^! ■ i ©utmteb from tlie tmtt of (igia-iaee) ^^t^'«'f*..^^^ o MBL/WHOI Library c jf^O'" 1 9i:an\eM. G. Kn'.'^^t' - o. cj. = ^= m = ^= m ^ ^=i CO ^ ^— r^ o= ^^ JD X 1 n = (-1 CD -J ^^S S— — r-q ^s = CD SSS ^s, m ^ a ^^~ s= (=> MOLDS, YEASTS, and ACTING MYCETES ^^n net s MOLDS, YEASTS, and ^^^ ACTINOMYCETES A Handbook for Students of Bacteriology SECOND EDITION CHAREBS E. SKINNER, Ph.D Assistant Professor of Bacteriology University of Minn^ota, Minneapolis, Minnesota CHESTER Mi .D Principal Mycologist, Divisiert'of Infecti^tJfDiseases National Institute of>^ealth, BetJj^oa^'Marylanc HENRY M. TSUCHIYA, Ph.D Research Associate, Division of Microbiology ^Hormel Institute, University of Minnesota Austin, Minnesota New York: JOHN WILEY & SONS, Inc London: CHAPMAN & HALL, Limited Copyright, 1930 BY Arthur T. Henrici Copyright, 1947 BY John Wiley & Sons, Inc. All Rights Reserved This book or any part thereof must not be reproduced in any form without the written permission of the publisher. SECOND edition Second Printing, June, 1947 PRINTED IN THE UNITED STATES OF AMERICA PREFACE TO THE SECOND EDITION Molds, Yeasts, and Actinomycetes by Henrici, published in 1930, has been out of date for a number of years. The late author made a start on a revision but had to abandon the task because of failing health. We undertook the revision very largely out of respect and regard for Dr. Henrici. We were aided by beginnings of a revision, which, however, were entirely concerned with the first three chapters. Chapter II was prepared by Henrici and is inserted almost ex- actly as he wrote it. However, scattered notes in his personal marked copy and especially the memory of many conversations indicated the general nature of the revision which he hoped to make. Mere verbal changes, a modernization of nomenclature, a few deletions and insertions of new material were found insufficient. A complete rcAvriting was essential, and a considerable change in ar- rangement of topics seemed advisable. Two entirely new chapters dealing with material scarcely mentioned in the first edition have been added. Some deletions of portions of the old book, which ex- perience had shown were of limited value, were made. In spite of all this, however, we have attempted to produce a book in the spirit of the original book — namely a 'discussion of molds, yeasts, and actinomycetes for students of bacteriology. Like Henrici, we in- clude in the term students many individuals who have completed their formal education. It was for such students that Henrici called his first edition a handbook. Dr. Henrici was very insistent that, for their own good, bacteriol- ogists would do well to have some working knowledge of mycology. Inasmuch as most books on fungi are written for the botanist, the agriculturist, the phytopathologist, and others, the emphasis, in gen- eral, is often on those forms of least interest to the bacteriologist, and the forms of greatest interest to him are likely to be ignored or merely mentioned. Here the emphasis is reversed. The stress on medical and industrial applications of mycology has been retained in this edition and has been intensified. Moreover, Henrici was of the opinion that bacteriologists, whatever their special field, should have some knowledge of the morphology of fungi— classification, cytology, genetics, life cycles, and so on— so that they might better vi PREFACE TO THE SECOND EDITION understand and more intelligently discuss the many problems of the morphology of bacteria. He also maintained that an elementary knowledge of mycology, including a vocabulary, should be a special requisite for writers on variation and so-called life cycles in bac- teria. He frequently predicted that, if and when sexuality in bac- teria was demonstrated and proved, the persons responsible would be individuals wdth some knowledge of modern mycology. We, therefore, have not cut the discussions of life cycles in Chapter I and "sexuality" of yeasts in a later chapter. Rather we have, as Henrici explicitly intended, expanded them considerably. The in- creased knowledge in recent years in the very things of greatest importance to the bacteriologist, namely, "sexuality" in fungi and medical and industrial applications of mycology, has necessitated a larger book. This was foreseen by Henrici who at one time con- sidered the advisability of two separate volumes. Unfortunately, information on certain subjects was unavailable to us because of its restricted or confidential nature or because cer- tain published materials were not available in the United States during the war period in which the manuscript was prepared. We should like to ask the indulgence of our readers on such matters and we hope that it will be possible to rectify these omissions later. Although separated by half a continent, we have kept in touch with one another by correspondence and most parts of the text have been read and approved by the other two authors. Troublesome points in nomenclature and other slight differences of opinion were resolved by copious use of the mails. Although many suggestions from the other two authors were incorporated, in general Chapter I was prepared by Skinner from Henrici 's notes; Chapter II is Hen- rici's own; Chapter III was prepared by Skinner from Henrici's notes, and many additions, deletions, and rearrangements were made by Emmons; Chapters IV, V, IX, and XII were prepared by Skinner; Chapters VI, VII, X, and XIII by Emmons; and Chapters VIII, XI, and XIV by Tsuchiya. We hope that those who use the book will make comments and criticisms and write their suggestions for im- provement. We wish to express thanks to various individuals who have assisted us: to Dr. Louise Dosdall who read the rough draft of Chapter I and made many suggestions for materials to be included; to Dr- B. O. Dodge who read the same chapter in its nearly final form; to Dr. J. J. Christensen who went over the discussion on the Basidio- mycetes; to Dr. Charles Thom who made many suggestions for the revision of Dr. Henrici's chapter on Fungi Imperfecti, and to Dr. PREFACE TO THE SECOND EDITION vii Rhoderick Sprague who read the rough draft after revision ; to Dr. L. L. Ashburn who read Chapters VI and VII; to Dr. H. Koepsell who read portions of Chapter XI; and to Dr. S. A. AVaksman and Dr. Charles Drake who read Chapter XIII. Thanks are due Dr. L. G. Romell, Experimentalfaltet, Sweden, who in the face of great diffi- culties got through to us much information on Bjorkman's theory of mycorrhiza, and to Dr. W. P. Larson, "Chief" to Dr. Henrici and to two of us, who suggested the revision and encouraged