearance of gas production or may allow the gas to escape. Changes of temperature or of atmospheric pressure may also give erroneous results. Guilliermond modified the method by filling a Van Tieghem cell with a rela- tively large volume of liquid, and dissolving the sugar in a definite concentrar tion in a yeast decoction. This modification presupposes that the yeast may grow, and consequently the COg may be partly the result of respiration rather than fermentation in the strict sense of the word, especially as there may be dissolved oxygen in the liquid. The Fermentation Tube Method. — In this method, bent tubes of varying pattern, perhaps originally used by Einhorn in 1885 but introduced into mi- crobiology by Theobald Smith in 1890, are filled with a sugar solution and sterilized. The amount of liquid should be sufficient to slightly more than fill the long arm but not so much as to wet the plug. This is inoculated with the organism and set aside in an incubator until the organism has grown long enough to develop gas. If the long arm is graduated, the volume of gas pro- duced may be noted. Since the surface of the liquid in the long arm is not exposed to free oxygen, strict anaerobic conditions are maintained. Also, if the organism is strictly aerobic, it grows only in the short arm, and there is not enough growth in the long arm to show fermentation, even if the organ- ism is capable of producing it. On the other hand, since the carbon dioxide is quite soluble in the solution, small amounts are dissolved and do not show up. There is also the problem as to the source of the gas, whether it is from fermentation or respiration. Aichelburg (1932) reports that gas production in fructose shows on the third day while glucose shows on the first day. 64 MEDICAL MYCOLOGY The ideal method would be one in which the reaction was studied quan- titatively in special apparatus, a condition not attained except in purely physiologic researches (cf. Kluyver 1914). Since comparatively few organisms ferment hexoses to carbon dioxide and alcohol, in the majority of cases we are really interested rather in the ability of the organism to attack and utilize sugars than in their fermentative ability in the narrow sense of the term. Hence, a quantitative titration of the sugar medium for the presence of accumulated acid is equally useful. In fact, in the majority of "fermentations" mentioned for Monilia by Castellani, the production of acid rather than of alcohol is meant. Probably this has been done very roughly, since the author rarely mentions the indicator used and never the relative amount of sugar converted to acid in a unit of time by a definite number of organisms per cubic centimeter. Stelling-Dekker sug- gests that more quantitative methods are useful, especially in the case of a trisaccharide in which several organisms secrete raffinase which by hydrolysis splits the rafSnose to mellibiose and fructose and is able to ferment the fructose so produced but not to hydrolyze the mellibiose. Aichelburg (1932) reports that slight acidity shows after one week with inulin but only after two weeks with starch. For further consideration of the problems involved, especially of the chemical reactions, the reader should consult some of the standard works on biochemistry, or special monographs on alcoholic fermentation, such as that by Harden. Maltose, fructose, and glucose are quite regularly fer- mented by many species of yeasts while galactose, sucrose, and dextrin are more variable. Inulin, raffinose, and mannite are often attacked by certain species and should be tried in placing a strange Monilia. Lactose is attacked by comparatively few fungi, but when it is attacked the amount of fermenta- tion is apt to be large. Most of the other sugars are too rarely fermented and too expensive to be used in most routine work. Ashford has shown that ultraviolet light may destroy the normal fermenting power of Syringospora psilosis (Monilia psilosis). Sometimes characteristic deposits may be evident upon cultivation in connection with fermentation studies. Ashford 's laboratory usually tests fer- mentation on 1-4% concentration of the sugar in peptone water, although nutrient bouillon may be used. The pH is adjusted to about the neutral point, and changes of acidity or alkalinity are noted. Animal and Human Inoculations and Recovery of Organisms From Lesions. — The methods used for inoculations, both of animals and human volunteers, are too well known by the medical profession to need discussion here, and have little value in the hands of an experimenter without a good medical training. The necessity of a thorough sterilization of the skin cannot be too strongly emphasized, for mold spores from dust or clothing may be picked up as a contaminant and, in some cases, even be considered as the etio- logic agent. Perhaps the advice of Erwin F. Smith needs especial emphasis in this connection. "I now endeavor to repeat all my own experiments several times over and in the end I have a rounded out and better view than the one ISOLATION OF MICROORGANISMS 65 series only could possibly give me. Incidentally, I usually succeed in eliminat- ing some errors or half truths which appertained to the first experiment." BIBLIOGRAPHY Beijerinck, M. W. 1898. tJber Eegeneration der Sporenbiklung bei Alkoholhefen wo diese Funktion im Verschwinden begriffen ist, Centralbl. BoM. II 4: 657-663, 721-730, 1 pi. Benedek, Tibor. 1926. tJber directe mikrospische Beobachtung voa Eeagensglas Pilzkulturen und ihre Verwendungs mogliclikeiten, nebst Angabe von neuen Mikroskop Objekt Tisch-Klemmen fiir Mykologische Arbeiten, Derm. Woch. 82: 1-5. Dalmau, Luz Maria. 1929, 1930. Observations on mycologic technique with particular refer- ence to pathogenic fungi, Porto Eico Jour. Public Health & Trop. Med. 5: 302-311 [tr. M. Langeron, Remarques sur la technique mycologique, Ann. Parasitol. Hum. Corny. 7: 536-545, 1929]. Dekker, N. M. Stelling. 1931. Die Hefesammlung des " Centraalbureau voor Schimmelcul- tures. " Beitrage zu einer Monographie der Hefcarten. I. Die Sporogenen Hefen, Verhandel. K. Akad. Wetensch. Amsterdam. Afdeel. NatuurT:. 28:1; 1-547, illus. Engel, L. 1872. Les ferments alcooHques, These Paris 336: 1-62, 1 pi. Farley, David L. 1920. The use of gentian violet as a restrainer in the isolation of pathogenic molds. Arch. Derm. Syphilol. 2: 459-465, 2 figs. Gilchrist, T. Caspar & William Eoyal Stokes. 1898. A case of pseudolupus vulgaris caused by Blastomyces, Jotir. Exp. Med. 3: 53-78, Fls. 4-8. Gorodkova, A. A. 1908. O bystrom poluchenii spor u drozhzhevykh gribov, Bull. Jard. Imp. St. Peterslourg 8: 165-170. Grigorakis, Leonidas. 1933. Sur un nouveau milieu de conservation des dermatophytes pleomorphisme, caractere acquis, specificite tissulaire, C. E. Acad. Sci. 196: 60-62. Hansen, Emil Christian. 1883. Unders0gelser over alkoholgjaersvampenes fysiologi og morfologi, Meddelelser Carlsherg Laboratoriet 2: 29-102; 152-210; 220-256 [in French 13-59; 92-136, 142-157]. Ges theor. Abhandl. u. Garungsorganismen, 125- 169, 1911. Karrenberg, Karl Ludwig. 1926. Ein neues Rohrchen der Ziichtung von Dermatophyten und Bakterien, Derm. Zeitschr. 49: 248-252. — . 1927. Ein neuer Kolben zur Ziichtung und Aufbewahrung von Pilzkulturen, Derm. Woch. 85: 1706-1708. — . 1927. Die Eignung der von Griitz angegebenen Nahrboden zur Bestimmung und Weiter- ziichtung der Dermatophyten, Derm. Woch. 84: 434-439. — . 1933. Untersuchungen iiber Wachstum und Konservierung pathogener Hautpilze auf Hirnbrei nach v. Hibler (Vorlaufige Mitteilung, Arch. Derm. Syphilis 168: 438-475, 10 figs. *Klocker, A. 1924. Die Garungsorganismen ed. 3. *Kluyver, A. J. 1914. Biochemische Suikerbepalingen. Delft. *Kufferath, H. 1928. Ann. Soc. Zymologie 1: 214. * — . 1930. Ann. Soc. Zymologie 2: 33. Mallinckrodt-Haupt, Asta St. v. 1926. Vitalfarbung niit Indicatorfarben bei Hyphomyceten, I, Derm. Zeitschr. 46: 293-305. Maneval, W. E. 1924. A method of securing spores of yeasts, Bot. Gaz. 78: 122, 123. Saito, Kendo. 1923. Beschreibung von zwei neuen Hefearten nebst Bemerkungen iiber die Sporenbildung bei Torulaspora, Delbriicki Lindner, Bot. Mag. Tolcyo 37: 63-66, 2 figs. Shrewsbury, J. F. D. 1931. Giant colony culture, Jour. Path. Bact. 34: 283-285, PI. 15. Wagner, Felix. 1928. Der Einfluss der Zuckerarten und der Wasserstoffsionenkonzentration auf die Sporulation der Saccharomyceten, Centralbl. BaM. II 75: 4-24, 5 figs. Weiss, Charles & Francisco Landron. 1928, 1929. Immunological investigations of tropical sprue in Porto Eico, 1-3. Am. Jour. Trop. Med. 9: 83-95, 1929. 4. Biology of Monilia psilosis in relation to sprue, Jour. Infect. Dis. 43: 557-564, 1928. CHAPTER V MICROSCOPY BY MORRIS MOORE To one acquainted with the cultural characteristics of various groups of fungi, it is easy to recognize the larger groups, but for accurate diagnosis a microscopic study of the morpholog}' of the organisms is necessary. The fungus must be allowed to grow for a sufficient length of time to permit im- portant morphologic characters to develop. If the organism is pathogenic, all the precautions mentioned for transfer must be taken to insure that spores of dangerous microbes are not detached to float in the air and perhaps inoculate some one. The slide and cover should be thoroughly cleaned with acid alcohol and passed through a flame to remove any traces of fat. In dislodging the material to be studied, great care should be taken not to entangle unneces- sarily the mycelium. With yeastlike fungi this latter step is simplified, since it is necessary only to take a loopful of the culture without fear of entangling the mycelium as may occur with filamentous forms. The mounting of the material should be done carefully. A drop of dis- tilled water, alcohol, Amann's lactophenol preparation, or glycerin, either with or without a stain, is placed in the center of the slide. The fungus is then lifted carefully from the tube with a platinum or nichrome needle or spatula, dislodged into the drop, or pushed oft* by another needle if not pulled off by the surface tension of the mounting medium. Platinum is preferable to nichrome because of its rapidity in cooling, but nichrome is a harder metal and is better for thick, hard growths which resist elevation by the needle. The material is spread out as gently as possible in the mounting medium and a cover glass is lowered carefully on the preparation, avoiding the inclusion of bubbles of air. Alcohol has the advantage of killing the organism, and, hav- ing a low surface tension, does not dislodge spores badl3^ It also has a tend- ency to form fewer air bubbles, but it soon dries out and must be replaced quite promptly by Avater or water and glycerol (2 parts water and 1 part glycerol). This is done by placing a drop on the slide next the cover glass and allowing it to be drawn in under as the alcohol evaporates. Care must be taken, if glycerol is used, that it does not wet the top of the cover glass, as it will be difficult to remove later. The disadvantage in the use of glycerol alone lies in the fact that yeastlike or even filamentous forms may be cleared to such an extent that it is \ery difficult to make out the morphology of the organism. To avoid this, various dyes, such as methylene blue, crystal violet, or eosin, are incorporated either as an aqueous or alcoholic solution (usually about 1%) in amount sufficient to produce the desired intensity. Water does not evaporate rapidly, but, owing to its high surface tension, tends to tear 66 MICROSCOPY 67 away spores from their attachments and, in general, is rather unsatisfactory for filamentous fung-i, although it is very satisfactory as a mounting medium for most 3^east and yeastlike organisms. Some of the various formulae of laetophenol give good results, as does also lactic acid alone. The latter has the disadvantage of preventing the use of many dyes in staining. So far as I am aware, laetophenol was developed in French laboratories, the formula of Amann (1896) being phenol crystals 20 gm., lactic acid 20 gm., glycerol 40 gm., and distilled water 20 gm. These are dissolved with gentle warming and then is added anilin blue (a mixture of the tri-sulphonates of tri-phenyl pararosanilin [C.I. 706] and of di-phenyl rosanilin) otherwise known as cotton blue (C. B. Poirier). Other compounds, such as methyl blue, are also called cotton blue, but are said to be distinctly inferior for this purpose. Sartory (1924) recommends 0.5% dye to his laetophenol. Linder (1929) ad- vocates the same formula while Henrici (1930) adds only 0.05% of the dye. I have found that the dye added directly to the laetophenol gives a blue back- ground to the preparation. Consequently, I use a 1% aqueous solution of cotton blue, place a drop on a clean slide, inoculate with the fungus, lower a cover glass on the mount, and then allow a drop of laetophenol to be drawn in under as shown previously for glycerol. By this method, the excess dye is pushed to the edge of the cover slip and the laetophenol forms a clear back- ground for the blue fungus. Weston (1929) recommends the addition of a small quantity of nigrosin, water soluble, either aqueous, or the picric acid solution described by Curtis and Colley (1915) in order to stain nuclei as well. Since the dye varies in different samples, at present Weston has found no other way than to add some of the dye, try it, and then add more dye or more laetophenol until satisfac- tory results are obtained. Sartory also recommends a mixture of Sudan III 1 part, and lactic acid 1000 parts by weight. This is ground in a mortar with slow additions of small amounts of the lactic acid. The mixture is then heated in a flask on a water-bath until it is completely dissolved, cooled and filtered. One part of anilin blue is added, also 1-2 drops of tincture of iodine for each 10 c.c. of solution. This triple stain colors fatty bodies, amyloid compounds, and the fungus protoplasm. Before adding the cover slip the mount should be heated gently until vapors are given off. Spore Stains. — Maneval (1924) suggests the following stain for yeast spores. Spread a film of cells in a drop of water on a slide and dry in air, fix by passing through a flame 12-15 times; stain with hot carbolfuchsin 1-3 minutes; wash with water; destain with 5% sulphuric acid 2-3 seconds; wash with water and stain with methylene blue 3 seconds, Avash with water. In 1929, he suggested the following procedure : stain with carbolfuchsin or carbol-methylene blue; destain with 5% acetic acid; treat with 5% tannin for 2 minutes; wash and counterstain with methylene blue (after carbolfuchsin) or with safranin (after carbol-methylene blue). Old spores should be heated in a small amount of sterile water for 10 minutes on a hot water-bath before 68 MEDICAL MYCOLOGY making the smear. Hufschmitt, Sartory and Meyer (1931) advocate the Moeller method which is slightly different from Maneval's procedure. Treat smear from 10 seconds to 5 minutes in 1% sulphuric acid; wash, stain with carbolfuchsin, heating for 1 minute; differentiate with 5% sulphuric acid for 5 seconds ; wash, counterstain with aqueous methylene blue for 3 minutes. Buschke and Harry (1923) recommend the Schumacher method. Fix, stain for 1 minute in carbol-methylene blue, rinse with distilled water, stain for 11/2 minutes, while slowly moving the slide, with 1% phosphin (diamido- phenylacridin). Spores also stain with Ziehl-Neelsen acid-fast procedure if the sulphuric acid is replaced by 1% nitric acid alcohol. Maneval (1929) suggests the following modification of Gutstein's pro- cedure for staining vegetative cells. Fix smear with heat, stain with 5% tannin for 2 minutes, then with safranin or 1% methylene blue, or stain with carbol-methylene blue (5% carbolic acid plus 1% methylene blue) or methy- lene blue; treat with 5% tannin for 2 minutes, wash, and counterstain with safranin. It is often very easy to find spores just by making mounts in glycerin and using some dye, such as crystal violet, or lactophenol preparations. Most of the dye preparations will stain vegetative mycelium. Stains for Fungi in Skin. — Unna, Jr. (1929) advises the following modi- fication of the Pappenheim-Unna, Sr. method for staining fungi in skin. Fix in absolute alcohol, then run through the alcohols to xylol, and embed in paraffin. Cut sections about 10/a thick; stain with pyronine-methyl green (pyronine 9 parts, methyl green 1 part, 96% alcohol 90 parts, glycerol 100 c.c, 0.5% phenol to make 1000 c.c.) for 5-10 seconds; rinse in water; dry with absolute alcohol, and mount in balsam. The fungi will be rubin red, leuco- cytes green to blue green. (N.B. The cells of the basal homy layer of the epi- dermis will have red nuclei by this method.) Fungi in tissue can be stained easily by the usual iron-alum hematoxylin and eosin procedure. The fungus elements take the hematoxylin stain rather nicely, although some difficulty may be encountered in distinguishing spheri- cal cells or spores from tissue elements. The Gram method of staining for bacteria has been used with a measurable amount of success since fungi are, in general, gram-positive. The formula of Malcolm Morris (Mallory and Wright 1924, p. 175) for staining various parasites of the skin avoids the use of hydrate of potash. The skin is placed in ether or in a mixture of alcohol and ether, equal parts, stained for 5-30 minutes in a solution of 5% gentian violet in 70% alcohol; iodine solution, 1 minute ; anilin, or anilin plus 2-4 drops of nitric acid ; anilin ; xylol ; xylol and balsam. Stains for Fungi in Other Tissues. — A number of methods listed in Mal- lory and Wright (1924) for staining bacteria in tissue, as well as various types of cells, have been used successfully with fungi. Mallory 's anilin blue stain (p. 118) has been used very nicely for Cryptococcus histolyticus (Torula histolytica) in brain tissue. The Gram-Weigert staining method (p. 288) is in MICROSCOPY 69 general use. On p. 414, the authors list two methods for staining Actinomyces in sections, although alum-hematoxylin followed by a strong eosin solution will give good results, as will also the Gram method for paraffin sections, which is as follows : Stain in anilin-methyl violet for 5-20 minutes ; wash in normal salt solution or water; iodine solution (1:2:300) 1 minute; wash in water; absolute alcohol, several changes, until no more color is given off and the section is ap- parently decolorized; xylol; xylol and balsam. The so-called "clubs" do not stain with Gram's stain while the central portion of the granule, the thin filaments, do. Stains for Hair and Scrapings. — Adamson (1895) recommended clearing with 5-10% KOH and staining by the Gram method. Chalmers and Marshall (1914) suggest soaking scales in 40% KOH for some hours in a w^atch glass in an incubator at 40° C. Transfer specimens to watch glass containing 15% alcohol for 30 minutes, remove to slide, allow alcohol to evaporate, and dry over flame; stain with anilin-gentian violet for 30 minutes. Treat with Gram's iodine solution for 3 minutes ; decolorize with anilin oil for 30 minutes ; stain in concentrated alcoholic eosin for 1 minute ; wash off eosin with anilin oil or clove oil ; treat with xylol, and mount in balsam. Priestley (1917) recommends lactophenol (lactic acid 1 part, phenol 1 part, glycerol 2 parts, water 1 part) for clearing instead of 40% KOH; or chloral hydrate crystals 2 parts, lactic acid 1 part, phenol crystals 1 part, may be used. For staining he recommends treatment with chloroform to remove the fat; boil for 2-3 minutes with formic acid; wash for a few minutes in water, stain with Sahli 's methylene blue ; wash ; differentiate with alcohol, if necessary; dehydrate, and mount in balsam. Bachman (1920) recommends the following procedure : Place scrapings in a drop of water on a cover slip, tease thoroughly with a dissecting needle, dry over a flame but do not scorch. Stain for 2 minutes; decolorize in 95% alcohol for 15-30 seconds; immerse in distilled water 15-30 seconds; pour off excess, dry by heat, and mount in balsam. The spores and mycelium will be blue, the scrapings yellow. His dye is made as follows: saturated alcoholic gentian violet 2.5 parts, distilled water 17.5 parts, orange G solution 9 parts, acetic acid 1 part, 95% alcohol 5 parts. His orange G solution is orange G 2 parts, 95% alcohol 20 parts, water 80 parts. Decolorize with 10-20% KOH. The host is not stained, the fungus appears yellowish red. Unna's method is to rub the scales of the epidermis in a little glacial acetic acid between two slides. These are drawn apart and quickly dried over a flame. The fat is removed by means of alcohol and ether, and the preparations are stained in borax-methylene-blue. Instead of these slightly complicated methods, I have found that infected hairs can be cleared sufficiently to show spores and mycelium by mounting in a hydroxide solution, sodium or potassium, 10-30%. Sodium hydroxide is not quite as satisfactory as potassium hydroxide and a 20% solution works with sufficient rapidity to give good results without seriously macerating the hair. The hair may be immersed in ether to dissolve off' oil or fat, and slight heating 70 MEDICAL MYCOLOGY of the preparation may speed up the reaction. Skin scrapings are also cleared satisfactorily by this simple method. Henrici advocates a 25% solution of antiformin, such as is used in digesting- tuberculous sputum, in place of the sodium hydroxide solution. Stains for Sputum, Pus, etc. — Fungi may be found in sputum, pus, or exudates by making mounts in 20% potassium hydroxide, or by smears. The latter is not very satisfactory except for indicating the presence of mycelium or cells, since smearing tends to disturb the arrangement of the cells. There is usually a great amount of contamination unless one is able to open a fresh lesion which shows no fistulae or draining sinuses. Where the latter are present, biopsies are necessary to find the suspected fungi. Since in exudates the or- ganisms may be few in number, it may require several examinations to locate the parasite. The hydroxide tends to dissolve most of the tissue elements and leave the fungi as refractile bodies. In potassium hydroxide preparations of scales from inflammatory lesions Becker and Ritchie (1930) have indicated artefacts which rather closely re- semble yeast cells. These bodies vary in shape from spherical to ellipsoidal and have a highly refractive wall of varying thickness. Even appearances of sprouting and septal formation occur. These may be removed by treating material progressively with absolute alcohol, ether, absolute and 95% alcohol. The appearances known as mosaic fungus, reported by Greenwood and Rock- wood (1930) to be degenerate hyphal cells, is suggested by Becker and Ritchie (1930) to be a result of inflammatory changes in the tissues. Vital Staining of Fungi. — Dalmau (1929, 1930) reports a technic of vital staining which, while not original with her, deserves much wider use than it receives at present. It is essentially similar to some of the blood stains. The slides should be new, free from blemishes, and should be freed from fat by the use of cleaning solution. After neutralizing, they should be cleaned with a fat-free cloth. Immediately prior to use, all dust must be removed with a new camel's hair brush, washed with ether, and dried. On one slide place one or two drops of Janus green, neutral red or Scharlach R solutions (1 :2500) in ethyl alcohol and let them extend over the entire slide, which they Avill do if the latter is fat-free. Dry in the air. Place a drop of the liquid medium containing the fungus upon a cover slip and invert upon the slide containing the stain. Let settle and then rim the edges with vaseline. Examine at intervals. Dalmau (1930) reports successful staining of fixed material with the com- mon blood stains (Wright's, Giemsa, and Leishman). With a platinum loop mix some of the colony with a drop of clear blood serum, placed at the end of the slide. Before it dries spread it gently with another slide, thus produc- ing a thin film. Fix with methyl alcohol from 1 to 3 minutes. Stain with the above-mentioned blood stains and use neutral water for washing or diluting the stain. The stain should remain from 5 to 15 minutes. Differentiate for a few seconds only with acetic acid (1:1000). Wash wftll. The addition of serum prevents undue shrinkage of the cells. MICROSCOPY 71 Fixing Agents. — It is at times desirable to kill and fix material to prepare the fungus for staining and clearing. A number of fixatives are used, but most authors recommend either Plemming's weak killing agent which con- sists of two solutions which are mixed when ready to fix the material (A. 1% chromic acid 25 c.c, 1% acetic acid 10 c.c, water 55 c.c. ; B. 1% osmic acid 10 c.c.) or Flemming's strong agent (A. 1% chromic acid 45 c.c, glacial acetic 3 C.C; B. 2% osmic acid 12 c.c). Ninety-five per cent alcohol is used, but it is not so suitable for fine work since the material is frequently plasmolyzed. There are various chromo-acetic acid formulae used, but the reader is referred to the standard books on methods in histology for these. One of these for- mulae, Benda's fluid, has been used favorably with yeastlike fungi. It is a modification of Flemming's strong agent and works well for chromatin inves- tigations. One per cent chromic acid 16 c.c, 2% osmic acid 4 c.c, and glacial acetic acid 2 drops. One of the best, yet most expensive fixing agents I have found for celloidin sections of pathogenic fungi is Hermann's fluid : 1% platinic chloride 15 parts, glacial acetic acid 1 part, 2% osmic acid 2 parts. Fix for 6-12 hours; wash overnight. MerkeFs fluid gives good results in organs filled with reserves of food materials. ParaJSn Method. — There are two general methods for embedding material for cutting sections : the paraffin and the celloidin technic Recently, modifi- cations have been reported, but they have not been sufficiently tested to report here. The paraffin method is familiar to practically all technicians and is in general use, although not very satisfactory for agar cultures. The diffi- culty seems to lie in the fact that it may cause shrinkage of the material, and it does not penetrate the agar sufficiently for good embedding. Masses of mycelium scraped from the surface of the agar and embedded in paraffin may give favorable results, but it is often desirable to see the characteristics de- veloped in the substrate. Nitrocellulose Method. — The outstanding method of embedding agar cul- tures is the second procedure. Although its particular disadvantage lies in the fact that it is difficult to obtain very thin sections as with paraffin and also more difficult to make serial sections, although possible, its advantages surpass those of paraffin. Material if fixed properly will show no shrinkage. The agar substrate is penetrated much better, so that celloidin blocks can be made easily and sections, even if slightly thicker than paraffin sections, clear sufficiently to permit study of the internal structure which is usually broken up or distorted by paraffin. There are various products of nitrocellulose on the market, e.g., celloidin and collodion. They are sold as shredded or granulated products and are in- flammable, but not explosive. Schering's celloidin is in general use. Du- pont's parlodion or the product of Mallinckrodt is a purified pyroxylin which gives very good results in embedding and can be obtained as small strips sold in 1-ounce jars. Celloidin may also be obtained in tablet form with directions for making the dilutions accompanying the tablets. 72 MEDICAL MYCOLOGY Because of the particular advantages celloiclin has over paraffin in mak- ing sections of agar cultures for cji:ologic purposes, I shall list steps that I follow in my work. The general procedure is that improved by Jeffrey (1928) and recently reviewed by Wetmore (1932), but with some slight changes as are better adapted to this type of growth. When the organism is sufficiently developed on a suitable medium, the fixing agent, Hermann's fluid, is poured slowly down the side of the tube. After fixation for from 6-12 hours, depend- ing on the type of organism and growth (a surface growth requiring less time than a deep growth), the fixed culture is washed overnight in slowly running tap water. Care should be taken not to let the water run strongly or the sur- face mycelium and yeast cells will be washed away. The agar slant is now taken from the test tube, which is broken, and cut up into pieces or blocks approximately 1 sq. cm. or if large tubes are used, approximately 1 cm. by the diameter of the tube. These blocks are next dehydrated in the following alcohols, 2 hours in each: 15%, 25%, 35%, 50%, 70%, 85%, 95%, 100%. Celloidin may be used warm or cold. For best results it should be used warm. An oven is kept regulated at 45° C. The material is next transferred to a bottle with a collar, containing a solution of ether and absolute alcohol in equal proportions. The bottle is corked and secured by passing a wire around the collar and over the cork as shown by Wetmore. This is placed in the oven and allowed to lie on its side for 24 hours. The preparation is now ready to be run up in celloidin, which has been washed, thoroughly dried, and made up in 2, 4, 6, 8, and 10 per cent solutions in ether-alcohol. Higher percentages may be made, but are not necessary. The material is transferred to each suc- cessive dilution every 24 hours, tightly corked, and placed in the 45° C. oven. After the 10% solution, blocks are poured as with paraffin, care being taken not to form bubbles. The blocks are arranged carefully and then set in chloroform to harden for about 12 hours or overnight. When found to be sufficiently hard, the material is placed in 70% alcohol for a few hours to allow for some softening and then it may be stored in glycerol alcohol (95% alcohol in- definitely). The blocks may be cut with a razor blade to get rid of excess celloidin and to make a uniform block. In order to make sections, the preparations are mounted on small wooden blocks as described by Wetmore. Sections are then cut, as thin as possible, placed in 95% alcohol and then run down to dis- tilled water: 95%, 85%, 70% alcohol, distilled water, 5 minutes in each change, or else run down to the percentage alcohol of the stain used. The sections are now ready to be stained. There are various stains used, but I have found it best to use a 4% mordanting solution of iron-alum (ferric am- monium sulphate) for 10 minutes, washing 4 times with water so that the excess of mordant may be removed. Then 2 drops of Haidenhain's or Ehr- lich's hematoxylin are added to a watch glass of sections in water and allowed to stand overnight. The sections are then examined to see whether the mate- rial is sufficiently stained. If heavily overstained, they can be decolored with dilute iron alum. They should be slightly overstained, however, because the MICROSCOPY 73 higher percentage alcohols usually take out some of the dye. The sections are then again dehydrated, but a few drops of chloroform should be added to the absolute alcohol so that the celloidin will not be dissolved. Two changes in absolute alcohol and chloroform and then benzol to clear completely. The sections are then mounted in balsam, making sure that they are completely flattened out. A lead weight is placed on the cover slip and the slide placed in a warm oven for 2 or 3 days. Thus the excess balsam may be pressed out to allow the preparation to be examined with oil immersion. BIBLIOGRAPHY Adamson, H. G. 1895. Observations on the parasite of ringworm, Brit. Jour. Dermatol. 7: 201-211, 237-244. [Trans. Internat. Cong. Dermatol. 3: 555, 1897.] Amann, Jules. 1896. Conservierungsflussigkeiten und Einschlussmedien fiir Moose, Chloro- und Cyanophyceen, Zeitschr. Wiss. MiJcr. 13: 18-21. Bachmann, Kowland W. 1920. Spore identification in scrapings. Arch. Derm. Syphilol. 1: 50-54. Becker, S. William So Earl B. Kitchie. 1930. The role of yeasts in the production of super- ficial dermatitis, Arch. Derm. Syi)hilol. 22: 790-802. Bigot, A. 1924. Differents procedes de coloration des cryptocoques pathogenes en medecine veterinaire. Bull. Soc. Path. Exot. 17: 547-551. Buschke, A. & F. Harry. 1923. Beitrag zur Frage der Sporulations-fahigkeit parasitischer und pathogener Hefen, Derm.. Woch. 76: 357-360. Chalmers, Albert J. & Alexander Marshall. 1914. Tinea capitis tropicalis in the Anglo- Egyptian Sudan — the systematic position of the genus Trichophyton Malmsten, 1845, Jour. Trop. Med. Byg. 17: 257-265, 289-291, 3 pis. Cornbleet, Theodore. 1930. A reagent for demonstrating fungi in skin scrapings and hair, Jour. Am. Med. Assn. 95: 1743, 1744. Cfurtis, Otis F. & Eeginald H. Colley. 1915. Picro-nigrosin, a combination fixative and stain for algae. Am. Jour. Bot. 2: 89-92. Dalmau, Luz Maria. 1929, 1930. Observations on mycologic technique with particular refer- ence to pathogenic fungi, Porto Rico Jour. Puhlic Health and Trop. Med. 5: 302- 311. Ferrari, Angela. 1930. Un nuovo metodo per la colorazione del micelio, Atti 1st. Bot. B. Univ. Pavia IV, 2: 81-87. Greenwood, A. M. & Ethel M. Eockwood. 1930. The skin in diabetic patients, Arch. Derm. & Syphilol. 21: 96-107, 10 figs. Henrici, A. T. 1930. Molds, yeasts, and Actinomycetes, New York, John Wiley & Son, 296 pp. Hufschmitt, G., A. Sartory, E. Sartory & J. Meyer. 1931. Un cas de blastomycose cutanee a foyers multiples, Ann. Derm. Syphiligr. 7: 850-876. Jeffrey, Edward Charles. 1928. Technical contributions, I-V, Bot. Gaz. 86: 456-467, Figs. 1-3. Kater, J. McA. 1927. Cytology of Saccharomyces cereviciae with especial reference to nuclear division, Biol. Biill. 52: 436-448, £ pis. Kraus, Alfred. 1904. Zur Farbung der Hyphomyceten im Horngewebe, Zentralbl. Bakt., I, 37: 153-156. Lepik, E. 1928. Differential staining of Peronosporuceae, Phytopath. 18: 869-872. Linder, David Hunt. 1929. An ideal mounting medium for mycologists. Science 70: 430. Mallinekrodt-Haupt, Asta v. 1926. Vital farbungen mit Indikatorfarben bei Hyphomyceten, Derm. Zeitschr. 46: 263-305. Mallory, F. B. & J. H. Wright. 1924. Pathological Teclmiquc, Philadelphia and London, 666 pp. 74 MEDICAL MYCOLOGY Maneval, W. E. 1924. A method of securing spores of yeasts, Bot. Gas. 78: 122, 123. Pels, Isaac R. & S. Bayne-Jones. 1923. Elastic tissue simulating mycelial filament in skin scrapings, Arch. Derm. Syphilol. 8; 37-43. Priestley, Henry. 1917. Ringworm and allied parasitic skin diseases in Australia, Med. J. Australia [4] 2; 471-475, IS figs. Sartory, Antoine. 1924. Guide pratique des principales manipulations de mycologie para- sitaire a 1 'usage des pharmaciens, Paris, 341 pp. Smith, J. Lorrain. 1907. On the simultaneous staining of neutral fat and fatty acid by oxazine dyes, J. Path. Bact. 12: 1-4. Unna, Paul, Jr, 1929. tJber Piirbung von Fadenpilzen in der Oberhaut, Derm. Woch. 88: 314-321. Unna, Paul. 1S91. Die Farbung der Mikroorganismen im Horngewebe, Monatsch. Derm. 13: 225-237, 286-311. Weston, William H. 1929. A useful modification of Amann's medium, Science 70: 455. Wetmore, Ralph H. 1932. The use of celloidin in botanical technique, Stain Techn, 7: 37-62, 6 figs. CHAPTER VI BOTANICAL NOMENCLATUEE The problem of selecting a name for an organism is a very ancient one. Early plant names were simple nouns in the language in use by various bota- nists. As likenesses and differences were more clearly realized, adjectives were applied to distinguish between closely related groups. In the course of centuries these adjectives became attached to nouns in a definite and usually stable manner. During the period from the introdviction of printing to the middle of the eighteenth century, the noun gradually took on the generic con- cept, and the group of adjectives, the specific concept. As this usage became more prevalent, a binomial nomenclature was approached, until Linnaeus in his Species Plant arum of 1753 used it almost universally. The last half of the eighteenth century was predominantly one of ex- ploration and description of many new plants and animals. The same or- ganism was often named more than once by workers in ignorance of the pub- lications of others. Then the problem of which name to choose became in- creasingly urgent. In general, the principle of priority developed, by which the oldest binomial name for a group was chosen. During the nineteenth cen- tury, these problems became increasingly (difficult, and authors developed codes of rules for their own use. As a result of this, by the close of the nineteenth century varying practice for handling the same situation had grown up in various countries. In America we had two more or less divergent systems which caused much confusion, as one set of workers used one set of names for their plants while another group used different names for the same plants. An attempt to reach a compromise was made in the last decade of the century, but this was ineffective. At the International Congress at Paris in 1900, a committee was appointed to draft a code and report to the Congress at Vienna in 1905. This code, adopted after much discussion, forms the basis for our present code. It was extensively amended at Brussels in 1910 and at Cambridge (England) in 1930. The official edition of the Code with the 1930 amendments has not yet been published. The most active member of the editorial committee died shortly after the Congress. I have been unable to secure from the surviving member, information as to the probable time of publication. As this book goes to press, A. B. Rendle, Jour. Bot. Brit. For. 72 : Supple- ment 1-29, June, 1934) has published his version of the Code with the approval of the surviving member of the editorial committee, so that it is probable that Rendle 's version will not differ materially from the official version. In gen- eral, Rendle 's version has been reproduced in the following pages, but I have 75 7b MEDICAL MYCOLOGY corrected obvious lapsi calami and added examples of the application of the rules based on names of fungi familiar to medical men which illustrate the point as well as the examples given by Rendle. In this book, I have endeavored to follow the spirit of the International Rules, although it has been impossible to apply the letter of the law in some instances. INTERNATIONAL RULES OF BOTANICAL NOMENCLATURE* Chapter I. — General Considerations and Guiding Principles Art. 1. Botany cannot make satisfactory progress without a precise system of nomenclature, which is used by the great majority of botanists in all countries. Art. 2. The precepts on which this precise system of botanical nomenclature is based are divided into principles, rules, and recom- mendations. The principles (Art. 1-9, 10-14, 15-19 1) form the basis of the rules and recommendations. The object of the rules (Art. 19-74) is to put the nomenclature of the past into order and to pro- vide for that of the future. They are ahvays retroactive : names or forms of nomenclature contrary to a rule (illegitimate names or forms) cannot be maintained. The recommendations deal with sub- sidiary points, their object being to bring about greater uniformity and clearness in future nomenclature : names or forms contrary to a recommendation cannot on that account be rejected, but they are not examples to be followed. Art. 3. The rules of nomenclature should be simple and founded on considerations sufficiently clear and forcible for everj^one to com- prehend and be disposed to accept. Art. 4. The essential points in nomenclature are: (1) to aim at fixity of names; (2) to avoid or to reject the use of forms and names which may cause error or ambiguity or throw science into confusion. Next in importance is the avoidance of all useless creation of names. Other considerations, such as absolute grammatical correctness, regularity or euphony of names, more or less prevailing custom, re- gard for persons, etc., notwithstanding their undeniable importance, are relatively accessory. Art. 5. In the absence of a relevant rule, or where the consequences of rules are doubtful, established custom must be followed. Art. 6. Botanical nomenclature is independent of zoological no- menclature in the sense that the name of a plant is not to be rejected simplj' because it is identical with the name of an animal. If, how- ever, an organism is transferred from the animal to the plant kingdom, its validly published names are to be accepted as botanical nomen- clature in the form prescribed by the rules of botanical nomenclature ; and if an organism is transferred from the plant to the animal king- dom its names retain their status in botanical nomenclature. Art. 7. Scientific names of all groups are usually taken from Latin or Greek. When taken from any language other than Latin, or formed •This entire section set in narrow measure is reprinted from International Rules of Botanical Nomenclature adopted by the Fifth International Botanical Congress, Cambridge, 1930. Supplement to The Journal of Botany, June, 1934. Printed and published by Taylor and Francis. t Art. 19 is both a principle and a rule. BOTANICAL NOMENCLATURE 77 in an arbitrary manner, they are treated as if they were Latin. Latin terminations should be used so far as possible for new names. Art. 8. Nomenclature deals with: (1) the terms which denote the rank of taxonomic groups (Art. 10-14) ; (2) the names which are applied to the individual groups (Art. 15-72). Art. 9. The rules and recommendations of botanical nomenclature apply to all groups of the plant kingdom, recent and fossil, with cer- tain distinctly specified exceptions. Chapter II. — Categories of Taxonomic Groups, and the Terms DENOTING THEM (Art. 10-14, RcC. I, II). Art. 10. Every individual plant, interspecific hybrids and chimseras excepted, belongs to a species (species), every species to a genus (genus), every genus to a family (familia), every family to an order (ordo), every order to a class (classis), every class to a division (divisio). Art. 11. In many species varieties (variefas), forms (forma), and races or biological forms (forma hiologica) are distinguished; in parasitic species special fonns (forma specialis) , and in certain cul- tivated species modifications still more numerous ; in many genera sec- tions (sectio) are distinguished, in many families tribes (tribus). Recommendation I. In parasites, especially parasitic fungi, authors who do not give specific value to forms characterized from a biological standpoint, but scarcely or not at all from a morphological standpoint, should distinguish within the species special forms (forma specialis) characterized by their adaption to different hosts. Art. 12. Finally, if a greater number of intermediate categories are required, the terms for these subdivisions are made by adding the prefix sub (sub) to the terms denoting the categories. Thus subfamily (suh- familia) denotes a category between a family and tribe, subtribe (suh- tribus) a category between a tribe and a genus, etc. The classification of subordinated categories may thus be carried, for wild plants, to twenty-three degrees in the following order: Regnum vegetabile. Divisio. Subdivisio. Classis. Subclassis. Ordo. Subordo. Familia. Subfamilia. Tribus. Subtribus. Genus. Subgenus. Sectio. Sub- sectio. Species. Subspecies. Varietas. Subvarietas. Forma. Forma biologica. Forma specialis. Individuum. If this list of categories is insufficient it can be augmented by the intercalation of supplementary categories, provided that this does not in- troduce confusion or error : e. g., series and subseries are categories which can be intercalated between subsection and species. Recommendation II. The arrangement of species in a genus or in a subdivision of a genus is made by means of typographic signs, letters or numerals. Art. 13. The definition of each of these categories varies, up to a certain point, according to individual opinion and the state of the science; but their relative order, sanctioned by custom, must not be altered. No classification is admissible which contains such alterations : e. g., a form divided into varieties, a species containing genera. Art. 14. The fertilization of one species by another may give rise to a hybrid (hyhrida) ; that of a modification or subdivision of a species by another modification of the same species may give rise to a half- breed (mistiis). 78 MEDICAL MYCOLOGY Chapter III. — Names of Taxonomic Groups (Art. 15-72, Rec. Ill — L). Section 1. — General Principles: priority (Art. 15-17, Rec. III). Art. 15. The purpose of giving a name to a taxonomic group is not to indicate the characters or the history of the group, but to supply a means of referring to it. Art. 16. Each group with a given circumscription, position, and rank can bear only one valid name,* the earliest that is in accordance with the Rules of Nomenclature. Art. 17. No one may change a name (or combination of names) without serious motives, based either on more profound knowledge of facts or on the necessity of giving up a nomenclature that is con- trary to the Rules. Recommendation III. Changes in nomenclature should be made only after ade quate taxonomic study. Section 2.— The Type Method (Art. 18, Rec. IV-VII). Art. 18. The application of names of taxonomic groups is deter- mined by means of nomenciatural types. A nomenclatural type is that constituent element of a group to which the name of the group is permanently attached, whether as an accepted name or as a syno- nym. The name of a group must be changed if the type of that name is excluded (see Art. 66). The type of the name of an order or suborder is a family, that of the name of a family, subfamily, tribe or subtribe is a genus, that of a generic name is a species, that of the name of a species or group of lower rank is usually a specimen or preparation. In some species, however, the type is a description or figure given by a previous author. Where permanent preservation of a specimen or preparation is im- possible, the application of the name of a species or subdivision of a species is determined by means of the original description or figure. Note. — The nomenclatural type is not necessarily the most typical or repre- sentative element of a group; it is merely that element with which the name of the group is permanently associated. Recommendations : IV. When publishing names of new groups authors should indicate carefully the subdivision which is the type of the uew name: the type-genus in a family, the type-species in a genus, the type-variety or specimen in a species. This type deter- mines the application of the' name in the event of the group being subsequently divided. When describing new species, varieties or forms of parasitic plants, especially Fungi, the host plant of the type should be indicated. V. When revising a genus an author should state which species he accepts as the nomenclatural type. VI. In selecting a nomenclatural type for a genus of non-vascular Cryptogams, botanists should, where possible, choose a species that will fix the generic name as it is now commonly applied. *In genera and groups of higher rank the valid name is the earliest name pub- lished with the same rank, provided that this is in conformity with the Eules of Nomenclature and the provisions of Arts. 20 and 21. In subdivisions of genera the valid name is the earliest name published with the same rank, provided that this name and its combination with the generic name are in conformity with the Eules of Nomenclature. In species and groups of lower rank the valid name is the binarj^ or ternary combination containing the earliest epithet published with the same rank, provided that this combination is in conformity with the Eules of Nomenclature. BOTANICAL NOMENCLATURE 79 VII. The utmost importance should be given to the preservation of the orginal ("type") material on which the description of a new group is based. In micro- scopic Cryptogams the preparations and original drawings, in fleshy Fungi water- colour drawings and specimens suitably piepared or dried, should be preserved. The original account should state where this material is to be found. Section 3. — Limitation of the Principle of Priority: publication, start- ingf-points, conservation of names (Art. 19-22). Art. 19. A name of a taxonomic g-ronp has no status under the Rules, and has no claim to recognition by botanists, unless it is validly published (see Section 6, Art. 37). Art. 20. Legitimate botanical nomenclature begins for the differ- ent groups of plants at the following dates: — {a) Phanerogamae and Pteridophyta, 1753 (Linnaeus, Species Plan- tarum, ed. 1). (&) Muscineae, 1801 (Hedwig, Species Mnscorum). (c) Sphagnaceae and Hepatieae, 1753 fLinnasus, Species Pkmtarum, ed. 1). (d) Lichenes, 1753 (Linnasus, Species Plantarum, ed. 1). {e) Fungi: Urediuales, Ustilaginales and Gasteromycetes, 1801 (Per- soon, Synopsis methodica Fungorum) . (/) Fungi cfeteri, 1821-32 (Fries, Systema mycologicum) . (g) Algae, 1753 (Linnasus, Species Plantarum, ed. 1). Exceptions. — Nostocaceae homocysteae, 1892-93 (Gomont, Monographic des Oscillariees in Ann. Sci. Nat. ser. 7, Bot. vi. 91; vii, 263). — Nostocaceae heterocysteae, 1886-88 (Bomet et Flahault, Revision des Nostocacees heterocystees in Ann. Sci. Nat. ser. 7, Bot. iii, 323 ; iv, 344; v, 51 ; vii, 177). — Desmidiaceae, 1858 CRalis, British Desmidiaceae) . — Oedogoniaceae, 1900 (Hirn, Monographic und Iconographie der Oedogoniaceen in Act. Soc. Sci. Fen7i. xxvii, No. 1). {h) Myxomycetes, 1753 (Linnaeus, Species Plantarum, ed. 1). The nomenclature of Fossil Plants of all groups begins with the year 1820. It is agreed to associate generic names which appear in Linnseus's Species Plantarum., ed. 1 (1753) and ed. 2 (1762-63), with the first subsequent descriptions given under those names in Linngeus's Genera Plantarum, ed. 5 (1754) and ed. 6 (1764). Art. 21. However, to avoid disadvantageous changes in the nomen- clature of genera by the strict application of the Rules of Nomen- clature, and especially of the principle of priority in starting from the dates given in Art. 20, the Rules provide a list of names which must be retained as exceptions. These names are by preference those which have come into general use in the fifty years following their publication, or which have been used in monographs and important floristic works up to the year 1890. Note 1. — These lists of conserved names will remain permanently open for addi- tions. Any proposal of an additional name must be accompanied by a detailed statement of the cases for and against its conservation. Such proposals must be submitted to the Executive Committee, who will refer them for examination to the Special Committees for the various taxonomic groups. 80 MEDICAL MYCOLOGY Note 2. — The application of conserved names is determined by nomenclatural types, or by substitute-types where necessary or desirable. Note 3. — A conserved name is conserved against all other names for the group, whether these are cited in the corresponding list of rejected names or not, so long as the group concerned is not united or reunited with another group bearing a legitimate name. In the event of union or reunion with another group, the earlier of the two competing names is adopted in accordance with Art. 56. Note 4. — A conserved name is conserved against all earlier homonyms. Art. 22. "When a name proposed for conservation has been provi- sionally approved by the Executive Committee, botanists are author- ized to retain it pending the decision of the next International Botanical Congress. Section 4. — Nomenclature of the Taxonomic Groups according to their Categories (Art. 23-35, Rec. VIII-XX). § 1. Names of Groups ohove the Bank of Family. Recommendations : VIII. Names of divisions and subdivisions, of classes and subclasses, are taken from their chief characters. They are expressed by words of Greek or Latin origin in the plural number, some similarity of form and termination being given to those which designate groups of the same nature. IX. Orders are designated preferably by the name of one of their principal families, with the ending -ales. Suborders are designated in a similar manner, with the ending -ineae. But other terminations may be used for these names, provided that they do not lead to confusion or error. § 2. Names of Families and Suhfamilies, Tribes, and Subtrihes. Art. 23. Names of families are taken from the name or former name of one of their genera and end in -aceae. Exceptions : (1) The following names, sanctioned by long usage, are treated as exceptions to the rule: Palmae, Gramijieae, Cruciferae, Leguminosae, Guttiferae, Uinbelliferae, Labiatae, Compositae. Botanists are authorized, however, to use as alternatives the appropriate names ending in -aceae. (2) Those who regard the Papilionaceae as constituting an independent family may use that name, although it is not formed in the prescribed manner. Note.— To avoid disadvantageous changes in the nomenclature of families by the strict application of the Rules, and especially of the principle of priority, a list of names which must be retained as exceptions will be provided (Appendix II). Art. 24. Names of subfamilies (subfamiliae) are taken from the name of one of the genera in the group, with the ending -oideae, simi- larly for tribes {tribus), with the ending -eae, and for subtribes {sub- tribus), with the ending -inae. § 3. Names of Genera and Subdivisions of Genera. Art. 25. Names of genera are substantives (or adjectives used as substantives), in the singular number and written with an initial capi- tal, which may be compared with our family names. These names may be taken from any source whatever, and may even be composed in an absolutely arbitrary manner. Recommendation X. Botanists who are forming generic names show judgment and taste by attending to the following recommendations: — (a) Not to make names long or difficult to pronounce. (b) Not to dedicate genera to persons quite unconnected with botany, or at least with, natural science, nor to persons quite unknown. t BOTANICAL NOMENCLATURE 81 (c) Not to take names from barbarous languages, unless those names are frequently cited in books of travel, and have an agreeable form that is readily adaptable to the Latin tongue and to the tongues of civilized countries. (d) To indicate, if possible, by the formation or ending of the name the affinities or analogies of the genus. (e) To avoid adjectives used as nouns. (/) Not to give a genus a name whose form is rather that of a subgenus or section (e. g. Eutorula, a name given to a genus of Saccharomycetaceae Imperfectae. This, however, being legitimate, cannot be altered). (g) Not to make names by combining words from different languages (rioviina hybrida) . Art. 26. Names of subgenera and sections are usually substantives resembling the names of genera : e. g. Fraxinaster, Archieracium. Names of subsections and other lower subdivisions of genera are pref- erably adjectives in the pleural number agreeing in gender with the generic name and written with an initial capital, or their place may be taken by an ordinal number or a letter: e. g. Pleiostylae, Fimhriati, Bihracfeolata. Recommendations : XI. Botanists constructing names for subgenera or sections will do well to attend to the preceding recommendations and also to the following: — (a) To give, where possible, to the principal division of a genus a name which recalls that of the genus with some modification or addition. Thus Eu may be placed at the beginning of the generic name when it is of Greek origin, -astmrn, -ella at the end of the name when Latin, or any other modification consistent with the grammar and usages of the Latin language: e. g. Eucardamine (from Cardamine) , Drabella (from Draha). (b) To avoid giving to a subgenus or a section the name of the genus to which it belongs, with the ending -oides or -opsis: but on the contrary to reserve this ending for a section which resembles another genus and by then adding -oides or -opsis to the name of that other genus, if it is of Greek origin, to form the name of the section. (c) To avoid taking as the name of a subgenus or section a name which is already in use as such in another genus, or which is the name of a genus. (d) To avoid in co-ordinated subdivisions of a genus the use of names in the form of a noun together with those in the form of a plural adjective; the former should he used chiefly for subgenera and sections, the latter for subsections, series and subseries. XII. When it is desired to indicate the name of a subgenus or section (or other subdivision to which a particular species belongs) in connection with the generic name and specific epithet, the name of the subdivision is placed in parentheses be- tween the two (where necessary, the rank of the subdivision is also indicated) : e. g. Achorion (Sect. Lophophyton) muris. §4. Names of Species (hinary names). Art. 27. Names of species are binary combinations consisting of the name of the genus followed by a single specific epithet. If an epithet consists of two or more words, these must either be united into one or joined by a hyphen. Symbols forming part of specific epithets pro- posed by Linnaeus must be transcribed. The specific epithet, when adjectival in form and not used as a sub- stantive, agrees with the generic name. Recommendations : XIII. The specific epithet should, in general, give some indication of the appear- ance, the characters, the origin, the history or the properties of the species. If taken from the name of a person it usually recalls tlie name of the one who dis- covered or described it, or was in some way concerned with it. 82 MEDICAL MYCOLOGY XrV. Names of men and women, and also of countries and localities used as specific epithets, may be substantives in the genitive (Truchisi) or adjectives (Valeriana, lipsiense). It will be well, in the future, to avoid the use of the genitive and the adjectival form of the same epithet to designate two different species of the same genus. XV. In forming specific epithets botanists will do well to have regard also to the following recommendations: — (o) To avoid those which are very long and difficult to pronounce. (b) To avoid those which express a character common to all, or nearly all, the species of a genus. (c) To avoid using the names of little-known or very restricted localities, unless the species is quite local. (d) To avoid, in the same genus, epithets which are very much alike, especially those which differ only in their last letters. (e) Not to adopt unpublished names found in travellers' notes or in herbaria, attributing them to their authors, unless these have approved publica- tion. (/) Not to name a species after a person who has neither discovered, nor de- scribed, nor figured, nor in any way studied it. (g) To avoid epithets which have been used before in any closely allied genus. (h) To avoid specific epithets formed of two or more (hyphened) words. (i) To avoid epithets which have the same meaning as the generic name (pleonasm). § 5. Names of Groups below the rank of Species {ternary names). Art. 28. Epithets of subspecies and varieties are formed like those of species and follow them in order, beginning- with those of the high- est rank. "When adjectival in form and not used as substantives they agree with the generic name. Similarly for subvarieties, forms and slight or transient modifications of wild plants, which receive either epithets, or numbers, or letters to facilitate their arrangement. The use of a binary nomenclature for subdivisions of species is not admis- sible. It is permissible to reduce more complicated names to ternary combinations. Art. 29. The same epithet may be used for subdivisions of different species, and the subdivisions of one species may bear the same epithet as other species: e. g. Rosa JundzilUi var. leioclada and Bosa glutmosa var. leioclada, Viola tricolor var. hirta in spite of the existence already of a different species named Viola hirta. Art. 30. Two subdivisions of the same species, even if they are of different rank, cannot bear the same subdivisional epithet, unless they are based on the same type. If the earlier subdivisional name (ternary combination) was validly published, the later one is illegitimate, and must be rejected. The ternary combinations Biscutella didyma subsp. apula Briq. and Biscutella didyma var. apula Halacsy may both be used because they are based on the same type, and the one includes the other. The following is incorrect: Erysimum hieracifolium subsp. strictum var. longisiliquum and E. hieracifolium subsp. pannonicum var. longisiliquum — a form of nomenclature which allows two varieties bearing the same name in the same species. Recommendations : XVI. Eecommendations made for specific epithets apply equally to epithets of subdivisions of species. XVII. Special forms (forma specialis) are preferably named after the host species ; if desired, double names may be used : e. g. Puccinia HieracU f . sp. villosi, Pucciniastrum Epilohii f. sp. Aiieti-Chamaenerii. XVIII. Botanists should avoid giving a new epithet to any subdivision of a species which includes the type either of the specific name or of a higher sub- BOTANICAL NOMENCLATURE 83 divisional name. They should either repeat that epithet or use one of the customary- epithets, typicus, genuinus, originarvus, etc. E. g. Andropngon caricosus subsp. mollissimus var. mollissim'us Hackol; Arthraxon ciliaris Bcauv. subsp. Langsdorfii var. genuinus Hackel. XIX. Botanists proposing new epithets for subdivisions of species should avoid such as have been used previously in the same genus, whether for species or for subdivisions of other species. § 6. Names of Hybrids and Half-hreeds. Art. 31. Hybrids or putative hybrids between species of the same genus are designated by a formula and, whenever it seems useful or necessary, by a name. (1) Sexual hybrids. — The formula consists of the names or specific epithets of the two parents in alphabetical order and connected by the sign X. When the hybrid is of known experimental origin the fonnula may be made more precise by the addition of the signs 9 $ , the name of the female (seed-bearing) parent being placed first. The name, which is subject to the same rules as names of species, is distinguished from the latter by the sign x before the name : e. g. X Salix capreola {Salix anrita x caprea) . (2) Asexual hybrids (graft hybrids, chinueras, etc.). — The formula consists of the names of the two parents in ali)habetical order connected by the sign +. The name has a "specific" epithet different from that of the corresponding sexual hj'brid (if any), and is preceded by the sign +: e. g. + Solanum tubingense {Solanum nigrum + 8. Lycopersi- cum) . Art. 32. Bigeneric hybrids (hybrids between species of two genera) are also designated by a formula and, whenever it seems useful or necessaiy, by a name. The formula consists of the names of the two parents connected by a sign, as in Art. 31. The name consists of a new "generic" name usually formed by a combination of the names of the parent genera, and a "specific" epithet. All hybrids (whether sexual or asexual) between the same two genera bear the same "generic" name. (1) Sexual hybrids. — In the formula the connecting sign x is used. The name is preceded by the sign x : e. g. x Odoniioda Boltonii (Coch- lioda Noezlianax Odontoglossum Vuylstekeae) . (2) Asexual hybrids. — In the formula the connecting sign + is used. The name is preceded by the sign +. The "specific" epithet is differ- ent from that of the corresponding sexual hybrid (if any) between the same species. E. g. + Laburnocytisus Adami {Laburnum anagyroides +Cytisus purpurens) . Art. 33. Ternar.y hybrids, or those of a higher order, are designated, like ordinary hybrids, by a formula and, whenever it seems useful or necessary, bj^ a name. Such as are trigeneric or polygeneric are given new "generic" names usually formed by a combination of the names of the parent genera. Recommendation XX. Half-breeds, or putative half-breeds, may be designated by a name and a formula. Names of half-breeds are intercalated among the sub- divisions of a species, and are preceded by the sign x. In tlie formula the names of the parents are in alphabetical order. When the half-breed is of known experi- mental origin the formula may be made more precise by the addition of the signs 9 $, the name of the female (seed-bearing) parent being placed first. 84 MEDICAL MYCOLOOY Art. 34. When different hybrid forms of the same parentage (pleomorphic hybrids; combinations between different forms of a col- lective species, etc.) are united in a collective group, the subdivisions are classed under the binary name of the hybrid, like the subdivisions of a species under that of a species. Examples: x Mentha niliaca /3 Lamarckii (=: M. longifolia x rotundifolia) . The preponderance of the characters of one or other parent may be indicated in the formulae in the following manner : Mentha longifolia > x rotundifolia, M. longi- folia X < rotundifolia. The participation of a particular variety may also be indicated : e. g. Salix caprea x daphnoides var. pulchra. § 7. Names of Plants of Horticultural Origin. Art. 35. Forms and half-breeds among cultivated plants receive fancy epithets, preferably in common language, as different as possible from the Latin epithets of species or varieties. When they can be at- tached to a species, a subspecies or a botanical variety this is indi- cated by a succession of names : e. g. Pelargonium zonMe Mrs. Pollock. Section 5. — Conditions of effective Publication. Art. 36. Publication is effected, under these Rules, either by sale or distribution of printed matter or indelible autographs to the gen- eral public, or to specified representative botanical institutions*. No other kind of publication is accepted as effective : communica- tion of new names at a public meeting, or the placing of names in collections or gardens open to the public, does not constitute effective publication. Section 6. — Conditions and Dates of valid Publication of Names (Art. 37-45, Rec. XXI-XXIX). Art. 37. A name of a taxonomic group is not validly published un- less it is both (1) effectively published (see Art. 36), and (2) accom- panied by a description of the group or by a reference to a previously and effectively published description of it. Mention of a name on a ticket issued with a dried plant without a printed or autographed description does not constitute valid publica- tion of that name. Note. — In certain circumstances a plate or figure with analyses is accepted as equivalent to a description (vide Art. 43, 44). Art. 38. From January 1, 1935, names of new groups of recent plants, the Bacteria excepted, are considered as validly published only when they are accompanied by a Latin diagnosis. Art. 39. From January 1, 1912, the name of a new taxonomic group of fossil plants is not considered as validly published unless it is ac- companied by illustrations or figures showing the essential characters, in addition to the description. Art. 40. A name of a taxonomic group is not validly published when it is merely cited as a synonym. Art. 41. A group is not characterized, and the publication of its name is not validated, merely by mention of the subordinate groups included in it : thus the publication of the name of the order is not *The preparation of a list of representative botanical institutions is referied to tlie Executive Committee (see App. VII). BOTANICAL NOMENCLATURE validated by mention of the included families; that of a family is not validated by mention of the included genera ; that of a genus is not validated by mention of the included species. E. g. the generic name Ibidinm Salisbuiy (Trans. Hort. Soc. i, 291: 1812) was published merely with the mention of four included species: as Salisbury sup- plied no generic description, the publication of Ihidium was invalid. Art. 42. A name of a genus is not validly published unless it is ac- companied (1) by a description of the genus, or (2) by the citation of a previously and effectively published description of the genus under another name, or (3) by a reference to a previously and effectively published description of the genus as a subgenus, section or other sub- division of a genus. An exception is made for the generic names published by Linnaeus in Species Plantarum, ed. 1 (1753) and ed. 2 (1762-63), which are treated as having been validly published on those dates (see Art. 20). Note. — In certain circumstances a plate witli analyses is accepted as equivalent to a generic description (see Art. 43). Art. 43. The name of a monotypic new genus based on a new species is validated (1) by the provision of a combined generic and specific de- scription (descriptio generico-specifica) , (2) by the provision of a plate with analj^ses showing essential characters; but this applies only to plates and generic names published before January 1, 1908. Art. 44. The name of a species or of a subdivision of a species is not validly published unless it is accompanied (1) by a description of the group, or (2) by the citation of a previously and effectively published description of the group under another name, or (3) by a plate or figure w^th analyses showing essential characters; but this applies only to plates or figures published before January 1, 1908. Art. 45. The date of a name or of an epithet is that of its valid publication (see Art. 19, 36). For purposes of priority, however, only legitimate names and epithets published in legitimate combinations are taken into consideration* (see Art. 60). In the absence of proof to the contrary, the date given in the work containing the name or epi- thet must be regarded as correct. On and after January 1, 1935, only the date of publication of the Latin diagnosis can be taken into account for recent plants except Bacteria. For fossil plants, on and after January 1, 1912, the date is that of the simultaneous publication of the description and figure (or, if these are published at different dates, the later of the two dates). Botanists will do Avell in publishing to conform to the following recommendations :■ — ■ XXI. Not to publish a new name without clearly indicating whether it is the name of a family or a tribe, a genus or a section, a species or a variety; briefly, without expressing an opinion as to the rank of the group to which the name is given. Not to publish the name of a new group without indicating its type (see Eecom- mendation IV). XXII. To avoid publishing or mentioning in their publications unpublished names which they do not accept, especially if the persons responsible for these names have not formally authorized their publication (see Recommendation XV 85 •A legitimate name or epithet is one that is strictly in accordance with the Rules. 