its completion. We wish to thank Dr. Sprague, Dr. N. H. Huff, Dr. Kenneth Raper, and Miss Hazel Jean Henrici for original drawings and photographs; also the following holders of copyrighted figures and tables, each of which is credited to the author in the text: Macmillan and Company, Ltd., Dr. Fred J. Seaver, Phytopathology, Williams and Wilkins Co., Minnesota Agricultural Experiment Station, American Medical Asso- ciation, New York Botanical Garden, W. B. Saunders Co., United States Department of Agriculture, Dr. S. A. Waksman, Dr. R. L. Starkey, and Dr. J. R, Porter. C. E. Skinner c. W. Emmons H. M, , TSUCHIYA August, 1945 PREFACE TO THE FIRST EDITION The bacteriologist is continually confronted with molds, yeasts, and Actinomycetes, no matter what may be the particular field of application of the science in which he is engaged. Many of these are but contaminants, as Thom says, "noxious weeds" which plague him by overgrowing his cultures of bacteria. But an ever-increasing number of fungi are found to be causes of disease processes in man. and animals, while in the chemical transformations of the soil and spoilage of foods and other organic products of industry they are of equal importance with the bacteria. The industrial uses of these microorganisms have been known to a limited extent for some time, but promise to undergo extensive development in the near future. These fungi cannot, then, be ignored by the bacteriologist. The lower fungi are of interest, however, not alone because of their practical importance. Very significant and interesting facts are being discovered concerning their complicated life-cycles, par- ticularly with regard to the behavior of the nuclei, and their sexual relations, which will have an important bearing upon problems of variation and evolution in this group. Since it is becoming daily more clearly evident that at least some of the bacteria belong to the fungi, and there is a well-defined movement to trace similar cycles and relationships in the bacteria, it behooves the bacteriologist to make himself acquainted with at least the essentials of this newer knowledge. This book will, it is hoped, help fill the gap existing between the brief and inadequate discussions of the fungi found in current textbooks of bacteriology and the extensive monographs and tech- nical articles which treat of particular groups. The latter are too extensive to be easily used, too technical to be easily understood by the student of bacteriology who generally has no foundation in mycology. In this work I have tried to present sufficient information about fungi in general, and about those forms of importance to the bacteri- ologist in particular, so that the student will be prepared to use the more technical literature; to present such description and keys that will enable him with confidence to identify some of the commoner ix X PREFACE TO THE FIRST EDITION or more important species, and at least approximately place the others within their proper family and genus; and to furnish refer- ences to general works or specific monographs where detailed infor- mation on particular forms may be found. The book has grown out of a lecture course which has been offered for some years to advanced students in that ever-growing group that have planned their college work with the definite idea of be- coming bacteriologists. It is therefore a sort of textbook. But it will also, I hope, find a field of usefulness as a handbook to be kept in the clinical or technical laboratory for reference when problems involving yeasts, molds, or Actinomycetes are encountered. I feel strongly that bacteriology has distinctly suffered from a too sharp division of the science into categories according to particular fields of application. I have therefore attempted to deal equally with the medical and industrial applications of the subject. If the book seems to be somewhat overbalanced on the medical side, this is due to the richer literature available in that field. No claims are made for the work on the grounds of completeness or originality. It has been my task to select, condense, and where possible simplify, from the extensive available literature, such infor- mation as seemed important enough to be necessary, tangible enough to be useful, to the student of microbiology. The subject has been plagued by a tendency of its workers to create new species on the slightest provocation, to classify and reclassify, until a bewildering number of synonymous names are in use. My approach to the subject has been purely pragmatic. I have avoided discussion of debated problems of taxonomy except where such is necessary for an intelligent understanding of the literature. I have felt that to be useful the book must be kept within the scope of a small handbook, and must be made as simple as the subject matter will allow. I have therefore omitted discussions of numerous species and genera which are of no practical importance, or which are so uncommon that they are not likely to be encountered in routine bacteriological work. The literature cited includes, for the most part, only works of a general character or specific monographs in which extensive bibliog- raphies may be found, save that more recent contributions not found in such works have been included. I have as far as possible provided original illustrations. It has been necessary, however, to borrow illustrations of material that was not available to me. . . . Department of Plant Pathology of this institution. Dr. H. E. Michelson of the Division of Dermatology ... Dr. J. C. McKinley of the Division of Neurology ... Dr. F. W. Tanner ... Dr. P. PREFACE TO THE FIRST EDITION xi Tate ... Dr. Aldo Castellani ... Dr. W. J. MacNeal ... Dr. A. H. Sanford ... Dr. Lendner ... Dr. Thorn ... and Dr. Waks- man. ... I am indebted to my colleagues in the Department of Bacteriology for many helpful suggestions, and in particular to Dr. H. 0. Halvorson for his cooperation in preparing the section on alcoholic fermentation. To all of these I make grateful acknowl- edgment. Arthur T. Henrici The University of Minnesota April 1, 1930 CONTENTS CHAPTER PAGE I The Structure and Classification of the Fungi 1 Position of Fungi in the Plant Kingdom. Mycelium. Cells of Fungi. Oidia and Yeast-like Cells. Spores. Dikaryotic Mycelium. Homothallism and Heterothallism. Heterogamy and Isogamy. Basidiomycetes, Ascomycetes, and Phycomycetes; Life Cycles of Representative Fungi. Key to Classes and Subclasses of Perfect Fungi. II Variations in the Lower Fungi 35 Pleomoiphism or Degeneration. Microbic Dissociation. Sectors and Secondarj^ Colonies. Induced Variations. Variations in In- fection. Variations in Morphology and Physiolog^^ Reversible Variations. Genetics of Fungi. Practical Considerations. Rela- tion of Variations to Taxonom5^ III Methods for Studying Molds, Yeasts, and Actinomycetes 49 Culture Media. Isolation of Fungi. Methods of Study of Physio- logical Taxonomic Characters of Yeasts. Giant Colonies. Mor- phologJ^ Slide Cultui-es. Examination of Tissues and Exudates. IV Molds Belonging to the Phycomycetes 81 Entomophthorales. Families of Mucorales. Important G«nera. V Molds Belonging to the Fungi Imperfecti and the Ascomycetes 94 Position of Fungi Imperfecti. Vuillemin's Classification. Sac- cardo's Classification. Aspergillus. Penicillium. Cladosporium and Black Yeasts. Other Genera of Fungi Imperfecti. Neurospora. VI Fungus Diseases of Man and Animals — Gener.\l Considerations 119 Types of Mycoses. Host Specificity. Sensitization. Histology. Toxins. Immunity Reactions. Serology. Allergies. Skin Test- ing. Asthma. Treatment. VII Infections Caused by Molds 141 Coccidioidomycosis. Coccidioidin. Natural Habitat of Coccidioi- des. Dermatophytosis. Dermatophytes. Key to Principal Species. Principal Types of Dermatophytosis. Saprophytic Skin Fungi. American Blastomycosis. Blastomyces dermatitidis. South Amer- ican Blastomycosis. Histoplasmosis. Histoplasma capsulatum. Sporotrichosis. Sporotrichum. Chromoblastomycosis. Phialophora. Aspergillosis. M3'cetoma. xiii xiv CONTENTS CHAPTER PAGE VIII Biological Activities of Molds 215 Ecology of Molds. Nutritional Requirements of .Molds. Mold Fermentations. Metabolic Products. Mold Enzyme Preparations. Use of Molds in Food Products. Mold Spoilage. Molds in Textile and Wood Products. IX Morphology and Classification of the Yeasts and Yeast-like Fungi 264 Asexual Reproduction. Sexual Reproduction. Cytology. Classi- fication. Genera of Endomycetaceae. Species of Saccharomyces. Other Genera of Perfect Yeasts. Families and Genera of Asporog- enous Yeasts. Species of Cryptococcus. Candida. Geotrichum. Sporobolomyces. Rhodotorula. Other Genera of Imperfect Yeasts. Discussions of Nomenclature. X Pathogenic Yeast-like Fungi 305 Occurrence of Saprophytic and Pathogenic Yeast-like Fungi in Man. Cryptococcosis. Cryptococcus neoformans. Moniliasis. Candida albicans. Candida parakrusci in Endocarditis. XI Biological Activities of Yeasts 315 Ecology of Yeasts. Nutritional Requirements of Yeasts. Pasteur Effect. Manufacture of Yeast and Its Uses. Alcoholic Fermenta- tion. Industrial Applications of Alcoholic Fermentation. Glycerol Fermentation. Yeast Spoilage in Food Products. XII Classification, Morphology, and Biological Activities of the ACTINOMYCETES 349 Classification and Nomenclature. Morphology. Physiology. Ac- tinomyces. Nocardia. Streptomyces. Micromonospora. XIII Actinomycosis 371 Etiology. In Lower Animals. In Man. Culture of Actinomyces bovis. Mycetoma pedis. Acidfast Actinomycetes. Nocardia as- ter oides. XIV Antibiotic Substances 385 Penicillin. Streptomycin. Miscellaneous Antibiotic Substances. Index 397 CHAPTER I THE STRUCTURE AND CLASSIFICATION OF THE FUNGI The molds, yeasts, and actinomycetes are Fungi, a subdivision of the Thallophyta, one of the four divisions of the plant kingdom. A division in botany corresponds to a phylum in zoology. The Thal- lophytes are characterized by their growth in irregular plant masses not differentiated into roots, stems, and leaves like higher plants. Such a mass of plant tissue is called a thallus. The Thallophytes comprise the Algae and the Fungi. The former, being provided with chlorophyll, are capable of synthesizing their food from inorganic compounds by the energy of sunlight; the latter, being devoid of chlorophyll, must depend for their food upon organic matter syn- thesized by other organisms, growing as either saprophytes or para- sites. The lichens (Lichenes) have been considered a third subdivi- sion of the Thallophytes, because they are peculiar plants composed of algae and fungi growing together in symbiosis. Most modern authors classify them with the Fungi. The fungi are subdivided into the true fungi or Eumycetes, the bacteria or Schizomycetes, and the slime-molds or Myxomycetes. The relationships of the last group are not clearly understood; in fact some biologists believe that they should be classified with the protozoa in the animal kingdom. The relationships of the bacteria are also in some doubt, but the actinomycetes seem clearly to repre- sent a transition between them and the molds. The molds and yeasts belong to the Eumycetes. In this work where the word "fungi" is used without qualification, it will refer only to the Eumycetes. The fungi may be unicellular or multicellular. In some, as the true yeasts, the organism is ordinarily one-celled, though a few cells may be temporarily attached in irregular clusters when actively growing. Other fungi, however, may grow as one-celled organisms for a time, or in a particular environment, and become multicellular later or under changed conditions. Many of the fungi parasitic for man and animals show this dimorphism, growing in one form in the body, in another form on artificial culture media. Mycelium. The multicellular fungi are composed of cells arranged end-to-end to form filaments or hyphae. These filaments branch 1 2 STRUCTURE AND CLASSIFICATION OF FUNGI and rebranch and intertwine, sometimes even uniting or anastomos- ing, to form a structure called mycelium. This mycelium may form a loose meshwork as in the molds or a compact tissue as in the mush- rooms. Thus, although fundamentally the same in their finer struc- ture, fungi may show extreme variations in their external appear- ance, due simply to the degree of compactness of the mycelium. Such variations in external appearance have to some extent been used in the past as a basis for classification, but they are not always correlated with other characters and are unreliable. The same or- ganism may form a loose mold- like mycelium in some circum- stances, and a fleshy solid tissue in others. The true rela- tionships of the fungi are indi- cated by minute cell charac- ters, and more especially by their modes of reproduction. In some fungus tissues, how- ever, the cells are not arranged in filaments, but form com- pact masses of round or polyg- onal cells like those of higher plants. Such a tissue is called a pseudoparenchyma; its cells have their origin in mycelium. Sometimes such masses of fleshy tissue become quite dry and firm, developing thick, hard walls, and may have the property of main- taining vitality in a dormant state for long periods. In some fungi the