86 MEDICAL MYCOLOGY XXIII. When publishing names of new groups of plants in works written in a modern language (floras, catalogues, etc.), to publish simultaneously the Latin diagnoses of recent plants (Bacteria excepted) and the figures of fossil plants, which will make these names valid according to the Eules. XXIV. In describing new groups of lower Cryptogams, especially among the Fungi, or among microscopic plants, to add to the description a figure or figures of the plants, with details of microscopic structure, as an aid to identification. XXV. The description of parasitic plants should always be followed by the indication of the hosts, especially in the case of parasitic fungi. The hosts should be designated by their Latin scientific names and not by popular names in modern languages, the significance of which is often doubtful. XXVI. To give the etymology of new generic names and also of new epithets when the meaning of these is not obvious. XXVII. To indicate precisely the date of publication of their works and that of the placing on sale or the distribution of named and numbered plants when these are accompanied by printed diagnoses. In the case of a work appearing in parts, the last published sheet of the volume should indicate the precise dates at which the different fascicles or parts of the volumes were published as well as the number of pages in each. XXVIII. When works are published in periodicals, to require the publisher to indicate on the separate copies the date (year and month) of publication and also the title of the periodical from which the work is extracted. XXIX. Separate copies should always bear the pagination of the periodical of which they form a part ; if desired they may also bear a special pagination. Section 7. — Citation of Authors' Names for purposes of precision (Art. 46-49, Ree. XXX-XXXII). Art. 46. For the indication of the name (unitary, binary, or ter- nary) of a group to be accurate and complete, and in order that the date may be readily verified, it is necessary to cite the author who first published the name in question. Art. 47. An alteration of the diagnostic characters or of the cir- cumscription of a group does not warrant the citation of an author other than the one who first published its name. When the changes have been considerable, an indication of their nature and of the author responsible for the change is added, the words mutatis charact., or pro parte, or excl. gen., excl. sp., excl. var., or some other abridged indication being employed. Examples: Phyllanthus L. em. (emendavit) Miill. Arg.; Myosotis L. pro parte, K. Br.; Globularia cordifolia L. excl. var. (em. Lam.). Art. 48. When a name of a taxonomic group has been proposed but not published by one author, and is subsequently validly published and ascribed to him (or her) by another author who supplied the descrip- tion, the name of the latter author must be appended to the citation with the connecting word " ex. " The same holds for names of garden origin cited as "Hort." E. g. Capparis lasiantka R. Br. ex DC; Ges- neria Donklarii Hort. ex Hook. If it is desirable or necessary to abbreviate such a citation, the name of the publishing author, being the more important, must be retained. Where a name and description bj' one author are published by an- other author, the word ai^ud is used to connect the names of the two authors, except where the name of the second author forms part of the title of a book or periodical in which case the connecting word in is used instead. BOTANICAL NOMENCLATURE 87 Art. 49. When a genus or a group of lower rank is altered in rank but retains its name or epithet, the original author must be cited in parentheses, followed by the name of the author who effected the alteration. The same holds when a subdivision of a genus, a species, or a group of lower rank is transferred to another genus or species with or without alteration of rank. Examples: Mcdicago polymorpha L. var. orbicularis L., when raised to the rank of a species, becomes Medicago orhicuJaris (L.) All. Sorbus sect. Ari^i Pers., on transference to Py)'us, is cited as Pijrus sect. Aria (Pers.) DC. Recommendations : XXX. Authors ' names put after names of plants are ablireviated, unless they are very short. For this purpose preliminary particles or letters that, strictly speaking, do not form part of the name are suppressed, and the first letters are given without any omission. If a name of one syllable is long enough to make it worth while to abridge it, the first consonants only are given (Br. for Brown) ; if the name has two or more syllables, the first syllable and the first letter of the following one are taken, or the two first when both are consonants (Juss. for Jussieu, Eich. for Eichard). Wlien it is necessary to give more of a name to avoid confusion between names begiiming with the same syllables, the same system is to be followed. For instance, two syllables are given together with the one or two first consonants of the third; or one of the last characteristic consonants of the name is added (Bertol. for Bertoloni, to distinguish from Bertero ; Michx. for Michaux, to distinguish from Micheli). Christian names or accessory designations, serving to distinguish two botanists of the same name, are abridged in the same way (Adr. Juss. for Adrien de Jussieu, Gaertn. fil. or Gaertn. f. for Gaertner filius). "When it is a well-established custom to abridge a name in another manner it is best to conform to it (L. for Linnseus, DC. for De Candolle, St. Hil. for Saint- Hilaire). In publications destined for the general public and in titles it is preferable not to abridge. XXXI. When citing a name published as a synonym, the words ''as synonym," or pro synon. should be added to the citation. "When an author published as a synonym a manuscript name of another author, the word ex should be used to con- nect the names of the two authors: e. g. Myrtus serratus Koenig ex Steud. Nomencl. 321 (1821), pro synon., a manuscript name of Koenig 's published by Steudel as a synonym of Eugenia laurina Willd. XXXII. The citation of authors earlier than the starting point of the nomen- clature of a group is indicated, when considered useful or desirable, preferably be- tween brackets or by the use of the word ex. This method is especially applicable in mycology when reference is made to authors earlier than Fries or Persoon. Section 8. — Retention of Names or Epithets of Groups which are re- modelled or divided (Art. 50-52). Art. 50. An alteration of the diagnostic characters, or of the cir- cumscription of a group, does not warrant a change in its name, except so far as this may be necessitated (1) by transference of the group (Art. 53-55), or (2) by its union with another group of the same rank (Art. 56-57), or (3) by a change of its rank (Art. 58). Examples: The genus Myosotis as revised by E. Brown differs from the original genus of Liimffius, but the generic name has not been changed, nor is a change allowable. — Various authors have united with Centaurea Jacea L. one or two species which Linnaeus had kept distinct; the group thus constituted must be called Centaurea Jacea L. sensu ampl. or Centaurea Jacea L. em. "Visiani, or cm. Godron, etc. : the creation of a new name such as Centaurea vulgaris Godr. is superfluous. Art. 51. When a genus is divided into two or more genera, the generic name must be retained for one of them, or (if it has not been retained) must be re-established. When a particular species was 88 MEDICAL MYCOLOGY originally designated as the type, the generic name must be retained for the genus including that species. When no type was designated, a type must be chosen according to the regulations which will be given (Appendix I). Art. 52. When a species is divided into two or more species, the specific epithet must be retained for one of them, or (if it has not been retained) must be re-established. When a particular specimen was originally designated as the type, the specific epithet must be retained for the species including that specimen. When no type was designated, a type must be chosen according to the regulations to be given (Appendix I). The same rule applies to subdivisions of species ; for example, to a subspecies divided into two or more subspecies, or to a variety di- vided into two or more varieties. Section 9.^ — Retention of Names or Epithets of Groups below the Rank of Genus on transference to another Genus or Spe- cies (Art. 53-55). Art. 53. When a subdivision of a genus is transferred to another genus (or placed under another generic name for the same genus) without change of rank, its subdivisional name must be retained, or (if it has not been retained) must be re-established unless one of the following obstacles exists: (1) that the resulting association of names has been previously published validly for a different subdivi- sion, or (2) that there is available an earlier validly published sub- divisional name of the same rank. E. g. Saponaria sect. Vaccaria DC, transferred to Gypsophila, becomes Gypsophila sect. Vaccaria (DC.) Gren. & Godr. Art. 54. When a species is transferred to another genus (or placed under another generic name for the same genus), without change of rank, the specific epithet must be retained or (if it has not been re- tained) must be re-established, unless one of the following obstacles exists: (1) that the resulting binary name has been previously and validly published for a different species, (2) that there is available an earlier validly published specific epithet. When the specific epithet, on transference to another genus, has been applied erroneously in its new position to a different plant, it must be retained for the plant on which the group was originally based : e. g. the specific epithet of Pinus Mertensiana Bong, was trans- ferred to Tsuga by Carriere, who, however, erroneously applied the new combination Tsuga Mertensiana to another species of Tsuga, namely, T. heterophylla (Raf.) Sarg., as is evident from his descrip- tion: the epithet Mertensiana (Bong.) must be retained for Pinus Mertensiana Bong, when that species is transferred to Tsuga; the cita- tion in parentheses (under Art. 49) of the name of the original author, Bongard, indicates the type of the epithet, Tsuga Mertensiana (Bong.) Sargent, non Carriere. Art. 55. When a variety or other subdivision of a species is trans- ferred, without change of rank, to another genus or species (or placed under another generic or specific name for the same genus or species), the original subdivisional epithet must be retained or (if it has not been retained) must be re-established, unless one of the fol- lowing obstacles exists: (1) that the resulting ternary combination BOTANICAL NOMENCLATURE 89 has been previously and validly published for a subdivision based on a different type, even if that subdivision is of a different rank; (2) that there is an earlier validly published subdivisional epithet available. When the epithet of a subdivision of a species, on transference to another species, has been applied erroneously in its new position to a different plant, the epithet must be retained for the plant on which the group was originally based. Example: The variety micranthum Oren. & Godr. (Fl. France, i, 171: 1847) of Helianthermi/m italiouTn Pers., when transferred as a variety to H. penicillatum Thib., retains its varietal epithet, becoming H. penicillatum var. micranth^tm (Gren. & Godr.) Grosser (in Engl. Pflanzenreich, Heft 14, 115: 1903). Section 10. — Choice of Names when two Groups of the same Rank are united, or in Fungi with a pleomorphic Life-cycle (Art. 56, 57, Rec. XXXIII-XXXV). Art. 56. When two or more groups of the same rank are united the oldest legitimate name or (in species and their subdivisions) the old- est legitimate epithet is retained. If the names or epithets are of the same date, the author who unites the groups has the right of choos- ing one of them. The author who first adopts one of them, definitely treating another as a synonym or referring it to a subordinate group, must be followed. Recommendations: XXXni. Authors who have to clioose between two generic names should note the following recommendations: — 1. Of two names of tlie same date to prefer the one which was first accom- panied by the description of a species. 2. Of two names of the same date, both accompanied by descriptions of species, to prefer the one which, when the author made his choice, in- cluded the larger number of species. 3. In cases of equality from these various points of view to prefer the more correct and appropriate name. XXXIV. "When several genera are united as subgenera or sections under one generic name, the subdivision including the type of the generic name used may bear that name unaltered (e.g. Anarrhinum sect. Anarrhiniim), or with a prefix (An- thriscus sect. Eu-Anthriscus) , or a suffix (Stachys sect. Stachyotypiis). These pre- fixes or suffixes lapse when the subdivisions are raised to generic rank. XXXV. When several species are united as subspecies or varieties under one specific name, the subdivision whicli includes the type of the specific epithet used may be designated either by the same epithet unaltered (e. g. Stachys recta subsp. recta), or with a prefix (e.g. Alchemilla alpina subsp. eii-alpina), or by one of the customary epithets (typicus, origmarius, genuinvs, verus, veridicus, etc.), in- dicating that it is the type subdivision. Art. 57. Among Fungi with a pleomorphic life-cycle the different successive states of the same species {anamorphoses, status) can bear only one generic and specific name (binary), that is the earliest which has been given, starting from Fries, Sy sterna, or Persoon, Synopsis, to the state containing the form which it has been agreed to call the per- fect form, provided that the name is otherwise in conformity with the Rules. The perfect state is that which ends in the ascus stage in the Ascomycetes, in the basidium in the Basidiomycetes, in the teleutospore or its equivalent in the TJredinales, and in the spore in the TJstilaginales. Generic and specific names given to other states have only a temporary value. They cannot replace a generic name already existing and apply- ing to one or more species, any one of which contains the ''perfect" form. 90 MEDICAL MYCOLOGY The nomenclature of Fnngi wliicli have not a pleomorphic life-cycle follows the ordinary rules. Section 11.^ — Choice of Names when the Rank of a Group is changed. Art. 58. AVhen a tribe becomes a family, when a subg'enus or sec- tion becomes a genus, when a subdivision of a species becomes a species, or when the reverse of these changes takes place, and in gen- eral when a group changes its rank, the earliest legitimate epithet given to the group in its new rank is valid, unless that name or the resulting association or combination is a later homonym (see Art. 60, 61). E. g. the section Campanopsis R. Br. (Prodr. Fl. Nov. Holl. 561 : 1810) of the genus Campanula was first raised to generic rank by Schrader, and as a genus must be called Wahlenbergia Schrad. (Cat. Hort. Goett.: 1814). not Campanopsis (R. Br.) 0. Kuntze {Eev. Gen. ii, 378: 1891). Recommendation XXXVI. 1. When a sub-tribe becomes a tribe, when a tribe becomes a subfamily, when a subfamily becomes a family, etc., or when the inverse changes occur, the root of the name should not be altered but only the termination (-inae, -eae, -oideae, -aceae, -ineae, -ales, etc.), unless the resulting name is re- jected under Section 12 or the new name becomes a source of error or there is some other serious reason against it. 2. When a section or a subgenus becomes a genus, or the inverse changes occur, the original name should be retained unless it is rejected under Section 12. 3. When a subdivision of a species becomes a species, or the inverse change oc- curs, the original epithet should be retained unless the resulting combination is re- jected under Section 12. Section 12.— Rejection of Names (Art. 59-69, Rec. XXXVII). Art. 59. A name or epithet must not be rejected, changed, or modi- fied merely because it is badly chosen, or disagreeable, or because an- other is preferable or better known (see also Art. 69). Art. 60. A name must be rejected if it is illegitimate (see Art. 2). The publication of an epithet in an illegitimate combination must not be taken into consideration for purposes of priority (see Art. 45). A name is illegitimate in the following cases : — (1) If it was superfluous when published, i. e. if there was a valid name (see Art. 16) for the group to which it was applied, with its par- ticular circumscription, position and rank. (2) If it is a binary or ternary name published in, contravention of Art. 16, 50, 52 or 54, i. e. if its author did not adopt the earliest legiti- mate epithet available for the group with its particular circumscrip- tion, position, and rank. (3) If it is a later homonym (see Art. 61) (except as regards Art. 54 and 55). (4) If it is a generic name which must be rejected under Art. 67. (5) If its specific epithet must be rejected under Art. 68. Art. 61. A name of a taxonomic group is illegitimate and must be rejected if it is a later homonym^ that is, if it duplicates a name pre- viously and validly published for a group of the same rank based on a different type. Even if the earlier homonym is illegitimate, or is gen- erally treated as a synonym on taxonomic grounds, the later homonym must be rejected. BOTANICAL NOMENCI.ATimE Examples: The generic name Tapei7ia7ithus Boiss. ex Benth. (1848), given to a genus of Labiatae, is a later homonym of Tapeinanthus Herb. (1837), a name pre- viously and validly published for a genus of Amaryllidaceae ; Tapeinanthus Boiss. ex Benth. must, therefore, be rejected, as was done by Th. Durand (Ind. Gen. Than. 703: 1888) who renamed it TMispeinanta. — The generic name Amblyanthera Miill. Arg. (1860) is a later homonym of the validly published generic name Amblyanthera Blume (1849), and must, therefore, be rejected, although Ambly- anthera Blume is now reduced to OsbecMa L. (1753). — Astragalus rhisanthus Boiss. (Diagn. Fl. Or. ser. 1^ ii, 83: 1843) is a later homonym of the validly published name Astragalus rhisanthus Koyle (Illstr. Bot. Himal. 200: 1835), and it must, therefore, be rejected, as was done by Boissier, who renamed it A. cariensis (Diagn. ser. 1, ix, 57: 1849). Note. — Mere orthographic variants of the same name are treated as homonyms — see Art. 70. Art. 62. A name of a taxonomic group must be rejected if, owing to its use with different meanings, it becomes a permanent source of confusion or error. A list of names to be abandoned for this reason (Nomina amhigua) will form Appendix IV. Examples: The generic name Alsine L., being used by various authors for three genera of Caryophyllaceae (Stellaria L., Spergularia J. & C. Presl, Mirmiartia L.), has been a permanent source of confusion and error (see Sprague in "Kew Bul- letin," 1920, 308).— The name Eosa villosa L., 8p. Fl. ed. 1, 491 (1753), is re- jected, because it has been applied to several different species, and has become a source of confusion. Art. 63. A name of a taxonomic group must be rejected when its application is uncertain (nomen duhium) : e. g. Ervum soloniense L. (Cent. II. PI. 28: 1756) is a name the application of which is uncer- tain; it must, therefore, be rejected (see Schinz and Thell. in Vier- teljahresschr. Nat. Ges. Zurich, Iviii, 71: 1913). Recommendation XXXVII. Wlien the correct application of a nomen dubium has been established by subsequent investigation (of types, etc.), authors adopting it should, for purposes of precision, cite the name of the author who published the additional certifying evidence as well as that of the original author. It is also desirable to add the date of certification. Art. 64. A name of a taxonomic group must be rejected if the charac- ters of that group were derived from two or more entirely discordant elements, especially if those elements were erroneously supposed to form part of the same individual : e. g. the characters of the genus Schrehera L. (Sp. PI. ed. 2, 1662; 1763; Gen. PI. ed. 6, 124: 1764) were derived from the two genera Cuscufa and Myrica (parasite and host), see Retzius (Ohs. vi, 15: 1791). A list of names to be abandoned for this reason (Nomina confum) will form Appendix VI. Art. 65. A name or epithet of a taxonomic group must be rejected when it is based on a monstrosity. Art. 66. The name of an order, suborder, family or subfamily, tribe or subtribe must be changed when it is taken from the name of a genus which is known not to belong to the group in question — e. g. if the genus Portidaca were excluded from the family now known as Portulacaceae, the residual group could no longer bear the name Port- ulacaceae, and would have to be renamed. Art. 67. Names of genera are illegitimate in the following special cases and must be rejected : — (1) When they are merely words not intended as names: e. g. Anonymos Walt. (Fl. Carol, 2, 4, 9, etc.: 1788) must be re- jected as being a word applied to 28 different genera by Walter to indicate that they were without names. 9] 92 MEDICAL MYCOLOGY (2) When they coincide with a technical term currently used in morphology unless they were accompanied, when originally published, by specific names in accordance with the binary method of Linnffius. On and after Jan. 1, 1912, all new generic names coinciding with such technical terms are uncondition- ally rejected. (3) When they are unitary designations of species: e. g. Ehrhart (Phytophylacmm: 1780; and Beitr. iv, 145-150: 1798) pro- posed unitaiy names for various species known at that time under binary names : e. g. Phaeocephalnm for Schoenus fuscus, and Leptostachys for Carex leptostachys. These names, which resemble generic names, should not be confused with them, and must be rejected, unless they have been published as generic names by a subsequent author. (4) When they consist of two words, unless these words were from the first combined into one, or joined by a hyphen. Art. 68. Specific epithets are illegitimate in the following cases and must be rejected : — (1) When they are merely words not intended as names: e. g. Viola "qiialis" Krocker {Fl. Siles. ii, 512 and 517: 1790) ; Atriplex "nova" Winterl (in l7id. Hort. Bot. Univ. Pest. fol. A 8, recto et verso: 1788), the word "nova'' being here used in connection with four different species of Atriplex. (2) When they are merely ordinal adjectives being used for enu- meration. (3) When they exactly repeat the generic name with or without the addition of a transcribed symbol. (4) When they were published in works in which the Linnean system of binary nomenclature for species was not consis- tently employed. Art. 69. It cases foreseen in Art. 60-68 the name or epithet to be rejected is replaced by the oldest legitimate name, or (in a combina- tion) by the oldest legitimate epithet. If none exists, a new name or epithet must be chosen. Where a new epithet is required, an author may, if he wishes, adopt an epithet previously given to the group in an illegitimate combination, if there is no obstacle to its employment in the new position or sense. Section 13.— Orthography of Names (Art. 70, 71, Eec. XXXVIII- XLIV). Art. 70. The original spelling of a name or epithet must be re- tained, except in the case of a typographic error, or of a clearly unintentional orthographic error. When the difference between two generic names lies in the termination, these names must be regarded as distinct, even though differing by one letter only. This does not apply to mere orthographic variants of the same name. Note 1. The words "original spelling" in this Article mean the spelling em- ployed when the name was validly published. 2. The use of a wrong connecting vowel or vowels (or the omission of a connecting vowel in a specific epithet, or in that of a subdivision of a species) is treated as an unintentional orthographic error which may be corrected (see Eec. XLIV). BOTANICAL XOMENCLATURE 93 3. In deciding wlietlicr two or more sliglitly different names should be treated :is distinct or as orthographical variants, the essential con- sideration is wliether they may be confused with one anotlier or not : if there is serious risk of confusion, they sliould be treated as orthographic variants. Doubtful cases should be referred to the Executive Committee. 4. Specific and other epithets of Greek origin differing merely by having Greek and Latin terminations respectively are orthographic variants. Epithets bearing the same meaning and differing only slightly in form are (considered as) orthographic variants. The genetive and ad- jectival forms of a personal name are, however, treated as different epithets (e.g. Lysimachia Eemsleyana and L. Hemsleyi). Recommendations : XXXVIII. Wlien a new name is derived from a Greek word containing the spiritiis asper (rough breathing), this should be transcribed as the letter h. XXXIX. When a new name for a genus, subgenus or section is taken from the name of a person, it should be formed in the following manner: — (a) When the name of the person ends in a vowel the letter a is added (thus Ashbya after Ashby; Blaheslea after Blakeslee), except when the name already ends in a, when ea is added (e. g. Collaea after CoUa). (b) When the name of the person ends in a consonant, the letters ia are added (e. g. Magnusia after Magnus, Guilliera after Guillier), ex^ cept when tlie name ends in er, when a is added, e. g. Kernera after Kerner). (c) The syllables which are not modified by these endings retain their original spelling, even with the consonants fc and w or with groupings of vowels which were not used in classical Latin. Letters foreign to botanical Latin should be transcribed, and diacritic signs suppressed. The Germanic a, o, ii become ae, oe, ue ; the French e, e, e become generally e. In works in which diphthongs are not represented by special type, the diaeresis sign should be used where required, e.g. C'cphaelis, not CepJiaelis. (d) Names may be accompanied by a prefix or a suffix, or modified by ana- gram or abbreviation. In these cases they count as different words from the original name. Examples: Durvillea and Urvillea; Lapeyrousea and Peyrousea; Bouchea and Ubochea; Gerardia and Graderia; Thaxtera, Thax- teriola. XL. When a new specific or other epithet is taken from the name of a man, it should be formed in the following manner: — (a) When the name of the person ends in a vowel, the letter i is added (thus Caoi from Cao), except when the name ends in a, when e is added (thus Faverae from Favera). (&) When the name ends in a consonant, the letters u are added (thus Magrmsii from Magnus, Gttilliermoudii from Guilliermond) , except when the name ends in -er, when i is added (thus Thaxteri from Thaxter). (c) The syllables which are not modified by these endings retain their origi- nal spelling, even when the consonants k or w or with groupings of vowels which were not used in classical Latin. Letters foreign to botanical Latin should be transcribed and diacritic signs suppressed. The Germanic a, o, ii become ae, oe, ue, tlie French e, e, e become generally e. The diaeresis sign should be used where required. (d) When epithets taken from the name of a person have an adjectival form they are formed in a similar way (e. g. Geranium Eohertianum, Verbena Hasslerana). XLI. The same provisions apply to epithets formed from the names of women. When these have a substantival form they are given a feminine termination (e. g. Cypripedium JSookerae, Bosa Beatricis, Scabiosa Olgae, Omphalodes Luciliae.) 94 MEDICAL MYCOLOGY XLII. New specific (or other) epithets should be written in conformity with the original spelling of the words from which they are derived :ind in accordance with the rules of Latin and latinization. Examples: silvestris (not sylvestris) , sinensis (not chinensis). XLTII. Specific (or other) epithets should be written with a small initial letter, except those which are derived from names of persons (substantives or adjectives) or are taken from generic names (substantives or adjectives). XLIV. In the formation of specific (or other) epithets composed of two or several roots taken from Latin or Greek, the vowel placed between the two roots becomes a connecting vowel, in Latin i, in Greek o; thus menthifolia, salvUfolia, not Menthae folia, salviaefolia. When the second root begins with a vowel and euphony requires, the connecting vowel should be eliminated (e. g. lepidantha) . The connecting vowels ae should be retained only where this is required for etymological reasons (e. g. caricaeformis from Carica, in order to avoid confusion with car- iciformis from Carex). In certain compounds of Greek words no connecting vowel is required, e. g. hrachycarpus and glycyphyllus. Art. 71. When the spelling of a generic name differs in Linnffius' Species Plantarum, ed. 1, and Genera Plantanoii, ed. 5, the correct spelling is determined by the following regulations: — (1) If Linnffius subsequently to 1753-54 consistently adopted one of the spellings, that spelling is accepted, e. g. Thuja (not Thmja) . (2) If Linnfeus did not do so, then the spelling which is more cor- rect philologically is accepted, e. g. Agrostemma (not Agro- stema) . (3) If the two spellings are equally correct philologically, and there is a great preponderance of usage in favor of one of them, that one is accepted, e. g. Rhododendron (not Rhododen- drum). (4) If the two spellings are equally correct philologically and there is not a great preponderance of usage in favor of one of them, then the spelling that is in accordance or more nearly in accordance with the Recommendations is accepted, e. g. Ludwigia (not Ludvigia), Ortegia (not Ortega). Section 14. — Gender of Generic Names. Art. 72. The gender of generic names is governed by the following regulations : — (1) A Greek or Latin word adopted as a generic name retains the gender assigned to it by its author: e. g. Orchis (f.), St achy s (f.). (2) Generic names which are modern compounds formed from two or more Greek or Latin words take the gender of the last. If the ending is altered, however, the gender will follow it. Examples of names formed from Greek* words: The generic name Andropogon L. was treated by Linnaeus as neuter, but it, like all other modern compounds in which the Greek masculine word pogon is the final element (e. g. Centropogon, Cymbopogon, Rhisopogon), is now treated as masculine. Similarly all modern com- pounds ending in -codon, -myces, -odon, -panax, -stemon and other masculine words are masculine. The generic name Dendromecon Benth., Eomecon Hance and Ees- peramecmi E. L. Greene are treated as feminine, because they end in the Greek fem- inine word mecon, poppy: the fact that Bentham and E. L. Greene respectively as- ♦Examples of names formed from Latin words are not given, as these offer few difficulties. BOTANICAL NOMENCLATURE 95 cribed the neuter gender to the names Dendromecon and Hesperomecon is imma- terial. Similarly all modern compounds ending in -ackne, -carpha, -cephala, -chlamys, -daphne and other feminine words are treated as feminine. The generic names Aceras 11. Br., Aegiceras Gaertn. and Xanthoceras Bunge are neuter because they end in the Greek neuter word ceras ; the fact that Kobert Brown and Bunge respectively made Aceras and Xanthoceras feminine is immaterial. Sim- ilarly all modem compounds ending in -dendron, -nema, -stigma, -stoma and other neuter words are neuter. Names ending in -anthos (or anthus) and those in -chilos (or -chilus) ought strictly speaking to be neuter, since that is the gender of the Greek words anthos and cheilos. These names, however, have been with very few exceptions treated as masculine, hence it is agreed to assign that gender to them. Similarly those ending in -gaster, which should strictly speaking be feminine, are treated as masculine in accordance with botanical custom. Examples of compound generic names where the termination of the last word is altered: Hymenocarpus, Dipterocarpus and all other modern compounds ending in the Greek masculine carpos (or carpiis) are masculine. Those in -carpa or -carpaea, however, are feminine, e. g. CalKcarpa and Polycarpaea; and those in -carpon, -carpum or -carpium are neuter, e. g. Folycarpon, Ormocarpum and Pisocarpimn. (3) Arbitrarily formed generic names or vernacular names used as generic names take the gender assigned to them by their au- thors. Where the original author has failed to indicate the gender, the next subsequent author has the right of choice. Examples: Taonabo Aubl. (Hist. PI. Guiane, i, 569: 1775) is feminine; Aublet's two species were T. deniata and T. puncta,ia. — Agati Adans. (Fam. ii, 326: 1763) was published without indication of gender: the feminine gender was assigned to it by Desvaux {Jovrn. Bat. i, 120: 1813), who was the first subsequent author to adopt the name, and iiis choice is decisive. Section 15. — Various Recommendations (Rec. XLV-L). XLV. Wlien writing in modern languages botanists should use Latin scientific names or those immediately derived from them, in preference to names of another kind or origin (popular names). They should avoid the use of the latter unless these are very clear and in common use. XLVI. Every friend of science should oppose the introduction into a modern language of names of plants which are not already there, unless they are derived from Latin botanical names by means of some slight alteration. XL VII. Only the metric system should be used in botany for reckoning weights and measures. The foot, inch, line, pound, ounce, etc., should be rigorously ex- cluded from scientific language. Altitude, depth, rapidity, etc., should be measured in metres. Fathoms, knots, miles, etc., are terms which should disappear from scientific language. XL VIII. Very minute dimensions should be reckoned in /i (micromillimetres, microns, or thousandths of a millimetre) and not in fractions of millimetres or of lines, etc. ; fractions encumbered with cipiiers and commas easily give rise to mis- takes. XLIX. Authors should indicate clearly and precisely the scale of the figures which they publish. L. Temperatures should be expressed in degrees of the centigrade thermometer of Celsius. Chapter IV. — Interpretation and Modification op the Rules (Art. 73, 74). Art. 73. A small permanent International Executive Committee is established with functions including the following:— (1) Interpreting the Rules in doubtful cases, and issuing con- sidered "Opinions" on the basis of the evidence submitted. (2) Considering Nomina co7iservanda, Nomina amhigua, Nomina dubia and Nomina confusa, and making recommendations thereon to the next International Botanical Congress. 96 MEDICAL MYCOLOGY (3) Considering all proposals for the modification of the Rules and reporting thereon to the next Congress. (4) Reporting on the effects of modifications of the Rules accepted at the preceding Congress. Art. 74. These Rules can be modified only by competent persons at an International Botanical Congress convened for the express purpose. Modifications accepted at one Congress remain on trial until the next Congress, at which they will receive sanction unless undesirable conse- quences, reported to the Executive Committee, show need for further amendment or rejection. *Appendix I. — Regulations for determining types. *Appendix II. — Nomina conservanda familianim. Appendix III. — Nomina generica conservanda. *Appendix IV. — Nomina ambigua. *Appendix V. — Nomina dubia. *Appendix VI. — Nomina confusa. *Appendix VII. — Representative Botanical Institutions recognized under Art. 34. Appendix VIII. — Nomenclature of Garden Plants. ♦Drafts of these Appendixes will be prepared for submission to the next Inter- national Congress. CHAPTER VII MUCORALES In the Mucorales or Zygomycetes, the thallus is usually coenocytic, al- though in some of the higher forms it is secondarily divided into cells. Under certain conditions the hyphae may fragment into hyphal bodies, etc., which occasionally develop further as sprout mycelia. In an extremely unfavorable environment, small portions of hyphae develop into thick-walled chlamydo- spores. In most forms the reproductive structures are aerial. The haploid my- celium produces sporangia or conidia, the diploid mycelium produces zygo- spores. Sporangia are typical of the Mucoraceae and the Endogonaceae, conidia of the Entomophthoraceae, a group of parasites upon insects. The sporangia form nonmotile, endogenous sporangiospores. In the successively higher genera there is a tendency for reduction in the number of sporangio- spores produced by a sporangium until the sporangium is practically reduced to the level of a conidium which produces mycelium directly without the medium of a spore formation. The sexual act is essentially isogamous in spite of heterothallism. The higher Mucorales tend more and more toward heterogamy. The products of the sexual act, the zygospores, are primarily resting spores and have never been reported in many species. The relationships of the Mucorales are wholly obscure. Vuillemin (1886, 1912) and Lotsy (1907) connect this order through Basidioholus, a genus dividing its life cycle between the digestive tracts of beetles and frogs, to the Conjugales of the green algae and derive the other Entomophthoraceae and Mucoraceae from this genus. Davis (1903) considers in general terms the green algae, especially the isogamous forms, such as Cladophora or the Siphonales. As intermediate forms are wholly lacking and apparently such simple structures as the Mucoraceous sporangium are still unknown in other groups, phylogenetic speculation is unfruitful. The present tendency is to connect the Mucorales with the more primitive Phycomycetes. It is quite po.ssible that the Mucorales include phylogenetically heterogeneous organisms, which are only similar in the copulation of their coenocytic gametangia. In their classification the Mucorales are divided into two groups, one of which forms sporangia, the other conidia. The sporangia! group includes the large family of Mucoraceae and a small family, the Endogonaceae, consisting of small, truffle-like fungi usually growing under leaf mold or in the soil. The conidial group contains the Entomophthoraceae consisting of two tribes, the Basidioboleae found in the intestinal tracts of beetles and amphibia and the Entomophthoreae mostly parasitic on living insects, although capable of 97 98 MEDICAL MYCOLOGY continuing growth and reproduction saprophytically on dead insects. Only the Mucoraceae have been found parasitic on mammals. Basidioholus hominis has recently been reported but has been so poorly described that its relation- ships are still obscure. Mucoraceae. — This family is commonly saprophytic on plant or animal remains, more rarely parasitic on other Mucoraceae, on higher plants, or on animals. They play a large part in the decay of organic substances and a fow species have some economic importance because of fermentations, such as alcoholic fermentation by Mucor javanicns or starch hydrolysis by Rhizopns Oryzae (Wehmer 1907). Except in Haplos%)orangium, where the hyphae are early divided into multinucleate segments by septa, the thallus consists of branched coenocytic hyphae without septa ; in senescence or in the development of reproductive structures, septa are formed irregularly to cut off older vacuolate sections from the younger portions. Furthermore, the hyphae of some genera, in high concentrations of sugars and anaerobic conditions, break up into oidia which may develop further by sprouting ; this sprout mycelium may ferment sugars very much as the true yeasts. (Fig. 2.) In Mortierella and Syncephalis, hyphal branches may fuse where they come in contact with each other, so that the mycelium becomes an anastomosing network. In general, heterothallic species have no definite sexual dimorphism, the strains usually differing slightly in physiologic characters or the positive (female) strain being better developed. Only a few details are known concerning the internal structure of the hyphae. The hyaloplasm of Mortierella reticulata and Rhizopus nigricans (Moreau 1913) contracts into peculiar strands parallel to the hyphal axis. The nuclei are very small throughout (1-3/a in diameter). They divide simul- taneously, both directly and indirectly, in the same hyphal region. The hyphae generally spread out evenly within and upon the substrate. Bhizopus and Absidia have more or less well-differentiated stolons, each con- sisting of a node provided with appressoria or holdfasts, from which radiate new stolons. The appressoria of the forms parasitic on other Mucoraceae are further modified; thus in Mortierella Bainieri they grasp the host hyphae as claws or spirals, and in Piptocephalis Freseniana, they penetrate the interior of the hypha and there branch into a small tuft, as haustoria. Thick-Avalled hypnospores are formed under unfavorable conditions, while in Mucor sphaerosporus the mycelium may form true sclerotia. The hypno- spores usually arise endogenously ; multinucleate protoplasmic portions of varying circumference draw together and, inside the original hyphal mem- brane, surround themselves with a special thick wall (Fig. 3). The stipitate hypnospores (mycelial conidia or stylospores) of Mortierella (Fig. 8, d) and Syncephalis are cut off in scattered or racemose groups on short branches of the mycelium (H. Bachmann, 1900). Under suitable environmental conditions both hypnospores and sclerotia develop to new mycelia. Asexual reproduction takes place through sporangia with sporangiospores. The parts of the mycelium from which the sporangia develop swell consider- MUCORALES 99 ably, and their nuclei divide repeatedly. In forms with stolons, the sporangio- phores branch almost exclusively from the nodes and are then firmly attached to the substrate by the group of rhizoids, but in Ahsidia they may branch directly from the stolons midway between nodes. In the simpler species the sporangiophores are unbranched (Fig. 4, 7) ; in the more highly organized, they are forked, racemose, corymbose, cincinnal, etc. (Fig. 4, 3, 9). The tip of each branch swells up to a sporangium, allowing Fig-. 