mycelium is not divided into individual cells by crosswalls or septa. The entire mass of mycelium forms one large cell containing many nuclei. This absence of septa makes possible a flowing of protoplasm. Such a structure (described as being co- enocytic) also occurs in the filaments of certain green algae, and has been considered (together with other evidence) to indicate a rela- tionship between these algae and those fungi which possess it. Con- sequently the latter have been called Phycomycetes, or alga-like fungi. The other fungi possess a septate mycelium. In these, each cell, separated from the others by crosswalls, may contain one or more nuclei. In some of the Basidiomycetes and Ascomycetes the my- celium a^ pertain stages of its development is made up of cells, each with two nuclei. Streaming of protoplasm has been shown by Buller and others to occur in septate hyphae through central pores of the Fig. 1. a, Coenocytic mycelium; h, sep- tate mycelium. CELLS OF FUNGI 3 septa. Slow motion pictures of growing septate mycelia, however, show that the streaming is very much cut down as soon as the septa are formed. Tlius the three main classes of fungi may be recognized, in part at least, by the character of their mycelium: the Phycomycetes possess non-septate or coenocytic mycelium (Fig. 1) ; the Ascomy- cetes and Basidiomycetes septate mycelium. These characters, how- ever, are not the main ones upon which the classification of fungi is based, and are not absolutely constant, for the mycelium of both Ascomycetes and Basidiomycetes may be non-septate when the plant is very young, and the mycelium of Phycomycetes will develop septa in certain conditions, as for separating off spores. Cells of Fungi. The cells of fungi are much like those of higher organisms in their general characters. They possess a cell wall which is frequently of appreciable thickness. This was formerly thought to be composed of a substance similar to cellulose, though it does not give the microchemical reactions of cellulose, and was called "fungus- cellulose"; but more recent investigations indicate that it is chitin, or a mixture or compound of chitin and cellulose. Within the cell wall is the cell proper, or protoplast. It contains one or more nuclei, as indicated above. These are very small and not easily demonstrated, special complicated staining methods being required. The cytoplasm generally presents a granular or foamy appearance due to the accumulation within it of various reserve sub- stances in the form of granules or vacuoles. These may be of various kinds— fat, carbohydrate, and protein. The amount of this reserve material varies with the age of the mycelium ; in old portions of the thallus it may nearly fill the cell. Fat appears in the form of very highly rfefractile globules; it may be identified by staining with Sudan III. Carbohydrate is stored as glycogen as in animals, not as starch as in green plants. Protein is apparently stored in several forms. A noteworthy reserve substance almost peculiar to fungi is known as volutin or metachromatic material. It is identical with the material found in some bacteria designated in them, metachro- matic granules or polar bodies. It may appear either as granules or as vacuoles, the former sometimes floating in the latter. It is prob- ably a colloid which may exist in either the sol or the gel state. There has been considerable discussion regarding its true nature and function, which will be gone into in further detail in connection with the yeasts. It is generally considered a reserve material and recent studies indicate that it is a free mucleic acid differing slightly 4 STRUCTURE AND CLASSIFICATION OF FUNGI from the thymonucleic acid found characteristically in the nucleus itself. It is easily demonstrated by vital staining with neutral red. Growth and Differentiation of Mycelium. Although in many cases any part of a thallus may grow and give rise to a new individual if artificially transferred to a new medium, normally fungi are repro- duced by specialized cells called spores. These spores give rise to a new mycelium by germination, i.e., the protoplasm within the spore absorbs water and swells when in a favorable environment, bursts through the spore wall; and extends outward as a long filamentous process called a germ tube. Several of these may be formed from one spore. Growth is mainly at the apex of this germ tube. At first the mycelium is non-septate, but after it reaches a certain size, septa are formed in those varieties which possess them, beginning in the oldest part of the mycelium and proceeding toward the periphery. Thus in a mold which is still growing there may be no septa at the tips of the filaments of myce- lium. Several stages of spore germination are sho^\Tl in Fig. 2. With most multicellular fungi there can be seen a differentiation of the mycelium into two parts: a vegetative portion which burrows into the substrate, digests and absorbs it, and a reproductive portion which usually extends into the air and forms and discharges the re- productive bodies or spores. The reproductive mycelium and its spores are widely diverse in different kinds of fungi and serve to classify and identify them. Not infrequently one finds molds grow- ing on artificial culture media which do not form any reproductive bodies. They can develop only imperfectly on such a medium. Such molds are called Mycelia sterila, and usually cannot be identified with any degree of satisfaction. Oidia and Yeast-like Cells. In addition to reproduction by special- ized bodies or spores, many species of fungi can also propagate by the separation of cells from any part of the mycelium, including the vegetative. Such methods, however, are not peculiar to any class of fungi and do not serve to classify. The same type may be formed by widely diverse species. But in some species they occur so regu- larly or are so prominent that they may be of great value in iden- tification. Fig. 2. Germination of spores (conidia) of a species of Aspergillus. CHLAMYDOSPORES 5 These free cells may be formed by a segmentation of the mycelium into its component cells by a split through the septa. The resulting cells may be cylindrical in form (Fig. 119) as in Geotrichiim candidum, or may become rounded as in Mucor racemosus (Fig. 36). Free cells formed in this way are known as oidia. They may give rise to new free cells by division or by budding, or may give rise to mycelium. Free cells may also arise from the mycelium by either lateral or terminal budding, as also occurs with M. racemosus (Fig. 37) and with the various species of Candida (Fig. 