4. — Sporangia. 1, Ahsidia Truchisi (after Lucet & Costantin) ; 2, Pirella circinans (after Bainier 1883) ; 3, i, Circinella umhellata, showing dehiscence and columella (after Tieghem 1873); 5, Mucor Mucedo (after Brefeld 1872); 6, Rhisopus niger (after Bainier): 7, Rhisoims reflexus (after Bainier 1883) ; S, Rhizopus parasHicus (after Costantin 1900) ; 9, Sporodinia grandis (after Lendner 1908). the protoplasm of the swollen hyphal portion to migrate into it, and is finally abjointed (cut off by a septum). In the tribe, Mortierelleae, the septum is plane or slightly convex, in the Mucoreae it forms a dome projecting far into the sporangium. This dome is usually called a columella. It is generally smooth, cylindric or pyriform and remains attached to the stalk long after the spores are shed. In Piloholus roridus (Tieghem 1875) and Pilaira anomala 100 MEDICAL MYCOLOGY (Brefeld 1881) on dung-, the sporangiopliore swells under the sporangium to a large head. On the absorption of more water, the sporangiopliore bursts at the point of insertion of the columella, shooting off the sporangium and columella often to a height of one meter and with an audible sound. In the Mucoreae the sporangial wall consists fundamentally of cellulose which subsequently is so inerusted with calcium oxalate crystals that it be- comes fragile ; simultaneously the cellulose is hydrolyzed to a more soluble hygroscopic compound, so that it finally dissolves and the crystals scatter. In the majority of genera, only the base of the sporangial wall remains, form- Fig. 5. — Showing the development of the sporangium of Sporodinia grandis. (After Harper 1899.) ing a basal collar about the columella. In the Mortierelleae the wall is equally soluble, but the oxalate crystals are not formed. In Piloholus it is cuticular- ized, except at the base, and permanent. At first, most of the cytologic processes within the sporangium are the same (Fig. 5, i). The content of the young swelling is divided into a central zone, filled mainly by sap and penetrated by a few protoplasmic threads, and a rich peripheral zone, containing most of the nuclei (Fig. 5, 2). The border between the two is differentiated into a foamy protoplasmic layer permeated by narrow, flattened vacuoles. These fuse laterally and form between the vacuolate central portion and the protoplasmic periphery a cleavage cavity; its bordering surfaces are covered with a plasma membrane which is thickened MUCORALES 101 into a wall on the side next the stalk. The central portion remains sterile and becomes the columella (Fig. 5, 4). Its nuclei no longer show any membrane or nucleoli and degenerate, although occasionally fusions or amitotic divisions appear. The peripheral, spherical cap forms the fertile sporogenous layer. There are three types of spore formation. In Piloholus crystallmus (P. microsporus) and P. oediinis (Harper 1899) vacuoles divide the whole sporog- enous protoplasm into uni-, rarely multinucleate, portions, the so-called proto- spores, which round up, swell, and undergo several nuclear divisions before separating into multinucleate portions. These portions again round off, are surrounded by a membrane, and become 2-spored. "We shall find suggestions of this mode of spore formation in the Endomycetales. {Coccidioides and Profomyces, pp. 147, 157.) In Circinnella conica (Moreau 1913) and C. minor (Schwarze 1922) de- velopment is simpler ; here the protospores are surrounded by a membrane with- out further splitting and become spores directly. In most of the other genera, as in Sporodinia (Hai-per 1899), Phycomyces, Ehizopus, Mucor, Ahsidia, and Zygorhynchus (Swingle 1903, Moreau 1913, Green 1927), the protospore stage is omitted. The division of the nuclei in Phycomyces, probably also in the other genera, occurs simultaneously in the whole sporangium. The sporog- enous protoplasm splits directly into multinucleate, rarely uninucleate, por- tions Avhich round off, form a membrane, and develop directly into spores without further nuclear division. The unicellular sporangiospores are gen erally ellipsoid or spherical, hyaline or dully colored; resting free in the sporangium or embedded in a granular gel, probably developed from within themselves, which swells rapidly in water. At germination they swell con- siderably and develop into a mycelium through one or more germ tubes. The original sporangial type discussed above, represented by Mucor, Sporodinia, etc., is modified in higher forms. Either the individualization of protospores is retarded without being suppressed or the sporangia decrease successively in differentiation, size, and spore number until they appear and function as conidia. By retardation of spore formation we have a series from Choanephora to PiptocephaJis. Poorly nourished individuals of Choanephora cucurbitarum exhibit sporangiophores and sporangia like those of Mucor. On the ends of the brown, smooth sporangiospores are 2-3 hyaline processes from each of which as many as 20 hairs may arise. AVhere there is abundant food supply, the spores develop, either on swollen tips of vertical hyphae or on the short secondary branches, exogenously by budding not endogenously by cleavage. Spore formation is ontogenetically retarded and transferred from the interior of the sporangium to its surface. Exogenous spore formation appears more clearly in CunninghameUa in- vestigated by Moreau (1913). In C. echinulata and C. Beriholletiae, the ends of the sporophores swell to sporangia whose content is differentiated into a watery inner, and a rich outer, zone. The peripheral layer pushes out into small spherical sacs on short sterigmata, with 3-8 nuclei each. These sacs are ]02 MEDICAL MYCOLOGY ab jointed and transi'ormed into spores, corresponding in size and form to those of Mncor but borne on the outer, rather than on the inner, surface of sporangia. The series may be continued in Blakeslea trispora (Fig, 6). Under certain environmental conditions, e. g., saturated atmosphere, this species forms typical multispored sporangia with large pyriform columellas (Fig. 6, 1). These sporangia show a marked tendency toward degeneration: with a slight alteration of cultural conditions, they decrease in size and spore number, and Fig. 6. — Blakeslea trispora. Modifications of sporangia: 1, original form; Z, reduced form without columella ; 3-5, formation of exogenous sporangioles ; 6, sporangiospore from sporan- giole. {1-5 X260; 6 X720.) (After Thaxter 1914.) the columella shrinks or disappears. The resulting forms only distantly re- semble the original sporangium (Fig. 6, 2). Where conditions of growth are normal, the spore protoplasm migrates into protrusions, borne on spherical sterigmata (Fig. 6, 3). Meridianal fission divides each of these into 3 spores adorned with little apical tufts of hair (Fig. 6, 5) . The mature protuberance separates from its sterigma or with its sterigma from the sporangium and is disseminated (Fig. 6, 4). Here spore formation is further retarded; between MUCORALES 103 the differentiation of sporangial content into a sterile and a fertile zone and the individualization of single spores, the sporangium also develops into numerous "partial sporangia," each of which forms a .small number of sporangiospores. In Syncephalastrum the ability to form sporangia of the Mucor type has entirely disappeared, and the extramatrical partial sporangia have reached a higher stage of development (Fig. 7). Several palmately joined sterigmata develop into long cylindric tubes which receive as many as 20 nuclei each. 9 8)0 0® ^J'm SS ©% V Fig-. 7. — Syncephalastrum cinereum. Development of extramatrical partial sporangia. (X950.) (After Moreau 1914.) When these have reached their full length, their content splits into uni- or multinucleate portions which round off and are surrounded by walls. The spores are finally liberated by the dissolution of the sporangial wall (Thaxter 1897, Moreau 1913). In Syncephalis, after the destruction of the partial sporangium, the spores remain connected with the adjacent cufflike part of the sporangial wall; the spore wall itself remains thin and insignificant while the sporangial wall is thick and occasionally sculptured. In S. aurantiaca, the partial sporangia divide by septa into as many locules as there are spores. When these septa 104 MEDICAL MYCOLOGY split, the partial sporangia divide into oidial members, each of which contains a spore ; thus the spores are completely surrounded by a sporangial wall, in- separable from their own wall. The functionless sporangium remains in open connection with the sporifer- ous hypha ; it remains capitate and does not collapse until after the maturing of the partial sporangia. It may be regarded as a degenerate form only in so far as the sterile inner part is no longer separated from the peripheral spore protoplasm by the columellar wall. In Piptoceplialus, the sporangium loses its capitate form, and shrinks to a verrucose basal cell, with an apical partial sporangium which the degeneration of the basal cell frees from the sporiferous hypha (Brefeld 1872, Tieghem 1875). The partial sporangia break up into monosporous members whose sporangial wall is fused with the spore membrane. The sporangiophores, therefore, have become conidiophores, rec- ognizable as sporangiophores only through their phylogeny. In the Thamnidium-Chaetocladium series, the sporangiospores are nu- merically much reduced, their functions being assumed by sporangia which successively degenerate to conidia. In Tliamnidium elegans, the main axis possesses an apical multispored sporangium which has a columella. Under certain conditions, dichotomous branches terminating in sporangia are formed from the main axis. These sporangia, however, are smaller than the terminal ones, have no columella, become loosened as a whole from the sporangiophores, and contain few, generally 4, spores. Spores are liberated not by deliquescense but by disintegration of the sporangial wall. kSuch reduced sporangia are called sporangioles. The spores in both types of sporangia behave similarly as regards germination and further development. "When well nourished, the sporangioles persist through several generations and finally become as large and multispored as the sporangia. Conversely, with poor food supply a termi- nal sporangium may turn into a sporangiole, often containing but one spore. This line of development is continued through Chaetostylum Fresenii (Thamnidiuni chaetocladioides) . Here true sporangia are borne only with adequate nourishment, while Avhere the food supply is limited, the terminal sporangia abort. The terminal sporangia which have declined in these two species, disap- pear in Chaetocladium, where the sporangioles also degenerate. They become monosporous, the spore membranes fusing with sporangial walls. In Chaeto- cladium Jonesii this double nature of the spore wall is evident, for on germina- tion the sporangial wall separates from the spore. In C. Brefeldii, however, this differentiation disappears, and the germ tubes protrude directly from the wall. Again the sporangium has been transformed to a conidium. In another series, Mortierella and Haplosporangium show a similar degen- eration. The sporangium of Mortierella is separated from the sporangiophore by a septum (Fig. 8). Since its contents are not differentiated into fertile and sterile zones, the spores arise directly by cleavage of the whole proto- plasm. Their number is notably reduced, in some species to 2 or 4. Haplo- MUCORALES 105 sporangium bisporale (Thaxter 1914) exhibits this condition as a general rule. The sporangia remain very small and retain only 1 or 2 spores. The spore wall is delicate, the sporangial wall thick and sculptured. The same process of reduction as in the unrelated Thamnidium and Chaetocladium has occurred here and has led to the formation of 1- or 2-spored sporangioles, biologically functioning as conidia. Both sexual and asexual reproduction are known in most Mucoraceae. The type of reproduction in the homothallic forms depends mainly on con- ditions of nutrition ; the heterothallic forms require the presence of both sexes. Mycelia of one sex may be cultivated alone indefinitely without the appear- ance of normal sexual reproduction, which appears promptly whenever the opposite sex is brought into the vicinity. Fig. 8. — Mortierella niveovelutina. a, g, hyphal anastomosis ; h, spoiangiospore ; c, sporangia ; d, stylospores or aerial conidia ; e, chlamydospores ; /, sporangia attached to sporan- giophores. When the environmental conditions are favorable, the mutual approach of two sexually mature (in heterothallic forms also dynamically opposite) hyphae results in the formation of outgrowths toward each other. Each out- growth is cut off from the hypha close behind the tip by a septum laid down from the wall inwards. The tip cell is the gametangium, the hypha is the suspensor. As the homothallic forms are bisexual, apparently there occurs in their hyphae at sexual reproduction, a spatial sepai-ation of + and - energids. In some species, the copulating branches arise from ordinary hyphae, in others they are developed on special branches, the zygophores (Fig. 9). The two separating walls between the gametangia are gradually dis- solved from the middle toward the edge, and the zygote becomes a hypnospore 106 MEDICAL MYCOLOGY by the formation of a many-layered wall. This spore is called a zygospore. In the homothallic species when the copulating branch finds no mate, the gametangium surrounds itself with a thick many-layered wall and is called an azyg-ospore or, less properly, a chlamydospore. The same thing happens Fig. 9. — Zygospores. 1, Ahsidia glauca (after Lendner) ; 2, Mucor hiemalis (after Lendner 1908) ; 3, Parasitella simplex (after Burgeff 1924) ; -i, Zygorhynchus heterogamus (after Blakeslee 1913) ; 5, Rhizopus nigricans (after Bary) ; 6, Sporodinia ginndis (after Bainier 1882) ; 7, Phycomyces nitens (after Van Tieghem & Lemonier) ; 8, Spinellus fusiger (after Bainier 1882) ; 9, Mucor racemosus (after Bainier 1883) ; 10, Circinella spinosa (after Bainier 1882). if the cultures are placed in an unfavorable environment, such as high tem- peratures. In the heterothallic forms, similar phenomena may occur if the MUCORALES 107 copulating branches belong to two different species; in this case, they cease growing and transform the; ganietniigia (in case they liave ali'(!ady been cut off as such) into azygospores. This inconii)lete hybridization, however, does not seem to occur between all species, for, while it occurs between Phycoriiycea nitens + and Mucor Mucedo - and conversely, it does not between Phycomyces nitens and Rhizopus nigricans (Blakeslee 1904-1927), While both gametangia are usually of the same size and thus externally suggest isogamy, in individual species their size relationships show a notable tendency to heterogamy. Thus, in the homothallic ZygorJiyncJins Moelleri, the copulating branches are unequally developed. In the heterothallic Absidia Orchidis the gametangia are of unequal diameter, the resulting zygospores being conic. In Piptocephalis, the zygospore grows upward from the point of fusion so that it is borne upon the top of the copulation branch. In Syncephalis Fig. 10. — Development of Uie zygospore of Sporodinia yrandis. (After Keene 1914.) nodosa, one copulation branch coils around the other in a helix (Thaxter 1897) ; the zygospore does not arise at the point of fusion but comparatively distant, on the outer portion of the helix near the septum separating the gametangium from the suspensor. Cytologically all the; above-mentioned processes behave similarly. Sporo- dinia grandis has been more complete studied. Its young gametangia con- tain more than a thousand nuclei each (Fig. 10). While the separating walls between the gametangia are dissolved, the nuclei undergo almost simultaneous division ; their cytoplasm intermingles and their nuclei subsequently pair and fuse. Those without mates, especially those near the periphery, degenerate and disappear. Meanwhile at the surface a wall of several layers has been formed, the suspensors collapse, and the zygospores presently lie free upon their substrate. It is characteristic of this process that no dynamic differentiation occurs oetween the + and - energids in spite of their spatial separation. Thus, both 108 MEDICAL MYCOLOGY gametangia are cytologically equivalent, and fertilization is isogamous with reference to the nuclei. In Sporodinia, since there is no individualization of gametes, two coenocytic gametangia copulate and accomplish multiple fertiliza- tions. In ZygorrhyncJius Dangcardi, all but 4 gamete nuclei degenerate in the young zygote. The surviving four fuse in pairs very late after the endo- spore has been formed. A similar retardation of caryogamy has been observed in Phy corny ces nit ens (Burgeff 1915) in which the nuclei in zygospores 5 months old and ready to germinate, still lie in pairs. The wall of the zygospores in the more carefully studied species of Mucor, Sporodinia, and ZygorrhyncJius consists of 5 layers (Vuillemin 1904). The in- nermost layer is thin and granular ; it forms the transition from the protoplasm and to a certain extent is the mother layer. The next is thickest and is called the cartilaginous layer on account of its elasticity. This is covered by a thin sheath, the middle cuticular layer. The fourth or carbonaceous layer is fragile and brown or black ; the outermost cuticular layer is either pale and elastic, or dark and fragile, and often interrupted or fractured. The greatest modifi- cations in the various genera are shown by the surface of the carbonaceous layer which is verrucose or reticulate. The two outer layers are grouped as the exospore, the three inner as the endospore. In Absidia and Phycomyces the zygospores are loosely surrounded by branches from the suspensors (Fig. 9, 1, 7). In Mortierella these branches intertwine with the neighboring hyphae into a solid felt whose outer surface is cuticularized and brown. AVithin this tissue lies the zygospore. The zygospores germinate only after a long resting period. The exospore is ruptured, the endospore puts forth a germ tube which develops to a mycelium or, with insufficient nourishment, directly to a sporangium or a conidiophore. During germination, meiosis of the diploid nuclei occurs. Where the germ tube becomes the fundament of a sporangium (e. g., Phycomyces nitens, Burgeff 1915) meiosis occurs only in the latter which is called a "germ sporangium," and as we shall see later is the precursor of the ascus. The sexual relationships existing at meiosis have been more closely studied for three types. {Sporodinia, Mucor Muceclo, and Phycomyces nitens, Blakeslee 1904, 1906.) In Sporodinia the sporangiospores are liomothallic and the separation of the + and - energids occurs only in the formation of the copulation branches. In the heterothallic Mucor Mucedo the separation of the + and - energids occurs probably in the formation of sporangia ; i.e., the spores are all of one sex in one sporangium, either all + or all -. In the equally heterothallic Phycomyces nitens, the separation of sexes occurs only in the formation of spores. Even so, it is incomplete ; besides the + and - spores there are also unstable, neutral, bisexual spores in whose sporangia the separation into + and - spores is continued (Burgeff 1912). MUCORALES 109 Although some of the following characters are often unstable, they should be noted in the study of the Mucorales. The presence or absence of branching is often difficult to determine. Sometimes typical branching may be found in the small sporangiophores next the substrate, when it is not observed in the larger sporangiophores. If stolons arc present, note their arrangement and the disposition of the sporangiophores upon them ; also note the presence of holdfasts, etc. The nature of the medium influences the height of the spo- rangiophore, which should be determined only in cultures where optimum con- ditions of growth prevail. Malt gelatin (10%) or 10% gelatin to which has been added the residue of white wine from which the alcohol has been distilled, are suitable. The latter is known as Lendner's medium. Report the height of the spo- rangiophore from a colony cultivated at room temperature for at least 8 days, its diameter, the diameter of the sporangium (one of the larger ones), the height and diameter of the columella, the mean diameter of the spores (or their mean dimensions), and the diameter of zygospores and chlamydospores. The spo- rangial membrane may be diffluent, in which case, younger sporangia should be measured, or the sporangia mounted in a mixture of glycerol and water. If the membrane easily becomes fragmented, this should be noted. Note the presence or absence of a collar about the columella and the surface of the latter. Spore shape varies in the same sporangium. When a species is re- ported as having spherical spores, the majority are spherical, although oval or irregular spores may be present. Disregard variations in size unless they are extreme. To find hypnospores use cultures 2 weeks old or more on solid media or on liquid media with much sugar for sprouting cells. Note fermenta- tions in case the sprout cells are abundant. Classification. — There is still considerable disagreement among mycologists as to the subdivisions of this order. It is clearly divisible into three groups which the older mycologists considered families (Mucoraceae, Endogonaeeae, and Entomophthoraeeae). Some of the younger generation would elevate the old families to suborders, the latter two suborders containing a single family each, while the old tribes of the Mucoraceae are elevated to family rank (Fritzpatrick 1930). In any case only members of the tribe IMucoreae and Mortierelleae have so far been reported pathogenic and need be considered here. Since many of the saprophytic genera are difficult to define, and there are strong differences of opinion on synonymy, only the pathogenic genera which have been reported pathogenic to mammals are included in the fol- lowing kej^s. MUCORACEAE Mycelium coenoeytic, forming loose felted colonies ; sporangiophores erect, often variously branched ; sporangia usually with a columella (absent in Moriierella) ; sporangiophores abundant in the Mucoreae, in other tribes often reduced to a few spores in small sporangioles ; zygospores resulting from the copulation of gametangia. 110 MEDICAL MYCOLOGY Key to Pathogenic Genera Sporangium containing a columella; zygospore not surrounded by a layer of interwoven hyphae; sporangioles not formed, sporangial wall thin, not cutinized. Miicoreae. Sporangiophores arising directly from the mycelium, suspensors lacking outgrowtlis ; gametangia essentially alike. Mucor. Sporangiophores arising from aerial arching stolons which develop rhizoids at points of contact with the substratum. Sporangiophores borne on the arching internodes of the stolons between the nodes; sporangia pyriform ; zygospores, when present, vpith prominent circinate out- growths. Absidia. Sporangiophores arising in a fascicle from the node of the stolon; sporangia spherical. Bhisopus. Sporangium lacking a columella; zygospore where known enveloped by a thick layer of in- terwoven hyphae; sporangioles and conidia formed in some cases, when present isolated, not covering an enlargement on the sporangiophore or conidiophore ; sporangiophore erect, tapering upward, usually not branched. Mortierella. MUCOR Mucor Micheli, NovaPlantarum Genera 215. 1729; Linne, Species Plan- tarum 1185, 1753; Gray, Natural Arrangement of British Plants 1: 560, 1821; Fries, Systema Myeologicum 3: 320, 1829. Type species : Mucor Mucedo L. Mycelium abundant both in and on the substratum, lacking stolons and rhizoids ; sporangiophores occurring singly, erect, simple or occasionally branched, each branch terminated by a sporangium which is large, spherical, many-spored with an evanescent sporangial wall neither cutinized nor in- crusted ; columella always present, variable in shape ; sporangiospores spheri- cal to ellipsoid, with a thin, smooth wall ; zj^gospores borne on the mycelium, suspensors lacking outgrowths ; chlamydospores present in some species termi- nal or intercalary, smooth, hyaline ; oidia accompanied by fermentation found in the submersed mycelium. At present there is little conclusive evidence that this typically saprophytic genus is pathogenic for man. Most of the cases originally attributed to this genus were based on misidentification of the organism and belong elsewhere. For descriptions of species of this genus see the systematic accounts of A. Fischer (1892), Lendner (1908), and Povah (1917). Mucor Mucedo L., Species Plantarum 1185, 1753. The case of Fiirbringer (1876) should probably be referred to Absidia corymhifera. Mucor racemosus Fresenius, Beitr. z. Mycol. 12, 1850. Pleurocystis Fresenii Bonorden, Handb. Allgem. Mykol. 124, 1851. f Mucor scarlatinosus Hallier, Zeitschr. f. Parasitenk. 1: 117-184, 290-352, Pis. 3, 4, 1869. Chlamydomucor racemosus Brefeld, Unters. Gesammtegebiet der Mykol. 8: 223, 1890. MUCORALES 111 M^icor scarlatinosus Hallier, a very poorly described organism was sup- posed to have been isolated from a case of scarlatina. It was quite probably a contamination and has been refen-ed here as a possible synonym by A. Fischer, 1892. M. racemosus was reported by Bollinger (1880) from the respiratory tract of birds but was not pathogenic for laboratory animals, by Zurn (1876) in the nasal cavity of a sheep, and by Frank (1890)' in a tumor in a horse, but both determinations doubtful. Savoure (1906) reports that it was not pathogenic for rabbits. Mucor pusillus Lindt, Arch. f. exp. Path, u Pharm. 21: 272. PL 2, Figs. 1-6, 1886. [Saprophyte.] M. ramosus Jakowski, Gazetta Lekarska No. 34: 1888. [Centralbl. Bakt. II, 5: 388, 1889] not Lindt, I.e., p. 275. The fungus reported by Jakowski from the outer ear has been referred here by Vuillemin (1904), while the original author and Barthelat (1903) refer it to Absidia ramosa (Lindt) Lendner. Since there is so little conclusive evidence of pathogenicity, the reader is referred to the systematic accounts of A. Fischer 1892, Lendner 1908, and Povah 1917 for aid in determining cultures. ABSIDIA Absidia Tieghem, Ann. Sci. Nat. Bot. VI, 4: 350, 1876. Lichtheitnia Vuillemin, Bull. Soc. Myc. France 19: 119-127, 1903. Type: Absidia capillata Tieghem. Vuillemin (1903) divided this genus into six genera of which Lichtheimia contained the parasitic fungi so far described. The characters on which the separation was based seem comparatively trivial, and this segregation has not been followed by systematists of the group, although recognized by some medical men. Mycelium forming stolons, often branched, more or less curved producing rhizoids more or less branched at the surface of the substratum; sporangio- phores erect, usually in groups of 2-5 arising from the curved part of the stolon, not from the place of origin of the rhizoids ; sporangia pyriform, erect, with an infundibulifonn apophysis, membrane neither cutinized nor incrusted, diffluent, leaving a small collar at the base ; columella hemispheric, conic, or terminated by a single projection, continuous with the apophysis which is cutinized and of deeper color than the sporangiophore ; spores small, oval usually smooth, rarely echinulate, hyaline ; zygospores formed on the stolons surrounded by circinate filaments, cutinized, growing from one or both of the suspensors. This genus differs from Rhizopus by the development of the sporangiophores from the internodes, by the pyriform sporangia, by the columella continuous with the apophysis and by the suspensors provided with circinate filaments. 112 MEDICAL MYCOLOGY Key to Pathog-enic Species Growth good at ordinary temperature Spores mostly spherical, SA/x. in diameter; columella usually somewhat spinescent A. corymbifera Spores elongate or oval, 4-5x2-3/^; columella smooth A. ramosa Growth poor at ordinary temperature, optimum about 37° C. Sporangia 36-70^, columella 60^, spores ovoid 4x2-3ja; some growth at 51° C. A. Truchisi Sporangia 30-38ai, columella 26/x, spores 3.2x3.75;U,; no growth at 51° C. A. Begnieri Absidia corymbifera (Cohn) Saccardo & Trotter in Saccardo, Sylloge Fungorum 23: 825, 1912. Mucor corymbifer Cohn in Lichtheim, Zeitschr. Klin. Med. 7: 147, Pis. 6-8, 1884; Barthelat, Ann. Parasitol. 7: 25-30, 1904. Lichtheimia corymbifera Vuillemin, C. R., Acad. Sei. Paris 136: 516, 1903. Mucor corymbifera var. typica LicJitheimi Lucet & Costantin, Arch, de Parasitol. 4: 380, 1901. Absidia Lichtheimi Lendner, Mat. Fl. Cryptog. Suisse 3: 143, 144, 1908. Many cases in the literature dealing with bronchomycosis (Paltanf 1885, etc.). Lang & Grabauer (1923) discuss the clinical and pathologic aspects fully and summarize earlier cases. This fungus has also been reported from the ear by Huckel 1884, Siebenmann 1889, and Graham 1890. Mycelium white, then clear gray, completely covering the substrate; hyphae often up to 15/x in diameter, branched, hyaline under microscope. Sporangiophores resupinate, branching as a corymb, terminated by sporangia, occasionally a few small sporangia on short pedicels. Sporangia hyaline, pyriform up to lO/x in diameter with mean 45-60/x, small sporangia 10-20'/^, wall hyaline, smooth, diffluent, often with a basal collar; columella hemispheric, 10-20/x, smooth or sometimes papillate, smoke-gray or brownish continuous with the infundibuliform apophysis, spores nearly spherical 2-4/t, smooth, hyaline, occasionally up to 6ju,. Growth has been reported good on moist bread, potato, carrot, sugar media with slightly acid reaction, and Sabouraud agar; growth poor in liquid media; unfavorable conditions of humidity or lack of oxygen cause abundant pro- duction of gemmae; growth possible at 12-15°, optimum 36°, killed at 55° C. Absidia italiana (Costantin & Perin) Dodge, comb. nov. Lichtheimia italiana Costantin & Perin. Bull. Soc. Med. Chir. di Pavia 35: 1922. Lichtheimia italica Pollacci & Nannizzi I miceti patogeni dell'uomo e degli animali 3: No. 26, 1924; Perin, Arch, di Clin, e Patol. Med. 2: 5, Oct., 1923; Trattato Micopatol. Umana 1: 55-73, 1925. I have been unable to locate any of the original descriptions of this organ- ism. Some have reduced it to synonymy with A. corymbifera. The following notes are based on Perin (1925). MUCORALES 113 Isolated from sputum and from tissue fragments from the lungs. Patho- genic for laboratory animals. Sporangiophores branched, lacking septa, primary axes resupinate, sec- ondary fertile axes erect with only two or three branches ; rhizoids variable. Sporangia up to 66 x 58/*, columella 45/a broad, varying from hemispheric to conic, pedicel 20/x in diameter. Spores slightly ovoid, 3.2-4.2 x 2-2. 8/a. Colony on agar, white, cottony, gradually becoming grayish and finally brownish. Gelatin slowly liquefied. On liquid media, small flocci in the depths, a cottony pellicle above. Milk coagulated and acidified. Absidia ramosa (Lindt) Lendner, Mat. Fl. Ciyptog. Suisse 3: 144-146, 1908. Mucor ramosus Lindt, Archiv. f. exp. Path. & Pharmakol. 21: 269, 1886. Lichtheimia ramosa Vuillemin, Arch, de Parasitol. 8: 562-572, Figs. 1-4, 1904. Not Mucor ramosus Bulliard which is a synonym of M. aspergilJus Scopoli transferred by Link 1824 to Sporodinia. Perhaps not the organism of Jakow- ski (1888) which may be M. pusillus Lindt fide Vuillemin. Reported by Jakowski (1888) in human ear. Originally isolated on damp bread, found pathogenic to laboratory animals, and reported by Vuillemin from lesions and mucus of the nose in horses, also adenitis of lower jaw. Found in generalized infection in swine by M. Christiansen (1922) and in cases of abortion in cows by Bendixen & Plnm (1929). Sporangiophores branched as in A. Corymhifera, usually lacking cross- walls. Primary axes resupinate like stolons, but not recurved. Fertile axes little branched, with fewer umbels, especially compound umbels, than in A. corymtifera. Primary axes bear rhizoids frequently instead of terminal spo- rangia. Rhizoids variable. Sporangia much as in A. corymhifera, the diffluent membrane covered with fine granulations. Spores elongate, oval, or sub- cylindric, 4.8 x 2.8ju, (4.6 x 2.6, 5.2 x 3/x) brownish yellow. Columella very rarely conic, mostly ovoid, not spinescent, 57.5 x 4/x where it separates from the apophysis, 35/* in maximum diameter; blue, darkening with age. Distinguished from A. corymhifera by nonspinescent columella and sub- cylindric spores. Near A. diibia, intermediate between the subgenera Lich- iheimia and Tieghemella. Var. Rasti Lendner, Mat. Fl. Cryptog. Suisse 3: 146, 1908. Rising 1 cm. at the most above the substrate, mycelium constantly bluish gray, sporangia more abundant, present everywhere, larger, spores slightly more elliptic, often abnormal in shape. Var. Zurcheri Lendner, Mat. Fl. Cryptog. Suisse 3: 146, 1908. Rising to 4 cm. above the substrate, mycelium pure white, culture less vigorous, sporangia formed only at the surface, the deeper filaments remain- ing pure white, spores not abnormal in shape. Both varieties grow well on potato at 45° C. Absidia Regnieri (Lucet & Costantin) Lendner, Mat. Fl. Cryptog. Suisse 3: 146, 147, 1908. 114 MEDICAL MYCOLOGY Mucor Reg7neri Lucet & Costantin, Arch, de Parasitol. 4: 362-384, 1901; Barthelat, Ann. Parasitol. 7: 34, 35, 1904. Lichtheimia Regnieri Vuillemin, C. R. Acad. Sci. (Paris) 136: 516, 1903. Isolated from another stable with an environment similar to that of A. Truckisi, pathogenic for rabbit. Mycelium lax, weak vegetative growth, of a uniform color of gray slightly tinged with blue. Sporangiophores in corymb or umbel, the outer rays longer, unequal, swollen below the sporangium so that the collar seems to divide the columella into two parts, 3-8/x, in diameter, sometimes up to 12.5 or even 19/*. Sporangial membrane smooth and transparent leaves little trace of a collar. Columella ovoid, pyriform, with a clear, brown color which ex- tends a certain distance down the sporangiophore, frequently small W.lfi, sometimes 23/* and rarely up to 35/*. Spores usually round, mean 3.2 to 3.75, some 2.5/*. Besides the typical spores some are avoid (3.8 x 5, 3.2 x 2.9/*) some are irregular to almost polyhedral. Zygospores unknown. The above characters were obtained at 25° C. on solid media. Below 20° growth normal, sporangia abundant, becoming 30-38/* and pedicels 3.8 to 6.5/*. At 51-52°, slight or no growth ; pedicels simple, sporangia 19/*, spores few. Killed at 55-56°. Absidia Tnichisi (Lucet & Costantin) Lendner, Mat. Fl. Cryptog. Suisse 3: 146, 1908. Mucor Truchisi, Lucet & Costantin, Arch, de Parasitol. 