116). These may also form either new free cells (by budding) or mycelium, depending upon their environment. Such free cells differ from spores in that they are not equipped for maintaining life in a dormant condition for long periods of time. They do serve, however, to multiply rapidly and disseminate the species, especially in liquid media. They are to be looked upon as "growth forms" rather then specialized reproductive bodies. The conditions which determine their appearance are quite diverse for different species. Thus in Mucors they are formed only under semi- anaerobic conditions, whereas in Candida they are predominant in aerobic cultures in sugar media. These oidia or free cells bear a great resemblance to and may be readily mistaken for true yeasts, especially when they multiply by budding. In fact, as will be shown later, yeasts are probably de- scended from more complexly organized fungi, which have perma- nently lost the power to produce mycelium and maintain only the one-celled growth form. But, as pointed out above, yeast-like cells may be formed under certain circumstances by representatives of all the great classes of fungi, Phycomycetes, Ascomycetes, and Basidio- mycetes. Similarly we may trace, with the true yeasts, evidences (in their spores) of relationships to at least two of these classes, Ascomycetes and Basidiomycetes. Yeasts, then, are a heterogeneous group of fungi which maintain a unicellular growth form, and are not an independent class of organisms. Their classification together is only for convenience and does not imply a true systematic rela- tionship. Chlamydospores. With many species of fungi we may find a cell here and there in the mycelium (including the submersed or vegeta- tive portion) which becomes differentiated from the others by in- creased size, due to the storage of much reserve foodstuff, and by a markedly thickened cell wall. These are known as chlamydospores. They are particularly adapted for maintaining vitality through long 6 STRUCTURE AND CLASSIFICATION OF FUNGI periods of dormancy. They remain intact and viable after the re- mainder of the mycehum has died and disintegrated (see Fig. 36). Like the oidia and the yeast-like cells, chlamydospores are not peculiar to any class of fungi, but may be formed by quite diverse species. Moreover, they are particularly likely to develop in just those species that frequently form free vegetative cells, and one may often find all transitions between the actively vegetative yeast-like cells and the dormant chlamydospores. Similarly, one may find all transitions between the vegetative oidia formed in the submersed mycelium of an organism and the conidia or true spores formed by its aerial mycelium. These transitions have led to much confusion in classification and nomenclature. These transitional types of growth or reproduction are particularly frequent in many of the lower forms of fungi important to the bacteriologist. Spores. The spores proper are very constant in their characters and mode of formation and are therefore largely relied upon for classification and identification. They may be either sexual, i.e., formed either directly or indirectly after the fusion of nuclei from two similar or dissimilar cells, or asexual, i.e., by the division of a single cell without fusion of nuclei. Some fungi reproduce by only one method, some by both, the latter forming non-sexual spores at one stage of their life history and sexual ones at another stage. In some parasitic fungi, the rusts, two different hosts may be necessary for the life cycle, and several kinds of spores may be produced in succession in each host, so that the cycle becomes complicated. Asexual Spores. Asexual spores are usually formed in great abun- dance, are ordinarily capable of dormancy, and serve to disseminate the species. At times they are enclosed in a slimy fluid, which may attract insects that carry them to a new habitat. More often they are dry and, being of light weight and small size, may be spread widely by the wind. Mold spores are abundant in the atmosphere. Asexual spores may be divided into two groups according to their mode of formation: endogenous and exogenous. In one large class of fungi, the Phycomycetes, the asexual spores are surrounded by a membrane during their formation. A cell at the tip of a filament of mycelium is cut off from the rest of the filament by a crosswalk The multinucleate protoplasm in this cell now sepa- rates into a number of small portions each of which develops a mem- brane. These small bodies are sporangiospores and the original cell wall forms a sac, the sporangium, to contain them. The filament which bears this sporangium is a sporangiophore. When the spo- rangiospores are mature, the sporangium either ruptures from internal ASEXUAL SPORES 7 pressure or it is dissolved by secreted enzymes, and so the sporangio- spores are set free. If sporangiospores are ciliated they are called zoospores. See Figs. 23 and 31. Exogenous spores, either unicellular or multicellular, may be formed from the mycelium in several ways, but in all cases they are born free, not contained within membranes. Collectively they are called conidia and the stalks of mycelium which bear them are conidiophores. The term sporophore means a differentiated portion of mycelium which bears spores. It is a more general term than sporangiophore, conidiophore, etc. In recent years many mycol- ogists have distinguished more carefully between the different sorts of conidia, especially in the class of Fungi Imperfecta Such distinc- tions were first clearly made by Vuillemin, who introduced new names to indicate the various sorts. Vuillemin divided exogenous spores into two main divisions, thal- lospores and "conidia vera." The true conidia, in contrast to the thallospores, are produced by an abstriction of the hyphae. The thallospores are further divided into arthrospores, produced by the disarticulation of a filament of septate mycelium into its component cells, and blastospores which are produced by budding from the ends or sides of the filaments of mycelium (Fig. 118). These two sorts of thallospores thus correspond to the two sorts of unicellular growth forms previously described as oidia and yeast-like cells. The distinc- tion between thallospores and unicellular growth forms is not clear, but in general the spores are formed on aerial mycelium, are dry, and are capable of remaining dormant and of being distributed by the wind, whereas the unicellular growth forms are produced by the submerged mycelium, are moist, and are capable of continued growth as unicellular bodies. But one may find in a single culture of, for instance, Geotrichum candidum all transitions from submerged oidia to aerial arthrospores, and recent authors have tended to avoid such distinctions, referring for example to the yeast-like growth forms of Candida albicans as blastospores and all the reproductive bodies of Geotrichum as arthrospores or oidia. We shall, in general, follow this usage. True conidia were subdivided by Vuillemin according to whether they were borne on what he interpreted as undifferentiated hyphae as in Sporotrichum, or on well-defined conidiophores as in most Fungi Imperfecta See Figs. 45 and 52. Among the latter he distinguished those which were borne on terminal, differentiated, bottle-shaped cells of the conidiophores, the phialides or sterigmata. Conidia borne on phialides have occasionally been called phialospores. See Fig. 52. 8 STRUCTURE AND CLASSIFICATION OF FUNGI Vuillcmin recognized two further types of conidia. Hemispores were considered to be transitional between arthrospores and true conidia. Aleuriospores (in French, "aleuries") w^ere differentiated from true conidia by the fact that they are not set free when mature, but are only liberated upon the disintegration of the mycelium that forms them. They are analogous to lateral chlamydospores. Recent workers have not recognized either of these as valid distinctions from true conidia. Fungi may form more than one type of conidiura from a single thallus. Thus in the dermatophytes some species form small uni- cellular conidia of the type which Vuillemin called aleuriospores, and large multicellular fusiform conidia which have been called spindle- spores. See Fig. 79. Certain other fungi may produce apparently the same kinds of conidia from different types of conidiophores. Some of the fungi isolated from cases of chromoblastomycosis have been found to form conidia from such different types of conidiophores. See Fig. 103. Since the classification of the molds is largely depend- ent upon the characters of the sporophores and the spores, such mul- tiple types of conidia and conidiophores lead to a great deal of confusion. In recent literature dealing with fungi which produce two sorts of conidia, they have often been classified according to size, as micro- conidia and macroconidia. Thus the bodies formerly referred to as aleuriospores in the dermatophytes are now often called microconidia or simply conidia ; the spindle-spores are referred to as macroconidia. In some fungi it has been shown that the microconidia are sex cells or spermatia, but this is not generally true. There are also so-called "conidia" which are actually sporangia containing one or only a few sporangiospores. They are seen singly or in chains and resemble closely true exogenous conidia. Actually their method of formation and the fact that the spores themselves, or small groups of them, are surrounded each by a membrane, show their true nature of sporangia. They are usually called conidia but sporangiola is a better term for them (Fig. 42). Sexual Spores. Spores resulting from the fusion of nuclei and subsequent reduction division are produced in most of the lower fungi less frequently and less abundantly than are the asexual spores. Here the functions of sex, concerned with heredity and variation, are to some extent separa.ted from the functions of multiplication and distribution of the species, which is carried on mainly by the conidia or sporangiospores. Often these "sexual spores" may be produced HOMOTHALLISM AND HETEROTHALLISM 9 only in some particular habitat, or in the presence of some environ- mental factor which is not at all necessary for asexual reproduction. Sexual spores result from the fusion of two nuclei containing chro- mosomes in the haploid, or Ix number. The fusion of the gametes, followed by a fusion of the nuclei, leads to the diploid, or 2x chromo- some number. Immediately after fusion of cells or at some later stage in the life history of the organism, a reduction division leads to a segregation of the "sexes" and to the haploid state of the nucleus. In all this the fungi behave in many respects like other plants and animals that reproduce sexually. Some of the fungi, however, ex- hibit certain special features. In some cases fusion of the gametangia, that is, the structures which contain the gametes, is not followed by an immediate fusion of the nuclei. From the fused cells there may arise an extensive mycelium wdth binucleate cells. The paired nuclei divide simulta- neously and one nucleus from each parent remains in the old cell and one of each goes into the new cell. This is known as conjugate nuclear division. In some cases the pairs of nuclei are not separated by septa until just before maturity. The nuclei, however, continue to divide separately and are found in pairs, each nucleus of the pair being of opposite "sex" to the other. The mycelium with paired nuclei of opposite "sex" which divides conjugately is known as the dikaryon or is described as dikaryotic. A mycelium such as fused to produce this dikaryon is often called the haploid mycelium. A hap- loid mycelium consists of uninucleate or multinucleate cells, all nuclei in the mycelium being haploid and identical genetically. All the nuclei were derived ultimately from the same uninucleate haploid spore. This dikaryon may give rise to binucleate spores which, when they germinate on the proper substrate, give rise to mycelium wdth paired nuclei again. Eventually the nuclei fuse in special organs and this fusion is followed immediately or after a period of dormancy by a reduction division. In many yeasts this diploid cell proliferates for a time before reduction division. Tracing the nuclear fusions and segregations, the diploid or the dikaryotic and the haploid stages of the fungus through its life history, has become an important part of modern mycology. It would take us too far afield to discuss this in detail. The reader is referred to monographs of Gaumann and of Kniep and to current botanical journals for further information. Homothallism and Heterothallism. In some fungi the gametes may arise from the same thallus or plant mass and are said to be homothallic. In many cases the cells which fuse must be derived from separate thalli, usually not of the same sex, and the process is 10 STRUCTURE AND CLASSIFICATION OF FUNGI called one of heterothallic conjugation. Heterothallism was first dis- covered in fungi by Blakeslee. It is widespread among the fungi and has been important in studying their life cycles, heredity, and varia- tions since it makes it possible to carry, in many cases, the sexes