4: 362-384, 1901. Barthelat, lUd. 7: 31-34, 1904. Mucor corymhifer Cohn, var. Truchisi Sartory, Champ. Paras. Homme Anim. 91, 1921. Isolated from a hoi-se infected with Trichophyton minimum. Pathogenic for rabbits. Mycelium lax and in general vigorous, whitish or very light gray, becom- ing darker in old cultures on solid media. Sporangiophores in corymbs or in an umbel with branches of unequal length, the outer usually longer, swollen below the sporangium so that the collar seems to divide the columella into two parts, 2-7/* thick, secondary axes 55-195/*; sporangia spherical with trans- lucent membrane, smooth, about 35/* in diameter, spores regularly ovoid to slightly elongate, mean 4 x 2.5/*, smaller 3.75 x 2.5/*, and larger 4.5 x 3/*. Columella pyriform, brown at the base, becoming lighter toward the apex; mean breadth 20-26/*, smaller 4-15, larger 30/*. Zygospores unknown. At low temperature, 10-18°, mycelium little developed, fine and delicate ; fructifica- tion little developed, pedicels always simple, sporangia up to 70/t, and spo- rangiophores 14/* in diameter. At 51-52° C. for 5 days, growth rich, fruiting abundant. Sporangia 26/*, columella 17-19/*, pedicels 5-6/*, spores 5 x 2.5- 3/*. At 53° for 17 days growth still noticeable; killed at 55-56°. Growth very good on raw potato. Absidia cornealis (V. Cavara & Saccardo) Dodge, n. comb. Mucor cornealis V. Cavara & Saccardo in V. Cavara, Ann, di OttalmoL 42: 650-674, 1 pi, 1913, MUCORALES 115 Producing lesions in the cornea, Italy. Mycelium loosely interwoven, white, cinereous-plumbeous on milk, bread, and potato; growth abundant at 37° C, very slow at 15° C, and at 51° C. ; sterile hypliae large, branched, 14-15/* in diameter, hyaline, corymbose or racemose branched at the tips ; branches of sporangiophores either alternate or opposite, simple or dichotomous, 80-300 x 7-8/i., leaving the main axis at about an angle of 45° slightly enlarged at the tips; sporangia spheric or sub- spheric with a thin membrane 40-44/t in diameter (rarely up to 50-55/a or as small as 15-22/a), columella obovate, pyriform, more or less light fuscous, 22-24/x broad ; spores thin-walled, hyaline becoming light yellowish, spherical, 4-4. 5/a, rarely ovoid, zygospores unknown. RHIZOPUS Rhizopus Bhrenberg, Nova Acta Acad. Leopold. 10: 198, 1820. Bhizomucor Lucet & Costantin, Rev. Gen. Bot. 12 : 81, 1900. The type species is Rhizopus nigricans Ehrenberg. The type of Rhizomucor is R. parasiticus Lucet & Costantin. Aerial mycelium of creeping stolons, with holdfasts at the nodes which at- tach the hyphae to the substrate. Sporangiophores arising in groups at the nodes, sometimes solitary, enlarged above into a columella, as in Absidia. Spo- rangia white at first, becoming black ; spherical or nearly so with base slightly flattened ; membrane not cuticular, uniformly incrusted and entirely diffluent without leaving a basal collar. Columella hemispheric, often flattening after dehiscence, suggesting the pileus of a mushroom. Spores spherical or ovoid, even angular, hyaline or brownish, cuticular walls, smooth or striate, rarely spinulose. Zygospores without covering from outgrowth of suspensors, form- ing in the substrate and on the stolons. Suspensors straight, swollen, without appendages. Key to Pathog-enic Species Spores irregular, angular, subspheric, oval. B. parasiticus. Spores spherical, smooth or echinulate, but not angular. Columella conici or subcylindric (black tongue). E. mger. Columella ovoid or pyriform, pathogenic for rabbit. Clilamydospores not produced. E. rhizopodiformis. Chlamydospores present. E. equinus. Rhizopus equinus Costantin & Lucet, Bull. Soc. Myc. France 19: 200, 1903. Isolated from a horse, pathogenic for rabbit. Found in generalized in- fection in swine by M. Christiansen (1922) and in bovine fetal membranes and fetus by Theobald Smith (1920) who referred his species to R. rhizo- podiformis. Mycelium at first white, then gray after the formation of sporangia. Spo- rangiophores at first isolated and without rhizoids, straight or curved, later in bouquets, frequently provided with rhizoids, cutinized, pale ochraceous ; 50-220/i, sometimes up to 600/x long, 3-12.3/a in diameter. Sporangia 30-115/x in di- 116 MEDICAL MYCOLOGY ameter. Columella 45-51 x 31-41ju. Spores round, sometimes slightly an^lar, smooth, 4/A. Chlamydospores eitriform, 30 x 25 or 40 x 26/a, or spherical, 20^16 in diameter, forming ordinarily on the mycelium. Zygospores unknown. Var. annamensis, P. N. Bernard, Bull. Soc. Myc. France 30: 230-232, PI. 14, 1914; Bull. Soc. Path. Exot. 7: 430, 1914. Isolated from sputum of an Annamite, aged thirty-two, 32 in 1911; sputum blackish, as if mixed with carbon grains. Cough with a little dyspnea. Whole left lung infected. No previous history except bronchitis, with com- plete recovery. Hospitalized for cough in fall, 1910, worse July, 1911, but alive in December, disease seemingly arrested and localized. Intravenous inoculations in pigeons, no effect ; nor intraperitoneal, in guinea pig ; rabbit succumbed by both methods, but not by subcutaneous inoculation. Organ- ism recovered. Isolated sporangiophores without rhizoids 72-450'/a x 8-12/^ usually 150 x 12/x. Sporangiophores in pairs near each other, pedicels short 78-210 x 8 - 12/A, 30-45/x between pedicels, without rhizoids. Sporangiophores with rhi- zoids singly or in pairs, the latter 138-144 x 8-9/a, occasionally pedicels up to 420-780/i long. Sporangia oblate spheroid, 48-84/a. Columella 18-48|U high X 24-52/i broad. Cutinization from pedicels to rhizoids and stolons, less ac- centuated on the columella. No collar after sporangial dehiscence. Inter- calary chlamydospores 36 x 24/* eitriform ; or spherical 30-42/* in diameter ; ovoid 60 x 48-42 x 30/t, numerous even in aerial portions. Spores round, smooth, 4/t, not cutinized. Growth good on Sabouraud agar for several months, then suddenly stopped fruiting on carrot and potato glucose; optimum 37-39° C, tube filled with mycelium in 3-4 days; sporangia in 5 days; no growth at 5° C. ; killed at 100° moist heat in 15-20 minutes. Rhizopus niger (Ciaglinski & Hewelke) Barthelat, Mucorinees patho- genes et les Mucormycoses 55, 56, 1903; Arch, de Parasitol. 7: 46, 47, 1903. Miicor niger Ciaglinski & Hewelke, Zeitschr. Klin. Med. 22: 626, 1893. Isolated in cases of black tongue ; not pathogenic for guinea pigs or rab- bits. Also found later by Sendziak (1894). Stolons provided with numerous rhizoids, forming a snow-white layer. Sporangiophores erect, straight, fasciculate, terminated by spherical sporangia, which become black at maturity. Columella at first cylindric 2-3 times as long as wide, later enlarging and becoming hemispheric ; after the dehiscence of the sporangium assuming the appearance of an open umbrella. Spores oval, smooth, gray, black in mass. Growth good on potato and in bread gelatin, optimum 25-27° C, growth ceases at 37° C. Rhizopus parasiticus (Lucet & Costantin) Lendner, Mat. Fl. Cryptog. Suisse 3: 115, 1908. Bhizomucor parasiticus Lucet & Costantin, Rev. Gen. Bot. 12: 81, PI. 3, 1900; Arch, de Parasitol. 4: 384-408, 1901. [See p. 394.] MUCORALES 117 Mycosis of lung of coiintiy woman, final recovery after several months of treatment with arsenic. See Arch, de Parasitol. 4: 386-389, 1901, for case history and results of inoculations. Nonpathogenic in subcutaneous inocu- lations. Mycelium gray (lead color) to mouse gray then grayish brown to yellow. Stolons and rhizoids irregular. Sporangiophores branched in simple clusters, or in corymbs 12-14/x in diameter, 1-2 cm. long ; sporangia 35-80/x,, with mem- brane covered with fine crystalline needles; columella ovoid, pyriform, cutin- ized slightly brownish, 30-70//, long by 24-56/a in diameter; lateral sporangia similar but much smaller ; pedicels rarely ramifying a second time ; spores irregular or reniform, smooth, 4 x 2.5/x. Zygospores unknown. Growth on most media very good, but less on peptone broth and on very acid or alkaline media, poor on coagulated sera, amniotic liquid, white of egg, cider, apples, or pears, the latter, however, is good if glycerin or glucose is added. Growth more rapid on solid than on liquid media. [Very full description given in Arch, de Parasitol. 4: 384-408, 1901.] Optimum about 37° C. ; growth starts at 22°, at 51-52° very abnormal vegetative growth but no spores. It needs much oxygen. Rhizopus rhizopodiformis (Cohn) Zopf, Die Pilze 317, 1890. Mucor rhizopodiformis Cohn in Lichtheim, Zeitschr. Klin. Med. 7: 148, 1884. Rhizopus Cohnii Berlese & de Toni in Saccardo, Syll. Fung. 7: 213, 1888. Pathogenic for the rabbit Avhen injected into peritoneum and veins. Ziegenhom (1886) was unable to modify pathogenicity of spores. T. Smith (1920) reports this organism on membranes and in lungs and digestive tracts of fetus but probably it was R. equinns. Mycelium white, then mouse-gray, rising as a spider web above the sub- strate. Stolons forming rhizoids at point of contact with substrate, in brown- ish bouquets. Sporangiophores isolated or grouped, erect or incurved, short, 120-125/i, not branched, with brownish membrane enlarging in an apophysis. Sporangia spherical, 60-110/t usually about 66/a, blackish at maturity, smooth with incrusted membranes. Columella forming with the apophysis an ovoid or pyriform organ, 50-75/i broad, membrane smooth and brownish. Spores usually spherical, small, 5-6/x, without angles, smooth, hyaline. Zygospores and chlamydospores unknown. Cultural characters are similar to those of Absidia corynibifera (p. 112). Optimum temperature 37-38° C, sporangia after 48 hours, mycelium changing from white to gray. At 12-15° spores germinate on third day, sporangia on fourth or fifth day. At 45° the mycelium is arrested and spores are killed at 68°. Doubtful Position Rhizomucor septatus (Bezold in Siebenmann) Lucet & Costantin, Arch, de Para.sitol. 4: 362, 1901; Barthelat, Mucorinees pathogenes et les mucormy- coses 52, 1903. Mucor septatus Bezold in Siebenmann, Die Schimmelmycosen 97. Wies- baden, 1889. 118 MEDICAL MYCOLOGY Mycelium colorless, sporangiophores brown, in branching cluster, some- times terminating in an umbel, with small rhizoids at the base with a mecii diameter of lO/i,; secondary pedicels, 3-4 in number, are short with crosswalls at point of branching; sporangia pale grayish brown, spherical, transparent membrane, smooth or slightly papillate, 32/x, in diameter ; columella also brown, spherical or slightly ovoid, mean diameter of 27/*; spores spherical or ovoid, smooth, clear yellowish or brownish, 2.5 - 4/a. Referred to Mucor racemosus by A. Fischer, and Lendner. It also resembles M. hifidus Fresenius. It differs from R. parasiticus in smooth sporangial wall devoid of crystals, sporangia smaller, and sporangiophores always septate. MORTIERELLA Mortierella Coemans, Bull. Acad. Sei. Belgique II, 15: 536, 1863. The type species is Mortierella polycephala Coemans. Mycelium within the substrate or forming a closely appressed weft over its surface, not typically aerial; sporangiophores erect, simple or branched, usually tapering to a delicate tip just below the sporangium, often swollen below; sporangia spherical without columella, wall soon disappearing; stylo- spores unicellular, spherical, echinulate, suggesting sporangia with a single spore; zygospores enveloped by a thick layer of densely woven hyphae which arise just below the gametangia and tend to obscure the details of conjugation. Mortierella niveovelutina Ciferri & Ashford, Porto Rico Jour. Publ. Health & Trop. Med. 5: 134-143, 1 pi, 1929. Isolated from a Porto Rican with a patch of inflamed papules covering the antero-extemal aspect of the right thigh and extending around behind, ending below at the insertion of the adductor muscles. At first glance the appearance reminded one of psoriasis, but when fading, the eruption became discrete and parts once thickly studded with red nodules disappeared without leaving a trace of the former thickly infiltrated red nodular area. Intense pruritus present. Healed by the application of salicylic acid, ichthyol, and sulphur ointment. Colony white, velvety ; mycelium of highly branched hyaline hyphae with apical branches normally bifurcate, occasionally Avith three forks, continuous or later scantily septate, with frequent and complex anastomoses, without rhizoids, 2-3/i. in diameter; hypnospores in chains in liquid media, less abun- dant in solid media, from spherical to elongate with a smooth membrane ; stylospores or aerial hypnospores normally very abundant, singly or in chains of up to 12 cells, generally 2-3/* in diameter with a smooth epispore ; sporangio- phores 30-80/1 long, straight erect, of uniform diameter, never branched, con- taining an apical septum immediately beneath the sporangium ; a single spo- rangium for each sporangiophore, approximately spherical, 30-90/1 in diameter normally about 60/i, irregularly dehiscent with smooth, diffluent membrane, in part more or less firmly fixed in the sporangiophore ; numerous spores in each sporangiophore (15-20 or more) elliptico-apiculate, with the extremities more MUCORALES 119 or less pointed, 3.0-3.5 x 4-4.5/a, producing one or two germ tubes, simple then repeatedly dichotomous ; zygospores not seen. Ferments glucose feebly ; as- similates well the monohexoses, peptone and ammonium sulphate. Growth good on bread and liquid media, such as Raulin's fluid, Difco malt extract, and Lendner's dealcoholized white wine agar. Growth slow on many other media reported. Mortierella sp. Costantin, Bull. Soc. Myc. France 8: 57-59, 1892. Isolated from a cat. Too poorly described for identification. BIBLIOGRAPHY Barthelat, G. J. 1903. Les mucorinees pathogenes et les mucormycoses, These de Paris. 127 pp., 13 figs.; Arch, de Parasitol. 7: 1-116, 13 figs. Bernard, P. Noel. 1914. Sur un Ehizopus pathogene de I'homme: RMzopus equinus Lucet & Costantin var. annamensis. Bull. Soc. Path. Exot. 7: 430-437. — . 1914. Sur un Ehizopus pathogene de I'homme, Bull. Soc. Myc. France 30: 230-232, PI. 14. Bodin, Eugene & Pierre Savoure. 1904. Eecherches experimentales sur les mycoses internes. Arch, de Parasitol. 8: 110-136. Casagrandi, Carmelita. 1931. Sulla presenza di Basidioboli nell'uomo, Biv. Biol. 13: 1-8, 1 fig. Also Boll. Soc. Internas. Microbiol. Ses. Ital. 9: 63, 64. Cavara, Vittoriano. 1913. Una forma nuova di cheratomicosi (cheratomicosi mucorina), Ann. Ottalmol. 42: 650-674, 1 pi. — . 1913. Sull'importanza patogena per I'occhio di alcune specie di Mucor, Ann. Ottalmol. 42: 729-771. Christiansen, M. 1922, Mycoses generalisees chez le pore, determinees par des mucorinees, C. B. Soc. Biol. 86: 461-463. — . 1922. Generel mucormykose hos svin, K. Veterinaer og landtoh^jsTcole. Aarsshrift. 1922: 132-190, 2 pis., 11 figs. Ciaglinski, A. & O. Hewelke. 1893. tJber die sogenannte Schwarze-Zunge, Zeitschr. Klin. Med. 22: 626-632, 6 figs. Ciferri, Eaffaelle & Bailey K, Ashford. 1929. A new species of Mortierella isolated from the human skin, Porto Bico Jour. Puilic Health. & Trop. Med. 5: 134-143, 1 pi. Cohnheim. 1865. Zwei Falle von Mycosis der Lungen, Arch. Path. Anat. Physiol. Klin. Med. 33: 157. Costantin, Julien. 1892. Note sur un cas de pneumomycose observe sur un chat par M. Neumann, Bull. Soc. Myc. France 8: 57-59. Costantin, Julien & Adrien Lucet. 1903. Sur un Ehizopus pathogene. Bull. Soc. Myc. France 19: 200-216, Pis. 9, 10. Dessy, G. 1933. La chemiotherapie des mycoses. III. Mucoromycose. IL Experiences in vivo. Boll. Ses. Ital. Soc. Internas. Microhiol. 9: 201-206. Ernst, H. C. 1918. A case of Mucor infection. Jour. Med. Bes. 34: 143-146, PI. 3. Frank. 1890. [Aspergillus fumigatus, Mucor racemosus], Deutsche Zeitschr. Thiermed. Vergl. Path. 16: 296, 297. Gasperini, Gustavo. 1927. II dinamismo citoplasmatico nelle Mucorinee sottoposte a varie azioni e singolaramente a quelle degli elettroliti, Ann. Ig. 37: 193-231, 3 pis. Gortner, E. A. & A. F. Blakeslee. 1914. Observations on the toxin of Ehizopus nigricans. Am. Jour. Physiol. 35: 353-367. Graham, Harry. 1890. Mucor corymbifer in the external auditory meatus. Lancet 2: 1379. Herla, Victor. 1895. Note sur un cas de pneumomycose chez I'homme, Bidl. Acad. Boy. Med. Belgique. IV, 9: 1021-1031, 1 pi. ]20 MEDICAL MYCOLOGY Hiickel, Armand. 1884. Zur Keniitnis der Biologie des Mucor corymbifer, Beitr. Path. Anat. Allg. Path. 1: 115-131, PI. S. Korte, W. E. de. 1923. A paramycetoma (?) of the forearm, Jour. Path. Pact. 26: 189-192. Lang, F. J. «& F. Grubauer. 1923. tJber Mucor- und Aspergillusmykose der Lunge, Arch. Path. Anat. Phys. [Virchow] 245: 480-512, 17 figs. Leinati, Fausto. 1928. Micosi rare in animali. Osservazioni cliniche sperimentali, Atti R. Accad. Fisiocrit. Siena X, 3: 83-92, 3 pis. — . 1929. Ulcere micoticlie sperimentali dello stomaco, Atti R. Accad. Fisiocrit. Siena X, 4: 147-223, ^3 figs. Lichtheim, L. 1884. tJber pathogene Mucorineen und die durch sie erzeugten Mykose des Kaninchens, Zeitschr. Klin. Med. 7: 140-177. Lindt, Wilhelm. 1886. Mittheilungen iiber einige neue pathogene Schimmelpilze, Arch. Exp. Path. Pharm. 21: 269-298. Lueet, Adrien & Julien Costantin. 1899. Sur une nouvelle Mucorinee pathogene, C. B. Acad. Sci. 129: 1031-1034. — . 1900. Ehizomucor j)arasiticus, espece pathogene de I'homme, Rev. Gen. Bot. 12: 81- 98, PL 3. — . 1901. Contribution a 1 'etude des mucorinees pathogenes. Arch, de Parasitol. 4: 362- 408. Macfarlan, Douglas. 1924. Fungus of the tongue. Laryngoscope 34: 720-722. Martins, Cesar. 1928. Mycose pulmonaire a Ehizomucor parasiticus, C. R. Soc. Biol. 99: 957, 958. Motta, Eoberto. 1926. Contributo alio studio delle micosi del condotte uditivo esterno (Studio clinico e micologico), Atti R. Accad. Fisiocrit. Siena. IX, 17: 603-631, 9 figs. Paltauf, Arnold. 1885. Mycosis mucorina. Ein Beitrag zur Kenntnis der menschlichen Fadenpilzerkrankungen, Arch. Path. Anat. Physiol. [Virchow] 102: 543-565, PI. 6. Parisot, Jacques & Pierre Simonin. 1922. Mycose pulmonaire associee; reactions biologiques et recherches exp^rimentales, Rev. Med. de l-Est. 50: 8-11. Pena Chavarria, Antonio & Janet H. Clark. 1924. The reaction of pathogenic fungi to ultraviolet light and the role played by pigment in this reaction, Am. Jour. Hyg. 4: 639-649, 6 figs. Podack, Max. 1899. Znr Kenntnis des sogenannten Endothelkrebs der Pleura und der Mucormykosen, Deutsch. Arch. Klin. Med. 63: 1-78. Eedaelli, Piero. 1924. Contributo sperimentale alio studio della Moniliasi e della Lichtheimiasi, Bif. Med. 40: 665, 666. Savour^, Pierre. 1905. Eecherches experimentales sur les mycoses internes et leurs parasites. Arch, de Parasitol. 10: 5-70. Sendziak, Johann. 1894. Beitrag zur Aetiologie der sogenannten schwarzcn Zunge, Monatschr. Ohrenheillv. KehlJcopf-, Nasen- u. RachenkranTch. 28: 112-119, 228. Smith, Theobald. 1920. Mycosis of the bovine fetal mem.branes due to a mould of the genus Mucor, Jour. Exp. Med. 31: 115-122, 1 fig. Spillmann, L. So Ph. Lasseur. 1922. Un cas d 'epidermomycose, Congr. Derm. Syphiligr. Langue Frang. 1: 42, 43. *Stange, G. 1892. Experimenteller Beitrag zur Pathogenitat der Mucorineen, Inaug. Diss. Dorpat. Vuillemin, Paul. 1903. La serie des absidiees, C. R. Acad. Sci. 136: 514-516. — . 1904. La Lichtheimia ramosa (Mucor ramosa Lindt) champignon pathogene distinct du L. cormybifera, Arch, de Parasitol. 8: 562-672, Figs. 1-4. Ziegenhorn, Otto. 1886. Versuche iiber Abschwachung pathogenen Schimmelpilze, Arch, Exp. Path. Pharm. 21: 249-268. CHAPTEE VIII ASCOMYCETES The Ascomycetes are those fungi in which meiosis occurs in characteristic sporangia with endogenous spore formation. These sporangia are called asci and their spores, ascospores. Their thallus is generally well developed; its hyphae (in contrast to those of the Phycomycetes) are regularly divided by septa into uni-, bi- or, rarely, multinucleate cells. Under certain environ- mental conditions, they may continue growth by sprouting ; in the yeasts and a few other forms, only the sprout mycelium is known. The imperfect forms reach the culmination of development in this group, especially among the pathogens of the higher plants. Besides oidia, hypno- spores, etc., the most varied types of conidia are found, often produced in highly specialized organs which at times approach the perfect (sexual) forms in complexity. In certain families, several imperfect forms may be produced successively or even simultaneously in the same species, a condition usually referred to as polymorphism. In case the perfect stage is unknown, these im- perfect stages are given a name and classified among the Fungi Imperfecti. The sexual organs of the primitive groups with which we are concerned more or less resemble those of the Phycomycetes, especially those of the Muco- rales. In the most primitive family we have gametes differentiated and set free to copulate in pairs. These produce a diploid ascogenous hypha. These conditions approximate those in the Oomycetes, although the ascogenous hypha and ascus seem to be a new development. Also in the Ascoideaceae we have a proliferation of the gametangium or ascus which is suggestive of the Oomycetes. Aside from these very primitive forms there are simple isogamous or heterogamous copulation branches very much as we found in the Mucorales. In the higher groups, there is an extensive functional and morphologic differentiation, the male being differentiated as an antheridium and the female as an ascogonimn. A unicellular antheridium approaches a unicellular ascogo- nium and is surrounded by the filamentous end of the ascogonium, known as the trichogyne. In the Plectascales, the only group of interest to medical men, there is not a great differentiation of trichogyne from the ascogonium. In most groups plasmogamy has lost its obligatory character and becomes facultative. Morphologically this functional disturbance first affects only the antheridia ; these disappear and amphimictic fertilization is replaced by many deuterogamous processes. (For details, see Gaumann & Dodge 1928.) Grad- ually this functional degeneration extends to the female organs which also disappear in many groups. Eventually no sexual organ is formed and plas- mogamy becomes pseudogamous. In conjunction with this degeneration, there is a shifting in the signifi- cance of the sexual organs for the formation of fructifications. In the lower 121 122 MEDICAL MYCOLOGY groups the fructification is initiated by the formation of the sexual organs. In many of the higher forms, external stimuli cause the fructifications to de- velop independent of sexual organs which are later formed within the fruc- tification. Plasmogamy is not followed directly by caryogamy but one or several dicaryons are formed. In the lower forms, the dicaryon migrates directly into an ascus which is the product of the plasmogamy. In the higher forms, plasmogamy is increasingly retarded and the fertilized gametangium develops into one or more hyphae. These take up the dicaryon and by conjugate divi- Fig-. 11. — Pyronema confluens. 1, Two antheridia arising from a dichotomous hypha, a trichogyne is in contact with each. 2, An ascog-onium showing fusion in pairs of the sexual nuclei. S, An older stage, showing the beginning of ascogenous hyphae. i, Young ascogenous hypha. 5, An older hypha in which wall formation is in progress. 6, Older hvphae in which the binucleate cells are building out to form croziers. (i and 3 X660, 2 Xl.060, Jf-S Xl,230.) (After Gwynne Vaughan & Williamson 1931.) sion, branch and form asci. Such dicaryotic hyphae are therefore called ascogenous hyphae; biologically they offer the advantage that one gametan- gium can create a number of asci. In most of the higher Ascomycetes, the asci develop from croziers at the end of the ascogenous hyphae. Each of the ascogenous hyphae arising from the ascogonium contains a number of dicaryons and develops by repeated forking, more or less vertically toward the top of the future fructification ASCOMYCETES 123 (Pig. 11, n) . Subsequently it divides by septa so that in the vicinity of the ascogonium the cells contain 2-8 dicaryons and farther away only one (Fig. 11, 4). A cell with only one dicaryon puts forth a lateral process whereby the nuclei are rather far separated (Fig. 11, 6) ; shortly the process bends around into a crozier, and the nuclei begin to divide conjugately (Fig. 12, 1). The spindles lie approximately parallel to each other. After the division, the crozier is ab jointed from both the tip and stipe cells which contain one nucleus each (Fig. 12, 2). In the simplest case the nuclei of the crozier fuse to a diploid nucleus, the primary ascus nucleus, and the crozier develops an ascus (Fig. 12. 3). In another type, the crozier develops a new crozier which in turn may develop still another. In any case it is only the terminal crozier that develops mi m wmm Fig. 12. — Pyronema confluens. 1, Older ascogenous hypha with a new ascogenous hypha budding out to form croziers, the tip cell uninucleate. 3, A crozier, showing fusion in the ascus cell. S, Prophase in the two nuclei of a crozier, each showing twelve chromosomes. 4, First mitotic telophase in the ascus with twelve whole chromosomes going to each pole. 5, Metaphase of the second division in the ascus, showing six chromosomes. 6, Third division in the ascus ; the lower nuclei are in the late metaphase, the next shows the anaphase, and that nearest the apex an ea.rly telophase in which six chromosomes can be counted at the pole. 7, Mycospliaerelln Fragariae, a tvpical peritheciuni. 8, Ascospores. (/ and 2 Xl,230 : S-6 XI, 760; 7 X360 ; S X800.) (After Gwynne Vaughan & Williamson, 1931 and Klebahn 1918.) an ascus. In a third type, the dicaryon of the crozier divides without form- ing any new crozier. The original crozier develops a branch which later may form a new crozier whereon an scus may arise directly; or caryogamy may again be retarded with the result that a tuft of croziers is formed. Occa- sionally the stipe and tip of the crozier fuse, the stipe nucleus generally migrating into the tip cell. This proceeds to develop a binucleate branch which gradually forms a crozier that may develop an ascus by fusion of its nuclei or repeat crozier formation. 124 MEDICAL MYCOLOGY In the higher Ascomycetes, there are, in addition to the crozier type, a whole series of other developmental forms of ascogenous hyphae which need not concern us at present. As far as is known, the further development of the asci is the same in all Ascomycetes. The primary ascus nucleus, which has arisen from the fusion of the dicaryon (Fig. 12, 3), undergoes three divisions, at least one^ of which is meiosis ; the eight daughter cells cut out eight ascospores from the cytoplasm of the ascus by free cell formation. The cytoplasm not included in the spores is called epiplasm, which, besides nourishing the ascospores, pro- vides substances for the sculpturing of the spore walls. In certain forms, the number of nuclear divisions may be limited to two or may increase to sixteen, in the former case producing but 4 ascospores and in the latter 64,936. Where the ascospores are thick-walled, they usually possess a typical germ pore or a meridional fissure. In the latter case, the halves of the ascospore wall sepa- rate in germination like the two valves of a mussel. According to the Anglo-Saxon school, represented by Harper, B. 0. Dodge and Gwynne-Vaughan (nee Fraser) the nuclear fusion in the young ascus is not the first and only fusion ; but is preceded by another fusion in the ascogonium directly after plasmogamy. The ascogenous hyphae, accord- ing to this conception, do not contain haploid dicaryons but undivided diploid nuclei which only after the formation of the croziers come together as di- caryons. Because of this double fertilization, the primary ascus nucleus is tetraploid and contains 2x double chromosomes. At the first ascus division (Fig. 12, 4) meiosis occurs with each daughter nucleus containing 2x simple chromosomes. The second step is homeotypic (Fig. 12, 5), the 2x simple chromosomes are halved so that each daughter nucleus still contains 2x simple chromosomes. In the third step (brachymeiosis) (Fig. 12, 6) one-half of the undivided chromosomes migrates to each pole, so that each daughter nucleus of the third division contains x simple chromosomes. Although the cytologic reports are somewhat contradictory and in part may be interpreted by either hypothesis, the students of Continental Europe and some in America prefer the interpretation of Dangeard and Claussen. First imperfect forms, then sexual organs arise on the haplont. Between these sexual organs plasmogamy occurs, while the male and female nuclei pair as a dicaryon. These dicaryons migrate into the ascogenous hyphae and divide conjugately. The ascogenous hypha thus represents a special diploid phase, the dicaryophase, which ends with caryogamy (fusion) in the young asci. Caryogamy is followed directly by meiosis, usually producing 8 haploid ascospores. In the higher Ascomycetes this scheme of development is further complicated, since the haploid thallus proceeds to form fructifications on or in which the ascogenous hyphae complete their development. As in most red algae and in the sporophyte of the mosses, the dicaryophase is to a certain extent parasitic on the haplont and nourished by it. In the simplest case, these fructifications form an undifferentiated mass of tissue, a stroma on or in which the asci are formed. A fructification of this ASCOMYCETES 125 type is called an ascostroma. In the liigher forms the hyphal tissue of the stroma undergoes differentiation both in form and histologic structure, and develops the fructifications which furnish important characters for classifica- tion. Only one of these structures in its simplest form need concern us here. For a further consideration of the higher Ascomycetes see Gaumann & Dodge (1928) and the recent fundamental work of Nannfeldt (1932). The perithecium consists of a solid, often pseudoparenchymatous wall and a cavity in which the asci are borne (Fig. 12, 7). The more primitive types are usually spherical ; the asci lie irregularly in the interior and are only liberated by the decay of the perithecial wall. In the higher types, there are more elaborate mechanisms for spore dispersal. BIBLIOGRAPHY Gaumann, Ernst A. & Carroll William Dodge. 1928. Comparative morphology of fungi. New York, McGraw Hill Book Company, 701 pp., 398 jigs. Nannfeldt, J. A. 1932. Studien iiber die Morphologic und Systematik der nicht-lichenisierten inoperculaten Discomyceten, Nova Acta E. Soc. Sci. Upsaliensis IV, 8: 2:1-368, Pis. 1-20. CHAPTER IX ENDOMYCETALES The Endomycetales include those forms in which an ascus arises directly as the product of a sexual act, wherever this occurs. They comprise eleven families distributed among four diverging lines of degeneration. In the most primitive family, the Spermophthoraceae, the mycelium is nonseptate and coenocytic, the gametes are differentiated and freed from the gametangium, and a septate uninucleate secondary mycelium results. From this primitive family we have four diverging lines of degeneration, each line having a char- acteristic spore shape. In the Ashbyaceae, the mycelium, when present, may be septate, but the cells are usually coenocytic and have the elongate fusiform ascospore of the Spermophthoraceae (Fig. 13, 1-3). In the Ascoideaceae — Endomycetaceae line, the mycelium becomes uninucleate, the ascospores, which in the early stages of its development are fusiform, become cucullate (Fig. 13, 10), or the rim assumes an equatorial position, producing a saturnine spore (Fig. 13, 6). The position of the Pichiaceae is not clear. Here the ascospores are hemispheric or slightly angular (Fig. 13, 7, 8). They may pos- sibly be derived from GuiUermondeUa (Fig. 13, 5), a member of the Ashbyaceae, or more probably Hanseniospora, one of the Endomycetaceae with rough cucul- late spores, by the loss of the rim (Fig. 13). In the two remaining lines, the spores are ellipsoid or spherical and have probably diverged from the Spermophthoraceae through Dipodascus. One line retained strong evidence of sexuality, gradually losing it with extreme degenera- tion, producing its spores saprophj'tically, and early reducing the ascospore number to 8, 4, or fewer. This line is represented by the Eremascaceae and Saccharomycetaceae. The other line promptly discarded traces of sexuality, produced its spores in the host tissue, very rarely under saprophytic condi- tions, and retained the large number of ascospores of the Dipodascaceae. This latter line is represented by three strictly parasitic families, one comprising predominantly mammalian parasites, the other two plant pathogens. There is a small residue of species usually placed in the Saccharomycetaceae, in which the ascospores copulate in pairs. Their systematic jDosition is not clear. Guil- liermond has recently (1931) suggested that this group of species has been derived from the Taphrinaceae, the end member of the fourth line mentioned above. Key to Families Gametes fusiform, set free from the gametangium, copulating in pairs, producing an ascogenous hypha; ascospores fusiform. Spermophthoraceae. Gametes not set free, gametangial copulation the usual type, or the ascospores develop parthenogenetically. Ascospores fusiform to acicular. Ashbyaceae. 126 ENDOMYCETALES 127 Ascospores, cucullate, saturnine. Mycelium multinucleate, conidia produced, ascus many-spored, proliferating. Ascoideaceae. Mycelium uninucleate, degenerating to sprout mycelium; conidia not differentiated; ascospore number usually 4 or fewer; not proliferating. Endo'mycetaceae. Ascospores hemispheric or angular; sprout mycelium uninucleate; ascospores 4 or fewer. Pichiaceae. Ascospores ellipsoid or spherical. Mycelium multinucleate, ascus resulting from copulation of two hyphal tips. Ascus many-spored. Dipodascaceae. Ascus 8- or 4-spored. Eremascaceae. Mycelium uninucleate, usually sprout mycelium, asci resulting from copulation of two cells or from parthenogenesis or apogamy. Saccharomycetaceae. No trace of copulation; asci many-spored, rarely reduced to 8; mycelium often scanty in the tissue but then developing readily in culture; asci usually thick-walled, often differentiated as a resting spore, usually abundant in host tissue, rare in culture. Ascospores developing directly and filling the ascus. Coccidioideaceae. Ascospores developing in tetrads about the wall of the ascus. Protomycetaceae. Ascospores developing directly, but reduced in numbei', not filling the ascus, mycelium developing in host tissue. Taphrinaceae. Fig. 13. — Spore shapes in the Endomycetales. J, Nematospora; S, Coccidiascus; S, Mono- sporellaj ■'/, Schwanniomyces ; 5, Nadsonia; 6, Williopsis ; 7, S, Pichia; 9, Guilliermondella ; 10, Endomyces ; 11, Saccharomyces. (After Guilliermond 1928.) Spermophthoraceae. — In 8'permophthora Gossypii on cotton, the mycelium is nonseptate and coenocytic. It produces gametangia with numerous fusiform gametes (Fig*. 14, 1). After the dehiscence of the g-ametangium, the gametes fuse in pairs, and the resulting zygote germinates immediately by a septate, uninucleate, diploid mycelium (Fig. 14, 2-7). The asci are borne directly on this mycelium without crozier formation (Fig. 14, 8). The ascospores are fusiform, but smaller and shorter than the gametes. The differentiation of gametes is suggestive of remote derivation from tlie Oomycetes rather than from the Zygomycetes or Mucorales, as suggested by Gaumann (1926). In neither group is there any structure in any way comparable to the diploid ascogenous mycelium of this family. 128 MEDICAL MYCOLOGY Ashbyaceae. — This family has been little studied cytologically. In Pied- raia Hortai, forming hard nodules on human hair, the mycelium is thick-walled, septate, and more or less agglutinated into a solid mass surrounding the repro- ductive structures (Fig. 15, 1, 2, 12). Young cells contain up to 8 nuclei, but mature cells are mostly uninucleate, although one cell figured by Horta (1911) suggests a binucleate condition. The functions of gametangium and ascus, performed by distinct structures in the Spermophthoraceae, are performed by a single stinicture, usually called the ascus, which arises as the terminal cell of a hypha. The gametes are differentiated within this structure but unite in pairs without being set free (Fig. 15, 3-11). The resulting zygotes then Fig. 14. — Spermophthora Gossypii. 