in separate pure cultures and to make crosses at will. Hybrids may frequently be formed and certain fungi are being used by geneticists. Their rapid growth and fructification and the fact that they can so readily be grown in absolutely pure culture, under conditions con- trolled at will, give fungi a decided advantage over seed plants, fruit flies, etc. Heterogamy and Isogamy. In some cases the cells which fuse are morphologically differentiated into male and female elements. In some primitive forms, a passive egg cell may be fertilized by a motile sperm cell. In higher forms the smaller or male cell is designated an antheridium, the larger or female cell an oogonium. In many fungi, however, the gametangia are not morphologically distinguish- able. Being exactly alike in appearance, they cannot be called male and female, but are designated + and — . Where the sex cells are differentiated, reproduction is raid to be heterogamous ; when they are alike the process is isogamous. Isogamy is often associated with heterothallism, in which case the whole thallus is + or — . Instead of the term sex, sign is sometimes used where there is no morpho- logical differentiation into oogonia and antheridia. Multipolar Sexuality. Sex in the fungi is still further complicated by the occurrence in some species of more than two "sexes." In some of the Basidiomycetes one may find that of the four basidio- spores, the nuclei of the mycelium from one may pair with the nuclei in the mycelium from only one of the other three; thus mycelium from basidiospore 1 may conjugate with 2 but not with 3 or 4, while 3 will conjugate with 4 but not with 1 or 2: Here there are obviously not two but four "sexes." However, in some species only two types of spores are produced from a single basidium. Mycelium from spore 1 or 2 will mate with mycelium from spore 3 or 4, however the hyphae may anastamose from any mycelium of the same species: 1 and 1, 1 and 2, 3, or 4, etc., but the nuclei will pair and divide con- jugately only when the proper "sexes" come together. The mycelium from a single spore in some species may fuse and divide conjugately with mycelia from any one of the four spores from another individual of the same species collected from some distant area. By matching spores from many collections, a large number of races or strains capable of fertile conjugation may be obtained. A somewhat similar situation is found in some of the ciliated protozoa. Such a condition, LIFE CYCLE OF A GILLED MUSHROOM 11 where apparently more than two "sexes" occur, has been designated multipolar sexuality, and the different strains capable of conjugation have been called mating types rather than sexes. The term mating type has come, to a considerable extent, to replace the term sex in discussions of the Basidiomycetes. Classes of Fungi. Instead of recognizing only one class of true fungi, the Eumycetes, most mycologists recognize four classes, which together with the Schizomycetes and the Myxomycetes make up the subdivision Fungi. They are Basidiomycetes, Ascomycetes, Phyco- mycetes, and Fungi Imperfecti. This last is an artificial class created to include the fungi whose perfect stage, that is, the stage involving sexual reproduction, has not been observed. The first three classes are divided upon the basis of their sexual reproduction. In the Basidiomycetes the sexual spores, basidiospores, are exogenous and typically four in number. In the Ascomycetes the sexual ascospores are endogenous and typically eight in number. The Phycomycetes form sexual spores which are in some cases single and in others multi- ple. There are also other differences between these three classes. Many modern mycologists divide the Phycomycetes into two or three classes, each of equal rank with the other three classes. Basidiomycetes. The class Basidiomycetes comprises in part the large, fleshy fungi, as the mushrooms, the puff balls, and the bracket fungi which grow upon trees. These are the Horaobasidiomycetes. Some species are known to form oidia in cultures, and some form conidia on either the mononucleate or binucleate mycelium or both, but mostly they reproduce by only one type of spore, the basidio- spore. What we call a mushroom consists of only the spore-forming or reproductive part of the plant. There is an extensive vegetative mycelium penetrating the substrate. Life Cycle of a Gilled Mushroom. The stalk of a mushroom is composed of numerous filaments of mycelium arranged in a compact bundle. These terminate in the gills in the form of swollen or pear- shaped tips, the basidia. In most species each basidium gives rise to four basidiospores attached each to a minute stalk, the sterigma (Fig. 3). When these basidiospores germinate they give rise to a haploid mycelium which sooner or later becomes uninucleate, i.e., divided by septa into cells with but one nucleus each. In hetero- thallic species, if mycelia from two mating types come in contact the hyphae may anastamose and the nuclei pair. From this point on, the paired nuclei divide conjugately and an extensive binucleate mycelium, in many species with clamp connections at the septa, is 12 STRUCTURE AND CLASSIFICATION OF FUNGI formed (Fig. 4a). Progeny of the original paired nuclei, many- nuclear generations removed, fuse in the basidia. Conjugate nuclear division of many species of Basidiomycetes is accomplished by the formation of clamp connections. From the terminal binucleate cell, a hook bends back (Fig. 45), each nucleus divides (Fig. 4c), and two septa are formed, one cutting off a single nucleus in the hook and the other cutting off another nucleus of op- posite mating type in the penultimate cell, but leaving one of each mating type in the terminal cell (Fig. 4(i) . The hook and the penul- timate cell then anastamose and each cell now has two nuclei of AT« Fig. 3. Section through the margin of a gill of a mushroom (Coprinus sp.) showing: a, basidia; b, sterigmata; c, basidiospores. opposite mating type (Fig. 4e). This continues throughout the bi- nucleate stage of the organism. Eventually nuclei in the terminal cells fuse and so form a diploid nucleus (Fig. 4^). This nucleus undergoes a reduction division and divides again (Fig. 4/i). The four nuclei, now again haploid, migrate through projections (sterig- mata) of the now swollen tip cell (basidium) and are cut off to be- come the four haploid basidiospores (Fig. 4i). These basidiospores usually are mononucleate and haploid, of different mating types and, when mature, are forcibly discharged and are capable of germinating to form the haploid mycelium again. Only haploid mycelium is