1, gametangium showing immature gametes within ; Z-%, stages of copulation of gametes, ascogenous filament with young ascus ; 8, asci showing as- cospores ; 9, germinating ascospores. (After Guilliermond 1928.) elongate to produce the 8 uninucleate, fusiform, or crescent-shaped ascospores with 2 (rarely 3) filiform appendages (Fig. 15, 13-16). The details of the cytology have not yet been reported. The ascospores germinate directly to mycelium which penetrates beneath the cuticle of the hair, forming a pseudo- parenchymatous palisade which eventually ruptures the cuticle and expands to produce the typical nodule. In Pieclraia veneznelensis, little is known of its life history, but the ascospore number is reduced to four and the filiform ap- pendages practically disappear. Langeron (1929) and Brumpt & Langeron (1934) suggest that Piedraia is related to the sooty molds which it resembles slightly in general appearance, but in the curious development of gametes and ENDOMYCETALES 129 Fig. 15. — Fiedraia Hortai. 1, S, sections of masses on hair, showing developing asci ; S-11, development of ascospores in ascus ; 12, mycelial mass on hair ; IS, 15, 16, ascospores ; 1^, ascospores emerging from ascus. 130 MEDICAL MYCOLOGY in the lack of ascogenous hyphae, it has nothing in common with that group, and is intermediate between the Spermophthoraceae and the Ashbyaceae. In Eremofheciuni (Fig. 16), the mycelium is multinucleate and rarely sep- tate. The genus has not been carefully investigated cytologically, but the gametangium (or ascus?) resembles that of Spermophthora in shape. The spore number is somewhat less. The spores are fusiform, rounded at one end, taper- ing to a long filiform appendage at the other. They are arranged in the ascus with the rounded ends in contact and the filiform appendages gathered in a fasicle at the poles, giving the whole spore mass the appearance of a huge nuclear spindle. This grouping suggests that figured by Horta (1911) for Piedraia. The protoplasm of the spore is much denser in the end opposite the appendage, and germination takes place only in the end of the spore with the dense protoplasm. No septum has been detected separating the spore into two cells. Yig 16. — Ashbya Gossypii. 1, mature spore; Z, S, germinating spores; i, mycelium; 5, 6, de- velopment of gametangium; 7, mature ascus with ascospores. (After Guilliermond 1928.) In Ashhya Gossypii, a parasite on cotton, the mycelium is septate but the cells are multinucleate. Sexuality has been lost, the asci developing partheno- genetically. The nuclei divide twice, forming the tetrads which precede spore formation. This is reminiscent of sporangiospore formation in Piloholus and will be encountered several times in other lines of this group. The number, both of nuclei and of nuclear divisions, is reduced and stabilized so that ordinarily either 8 or 16 spores are produced. The spores are usually rounded at one end and taper at the other into a long slender projection, sug- gestive of a flagellum, but without motility. In Nematospora, which is also parasitic on plants, degeneration has pro- ceeded further until sprout cells as well as mycelium are produced ; the spores are long fusifonn to acicular and reduced to 8 per ascus (very rarely 16, or ENDOMYCETALES 131 further reduced to 4 or 2 in N. Nmjpuyi) . The same fiagellar appendage is also present but usually shorter (Fig-. 17). Two other little known genera may either beh)ng here or in the next family. In (Joccidiascus mycelium is absent, the asci develop following isogamous copu- lation, and the spore number is fixed at 8 (Fig. 13, 2). This species has been found in the digestive tract of Brosophila but has not been cultivated. In Mono- sporella both mycelium and copulation are absent. The ascus produces a single acicular spore parthenogenetically (Fig. 13, 3). The members of this genus have also been found in the digestive tract of invertebrates and not cultivated. Fig. IT. — Nematospora Coryli. 1, mature ascospore ; 2, 3, geiminating: ascospores ; i, 5, sprout celLs ; b", developing ascus; 7, mature ascus. (After Guilliermond 1928.) PIEDRAIA Piedraia Fonseca & Area Leao, Inst. Oswaldo Cruz, Suppl. das Mem. 4: 124-127, 2 pis., 1928. Trichosporon Behrend, Berliner Klin. AVoch. 27: 464-467, 1890. TriclwHporum Vuillemin, C. R. Acad. Sci. 132: 1369-1371, 1901. Arch, de Parasitol. 5: 38-66, 12 figs., 1902, non Fries 1825, 1849. Fries, Syst. Orb. Veg. 306, 1825, published a new genus Trichosporum which is spelled Tn'chosponum in the index. No species was attributed to this genus at the time. I can find no mention of the genus in his Sy.stema mycologicum 132 MEDICAL MYCOLOGY 1832. Its first place of effective publication may be considered to be his Summa Veg. Scand. 2 : 492, 1849, where he treats twelve species, many of them f oi-ming the first section of liis Sporotrichum in the Systema. For spelling we are equally puzzled, as it is Trichosporum in the text and Trichosporium in the index. Saccardo and later authors have used the latter spelling. In any case Fries' use of the former precludes the possibility of using Trichosporum for another genus, and TricJiosporum of Vuillemin, Schachter, and later medical men, must be renamed. One might consider the retention of Trichosporon Behrend, since it differs by the last two letters, but the frequent interchange of spellings of genera, such as Microsporum and Microsporon, as well as the fact that the spelling Trichosporum has had the wider usage in the present century, makes it a permanent source of error and confusion, so that I am in favor of abandoning it altogether. Ponseca and Area Leao (1928) have reported asci and ascospores for Trichosporum Hortai and have transferred this species to Piedraia. No one has suggested asci in the European species while practically all investigators have noted these structures in the South American species whether they have called them asci or not. Also the lesions in the hair in the case of the European species are much more serious, causing irregular splitting of the hair, suggesting that the European species may belong in some other group of fungi, perhaps remotely related to the Gymnoascaceae or the Eremascaceae. Until more is learned about these imperfectly described species, we prefer to leave them as an appendix of doubtful species of Piedraia rather than to transfer them elsewhere. Piedraia is very imperfectly characterized. Mycelium thick-walled, septate, agglutinating into solid masses on the hair; asci 8-spored; ascospores large, fusiform with acute ends prolonged into filiform appendages. Piedraia Hortai (Brumpt) Fonseca & Area Leao, Inst. Oswaldo Cruz. Suppl. das Mem. 4: 124-127, 2 pis., 1928. Tnchosporum sp. Horta, Mem. Inst. Oswaldo Cniz. 3: 87-107, Pis. 5, 6, 1911. Trichosporum Hortai Brumpt. Precis Parasitol. 1913. Forming characteristic hard, black, adherent, small, spherical or long conic nodules on hair, Brazil. Hyphae septate, 8-12^ in diameter slightly brownish, thick-walled; asci not clearly seen in culture; ascospores fusiform (Fig. 22), curved, greenish yellow, each end acute and prolonged into an appendage about 30;u, long, the body of the spore being about 30 x 10/a. On Sabouraud agar, colonies small, dark brown, very adherent to the medium, velvety, margin somewhat lighter, finally becoming folded. After 10 weeks the whole colony is black. Growth much better on carrots where the lighter colored margin is lacking. Piedraia Sarmentoi Pereira f., Kev. Med. Cimrg. Brasil 38: 49-52, 6 pis., 1929; C. R. Soc. Biol. 104: 680, 1930. ENDOMYCETALES 133 Abundant on the hair of young people in the state of Rio Grande do Sul, Brazil. General microscopic appearance on hair similar to that of P. Hortai. Hyphae up to Tfi in diameter. Asci more spherical, up to 30/x long, 8-spored, ascospores 35-40 x 7-8fi, filiform appendages 7-8 rarely lO/x long. In cultures only terminal and intercalary chlamydospores seen. Growth rapid on Sabouraud agar, colonies white, low margins dentate, creamy then entirely black, fuliginous, easily detachable although penetrating the substrate deeply. Growth slow on potato, and pigment formed very late. On carrot, colony creamy white, pigment first appearing in spots, becoming diffused, fuliginous, cerebriform. Piedraia surinamensis Dodge, n. sp. TricJiosporum sp. Aars, Arch. Derm. Syphilol. 22: 401-409, 1930. Nodosities on hair commonly up to 500/*, rarely 1 mm. long, mostly about 100/A in diameter exclusive of the diameter of the hair, composed of thick- walled cells 4:-6fx in diameter. Asci occurring singly, 32-44 x 20/a; ascospores fusiform, 42 x 6^, with two, seldom three, filiform appendages at the ends. On maltose agar and honey agar, dark brown to black, hard, slightly velvety colonies. Piedraia colombiana Dodge, n. nom. Dematium sp. Juhel-Renoy & Lion, Ann. Derm. Syphiligr. Ill, 1 : 765-772, 2 pis., 1890. TricJiosporum giganteum Vuillemin, Arch, de Parasitol. 5: 38-66, 12 figs., 1902 non Unna 1895. Producing nodules on the hair in Colombia. Cells 4-5 X 5-6/x sometimes 8-12/xi long, yeastlike and proliferating when young, later forming a mycelium, in old age again becoming more or less yeastlike. Coils were observed in cultures, but it is not certain whether they were functional antheridia and ascogonia. [Malcolm Morris (1879) had pre- viously noted asci with spores, but he does not describe them in sufficient detail. Peiia Chavarria & Rotter (1983) describe the ascocarps, asci, and spores in some detail but fail to give measurements of the ascospores.] Brumpt & Langeron (1934) studying a case from Medellin, Colombia, state that the asci were 50 x 30/a, containing 8 ascospores ; spores thick-walled, 40 x 6-9/* with very short filiform appendages, 4-5/t long. On Sabouraud agar, colonies white at first, becoming yellowish, cerebri- form, not penetrating the substrate and easily separable. In old cultures the cerebriform appearance is lost. On maltose agar, colonies darker and more adherent. On sugar beets and carrots, growth good. Gelatin liquefaction begins within 8 days. Piedraia venezuelensis Brumpt & Langeron, Ann. Parasitol. Hum. Comp. 12: 155-158, Figs. 27-32, 1934. Infected hairs sent by Machado of Caracas from a case of 2 years' dura- tion, apparently the first case reported from this region. 134 MEDICAL MYCOLOGY Nodules on the hair, large ovoid or fusiform ; asci 35-40/* long, only 4- spored; ascospores 25-30 x 10-14/* very thick-walled, lacking filiform append- ages but ending in long slender points up to 10-12/* long, often somewhat curved. Not cultivated. Species of Doubtful Position The following species are rather imperfectly described and may belong elsewhere, although they were placed here by the original authors. In some cases I have been unable to locate the original description and know the name only by a brief mention in the literature. Trichospomm Beig-elii (Rabenhorst) Vuillemin, Arch, de Parasitol. 5: 38-66, 12 figs., 1902. Pleurococcus Beigeli Klichenmeister in Rabenhorst, Hedwigia 6: 49, 1867. Sclerotium Beigelianum Hallier, Paras. Unters. 75, PI. 2, Figs. 24, 25, 1868. Zoogloea Beigeliana Eberth, Centralbl. Med. Wiss. 11: 307, 1873. Hyalococcus Beigeli Schroeter, Kryptog. Fl. Schlesien 3: 152, 1886. Chlamydotomus Beigeli Trevisan in Saccardo, Syll. Fung. 8: 1042, 1889. Micrococcus Beigeli Migula, Syst. Bakterien, 1: 193, 1900. Attacking the hair in wigs and switches, Germany. Vuillemin (1902) isolated what he called this fungus from the hairs of the moustache. The report of this fungus from pubic hair in Nigeria by Manson-Bahr (1932) probably should be referred to Favotrichophyton. Cells 3-5/t in diameter, spherical or angular by mutual pressure, surround- ing the hair in a spherical mass of gel ; sporangia mostly 1/* thick, containing 12-20 spores. Vuillemin describes the fungus isolated by him as follows: Cells 2.5-4/*, mostly 3-4/*, wall thick, held together by gelification of the outer layer of the wall. Larger chlamydospores in the interior of the stroma, 6/* in diameter. On maltose agar, gelatin, carrot, beet, and potato, a yellowish gray, humid colony, becoming convoluted, surface drying chalky white. Cells cylindric, 4-5/* in diameter, forming a more or less dichotomous mycelium about 2/i in diameter, producing hypnospores filled with reserves up to 12/* in diameter. Gelatin and serum not liquefied. Producing a pellicle on the surface of liquid media, growth suggesting- that of Geotrichum lactis. Trichospomm ovale Paoli, Giorn. Ital. Mai. Ven. Pelle 54: 566-572, 1913, non Unna ap. Vviillemin 1902. Isolated from infected hairs of the moustache with the usual trichosporic nodules, hair not breaking readily. Spores in nodules 3-4/i, hyphae present. In cultures, mycelium of branched, septate hyphae bearing terminal chains of spores often suggesting clostero- spores. Not pathogenic for guinea pig or rabbit. On Sabouraud agar, colony whitish, moist, radially furrowed, slightly velvety, becoming yellowish. On gelatin, colony similar but growth much ENDOMYCETALES 135 slower, gelatin liquefied in 13-15 days. On broth, grayish white pellicle settling after 6-7 days and replaced by another pellicle. From the brief description, the systematic position of this organism is uncertain. Many characters suggest much closer relation to the Favotricho- phyfon ochraceuni group rather than to the yeasts (near Mycotornla) . Clini- cally it seems very close to the European T. Beigeli, if it be not the same organism. Trichosporum ovcides Behrend, Berliner Klin. Woch. 27: 464-467, ld90. Trichosporum ovale Unna ap. Vuillemin, Arch, de Parasitol. 5: 38-66, 12 figs., 1902. ITrichosporum giganteum. Unna, Deutsch. Med. Zeitschr. 1895: 255-256, 1895, non Vuillemin 1902. Isolated from sycosis in Germany, forming brownish yellow nodes on the hair, leaving the hair shaft nearly intact. Spores ovoid, 2-4.5 x 1-4/x, sometimes in-egular. Colonies cerebriform, blackish brown on several media. Trachsler (1896) tried to separate T. ovoides and T. giganteum, but the differences between the strains are so slight that they are probably not sig- nificant. Trichosporum glycophile DuBois, Ann. Derm. Syphiligr. V, 1: 447-456, 1910. Causing irritation in female genitalia. The hairs which have been moistened with urine (patient was a mild diabetic) have nodosities and terminate in brushlike tips, often breaking a few millimeters from the skin. The mycelium proliferates between the fibrillae of the hair and terminates at the surface under a layer of sporiform elements. Not pathogenic for the guinea pig, rabbit, or rat. The following description of morphology based on cultures from 5% maltose agar, 5% peptone agar, or 2.5% maltose + 2.5% peptone agar. The yeast cells 5-6/x in diameter. The hyphae slender, septate, at long intervals, uniform at least in the terminal portion. As the hyphae become older, they become thicker, more tortuous, and end in a terminal swelling. Spores verticillate, regularly spaced. Chlamydospores large. On solid sugar media, abundant mycelium below the surface while on the surface a small gray colony scarcely covers the hair. On solid protein media, colonies yellowish, smooth, humid, proliferating on surface with cen- tral elevation and sometimes Avith little knobs, margin with a circular elevation connected with the center by furrows. Cross inoculations showed these two types of colonies to be the same organism. On Sabouraud agar, colonies smooth gray, or yellowish, growth rapid. On broth agar and peptone agar, yeast form prevails on the surface, growth slow, colony gray, smooth with a slight central knob, colony bordered by fine hyphae at the end of 3 weeks. On liquid media, grayish white pellicle, dry but not poAvdery, pellicle settling in 10-12 days and a new one forms. On potato, colonies gray yellowish, moist, rugose. 136 MEDICAL MYCOLOGY Gelatin not liquefied, but fine hyphae perpendicular to the line of the stab seen and a tuft of mycelium at the bottom. The systematic position of this species is not at all clear. Its behavior on the hair suggests relationships with the European species previously placed in Trichosporuni. On the other hand, this organism caused considerable irrita- tion of the genitalia while the so-called Trichosporuni of Vuillemin and others caused no irritation. Its morphology in culture would relate it to Mycotorula as has already been pointed out by Langeron & Talice (1932), but none of the other species of the group have been reported able to penetrate the hair. Trichosporuni equinum Fambach teste Fonseca, Rev. Med. Cirurg. Brasil 38: 256, 1930. Producing white nodules on hair of horse, becoming yellowish or darker. Trichosporum Foxi Castellani, 1908. Pichiaceae. — The systematic position of this family is not clear. It may have arisen from Nematospora or Ashhya or, more probably, as another line of degeneration from the Ascoideaceae. The most primitive member is Guillier- mondella which produces considerable mycelium with conidia. Asci 4-spored, either lateral or intercalary, follow isogamous copulation or may develop par- thenogenetically. Spores sickle-shaped [Dekker (1931) figures them as kidney- shaped very close to those of Pichia]. A somewhat more degenerate species, GuilUermondella Vuillemini (Endomyces albicans Vuill. non Johan-Olsen, En- domycopsis albicans Dekker) from a case of thrush, still retains both mycelium and sprout mycelium. The spore number is 4, or fewer. In Zygopichia and Pichia there are cylindric cells and some mycelium for- mation, forming a thick, dry pellicle on the surface of liquid media. In Zygopichia heterogamous copulation precedes ascospore formation, in Pichia the degeneration is complete, and the ascospores develop parthenogenetically. Both genera have kidney-shaped, hemispheric, or angular ascospores. Guilliermondella Vuillemini (Lindau?) Dodge, n. comb. Endomyces albicans Vuillemin, C. R. Acad. Sci. 127: 630-633, 1898; Rev. Myc. 21: 43-45, Pis. 189-190, 1899 not Johan-Olsen 1897 (excl. all syn. based on Oidium albicans Robin). Endomycopsis albicans Dekker (excl. syn.), Verhandel. K. Akad. Wetensch. Amsterdam, Afd. Natuurk. 28: 231, 1931. Endomyces Vuillemini Lindau?, Mikroscopische Pilze 1912 [cited as Landrieu by Castellani & Chalmers, Man. Trop. Med. ed. 2, 1913, but I am unable to verify this citation]. Isolated from a case of thrush. Yeast cells abundant at first, giving rise to mycelium with chlamydospores and finally producing asci, spherical or ellipsoid, 4-5/*, 4-spored, rarely 2-3- spored, axes of ascospores 3.8-3.5 x 1.75-2 x 1.2-lA/x, not staining with nuclear stains (i.e., the deeply staining bodies called by Vuillemin the ''globules internes" are probably nuclei, fide unpublished work of Morris Moore). The ascospores cling together in groups for some time after the asci have dis- appeared. ENDOMYCETALES 137 Ascoideaceae. — In Ascoidea rubescens in the slime flux of trees, the myce- lium is coenocytic, septate, and branched. The mycelium produces conidia either singly or in tufts. The gametangium is a large multinucleate cell. Varitchak has reported the degeneration of all but one pair of nuclei, which proceed to fuse. Walker (1935) was unable to confirm this statement and suggests that the "privileged sexual nuclei" of Ascoidea are degenerating nuclei which she has also seen in other parts of the fungus. By two or three successive divisions the spore initials are cut out of the mass as lenticular, uninucleate portions of the protoplasm. Finally, these spore initials contract and form the typical cucullate spores of this series. Not all of the protoplasm is used up in the process, some remaining behind as the epiplasm, as in other groups of Ascomycetes. The ascospores are extruded from the mouth of the ascus by the proliferation of the cell next below it. This proliferating cell proceeds to form another ascus at the same site. Plasmogamy occurs shortly after spore germination, but the occurrence of caryogamy is still uncertain. The spore sac appears similar to the gametangium of Spermophthora, but gametes are not differentiated and whether there is nuclear fusion in this organ is still questioned. In its later stages the organ behaves as a multi- spored ascus, although ontogenetically it bears little relation to such a structure. The proliferation of the basal cell into the mature organ sug- gests sporangial proliferation in the Saprolegniaceae, but the manner of spore formation is entirely different. It would seem likely that we are dealing with a stage of degeneration from an ancestral form like Spermoph- thora in which the gametangium has ceased to function as such and func- tions as an ascus with a shortening of the stages between gametangium and ascus and with a partial or complete elimination of the sexual act. The fact that the spore initials appear similar to the ascospores of Spermophthora and finally become cucullate as in the Endomycetaceae, suggests that it may be an intermediate stage in the phylogeny of the latter. Endomycetaceae.- — In this family the mycelium is usually uninucleate, soon degenerating to sprout mycelium, conidia are no longer differentiated, the ascospore has become reduced in number per ascus and has assumed a cucul- late or saturnine shape. So far as is known this family is saprophytic, although Hansenula has occa- sionally been isolated from sputum, and Pijper (1928) reports Hanseniospora from a case of onychomycosis. The strains of fungi from these isolations have not been reported pathogenic for experimental animals. It is possible that they may cause irritation in the mucous membranes or aggravate a condition primarily due to some other organism, but reports of the cases should be scrutinized very carefully before admitting them as pathogens. On the other hand, there is a large group of parasites previously referred to Endomyces, having spherical to ellipsoid, smooth spores, which is here treated in the Eremascaceae. 188 MEDICAL MYCOLOGY This family is characterized by the rim around tlie spore, producing a cucullate spore if the rim is on one side, or a saturnine spore if the ring is equatorial. Within the family there is a large isogamous series and a small heterogamous one. In the heterogamous series, which seems to be the more primitive, we have Endoniyces Magnusii {Magnusiomyces) from the slime flux of trees (Fig. 18). The hyphae are generally multinucleate (2-8). In growing hyplial tips this number may mount to 50 (Pig. 18, 1), in weak hyphae it may be as low as one. The hyphae divide easily into oidia ; Avliich are generally multinucleate, rarely uninucleate. In successive divisions the tendency is toward the uninu- cleate condition. Often their wall thickens and the oidia become hypnospores Fig. 18. — Endomyces Magnusii. 1, young- multinucleate hypha ; 2, older hypha ; 3-9, II, development of asci ; 10, hvpnospores. {1, 2, 10 xl.500; S-9, 11 X500.) (After Guillier- mond 1909.) (Fig. 18, 10) ; with the consequence that, under certain environmental con- ditions, a culture may disintegrate into hypnospores after a couple of weeks. With favorable conditions, oidia may develop to sprout mycelia, not by independent development of small outgrowths of the mother cell to sprout cells but by fission of the mother cell. Both daughter cells round oft' and develop to the size of the mother cell (ordinary cell division in contrast to sprouting). When the mycelium is ready to form asci, it divides into numerous short, slender branches with short cells containing few nuclei, often not more than one (Fig. 18, 3, and 4). A branch ends either in a very large cell full of reserves, the ascogouium, or in a narrow hyaline cell, often much twisted, the antheridium. The upper third of the ascogonium swells considerably and ENDOMYCETALES 139 collects the cytoplasm with 2 or 3 nuclei. At the beginning' of copulation, it bends over to meet the antheridium. In this stage, the swollen part contains only one nucleus, the others having^ migrated (Fig-. 18, 5). The narrow an- theridium contains, when young, 1-3 nuclei of which only one remains at the tip. In about three-fourths of the cases, copulation occurs. The antheridium approaches the ascogonium, swells slightly, and ab joints the apical uninucleate gametangium from the stipe cell. Meanwhile the uninucleate tip of the ascogonium is abjointed from its basal cell. Hereupon the walls separating the gametangia dissolve, with the development of tlie zygote to a 4-spored ascus (Fig. 18, 6-9). There often occur numerous variants of this usual course of development. Thus, the antlieridium may approach the ascogonium at the side instead of the tip; or both may be uninucleate without the abjnnction of basal cells; or the ascogonium may develop parthenogenetically. Fig. 19. — Endomyces decipiens. 1-3, stages of copulation; .'(-12, development of asci. (X 1,200.) (After Juel 1921.) The end member of the heterogamous series is Endomyces decipiens, found on fructifications of the mushroom, Armillario. mellea, producing its perfect stage on the lamellae. Sexual organs are almost entirely absent, copulation being heterogamous when present or very rarely isogamous (Fig. 19, 1-3). The asci are lateral on the hyphae. Although sometimes three nuclear divi- sions occur in the ascus, only 4 ascospores are formed (Fig*. 19, 4-12). In cultures the hyphae easily break apart into uninucleate oidia. Sometimes the tips of branches form thick-walled yellowish hypnospores instead of asci. The isogamous series begins with the genus Endomycopsis. In this genus the abundant sprout mycelium is apparently connected with the adaptation of these species to media containing starch and sugars. Wherever the sprout cells arise on aerial mycelium, their diameter is smaller and the Avail some- what thicker than in tlie submersed sprout cells. They are then ver}^ resistant and survive a long period in temperatures up to 55° C. Biolog-ically their significance seems to be that of hypnospores, and because of their exogenous formation they are usually called conidia. 140 MEDICAL MYCOLOGY In Endomycopsis fihuliger, although any two cells may form copulation branches which approach each other, even with the dissolution of the wall the 2 nuclei rarely fuse {Fig. 20, 1). Generally the copulation branches de- velop parthenogenetically, though the separating walls may be temporarily dissolved. In exceptional cases there may occur a pseudogamous anastomosis of two sprout cells, one changing to an ascus. (Fig. 20^ 2-4). In a large number of cases no copulation branches are formed, but the asci, like the sprout cells, arise as lateral outgrowths of the hyphal cells (Fig. 20, 6). These asci are three or four times larger than the ordinary sprout cells. Occasionally, they arise from ordinary swollen hyphal cells, or from swollen sprout cells. When they begin to appear, the formation of sprout cells slows up, but does not cease, with the result that hyphae may form sprout cells and asci simultaneously. One finds even young asci which continue to cut off sprout cells until the beginning of spore formation. Periods Fig-. 20. — Endomycopsis fihuliger. Development of asci. (XoOO.) (After Guilliermond 1909.) of vegetative growth and fructification are thus not sharply differentiated one from the other. Each ascus contains four cucullate spores. At germina- tion, these throw off the exospore and germinate either with a germ tube or with a sprout mycelium. Here sexuality is so completely weakened that only vestiges of the sexual organs remain. In a large number of cases where no copulation branches are formed, the asci arise directly from vegetative hyphae or sprout cells. In the remaining forms of the isogamous series, copulation branches de- velop less frequently and the asci arise parthenogenetically without fusion. Growth of mycelium by sprouting increases proportionally. In two Chinese species, E. Lindneri and E. Hordei, the copulation branch no longer changes directly to an ascus but develops to a short, occasionally branched mycelium where asci arise by swelling of the hyphal cells (Fig. 21, 1, 2) . In most cases the asci are formed directly from the sprout cells without this detour. ENDOMYCETALES 141 In other species which terminate the isogamous series, E. javanensis and E. capsularis, the copulation branches have entirely disappeared (Fig. 21, 6-8). According to the conditions of environment, either the hyphal or the sprouting condition may prevail. By swelling of the terminal cells of the hyphae or by lateral sprouting (sometimes also intercalary), there arise 4- spored asci. Each of these spores is divided into two unequal parts by annular thickenings (Guilliermond 1909). Neither species is possessed of much fer- mentative ability. From Endomycopsis two lines diverge. In Hansemda the ascospore is cucullate, the mycelium has disappeared, although the cells are quite elongate and sometimes in chains. Two of the sprout cells form copulation tubes to- Fig. 21. — Endomycopsis Lindneri. 1-5, E. capsularis ; 6-8, development of asci. (1, 2, 6-8 X470 ; 3-5 X500.) (After Mang-enot 1922 and Guilliermond 1909.) ward each other, the nuclei migrate into the bridge and fuse, the diploid nucleus divides, both daughter nuclei migrate back into the cells, there divide a second time, and develop two ascospores in each fusion cell. In Hansenio- spora the cells of the sprouts mycelium are mostly citriform, sprouting only from the poles. Copulation has not been observed and apparently the asco- spores develop parthenogenetically. Pijper (1928) reports H. Guilliermondii from the nails in a case of onychomycosis. In the other line diverging from Endomycopsis, the ring about the ascospore occupies an equatorial position. In Williopsis saturnus we have vegetative conditions very much as in Hansenula, where this species was formerly placed. M2 MEDICAL MYCOLOGY Copulation is reported to occur between ascospores before germination. The end member of this series is Schwarmiomyces, where the spore is rough as well as saturnine, the spore number is usually 1 per ascus, very rarely 2. Projec- tions simulating copulatory canals are produced, but the asci develop par- thenogenetically. Key to Genera IVEycelium present and well developed, no assimilation of nitrate. Copulation heterogamous ; no sprout mycelium, although the cells of the mycelium break apart into arthrospores; a thin pellicle on malt, also on ethyl alcohol. Endomyces. Copulation isogamous, sprout mycelium also present, pellicle thick, often gelatinous, on malt, none on ethyl alcohol. Endomycopsis. Mycelium absent although a pseudomycelium of sprout cells may be formed. Ascospores cucuUate. Yeast cells elongate, or ovoid ; copulation present ; nitrates assimilated ; thick, wrinkled pellicle on malt, often dry and powdery, fermentation of sugars positive, pellicle on ethyl alcohol. Hansenula. Yeast cells citriform, sprouting only from the poles, no copulation, nitrates not assimilated, no pellicle on malt, only weak fermentation of glucose, no growth on ethyl alcohol. Hanseniospora. Ascospores saturnine, yeast cells ovoid or spherical, copulation of ascospores before germination reported, nitrates assimilated; thick, wrinkled pellicle on malt, fermentation of sugars positive, pellicle on ethyl alcohol. Williopsis. Ascospores saturnine and rough, yeast cells ovoid, copulatory canals formed but not functional, nitrates not assimilated, usually only a thin ring on malt, occasionally a slimy pellicle; fermentation of sugars positive, very poor growth on ethyl alcohol. Schwanniomyces. HANSENULA Hansenula Sydow, Ann. Myc. 17: 44, 1919. Willia Hansen, Centralbl. Bakt. II, 12: 529, 1904 not Willia Miill. The type species is Hansenula anomala (Hansen) Sydow. Yeast cells ovoid to elongate, multiplication by sprouting, the sprout cells often clinging together in chains. On liquid media containing sugar a thick pellicle formed, dry and dull from the included air; fermentation of sugars positive, aesculin hydrolyzed, nitrate assimilated, good growth with pellicle formation on ethyl alcohol; ascospores eucullate, 1-4 per ascus. Hansenula anomala (Hansen) Sydow, Ann. Myc. 17: 44, 1919. Saccharomyces anomalus Hansen, C. R. Trav. Lab. Carlsberg 3: 44, 1891. Ann. Micrographie 3: 467-474, 1891. Willia anomala Hansen, Centralbl. Bakt. II, 12: 529, 1904. Willia anomala var. Beauverie and Lesienr, Jour. Pliysiol. Path. Gen. 14: 991, 992, 1912. Saccharomyces Beauveriei Froilano de Mello, Arq. Ilig. Pat. Exot. 6: 246, 1918 [based on case of Beauverie & Lesieur 1912]. ENDOMYCETALES 143 Reported occasionally from sputum but in none of the cases has patho- genicity been clearly shown. Beauverie & Lesieur (1912) reported a case of phthisis with the organism in the mucopurulent sputum. Grigorakis and Peju (1922) also report a case. Shrewsbury (1930), in a monograph on the genus, reports his strain 209 isolated from sputum from a case of chronic bronchitis along with Monilia, and an unidentified yeast. Shrewsbury was unable to find any pathogenicity for his strain on experimental animals. Pseudomycelium formed on many media, especially in pellicles on liquid media. On carrot at 26° C. in 6 days, cells spherical or ovoid, 3-8 x 2.5-6/*. Sporulation on carrot juice in the pellicle after 55 hours, cells 3-8/a in diameter. Asci spherical, ascospores typically cucullate. Colonies cream white, smooth, and shining. Usually developing a strong odor of fruit ethers. Hansenula bispora (Mattlet) Nannizzi, Tratt. Micopatol. Umana [Pol- lacci] 4: 134, 1934. Saccharomyces (Willia) Mspora Mattlet, Ann. Soc. Beige Med. Trop. 6: 32, 33, 1926. Isolated from stools of patients with various degrees of dysentery. Patho- genicity not proved; animal experiments not yet reported in detail. In potato decoction at 37° C. after 3 days, spherical or ovoid cells con- taining a vacuole and granulations, 2-10/*, budding, larger cells 6-7/* or rarely pyriform 11 x 7/*, thick-walled granular with little or no vacuole, other cells elongate, swollen at one end, the smaller tip of one against the swollen tip of the other and showing isogamous copulation. After about 10 days, oil droplets abundant in most of the cells or occasionally droplets unite into one large drop, copulation forms rarer. Little change in appearance after 30 days. On Gorodkova agar asci appear after 5 days, thick-walled, containing 2 cucullate ascospores 6 x 3/*. On Sabouraud agar colonies white dull, circular with even margin, in age the center becomes yellow and a few radiating folds develop. On gelatin stab, slight liquefaction with some gas formation. In potato decoction, abundant deposit of yellowish white clots which dissolve in the liquid on shaking. Optimum temperature 37° C. Litmus milk slightly acid. Acid and gas on glucose, fructose, maltose, galactose and sucrose; very slight acid on dextrin, no action on lactose and mannite. HANSENIOSPORA Hanseniospora Zikes, Centvalbl. II, 30: 145, 1911. Yeast cells citriform or elongate ovoid, vegetative multiplication by bipo- lar sprouting; no pellicle on malt, spores spherical at first, then cucullate (ability to form spores easily lost on cultivation), only glucose slightly fer- mented, nitrate not assimilated, practically no growth on ethyl alcohol. Hanseniospora Guilliermondii Pijper, Proc. Sect. Sei. K. Akad. Wetensch. Amsterdam 31: 989-992, 2 figs., 1928. Isolated from onychomycosis of European woman in Pretoria; patho- genicity quite probable but not proved. 144 MEDICAL MYCOLOGY Growth good at both room temperature and at 37° C. On fluid media cells citriform or ellipsoid, 5.2 x 2.4/*. In old cultures a slight tendency to- ward "mycelium" formation. Vegetative development by sprouting. No trace of sexual process, spores normally 4 per ascus, cucuUate, becoming spherical on germination. A tubelike protuberance is put out which leaves the spore and takes on the characters of the ordinary* vegetative cell. While sexuality has disappeared there is evidently some physiologic differentiation of the spores. After mordanting with chromic acid, staining with carbol- Fig. 22. — Dipodascus albidus. 1, 2, young copulation branches not yet abjointed ; S, h< diploid nucleus in female copulation ; 5, first step in division of diploid nucleus ; 6, later stage ; 7, 8, later stages of young ascus ; 9, upper portion of nearly mature ascus, the dark points in- dicating degenerate nuclei. (i, 2, 5-9 X900 ; 3 X800 ; 4 X600.) (After Dangeard 1907, Juel 1902.) fuchsin, decolorizing with sulphuric acid, and counterstaining with methylene blue, the equatorial pair of spores were acid-fast while the polar pair were blue. Giant colonies on malt agar, grayish brown, edge lobulated, surface smooth showing very delicate concentric rings corresponding to the days of growth, no radial lines, a central knob present from the beginning and later secondary knobs appear at various places. ENDOMYCETALES 145 Acid formed in raffinose, sorbite, and dextrin and a small amount of gas in glucose and fructose. No action on any of the other common sugars and glucosides. Malt gelatin liquefied slowly. On all liquid media, growth took place at the bottom only, no pellicle formed, even after many months. Dipodascaceae.- — In Dipodascus, the mycelium is septate, but the cells are coenocytic. The copulation of two hyphal cells produces gametangia (Fig. 22, 1-2). Fusion of the gametangial nuclei occurs without differentiation of the gametes. The formation of diploid mycelium has disappeared and the ascospores are formed in the gametangium without differentiation of an ascus (Fig. 22, 3-7). From this stage we have two main lines of divergence, one through Eremascus to the true yeasts, and one through Pericystis to the Coc- cidioideaceae. A third possible line has ended blindly in Actonia, a genus whose life cycle is not well known. In Actonia tropicalis, the mycelium is septate and may multiply by sprout- ing. A round gametangium (sporangium) forms at the end of a filament and develops small motile gametes (zoospores), which are apparently forced out of the gametangium by the invagination of the basal wall. "Whether this phenomenon is comparable to the proliferation of Ascoidea or is related to columellar formation in the Mucorales is uncertain. The further fate of the gametes ("zygospores") was not described. Whorls of sprout cells are some- times produced on the hyphae. After some weeks on Kaulin's medium, asco- spores (?) are formed. "At the end of a filament a round body appears with a well-marked external capsule and a small green body in its center. This body becomes flat and disklike, and from it four ascospores bud off. The four ascospores are surrounded by a limiting membrane which is extremely diffi- cult to see because of its transparency. The greatest care has to be taken not to rupture the membrane. "When rupture does occur, the membrane is coiled up under the parent cell and appears to be double" (Acton 1919). This, if correctly reported by its author, is quite similar to the peculiar spore for- mation we find in Paracoccidioides and reminiscent of partial sporangial forma- tion in the higher Mucoraceae. "When the morphology and cytology of this organism is better known, perhaps it will be found related to Paracoccidioides and Histoplasma rather than to the Dipodascaceae. In Pericystis alvei (Betts 1912, Claussen 1921) the mycelium is differen- tiated sexually, and copulation is heterogamous. Its cytology is unknown, but the ascus is spherical and contains many spores, suggestive of conditions found in the Coccidioideaceae-Taphrinaceae line. ACTONIA Actonia Dodge, n. gen. Mycelium cellulis curtis, crassis gemmiparis; zoosporangia terminalia, zoosporis motilibus, sphericis, e zoosporangiis ab basis invaginatione ejectis;, conidia verticillata, ovoidea gemmipara ; hypnosporae endogenae in hyphis veteribus; asci terminales, spherici, tetraspori. 146 MEDICAL MYCOLOGY Mycelium of short, stout cells, often multiplying by sprouting; zoospo- rangia terminal, producing motile, spherical zoospores which are ejected from the sporangium by the invagination of the basal wall ; conidia in whorls, ovoid, germinating by sprouting; endogenous hypnospores formed on old mycelium. Asci terminal, spherical, 4-spored. Type species is Enclomyces tropicalis Acton non Castellani. If the observations of the author are correct, this is a very curious fungus with a life cycle quite different from any other genus of fungi known, and the only place where motile gametes or zoospores have persisted in the Ascomycetes. It is possible, however, that flagelliform appendages of gametes similar to those in Spermophtliora were observed, or else that a contamination from some member of the Phycomycetes has been confused with some asco- genous fungus. Actonia tropicalis (Acton) Dodge, n. comb. Endomyces iropicalis Acton, Indian Journ. Med. Kes. 6: 591-600, 1919; not Endomyces tropicalis Castellani, Centralbl. Bakt. I, 58: 236-238, 1911. Monilia Actoni Vuillemin, Champ. Paras. Homme Anim. 84, 1931 nom. nud. Producing small creamy patches on the tonsils and uvula in throats of soldiers in Mesopotamia. The patches are difficult to remove but leave no raw bleeding surfaces. There is diffuse inflammation of the uvula, pillars of the fauces, and the posterior pharyngeal wall. In debilitated persons it may extend to the bronchi and bronchioles, causing fatal bronchopneumonia. Both sprout cells and mycelium present ; gametes produced in spherical sporangia. Their development is not altogether clear from Acton's descrip- tion, some of the phenomena suggesting proliferating sporangia of Ascoidea or of the Saprolegniaceae. Chlamydospores present. Ascospores budded off the ascus somewhat as in Paracoccidioides. On 1% sucrose agar, colony ivory white with raised crenate edges; be- coming creamy yellow and sticky in a few daj's, mycelium also penetrating the agar. On litmus milk, groAvth scanty, acid on the third day without coagu- lation. On Kaulin's solution, slight cream colored growth at the bottom of the tube. Ascospores in 2-6 weeks. On carrot, growth luxuriant, sticky, brown. From the stage attained by the Ascoideaceae two other lines of develop- ment diverge. One line has retained the large number of spores in the ascus, developed the ascus as a thick-walled resting spore, with a tendency to delay spore formation until the protoplasm has slipped out of the ascus. The nuclear history of most members of this line is so little known that there is doubt -j.- to whether sex has been retained, although the large nucleus (in some species) in the very young ascus suggests that a fertilization has taken place and the occurrence of spores in tetrads during one stage of development, in several genera, suggests a reduction division. Finally spore number is reduced in some species of the Taphrinaceae to 4 or 8, but so many intermediate forms exist and the number is so inconstant that it has been abandoned as a generic character. Along with this goes the elimination of the thick-walled resting ENDOMYCETALES 147 stag-e of the young- ascus. The collection of asci into more or less definite fructifications or ascocarps within the host tissue has been attempted in the end member (Taphrinaceae) where, in some species the clavate asci form a palisade layer under the epidermis of the host. The other line has early reduced the number of ascospores to a small, definite number, 8, 4, 2, or 1 and, perhaps owing to their habitats, have gradually diminished the amount of mycelium until in the Saccharomycetaceae, true mycelium has disappeared. For further discussion of this line, especially the steps in the gradual disappearance of sexuality, see Chapters XI and XII. Coccidioideaceae.- — This family has four genera causing more or less similar lesions in man and in experimental animals, with no saprophytic species so far recorded. Three of the genera are monotypic, from widely separated and rather restricted localities. Coccidioides is mostly restricted to California, Uruguay, and Argentina. Paracoccidioides to Brazil, while Rhino- sporidium has been reported from Argentina, India, and the Mississippi Valley. Histoplasma is known in the ^Mississippi Valley and in Panama. The large indefinite number of ascospores (Fig. 23, 19) suggests condi- tions found in the Ascoideaceae, but no conidial stage is knoAvn. Along with increasing specialization for strict parasitism in this family, sexuality has ap- parently disappeared without a trace.* The mycelium is septate and multinu- cleate as in the preceding family. In the host, however, it tends to less and less development until occasionally^ in Coccidioianisni as iMarcone and Tokishi<>'e. In 3 906, 8an Felice succeeded in g-rowing mierocolonies, but was unable to keep them alive very long, although he was able to reproduce the disease. In spite of these seem- ing successes, the next few years saw several hypotheses that the organism was a protozoon, the authors explaining the presence of hyphae in the cultures of earlier workers as contaminations in spite of the observations of San Felice who controlled his studies by microscopic examination. In 1916, Lindner and Knutli renamed the organism Monilia co.psidata. During- the decade of the World War, Boquet and Negre and their coworkers made a very thorough study of the problem and developed methods for its cultivation. The last decade has seen their work confirmed by several German workers. In the tissues, cells are spherical or ovoid, or acuminate at the two poles, sprouting, 3-5 x 2.5-3.5(U in diameter, membrane of variable thickness, granular. Occasionally elongate cells seen or larger cells, 5-7/t in diameter. In cultures, hyphae 2/i, in diameter, septa about 10-20/u apart, finely granu- lar, no oil globules, branched. Yeast cells pyriform, thin-walled, at first, becoming thick-walled after the cell separates from the parent cell, containing oil globules which may be large as the cell increases in size up to 8-12^ or even 15-16//,. Thick-walled hyphae formed from these yeast cells, 3-4// in diameter, with septa 10-18// apart and oil globules present. Chlamj^dospores from thick-walled mycelium, usually terminal, slightly polyhedral, 10-18//, protoplasm granular without oil globules. In old cultures the mycelium breaks up into somewhat irregular, thick-walled arthrospores. Asci 4-spored. [The ascospores figured by Tokishige were undoubtedly drops of oil.] Everbeck (1926) reports ascospores, thick-walled, ovoid, 3 x 2//, regularly produced after 14 weeks. In tissues from animal inoculations, yeast cells 3-4//, or, when actively sprouting, 5-6//, in groups of 10-15, and some thick-walled hyiDhae. The anti- bodies appear about the twentieth day of the disease and remain a long time after cure, so that it is very difficult to inoculate a horse which has had the disease. For isolation and early subcultures Boquet & Negre report best results with the following medium : Macerate 400 gm. horse dung in 2,000 c.c. of water for 24 hours in a cool place ; strain through cheesecloth, squeeze, filter, and add 10 gm. peptone and 18 gm. agar for each 1,000 c.c. of filtrate. Sterilize for 30 minutes at 120° C., add 40 gm. glucose per 1,000 c.c, tube and sterilize for 20 minutes at 115° C. For isolation add 20 drops of the following solution to the tubes after inocu- lation : chop 100 gm. lymphatic ganglions of the horse, macerate for 24 hours in 500 c.c. water, strain through cheesecloth, squeeze, filter, add 20 gm. glucose, tube and sterilize for 30 minutes at 115° C. Moisten cultures with this liquid as they begin to dry out. Incubate at 25-30° C. On dung agar, after 4-6 weeks, colonies appear on surface, as small, round, elevated, grayish white, slightly velvety, the size of a pinhead. The colonies 172 MEDICAL MYCOLOGY grow in height and at the periphery and turn brown, becoming contorted and scattered with small white, velvety points, with a white velvety area at the periphery which is festooned. Colonies hard, compact, adherent to the agar. On Sabouraud agar moistened with maceration of ganglia, the young colonies appear as on dung agar, the older colonies more folded and yellowish white, sandy, somewhat darker in age. Time of incubation shorter on suc- cessive subcultures (first in 1 month, fifth in 3 weeks, tenth in 10 days). Be- tween the fifth and tenth subcultures, organism is inoculable into ordinary agar, glucose agar, malt agar, with same aspect as on Sabouraud agar, but less abundant and less velvety growth. On carrot and potato, colony elevated, slightly folded, brownish gray, darkening in age, moist, smooth. On g-elatin, white velvety colonies, medium rapidly liquefied. On milk growth very slow, milk coagulated. Mycelium produced in the water of condensation of horse or sheep serum with 6% glycerol, horse serum agar, ordinary peptone agar, or glucose bean (2% agar with 20 drops of bean decoction added). The mycelium is oidiform, irregular with chlamydospores. While liquid media are not suitable for isolation or early subcultures, growth is possible after a time on peptone (1%) and glucose (5%), if the inoculum is floated on the surface and the culture incubated at 35° C. There is formed a thick whitish pellicle composed of yeast cells, spherical or ovoid, overlying the limpid liquid. Finally mycelium appears in the pellicle. If 0.5% agar is added to increase the viscosity, the pellicle is thick, folded, snow white, composed of yeast cells and, especially, thick-walled hyphae similar to those on Sabouraud agar. The optimum temperature is 37° C, but the medium dries out too rapidly; growth at this temperature is about twice as fast as at 25-30° C. At the higher temperature, the colonies are softer, more spongy, with more velvet, and not so adherent to the substrate. The maximum temperature is 38-40° and the minimum 15-18° C. At lower temperatures growth is very slow in the early subcultures, becoming more rapid after the organism has become adjusted to artificial media. At room temperatures, the colonies are elevated, folded, white, powdery or velvety, composed of very thin-walled hyphae and some sprout cells. After some weeks, short thick-walled hyphae are seen and the septa are more evident. In liquid media development is very slow at 20-25° C. If a culture is removed from 35-36° to 20-25°, it produces a grayish folded pellicle, the yeast cells cease budding and produce slender thin-walled hyphae as on glucose. If the culture is removed from 20-25° to 30-36°, the number of yeast cells increases. In low oxygen tension (under a layer of oil) growth is very slow, large irregular cells 12-15/a in diameter, thick-walled, granular, often in chains of 5-6 cells with large oil globules. Citric acid up to 1 :2,000 favors growth, producing a grayish folded pellicle or small round white colonies floating on or in the medium. Mycelial forms at low tempera- tures similar to those seen in pus. Bierbaum (1919) found slow growth on slightly alkaline horse meat agar with 2% glucose, 2.5% glycerol, and 3-4 c.c. EREMASCACEAE 173 sterile horse serum. Lange (1921) used egg medium with 2% glucose and 1% glycerol, reporting colonies small, brownish yellow, dry, center elevated, margin flattened, edge like a rampart, confluent near water of condensation. No fermentation of sugars, only glucose and sucrose utilized. Ammonia produced from peptone. Milk coagulated, gelatin rapidly liquefied. Zymonema Molardi (Salvat & Fontoynont) Dodge, n. comb. Endomyces Molardi Salvat & Fontoynont, Bull. Soc. Path. Exot. 15: 311- 320, 1922. Endomyces Molardi Fontoynont (nomen nudum) Bull. Mem. Soc. Chirurg. Paris 48: 439-442, 1922. Isolated from lesion on leg of man inoculated by scratching, (?) at first a fistula, then an ulcer, with depigmentation. Lesion finally cured by ex- ternal applications of methylene blue for about one month. Inoculation on thigh of a guinea pig caused abscess which healed spontaneously. Intraperi- toneal injection caused a nephritis which was fatal in 34-44 days. Organism recovered from the blood. Hyphae occasionally 2-3 fields long, 2.5-3.5/i. in diameter, irregularly septate or fragmented, appearing- nearly empty, tinted slightly pale rose by Gram's method. Yeast forms visible. Hyphae 2-6fx in diameter, contracted as septa, thick-walled, very rich in glycogen. Filament may terminate in a simple cell, a group of short, thick-walled, nearly round cells, one to two times as thick as the penultimate cell; or it may end by a spherical cell, 8-22/t in diameter, thick-walled, staining with aqueous eosin (chlamydospore). These chlamydo- spores appear occasionally in groups or short chains. Ascospores appear on glycerol agar or in liquid of glycerol-carrot. Asci spherical or ellipsoid, 4- spored, rarely 8-spored. On glycerol agar, growth rapid, colony thick, creamy, ivory white, ele- vated with little deeper yellow craters in the middle and never reaching the side of the tube. On Sabouraud maltose, growth as on glycerol but colony dirty, yellowish white. On Sabouraud glucose, growth less abundant, with slightly elevated polycyclic plateau, center creamy, white, glistening, then opaque. On fifteenth day fine radiations from central cone. On carrot with glycerol, from streak, growth appears like white porcelain, is thick, glistening, creamy, composed of a mass of granular colonies. Hyphae on walls of tube in fifth month, the whole surface is invaded and becomes crateriform, moist below, dryer above. Growth on potato and turnip with glycerol same as for carrot. Growth from gelatin stab appears like inverted fir tree. On surface plateau 8 mm. in diameter, three zones: (1) outer, 1 mm. broad, fine radiat- ing lines, (2) higher, 3 mm. wide, abrupt drop to outer zone, (3) uppermost, 3 mm. broad, granular. On horse serum, growth is meager, old ivory in color. On solid media, only yeastlike cells appear. In liquids a slight pellicle and very fragile ring appears, sediment is flocculent and abundant, liquid remains clear. On lactose bouillon, growth is good ; even after 5 months lactose is not exhausted. Medium gradually becomes acid. Growth in glycerol bouillon 174 MEDICAL MYCOLOGY poor. In Raulin's liquid, growth is fair, white sediment, liquid clear. In Gedo- elst liquid, growth very good. Coagulated serum not liquefied. Gelatin not liquefied in 30 days. Zymonema crateriforme (Iludelo, Sartory & Montlaur) Dodge, n. comb. Endomyces crateriforme Hudelo, Sartory & Montlaur, C. R. Acad. Sci. 170: 1086-1088, 1920. Saccharomyces, sp. Hudelo, Sartory & Montlaur, Bull. Sci. Pharmacol. 25: 352-357, 1918. Isolated from a lesion in the armpit of a young woman. Lesion oval, 3 cm. in diameter, erythematous, scaly, reminding one of dry seborrheic eczema. Nonpathogenic to rabbits and guinea pigs, by either subcutaneous, intraperi- toneal, or intravenous injections. On scarification an atypical, evanescent lesion results. Submerg'ed mycelium shows yeastlike budding cells, 9-11/a, or oidial cells, 15-16/A long. Conidia sprouting from aerial mycelial branches on some sort of sterigmata are variable but usually ellipsoid or ovoid, 5-7 x 6-9//.. Asci appear on solid media and old cultures after 80 hours at 22-23° C. These are usually terminal, rarely intercalary, 4-5/x in diameter, containing 4 spores each. Spores 3-3.5 x 1.75-2/a, with single smooth membrane. On Sabouraud agar, colony is small and white, becoming- creamy white, center yellow and irregularly crateriform, finally crackled, appearing- like small sponge. On carrot or potato, colony broader, thicker with several small craters, very much folded, cream white, covered with conidia. On broth gelatin, colony not fully developed before liquefaction on seventh day. After 18 days on malt extract, colony appears as thick, dry, folded mat covered with conidia, sediment of yeast cells, feeble fermentation with slightly aromatic odor. Growth similar on beef broth, peptone + glycerol + glucose broth, normal Raulin's solution with g-lucose, lactose, galactose or maltose, or on prune decoction. Sucrose, glucose, fructose fermented, not rafSnose, lactose, galactose, or maltose. Starch paste not liquefied. Milk not coagulated in 34 days. Gelatin liquefied. Zymonema albicans (Okabe) Dodge, n. comb. Endomyces albicans Okabe, Centralbl. Bakt. I, 111: 181-187, 1 pi., 1929, non aliorum. Isolated from 49 cases of thrush, in Japan, and pathogenicity of this strain proved. ["Monilia Candida," a filamentous species with strong- fermentation of sucrose and a strain wdiich completely failed to ferment, also isolated from some of the above cases, but proved to be nonpathogenic] Yeast cells spherical or ovoid, 4-6/a in diameter, with giant cells in old cultures reaching 15-20/x, after a time elongating and forming mycelium. Mycelium 3-4/i, in diameter, cells 20-30/x long, rarely up to 80/x. Chlamydo- spores pyriform, spherical or ovoid, 7-12/i, in diameter. Asci in 45 strains 1-spored, rarely 2-4-spored, 5-6/a in diameter; ascospores spherical to ellipsoid, 3.5-4 X 2.5-3/i., thick-walled, asci found only in the yeast stage, not on the mycelium, produced only on koji extract agar after 4-6 months. EREMASCACEAE 175 On koji extract agar, colony thin, milky white, smooth, surface moist with sharp borders, becoming thicker, duller, yellowish white ; on drying', becoming brownish yellow witli an ashy gray powder, mycelium very scarce. On 10% sucrose peptone agar, mycelium produced, colony verinicose and folded. No pel- licle on liquid media, powdery sediment. On sucrose peptone solution, mycelium floating as woolly masses hanging from the ring. By Lendner method, fermen- tation with glucose, levulose, mannose, maltose shows strong acid and gas ; dextrin and galactose show slight acid and gas, soon disappearing; no action on other sugars tried. Growth better at 37° C, than at 25° C. ; growth pos- sible in ice box or at 40°. Zymonema bonaerense (Greco) Dodge, n. comb. Endoniyces bonaerensis Greco, Argentina medica 1908 ; Origine des Tumeurs. 123-156, 412-421, Figs. 54-68, 225-231, 1916. Producing small miliary abscesses and plaques near eye. Pathogenic for rabbit, forming subcutaneous abscesses. Yeast cells 4-6 x 2-4/^, ovoid, hyphae mostly 2-4^ in diameter, occasionally much thicker and shorter. Asci 4-12/x spherical, spores 1-2/a. Colonies milk-white, shining-, discrete, or becoming confluent. On solid media after about a month, hyphae beg-in to show in the media. No gas but an ethereal odor in some cultures. On Sabouraud agar, growth at 20° and 37° white, creamy, a little thicker in the center, with filaments at the periphery. On beef agar and glycerol agar, growth less, colony flatter, more opaque, less shining white. On potato, colony creamy, very moist, white, later more opaque and grayish. On drying, small secondary yeast colonies form on surface of mother colony. Potato glycerol shows better growth, dryer, mammillate or slightly crateriform, slightly yellowish and cheesy in appearance, medium darkened. On carrot, colony creamy, spreading, white margin toothed. On liquid media no discoloration of media, no pellicle, but sediment produced. Sugars not fermented. Milk coagulated very slowly. Colony on gelatin stab resembles a test tube brush, without radial filaments, no liquefaction. The figures and description are not altogether convincing as to presence of ascospores, and I have hesitated to transfer it to this genus, but it is quite evidently not Endoniyces. Zymonema album, Dodge, n. sp. Monilia sp. Bianchi & Niilo, Bol. Inst. Ch'n. Quirurg. Univ. Buenos Aires 4: 531-538, 6 figs., 1927. Producing bronchomj^cosis. Pathogenic for guinea pig. Spherical yeast cells about Sfi in diameter; ovoid cells 3-5 x 2/*. Cells 10-12;u, in diameter, thick-walled with four deeply staining bodies which may be ascospores. In old cultures on glycerol, carrot, and potato, there are slender mycelium and large chlamydospores. This mycelial form continues on sub- culture. Colonies hemispheric, creamy white, moist, finally becoming confluent with a dryer, whiter margin. Optimum temperature 37°, but fair growth at 20° C. 176 MEDICAL MYCOLOGY On potato glycerol, medium, darkened; serum not liquefied, milk coagulated in 24 hours. In Raulin's solution, sediment, but not turbidity. Drigalski- Conradi slightly acid in 4 days. On neutral red agar, gas in 24 hours, color change in 4 days. No indol. No fermentation of sugars. Acid produced from glucose, fructose, galactose, lactose, and arabinose, no action on mannite. sucrose, raffinose, and inulin. Zymonema bucalis (Niiio & Puglisi) Dodge, n. comb. Monilia hucalis Niiio & Puglisi, Semana Med. 34: 222-229, 1927. The lesions began as perleche, extending gradually over the buccal mucosa which became covered by white plaques, slightly adherent, not continuous, suggesting clots of curdled milk, extending into the tonsillar crypts, produc- ing a burning sensation. Yeast cells and hyphae 2.5-3/x, little branched. In cultures yeast cells 3-7 X 2-5/x; asci spherical, 10/a in diameter, with 2-4 ascospores. Hyphae de- veloping from thick-walled cells, flexuous, little branched, 1.5-3/a in diameter; blastospores lateral; large terminal ehlamydospores present. Colonies at 37° C. on Sabouraud agar, potato-8% glycerol, carrot, and potato agar, circular, confluent, moist, white, little elevated. On liquid media (broth, potato decoction, 2% peptone solution and maltose-peptone solution) turbidity, with the formation of white clots, which settle to the bottom. Drigalski-Conradi medium becomes red, then decolorized; litmus sugar agars become red, then decolorized, and finally become blue. Acid on glucose, sucrose, and lactose, not on mannite. Sucrose not inverted, starch digested. Sugars not fermented. Milk coagulated on the third day, no liquefaction of gelatin nor of coagulated serum. Zymonema Cruzi (Froilano de Mello & Paes) Dodge, n. comb. Endomyces Cruzi Froilano de Mello & Paes, Arq. Hig. Pat. Exot. 6 : 51-60, 1918. Isolated from sputum of patient suffering from bronchitis and asthma. In sputum cells 4-8 x 2-4/^, rarely spherical, with refringent granules. No hyphae or spores present. On potato, cells ovoid or spherical, pseudomycelia 50-80/A long', simple or branched. Spherical or reniform asci present, 2-4- spored. On simple agar, spherical cells in chains of 8 to 16, no asci. On 1% agar + 6 drops of 10% NaOH, mycelial filaments definitely septate, 100/A long with terminal ehlamydospores or lateral conidia at the septa, and arthrospore formation. On 10 gm. agar + 5 drops 10% NaOH, some yeast cells, mycelium, terminal ehlamydospores. On 10 gm. agar + 4 drops 10% NaOH, feeble development, yeast cells, no true mycelium, asci abundant. On simple agar, feeble development, small milk-white colonies. On mal- tose agar (Sabouraud), colony white, then yellowish, verrucose. Colonies on potato, circular, elevated, wax color, opaque. Broth becomes cloudy after 72 hours with flocci in suspension, forming a filiform sediment. On Raulin liquid, thin whitish pellicle becoming thicker and chalky spotted, with small white EREMASCACEAE 177 points above and below with streamers 1-2 cm. long protruding into the liquid, finally breaking off and forming filiform sediment at bottom of the tube. Glucose, maltose, and sucrose fermented. Zymonema Alvarezsotoi (Mazza & Niiio) Dodge, n. comb. Monilia Alvarezsotoi Mazza & Niiio, apud ]\Iazza, Niiio, Quintana & Bem- aseoni, Bol. Inst. Clin. Quiriirg. Univ. Buenos Aires 6: 180-214, 38 figs., 1931. Generalized mycosis of native of Argentina. Fungus isolated from caseous lumps in feces, from lumps in the sputum and from the urine. Patient finally succumbed. Pathogenic to white rat, guinea pig, in intraperitoneal injection, to rabbit intravenously or applied to the mucous membranes. In one rabbit subcutaneous injection resulted in subcutaneous nodules which were surgi- cally removed. The animal recovered completely. On Sabouraud solid media, yeast cells mostly round, variable in size, some sprouting. No hyphae or asci observed. On coagulated human serum, after one month, many yeast cells, mostly round, 1-10/a in diameter. Rare ovoid forms. Few hyphae, composed of chains of fusiform, granular cells of vari- able size. Lateral branches at septa. Occasionally two large cells, approxi- mately of equal size, and with thick membrane, are joined by a narrow neck [isogamous copulation?]. Morphology in Gougerot gelatin slightly different. Hyphae predominate. These are variable in size, flexuous, composed of chains with lateral ramifications. Some spherical or ovoid cells are united directly to the hyphae or by little pedicels [immature asci?]. There are intercalary or terminal swellings resembling chlamydospores. Some cells are cylindric with rounded ends, 10 x 2/i,, vacuolate. In some filaments, internal spores like oidia. Optimum temperature for growth 30-37° C, no growth at 50° C. Gram-positive or negative. With Leishman stain, protoplasm sky blue, chromatin granules garnet. On Sabouraud glucose agar, colony rugose yellowish. Same on Sabouraud maltose. Good growth on agar and carrot. On potato, growth also good with darkening of medium. No growth on Drigalski medium. On potato, with 8% glycerol, good growth in 24 hours with darkening of medium. Colony white, humid, covering the whole surface. Pellicle on top of liquid and deposit at bottom of tube. On carrot, with 8% glycerol, abundant growth in 24 hours, colonies white, humid, covering whole surface of medium. Some days later on upper part of edge some white, agglutinated hyphae appear. Pellicle on surface of liquid ascending walls of tube. On Gougerot gelatin stab, rugose, caramel-colored pellicle on surface of medium, presently darkening on top. On plain gelatin stab at 15° C, dendroid growth along whole length of stab. On gelatin streak, good growth, best at the bottom where some lateral arbor- escences appear. Along coagulated human serum stab, growth slow and dend- roid. In plain broth and Sabouraud broth, turbidity and sediment in clots. In acid Raulin's solution, sediment in clots without turbidity. Starch paste unaltered. No fermentation of sucrose, rafiinose, lactose, maltose, mannite, sorbite, dextrin, or inulin. Slight acidity but no fermentation with glucose, 178 MEDICAL MYCOLOGY fructose, and galactose. No indol formation. Milk not coagulated. Coagu- lated human serum slowly liquefied. No effect on gelatin in 30 days. While no asci or ascospores have been reported in the following species, its pathogenicity, its cultural characters, and morphology all place it in Zymonema rather than Mycoderma, very close to Z. dermatitidis. Zymonema Harteri (Verdun) Dodge, n. comb. Cryptococcus Harteri Verdun, Precis ParasitoL, 1912. Atelosaccharomyces Harteri Beurmann & Gougerot, 1913. Parasaccharomyces Harteri Froilano de Mello, Paes & Sousa, Arq. Hig. Pat. Exot. 6: 33, 1918. Mycelohlastanon Harteri Ota, Jap. Jour. Derm. Urol. 28: [4] 1928. Fig-. 35. — Zymonema Harteri. (After Pollacci & Nannizzi 1926.) Monilia Harteri Vuillemin, Champ. Paras. Homme. Anim. 85, 1931. Mycotorula albicans Langeron & Talice, Ann. ParasitoL Hum. Comp. 10: 47, 1932, jyro parte. Torulopsis Harteri Almeida, Annaes Fac. Med. Sao Paulo 9: 10, 1933. Isolated from a case of generalized blastomycosis presumably contracted in Cochin China. The organism has evidently invaded the intestinal tract, liver, lungs and finally produced subcutaneous nodules. [Case of Harter, C. K. Soc. Biol. 64: 241-242, 1908; De la blastomycose humaine. These Fac. Med. Univ. Nancy 20: 1-222, 1909.] Pathogenic for rabbits and mice. Medi- cation with KI ineft'ective. Sprout cells ovoid or ellipsoid, 4-6 x 3-5/x, somewhat larger in liquid media, with some spherical cells in old cultures. On solid media elongate cells EREMASCACEAE 179 8-15 X 5-6/A appear. Hyphae about 2/x in diameter with occasional swellings up to 3/* seen in Raulin's liquid. Chlamydospores 5-8;u, with very thick walls seen on carrot. Asci not observed (Fig. 35). Growth over a Avide temperature range between 10 and 55° C, with good growth at 37° C, and at room temperature. On glycerol agar, growth abun- dant, white, smooth, velvety in the depths ; cells ovoid, with very large vacuoles in old cultures. On sucrose or glucose agar, growth creamy, abun- dant, shining with only ovoid cells. On ordinary agar, growth poor, granular with little penetration of the substrate. On carrot and turnip, growth creamy, white, smooth or granular, becoming mamillate, or even crateriform in old cultures, developing hyphae ; cells at first ovoid, soon becoming elongate and mycelial. On potato, growth slow, grayish, soon dry. On gelatin, colonies white, granular, with dendroid growth in the medium ; cells elongate or ellipsoid in the depths but no hyphae, ovoid at the surface. On blood serum, colony grayish white. In liquid media, floccose growth which slowly settles without producing ring or pellicle. Milk not coagulated, gelatin not liquefied after 8 months. No fermentation, sucrose only slightly inverted. I have been unable to locate the original descriptions of the following species and know them only from secondarj^ sources. Endomyces pulmonalis Senez, Boletin del Lab. de Bact. Tucuman (Argen- tina) 1: 58-60, 1918. [Reviewed in Bull. Inst. Pasteur 17: 636, 1919; Perin, Micosi polmonari 94-95, 1925; Pollacci & Nannizzi, I, Miceti Patogeni 4: No. 34, 1925.] Isolated from sputum of patient suspected to have tuberculosis. Creamy white colonies, asci ovoid, 4-spored, 10/a in diameter. Zymonema histosporocellularis Haberfeld, Tesis, 108 pp., 21 figs., Sao Paulo, 1919. Mycoderma histosporocellularis Neveu-Lemaire, Precis Parasitol. Hum. 70, 1921. This species is said to be a synonym of Paracoccidioides hrasiliensis (Almeida 1933). OLEINA Oleina Tieghem, Jour, de Bot. [Morot] 1: 289-292, 1887. Raquet mycelium Avell developed, intercalary chlamydospores present ; asci spherical, either intercalary or lateral, no trace of sexuality observed ; ascospores, 8 per ascus, varying from ellipsoid to spherical. No type species was designated. 0. nodosa, (Fig. 35, 3) was found grow- ing on fresh cartilage which was floating in olive oil during some studies of saprophytic fungi growing in oil. In this species the asci are intercalary, the spores ellipsoid, 4 x 6/*. 0. lateralis (Fig. 35, i), in which the asci are lateral and the ascospores spherical, 5/i. in diameter, was found on a bit of water- soaked cotton floated on the olive oil in similar experiments. It was cultivated 180 MEDICAL MYCOLOGY also on cartilage and maintained its distinctive characters. Yeast cell stage unknown, probably not normally present, as the author states that the chlamydospores germinate directly by germ tubes. The normal habitat of this genus is rather problematical. The cultures were made before the technic of pure culture had been highly developed, so that probably the cartilage had not been sterilized and the organism may have been present in it, or it may have been present in the surrounding oil. It is to be hoped that these organisms may be again encountered and studied. There is no trace of sexuality observed, so that this may represent an end member of a series. The relationship of Octomyces Froilano de Mello & Gon- zaga Fernandes is also problematical. OCTOMYCES Octomyces Froilano de Mello & Gonzaga Fernandes, Arq. Hig. Pat. Exot. 6: 237-242, 1918. Mycelium septate, sprout mycelium also present ; asci 1-, 2-, 4-, and 8- spored ; chlamydospores terminal. Type species Octomyces Bettencourti Froilano de Mello & Gonzaga Fern- andes. The type was originally isolated from a contamination at Nova Goa, so that nothing is known of its normal habitat. This genus may be considered as a synonym of Oleina, but it differs in the absence of raquet mycelium, the presence of sprout mycelium, and in the terminal rather than intercalary chlamydospores, which seem to be produced only in liquid media. Both genera have been rather poorly described and need much further study before their systematic position will be known. Neither seems to have a well-developed sexuality, such as found in Eremascus and Zymonema. Octomyces Bettencourti Froilano de Mello & Gonzaga Fernandes, Arq. Hig. Pat. Exot. 6: 237-242, 1918. Isolated from contamination in a Petri dish, at Nova Goa. Mycelium septate, yeast cells spherical with brown granules, asci ellip- soid, spherical, and lanceolate, 1-, 2-, 4-, and 8-spored. Chlamydospores terminal, yeast cells not sprouting. On glucose and maltose agar, colony moist, dirty white streak, same morphology as on potato. On potato, colony dry, veiy wavy, margins indented. On carrot, culture dry, very wavy, dirty white, granular in appearance. In broth, easily dissociable pellicle and sediment, no turbidity, with no fermentation, acid with dextrin, fructose, maltose, no action with lactose, mannite, or glucose. Octomyces Etiennei (Potron) Dodge, n. comb. Saccharomyces Etiennei Potron, Rev. Med. de I'Est. 45: 814-826, 841-855, 3 figs., 1913. EREMASCACEAE 181 Isolated from severe pleiiropiilmonary infection and bronchopneumonia with abundant j^easts appearing in the sputum. Producing local pyogenesis in rab- bit and guinea pig. Cells spherical, rarely ellipsoid, mostly 3-9 x 1.5-4.5/i. When actively bud- ding, cells are 15-18 x 4-5/i., with the formation of pseudomycelium but no true mycelium on turnip, liver decoction, and glycosuric urine. In other media, especially carrot and potato, cells separate early. On turnip, filamentous forms found, spherical cells about 6/* in diameter, asci abundant. Irregular allantoid forms found, also durable cells. Elongate forms on glycerol arti- choke as well as on turnip. Budding forms abundant in liquid media, espe- cially in the pellicle of old cultures. No trace of copulation before the formation of the ascospores, which are 4 per ascus and measure 2-2. 5/a. Asci 7-8 x 5ft. The ascospores swell and begin budding, still without any trace of copulation. No especial cultural conditions seem necessary for ascospore formation. On Sabouraud agar, colonies white, punctiform, rapidly confluent, forming a white, creamy, shining, flat surface elevated more than 1 mm.; margins crenulate. Colonies on potato grayish white, punctiform, spherical, very much elevated above the surface. As each colony grows rapidly, it becomes acumi- nate in appearance and is confluent with neighboring colonies. When the medium dries, the colonies become chalky white. On carrot, development is more rapid than on potato. Colonies white, rapidly confluent into a varnished, creamj^ surface. On turnip, growth at first similar to that on potato, then colony becomes prominent, surface mammillate, pebbled, remaining grayish white as the medium dries out. On artichoke, growth is slow but otherwise resembles that on carrot. Glycerol, on vegetable media, somewhat inhibits growth. On gelatin, combined with Lasseur's medium, colonies grayish white, develop slowly and are very slowly confluent, if at all. On liver decoction gelatin, grayish white, elevated, punctiform colonies. Development is rapid and abundant. On gelatin, combined with normal Raulin solution, colonies grayish Avhite, rapidly confluent to give the appearance of shagreen. In pepto- glycerol broth, development is slow, slight turbidity at first, with deposit of flocci at the bottom. Lasseur's medium is not especially favorable, develop- ment is as in pepto-glycerol broth. On Courmont's medium with glucose, development is rapid, sediment abundant, pellicle thick; with galactose and lactose, sediment even more abundant ; with sucrose, sediment less marked, pellicle feeble ; with maltose, sediment very marked, pellicle conspicuous ; with inulin growth, slow at first, with pellicle and sediment finall}^ appearing; with starch deposit, development slow. Grown in Sartory's mutton liver decoc- tion, organism causes grayish floccose sediment, leaving liquid clear. In glycosuric urine, containing 10 gm. glucose per liter, abundant deposit ap- peared, liquid clear with some gas evolution, then appearance of pellicle. Ring also after glucose had fermented. In normal Raulin 's solution, a powdery white deposit appeared on the walls and bottom of container. Fermentation occurred with glucose, fructose, galactose, lactose, maltose, and sucrose. Slight 182 MEDICAL MYCOLOGY action on dextrin, none on inulin, starch, or mannite. Milk very slowly coagu- lated, on the tenth day, with the evolution of C0_, gas. Curd not digested. Gelatin not liquefied. BARGELLINIA Bargellmia Borzi, Malpighia 2: 469-476, 1889. Hyphae very slender, hyaline, irregularly branched, septa remote ; asei solitary, terminal, spherical, membrane thickened, minutely tuberculate sca- brous, more or less brownish, indehiscent, spores spherical or subspheric, soli- tary, rarely 2 per ascus, wall thin, content oleaginous. This genus seems not to have been seen since its original isolation. It was not figured. Some characters suggest that it may be based on a misinterpreta- tion of Hemispora, in which the oil globules have been mistaken for ascospores. Until it is found again and more carefully studied, it should be regarded as doubtful. Bargellinia monospora Borzi, ]\Ialpighia 2: 476, 1889. Isolated from the external auditory conduit in catarrhal otitis. Hyphae subequal, 2-4;* in diameter, with distant septa ; asci more or less distant and indehiscent, 8-12/a. Spores spherical or nearly so, solitary or 2 per ascus, smooth, guttulate, 5-7/u, in diameter. HEMISPORA Hemispora Vuillemin, Bull. Soc. Myc. France 22: 125-130, PI. 7, 1906. Trachyiora Saccardo, Syll. Fung. 4: 262-263, 1886 [as subgenus only]. 8pore7idonema Ciferri & Redaelli, Jour. Trop. Med. Hyg. 37: 167-170, 1934; Redaelli & Ciferri, Atti 1st Bot. R. Univ. Pavia IV, 5: 145-198, 1934 not Desmazieres 1827 nor Oudemans 1885. The type species is Hemispora stellata Vuillemin. In tissues, yeastlike cells; in cultures, hyphae hyaline, septate, producing chlamydospores and conidia (blastospores?) ; asci in chains without trace of sexuality, containing a single echinulate spore. The morphologic interpretation of structures in this genus has long been puzzling. Vuillemin considered the structures here called asci as hemispores or deuteroconidia, representing an intermediate phylogenetic stage between arthrospores and true conidia (limited by Vuillemin to the types produced by phialides). More careful cytologic studies by Moore (1934) have shown that in H. coremiformis the asci are borne in chains at the ends of branches. In the early stage they resemble a chain of arthrospores or conidia on the end of a conidiophore. Then a densely staining structure develops within the cell walls which eventually forms an echinulate spore wall. The ascus wall then degenerates, leaving the aseospore free. The abundant formation of coremia in this species suggests that on equally careful cytologic study, Briosia, a saprophytic genus of the Fungi Imperfecti, might belong here. EREMASCACEAE 183 The species placed in this genus by Castellani belong elsewhere. Ciferri & Redaelli (1934) and Redaelli & Ciferri (1934) have attempted a compara- tive study of several organisms referred to this species, but since none of their organisms seem to agree with the morphology originally described by Vuil- lemin, it is doubtful whether they had the same organism as Vuillemin in his original paper. Among others they had a culture which originally came from Vuillemin, but there were no data to show that it was the strain upon which his original description was based. Their only strain which at all resembled H. stellata was isolated along with Aspergillus and may have also been parasitic on the latter. This strain differed more widely from their other strains than the other strains differed among themselves. If the name H. stellata, should be found to apply to a parasite of Aspergillus sp., then the determination by be found to apply to a parasite of Aspergillus sp., then the determination by cold abscesses, would remain in doubt. Hemispora stellata Vuillemin, Bull. Soc. Myc. France 22: 125-130, PI. 7, 1906. Fig-. 36. — Hemispora stellata. (After Vuillemin 1906.) Sporendonema epizoum Ciferri & Redaelli, Jour. Trop. Med. Hyg. 37: 167-170, 1934; Redaelli & Ciferri, Atti 1st. Bot. R. Univ. Pavia IV, 5: 145-198, 5 figs., 1934, excl. syn., quoad "ceppo Pun." Originally described as a parasite of Aspergillus repeiis forming a hyphal mat on the surface of a jar of preserved pears. Subsequently reported from cases of osteoperiosteitis by Gougerot & Caraven (1909, 1910) and from cold abscesses on penis by Beurmann, Clair & Gougerot (1909). Vuillemin identi- fied the organisms in these cases. More recently Fonseca & Area Leao (1927) report it from a sporotrichoid lesion on the arm. Cultures from lesions Avere pathogenic for rabbits, producing periosteitis. Torula epizoa Corda, in Sturm, Deutschl. Fl. Ill, 3: 97-98, PI. 45, 1829, upon which Ciferri & Redaelli base their specific name, was isolated from tallow, and judging from Corda 's figures is not related to the organism under consideration. I find no mention of the species in Corda 's later publications. Arthrospores in chains up to 30 or more, subspheric, 2.6-3.5yu. with a fuliginous granular wall except on the facet of insertion, occasionally elongate and barrel-shaped (Fig. 36). Hyphae 2-3/t in diameter, irregularly septate. 184 MEDICAL MYCOLOGY Colonies white, 0.5-2.5 mm. in diameter covered with conidiophores mak- ing little brown star-shaped spots. On sugar media, colony blackish brown, at first smooth and mammillate or irregular and coarsely convoluted, becom- ing powdered with ochraceous spores. Aerobic, not liquefying gelatin. Fig. 37. — Hemispora coremiformis. 1, hypha showing septation in lactose broth ; i, S, 5, 31, 37, mycelium showing variation on various media ; i, 17, hemispores ; 6, 7, 12-H, SJf, 33, 35, huge terminal cells (chlamydospores [?]) on various media; 8, young filament; 9^ 10, ampuUi- form cells on potato glucose agar; 11, deuteroconidia ; 15, 16, 18-2S, 25, 26, 32, Si, cells on wort agar, showing secretion of a gel ; 27, 28, terminal hemispores ; 29, ascogenous hypha showing- single echinulate ascospores. SO, Adjoining echinulate ascospores formed from hemispore. 3'/. coremium. 56^ hyphae from coremium. 38, Series showing development of echinulate ascospore and disintegration of the ascus. (1-S3, 35-38 X600 ; Si X500.) (After Moore 1935.) Hemispora coremiformis Moore, Ann. Missouri Bot. Gard. 21: 1934 [case of Rotter & Pena Chavarria, Arch. Schiffs Tropenhyg. 38: 414, 415, Fig. 10, 1934] . EREMASCACEAE 185 Isolated from lesions of the skin following scratching a bee sting with soiled hands in Costa Rica. Surface of the lesion is slightly raised and edges irregular, brass red, tissues infiltrated but not painful to touch. Subsequent lesions developed on edge of ear, angle of the jaw and clavicular lesion. The patient was treated with iodine and tartar emetic intravenously and anti- septics locally, with healing in 2 months. In cultures growth wholly of yeastlike cells and arthrospores for the first half year, then hyphae 2-4/a in diameter developed. Intercalary chlamydo- spores spherical 6-10/a or ellipsoid 7-9 x 12-15 fj. ; conidia 4-6/a in diameter. Asci spherical to clavate, at first terminal, in chains, apparently without sexuality; ascospores brown, echinulate 3-6/* in diameter (Fig. 37). On the more acid agars, growth slow, entirely submersed. On wort agar, colony vermiculate, light cinnamon, cells with sheath. On malt extract agar, colony buff to yellowish, vermiculate at center with radial folds and furrows. On Sabouraud agar, colony cerebriform at center surrounded by a ring of coremia, flatter toward the margin, buff to amber. On potato glucose agar, center acuminate surrounded by a cerebriform area from which radiate many furrows, buff. On nutrient agar, colony flat, center slightly elevated, margin irregular giving a stellate appearance. On glycerol agar, colony similar to that on potato glucose but covered with small coremia. On liquid media, no pellicle, flaky sediment. Sugars not fermented, no acid production, milk coagulated, and gelatin liquefied after 12 days. CHAPTER XI ENDOMYCETALES— EREMASCACEAE IMPERFECTAE The species to be discussed may or may not belong in the Endomycetales, since under certain environmental conditions the vegetative stages of many groups may assume a yeastlike appearance. However, when suitable media are employed, it is probable that ascospores will be formed in many species at pres- ent considered as imperfect. Judging from previous cases, we may anticipate that the ascosporic stage will place them definitely in the Eremascaceae. Since cultural studies seem essential in attempting to differentiate the species of a group where the morphology seems variable, the following pro- cedures, advocated by Redaelli and Ciferri (1929), by Talice (1930), and by Langeron & Talice (1932), may be considered as standard until better are pro- duced. They include most of the good features already advocated by Castel- lani during the previous two decades. The fungus is easiest isolated on carrot agar,* or Sabouraud glucose agar.f Spore formation is sought on Gorodkova agar. Cultures are incubated at 20°, 30°, and 37° C. From information gained from these cultures optimum temperature may be determined more accurately if desired. Comparative cultures may be made on the following media : Raulin, acid, or neutral solution, decoctions of carrot or potato, t malt extract solution (without hops), malt agar (2% agar), 2% glucose agar and corn meal agar (recommended by Smith & Sano, 1933) ; malt gelatin, carrot gelatin; glucose meat broth with 0.5% methylene blue, skimmed milk and peptone sugar broth (formula of the Committee of the Society of American Bacteriologists). Descriptions of the colonies on the above-mentioned media should be recorded after 1, 2, 4, 7, 10, and 30 days and even 60 and 90 days are often useful. A microscopic examination should be made on the second, fourth, tenth, thirtieth, sixtieth, and ninetieth days, on the latter days especial search being made for signs of copulation, and ascospore formation. Material should be examined from the bottom growth and pellicle in liquid media, and from the center and edge of the colony on solid media. The material may be mounted in glycerol, lactophenol, or Lugol's solution (water 11 c.c, potassium iodide 2 gm., iodine crystals 1 gm.). They may be stained Avith ZiehFs carbol- fuchsin, Loeffler's alkaline methylene blue, or Ehrlich's anilin methyl [gentian] violet (methods of the Committee of the Soc. Am. Bact.). The cells should *One kilogram of carrots is washed, triturated, and boiled in 1 liter of water for 3 hours. The solution is strained through cheesecloth, cooled. Altered through paper, made up to volume, and 20 gm. agar are added. tAgar 18 gm.. White's [W^itte?] peptone 30 gm., and glucose 40 gm., water 1 liter. JTalice recommends the following: Reduce 20 gm. of potato to pulp, suspend in 1,000 c.c. water, boil for 15 minutes, filter through cotton, replace water lost by evaporation, distribute to tubes, and sterilize at 120° for 20 minutes. It should be noted that this solution is about 1/10 the concentration usually employed in Thaxter's potato agar. The more concentrated solution is said not to give such good results. 186 EREMASCACEAE IMPERFECTAE 187 never be fixed by heat. A smear may be allowed to evaporate the excess water in air, then it should be fixed rapidly in alcohol and stained. Burri's India ink method and Mitsche & Harrison's collargol methods are useful. Since smears usually separate the cells of the filaments, they should be avoided as far as possible and the morphology compared with that obtained in hanging drops, using either liquid media or media containing 0.1% agar. Langeron & Talice suggest a very thin slant of agar, which is streaked all the way to the glass of the tube. For microscopic observation it is held as desired with two lumps of modeling clay on the stage and observed with an 8 mm. objective and an ocular of high magnification. In giant colonies, one must resort to one of the various devices for growing them, so that they may be uncovered for examination. These often give valuable morphologic details. Microcul- tures from hanging blocks of agar are useful. On liquid media, a little of the bottom deposit is lifted by a wide-mouthed pipette, and floated on a drop of liquid on the slide. The excess liquid is removed by blotting or by evapora- tion. It may be stained by the above-mentioned Lugol or by other suitable stains. Hanging drop cultures of dilute potato decoction should also be made. Here, after development has reached a suitable stage, the organism may be allowed to dry to the cover glass and stained if desired. It may be desirable to remove the lanolin which sealed the cover glass to the ring by wiping first with a dry cloth and then with a cloth moistened with toluene. Pigment formation should be noted. It is quite variable, depending on the composition of the medium, its density, the age of the culture, tempera- ture, light, etc. Fermentation should next be studied. Unfortunately, Castellani has em- phasized this to the exclusion of other characters, while others have failed to confirm his results with many strains, perhaps on account of the method used. Redaelli & Ciferri suggest the following list: arabinose, xylose, rhamnose, glucose, mannose, galactose, fructose, sorbite, dulcite, maltose, lactose, meli- biose, sucrose, trehalose, raffinose, starch, soluble dextrin, glycogen, and inulin. However, see remarks on Monilia Castellani, pp. 63, 64. The use of Lendner's microfermentation method is inadequate, unless all doubtful cases are studied more quantitatively. Experiments should be controlled carefully and repeated three or four times. The constancy of fermentative power has been ques- tioned (Bahr 1915), probably as a result of too great reliance on Lendner's method (see p. 63). While occasional cultures on a sugar which the fungus does not ferment will not alter its ability, repeated subcultures on that sugar may induce an irregular increase of abilitj'- to ferment that sugar Avhich is gradually but irregularly lost when again cultivated on the first medium. Mackie & Chitre (1928), in a study of the intestinal Moniliae of India associ- ated with sprue, show that many strains lose their ability to ferment certain sugars in subcultures on laboratory media and may regain this ability on pas- sage through experimental animals. Ability or nonability to ferment maltose was much more constant than that for any other sugars and may be used as a 188 MEDICAL MYCOLOGY character for the separation of species. The power to ferment other sugars is so variable (in their opinion) that it should not be used to separate species. A study of utilization of carbon sources is helpful, using Raulin's neutral solution, replacing the sucrose successively by glucose, maltose, lactose, man- nose, fi-uctose, inulin, starch and soluble dextrin, methyl alcohol, ethyl alcohol, glycerol, formic acid, acetic acid, oxalic acid, tartaric acid, and citric acid. In the case of the above-mentioned acids, the tartaric acid is replaced by the acid in question rather than the sucrose. Similarly sources of nitrogen are studied, substituting 1% potassium nitrate, potassium nitrite, ammonium car- bonate, urea, glycine, asparagine, and White's [Witte?] peptone for the nitro- gen source of the Raulin's neutral solution. Initial and ultimate hydrogen ion concentration should be recorded in these tests. In the liquid media, these authors suggest cultivation in graduated centrifuge tubes. After notes on the character of growth are taken, these tubes are centrifuged for 5 minutes at 2,000 revolutions per minute and the quantity of fungous cells at the bottom is taken as an approximate indication of the amount of growth. In the case of the sugars it is usually better to sterilize separately and add to the sterile Raulin's solution to avoid possible hydrolysis from the hydrogen ions of this solution. For the effect of different sugars on the morphology, see the inter- esting case of Blast odendrion intermedium (Fig. 38). Production of hydrogen sulphide (Kliger's method), hydrolysis of starch (Committee method), indol production (Ehrlich method modified by Gore) may also be tried, but so far have not yielded much information useful in classification. Finally parasitism and pathogenicity should be studied on experimental animals. Needless to say, this elaborate and ideal method has not been carried out for most organisms so far described in this group. It is often impossible to identify recently studied strains with older species in the literature, owing to the total lack of characters used by one author in the description by an- other. This is especially notable in the case of Castellani, who early aban- doned any mention of morphology and relied wholly on fermentation and enzyme reactions. The validity of fermentation reactions has been much dis- cussed in recent years (for general criticisms see pp. 63, 64), often without much apparent realization of the meaning of results or the limitations of the meth- ods employed (e.g., Castellani 1933). Under Castellani 's influence, very little attention has been paid to morphology until very recently, although we have occasional attempts to correlate morphology and fermentation reactions ; e.g., Fineman (1921) Nye, Zerfas & Cornwell (1928), Mackie & Chitre (1928). Re- cently the pendulum seems to be swinging the other direction, and we have Milochevitch (1929), Talice (1930), Shaw (1931), and Langeron & Talice (1932) emphasizing morphology very strongly, and searching for media which will produce normal mycelium rather than sprout mycelium, and in the case of myself and my students (Rewbridge, Dodge & Ayres 1929, Moore 1933- 1935 and much unpublished data) rather successful search for sexual or per- EREMASCACEAE IMPERFECTAB 189 feet stages has removed several organisms from the imperfect stages and placed them in the Eremascaceae. Since some of these researches have much biologic interest for those attempting to determine morphogenetic factors, they may be summarized in more detail. Marantonio (1893) working with a thrush organism found that sprouting occurred almost exclusively on solid media with occasional hyphae in old cul- Fig-. 38. — Blastodendrion intermedium. Showing the effects of various sugars on the morphology. 1, glucose ; 2, starch ; S, galactose ; Ji, maltose ; 5, lactose ; 6, raflflnose ; T, inulin ; 8, erythritol ; 9, mannitol ; 10, asparagin. (After Ciferri & Ashford 1929.) tures. On liquid media he found mycelium either in the pellicle or in the granular deposit that appeared without turbidity. The lower the pH, the greater the quantity of mycelium found. These observations Avere confirmed and extended to a large number of media by Concetti (1900). 190 MEDICAL MYCOLOGY Fineman (1921) working' with 17 strains isolated from cases of thrush and supposed to be Monilia albicans, finds the fermentation reactions constant. Mycelium develops in liquid media, in complex carbohydrate media, in media under low oxygen tension, and with low surface tension, Avhile the yeast form predominates on solid media, simple carbohydrates, abundant oxygen and high surface tension. Milochevitch (1929, also working with strains isolated from cases of thrush and supposed to be Monilia albicans, reports mycelium formed on media with higher surface tension and that the hydrogen ion concentration does not influence mycelium formation. He used a large number of animal tissues and extracts, and reported good growth on liver agar and blood, kidney and spleen agar, broth, kidney and lung broth. Growth was poor on peptone solution, brain, thyroid agar, urine, thyroid broth, ox gall and Raulin's solution. Talice (1930) undertook an extensive study of the media and conditions favoring the formation of hyphae, using a very wide variety of media and 30 strains of various species of Monilia. On solid media hyphae were produced in the first day or two; the yeast forms predominate afterward, hyphae being formed only in contact with the agar. In species Avhich seldom form hyphae, dextrin peptone media or glucose media give short periods of hyphal production, as also to a less extent do protein media. He found no advantage in semisolid media (0.1% agar) over liquid media in the production of hyphae. Hyphae develop best in liquid media, at least at first. Trying a large number of de- coctions, he concluded that he obtained the best growth with dilute potato decoction. In cultures which have been grown for a long time on solid media, as many as three transplants may be necessary to secure hyphae. Tessier (1890) reported that relatively high acidities favor hyphal production, but Talice states that this varies greatly with the species, probably accounting for some of the conflicting results by earlier workers. Lowered oxygen tension favors hyphal production to a certain point. Higher temperatures, as 37° C, produce the same results. Surface tension is important, as reported by Hahn & Junker and by Milochevitch, but again this varies with the species. The dictum of Roux & Linossier in the case of their strains of Monilia albica^is that complexity of morphologic structure increases with molecular weight of the sub- stances in the culture medium does not hold in this group. Talice regards the yeast form as senescent. Shaw (1931) suggests the morphology on dextrose ag-ar and gelatin stabs (i.e., the diameter of the hyphae, length of cells, size and position of monili- form clusters, and shapes of spores) is important in separating species and species groups. Pijper had previously noted that the creamy or membranous character of the giant colony is correlated with other characters, but Langeron & Talice first emphasized its fundamental importance. The most complete consideration of morphology so far produced is that of Langeron & Talice (1932). They emphasize the distinction between creamy and membranous colonies, the former producing abundant sprout mycelium while the latter do not, although the hyphae easily break apart in plane sec- EREMASCACEAE IMPERPECTAE 191 tions into arthrospores whose ends never become rounded. The creamy cul- tures are moist and shining at least in the first weeks, while the membranous colonies are dry and dull. The characters of these tAvo major groups may be distinguished as follows: CREAMY TYPE Earely folded, only when the growth is very rapid. Consistency of thick paste, easily adhering to the needle but never viscid, easily separating from the substrate. Yellowish or Ijrightly colored. Forming flocculent deposits in potato decoc- tion but no pellicle. Giant colony thick, convex, surface smooth, shining humid, uniform or with slight furrows, center often conic, margins lobulate. MEMBRANOUS TYPE Surface soon folded, soon velvety or studded with coremia. Consistency viscid, not adhering to needle, or if adhering drawing out in a long thread, more adherent to the medium. Dull grayish white. Forming less coherent flocculent deposits on potato decoction and usually a thick highly developed pellicle. Giant colony thick, dull, flat folded, fur- rowed, with coremia, margin not lobed. Two intermediate groups may be characterized in the creamy type. In Mycocandida, the thickness of the colony is variable, surface smooth or curdled, shining, or even iridescent, often transparent when young ; surface may be somewhat folded, j'-ellowish white, growth slower and colony diameter less than in the typical creamy type. In Blast odendrion colony thin and with deep radial furrows with a central eminence, surface smooth and dull. The sprout cell or blastospore is the fundamental element of the creamy group. In general, the shape is characteristic of the genus, but one may often find many variations in a given culture (Fig. 39). They may be characterized as spherical, short ellipsoid, long ellipsoid, ovoid, or long ovoid. (In examina- tion of material one should be sure that the cells have their longest axis ap- proximately at right angles to the line of vision, or a long ellipsoid cell may appear short ellipsoid or even subspheric.) Then we have an asjanmetrical form in Geotrichoides, an intermediate genus with membranous colonies. The pyri- form type (stalagmoide) often suggesting drops or tears is characteristic of Blast odendrion. Of the elongate types, cylindric and clavate are common. Sometimes they are somewhat irregular in development, producing allantoid and other irregular shapes. The various stages in the development of the cell have been clearly de- scribed by Shrewsbury for Hansennla (Willia) , and probably his obseiwations might be extended to the groups covered bj^ Langeron & Talice. The young cell is small, ovoid, spherical, or allantoid with a thin wall and a refractile, homogene- ous cytoplasm (Fig. 40, 1). Sprouting is active, the sprout cells being exactly like miniature mother cells. In each a small refractile corpuscle is visible near the center of the cell. The nature of this body is uncertain, as it could not be identified in fixed preparations and was not stained by vital stains. As growth progresses, the young (adolescent) cell enlarges and the cyto- plasm becomes more granular (Fig. 40, 2). Vacuoles appear, generally only 192 MEDICAL MYCOLOGY one per cell, but more may form in elongate cells. The vacuole soon enlarges to occupy about one-half the cell volume. Small, highly refractile bodies ex- hibiting active Brownian movement appear within the vacuoles. These are probably metachromatic corpuscles. Sprouting is still active and the sprout cells may or may not show vacuoles before separation from the parent cells. These adolescent (mote cells of Shrewsbury) cells correspond to the phase of maximum growth ; thereafter the cells begin to store up glycogen preparatory to production of sexual processes or of hypnospores. A B^ ■J©©©© 13O00f iilfii^wiiBM 10 11 Fig. 39. — Types of blastospores. 1, Structure: A, thick-walled; B, stained for glycogen; C, uniguttulate blastospores ; D, biguttulate blastospores ; E, showing refringent granules ; F, degenerating senescent blastospore. S, Spherical blastospores ; S, ellipsoid, short ; h long ellipsoid ; 5, ovoid types ; 6, asymmetric ; 7, stalagmoid or lacrimiform types ; 8, elongate cylindric types; 9, bacilliform types; 10, irregular bacilliform types; 11, still more irregular types from membranous cultures; 12, truncate types; 13, pyriform types; U, arthrospores. (After Lan- geron & Talice 1932.) As the culture ages, the adult cell (durable cell of Shrewsbury, Fig. 40, 3) appears and may persist unchanged for long periods. It is larger than the preceding types. It contains a large vacuole, usually empty but occasion- ally containing a single fat globule. Fat is stored generally in a single large globule at one of the poles. This globule is usually surrounded by a layer of I EREMASCACEAE IMPERPECTAE 193 protein. Some of these adult cells are transformed into hypnospores. The cell appears dark in color, often slightly larger than the other cells, the wall may not be thickened, but is usually darker in color. The cells often contain fat globules and small dark granules. Finally the degenerate, senescent, or dead cells (shadow cells of Shrews- bury, Fig. 40, 4) appear to be empty of contents, often with numerous fat globules in the vicinity of the ruptured cell. Other cells seem to be filled with fat globules. Perhaps the accumulation of fat reaches a stage where it cannot Fig. 40. -Showing' Shrewsbury's cell types in Hansenula. 1, young cell ; Z, S, adolescent cells ; 4, 5, adult cells ; 6-9, senescent cells ; 10, pseudomycelium. be utilized and causes the rupture of the wall. Occasionally these shadow cells may be artefacts caused by the mechanical rupture of young thin-walled cells which are filled with small fat globules. Sprouting may be from any portion of the cell in the true yeasts, but even among them it is often bipolar as it is in all members of the group under consideration. In very young, thin-walled cells before polarity is well estab- lished, sprouting may occur at other points. In thick-walled cells sprouting is almost always unipolar. In some genera verticils of sprouts may develop 194 MEDICAL MYCOLOGY from one pole, which by proliferation, prodnce dichotomously or polychot- omously branched chains of cells. When the branching is repeated in each cell, we have dense bushy masses forming the arhuscules of Ota. After a period of sprouting, some of the mature thick-walled cells begin to develop mycelium, very much as if a spore had been formed. The cytologic changes accompanying this process are unknown. A definite slender cylindric germ tube develops instead of a subspheric sprout cell. Sometimes the septation of the hypha follows promptly on its formation, at other times the septation lags until the hypha is very long and often multinucleate. The septa may be close or distant. After a time sprouting from the mycelial cells begins, producing the sprout conidia or blastospores. Sometimes these blastospores are borne in verticils, as in Mycotorula, or some of the members may be rudimentaiy and transformed into a hyphal branch, as in Mycocandida. The terminal portion of the hypha furnishes an important character. In Candida, the hyphae, instead of ending in a verticil as in Mycotorula, terminate in a chain of blastospores, which in turn may be branched but never verticillate. In Mycocandida and Blast odendrion each hypha ends in a single cell of variable length. In Mycotorida the hypha ends in a verticil or a dense tuft of blasto- spores. In Mycotoruloides the hyphal termination is a dense compound verticil. Hypnospores are often terminal. Thej^ appear on liquid media and in microcultures beginning to dry up. The contents of one or more cells migrate into the terminal cell where the cytoplasm appears dense and stains deeply with Lugol's solution. The hypnospores always germinate with a germ tube. Coremia are common in the group with membranous colonies (Fig. 3). Here the hyphae are collected into thick flexuous cords which rise perpendicu- lar to the surface and fray out at the top. They appear on all media, even on 2% glucose. Occasionally they are seen in some of the other groups where the blastospores are long and relatively slender, but in this case they are rare on 2% glucose. Besides coremia, on malt gelatin where the colony comes in contact with the glass, one often sees long pointed strands. These are also characteristic of the group with membranous colonies. The classification of this group presents exceedingly difficult problems. The earlier workers had very poor optical equipment and did not grow their organisms in culture. For the most part their descriptions are so brief and vague that it is very difficult to apply any of their names to organisms encountered at the present time. Since the same name early came to be used for entirely unrelated groups of organisms, we often have two or three distinct traditions for the application of a given name, the followers of each tradition claiming all the advantages of priority. To make the confusion worse, many authors have quoted incorrectly or cited dates from secondary sources. Frequently when one attempts to verify an original description, it is so different from that quoted that one can only conclude that the original description was not seen by the modern author. In the following discussions, I have attempted to present the various names in chronologic order, quoting from their original description, and tracing the various applications to various groups. It will thus be seen that practically none of the names published in the eighteenth and nineteenth centuries are legitimately available for members of this group, although many such names are in common use. There are only two alternatives, either we must abandon them altogether as has been done by Langeron & Talice, or else adopt by legislation in our code of nomenclature certain EREMASCACEAE IMPERFECTAE 195 new standard species whicli will conserve a name in one of its traditional uses and rename all the species which do not conform to the tradition selected. By either alternative the outlook is not bright for the medical man. To adopt the first would make a break with the past and involve a renaming of many of the species, and discarding the majority as unidentifiable on account of poor description. Unless this were formally legalized by an International Congress of Botanists, there would always be trouble from the legalist, the historian, and the publicity seeker by their puerile attempts to overturn existing nomenclature in favor of their own interpretation of some older name. If the second alternative is adopted, it must also be secured through the action of an International Congress of Botanists in which each faction would vote for the particular tradi- tion in the application of a name to which they were accustomed, with the deciding vote held by the systematists dealing with flowering plants who v.ould have no interest in, or knowledge of, the matter, and would decide it on national lines. By either horn of the dilemma, action by an International Botanical Congress is neces- sary and one is confronted by the practical problem as to which method to adopt, pending action by such a congress, which is apt to postpone resolutions for a generation; e.g., the action on bacterial nomenclature laid on the table at Brussels in 1910 for action at the next congress has not been acted upon yet. In view of the action of the last congress at Cambridge, England, in 19.30, in adopting the principle of the type species determination of the name, I have attempted to apply this principle strictly, and if the type species belongs in another genus with an older valid name, the genus name to which that type species belongs becomes a synonym of the earlier name. Where no type species can be definitely decided upon, I have adopted the view that it should be applied to the species which would produce the fewer new combinations by such applications. MONILIA Monilia Gmelin, Sy.st. Nat. 2: 1487, 1791. Gmelin segregated as Monilia various species previously placed in Mucor and Aspergillihs, iefining the genus as " Fila moniliformia in capitulum congregata." Most of the species belong to the genera Aspergillus and PenicilUum, although it is almost impossible to identify them with current species. Persoon took up the genus in Neues, Mag. Bot. 1: 121, 1794 (Dispositio 40, 1797) practically repeating Gmelin 's diagnosis but confining it to the erect species. He recognized four species, M. aurea, M. rosea, M. glauca, and M. Candida, M. rosea being described and figured by Batsch, the other three by Micheli. The latter belong in As