produced unless mycelia of opposite mating type come in contact, in which case cells may fuse to produce the binucleate mycelium again (Fig. 4a). The presence of clamp connections is thus an outward sign that the mycelium is binucleate and has arisen from the fused mycelium derived from two different spores (Fig. 4a). Thus the sexual act in such fungi is divided into two phases, a fusion of cells from mycelium arising from different spores early in the growth of the mushroom, and a fusion of the nuclei at a much later stage, when the plant is mature. LIFE CYCLE OF A GILLED MUSHROOM 13 This separation of the phases of sexual reproduction with a di- karyotic mycehum interpolated between the fusion of cells and the fusion of nuclei is characteristic of the Basidiomycetes. The Basidio- FiG. 4. Conjugate nuclear division and basidiospore formation in mushrooms. Diagrammatic. Explanation in text. Drawing by Hazel Jean Henrici. mycetes are isogamous and in many cases heterothallic in origin. In homothallic species paired nuclei appear to arise spontaneously in the mycelium from a single basidiospore and conjugate nuclear divi- sion may be accompanied with or without the formation of clamp 14 STRUCTURE AND CLASSIFICATION OF FUNGI connections. It is much more difficult to establish sexual relation in these species. In addition to the large, fleshy mushrooms and their allies (the subclass Homobasidiomycetes) certain more minute forms are in- cluded in the Basidiomycetes because of the mode of formation of their basidiospores. These Heterobasidiomycetes include largely plant parasites, the smuts (Ustilaginales) and the rusts (Uredinales) . Smuts. As an example of the Ustilaginales we may consider the causative agent of the corn (maize) smut, Ustilago Zeae. When a smut sporidium, which is uninucleate and haploid, comes in contact with the corn plant only a super- gJIg ^ "" ' .^il^^i ^^^^^ infection of fine haploid IdP^*;;. M^F^^ mycelium takes place. If myce- lia from two sporidia of opposite mating type come in contact, fu- sion of cells may take place and a heavy and extensive mycelium with paired nuclei which divide conjugatcly results. This dikary- otic mycelium eventually matures in special galls. Many of the cells become rounded, rough and dark in color, and in these cells, morphologically chlamydospores (each at first with a single pair of nuclei), the nuclei unite and the cells become uninucleate and diploid. These are the smut spores. They are disseminated in great numbers and are capable of considerable dormancy. On germination, reduction division takes place. Then septa are formed, dividing the promycelium, which is a short germination hypha growing from the smut spore, into three or four cells. From each of these cells of the promycelium, daughter cells repeatedly bud off. These are the haploid uninucleate sporidia mentioned above. (See Fig. 6.) The promycelium may be considered a basidium and the sporidia basidiospores. These sporidia are capable of multiply- ing by budding and can be cultured on nutrient media. They have a superficial resemblance to yeasts. It will be noted that both the mushroom and the corn smut have haploid uninucleate basidiospores which germinate to form a haploid mycelium. In both, mycelium of opposite mating type may fuse to produce a mycelium with paired nuclei, the dikaryon. After ex- FiG. 5. An ear of corn affected with smut. Photographed by Dr. N. F. Huff. SMUTS 15 AA^s^tk^fUffAT^ •*ttA: tv-.tj^ -r/A* f «:«/:/ . j CV.l.-.I!jrr,5;. «c^ ,j/'.«iW.»:rX7»scw. '•■"'«^>Kvi,.. a!.v*i;VVi!f-T.,T.T.y,c-».„.nSi,W,j, £ **'?'- ^S""- 'ijfljr ^P^W I* '4-*' ■''■^■'■•■^■.w.',;-j!„ic/ ..«»r,-iv^,-j "*^-. '••■^«<«/;„ 11 Fig. 6. Ustilago Zeae. 1, Smut spore, diploid nucleus; 2, reduction division has taken place ; 3, promycelium formed ; 4, one daughter nucleus divided ; 5, other nucleus dividing; 6, septa and sporidia are forming; 7, all four cells of basidium separated and sporidia forming from each; 8, sporidium budding; 9, many sporidia formed; 10, two sporidia (s) on surface of com plant have produced mycelia which penetrated plant; hyphae united to form mycelium with paired nuclei (n = nucleus of plant cell); 11, mycelium in plant came to surface of plant at a, and reentered at stomatal opening (clamp-connection at c). From W. F. Hanna, Phytopathology, 19, 415 (1929). 16 STRUCTURE AND CLASSIFICATION OF FUNGI tensive proliferation by conjugate nuclear division the pairs of nuclei, each pair in a separate cell, unite to form the only diploid cell in the life history. After reduction division in this cell (in both), the haploid uninucleate basidiospores are set free. These basidiospores are of different mating types (two to four to a basidium). The close relationships of the corn smut and the mushroom, so very different in gross appearance, is clear from their cytological life histories. Moreover in certain smuts the sporidia are discharged forcibly in the same manner as the basidiospores of the mushrooms. Rusts. In the rusts (Uredinales) the life cycle is still more com- plicated. The fungus {Puccinia graminis) causing black stem rust of wheat may be taken as an example. It requires two hosts, the common barberry and wheat, for its complete cycle. When a sporidium (or basidiospore), which is uninucleate, lodges on a barberry leaf, it may give rise to haploid mycelium. Such my- FiG. 7. Section through a portion of a pustule on the upper surface of a bar- berry leaf infected with Puccinia graminis showing pycnium, pycniospores (a), and receptive hyphae (b). Redrawn from figure of A. -H. R. Buller, Nature, 141, 33 (1938). RUSTS If celium may eventually form a spore-forming structure, the pycniura, on the upper surface of the barberry leaf (Fig. 7). In this structure stalks of mycelium cut off uninucleate spores, the pycniospores (spermatia, pycnidiospores). These are accompanied by a secretion which attracts insects, the latter serving to transfer the pycniospores to new barberry plants which, as will be shown, they cannot infect directly. The sporidium is uninucleate and the mycelium and the pycniospores derived from it are haploid. P. graminis is heterothallic. Fig. 8. Section through a "cluster cup" on the under surface of a barberry leaf infected with Puccinia graminis showing the formation of aeciospores. If two sporidia of opposite mating type infect the same barberry leaf near enough to each other to enable their mycelia to fuse, "cluster cups" or aecia form on the under side of the barberry leaf. The chains of aeciospores (aecidiospores) borne in these cluster cups are binucleate. As with the mushrooms and the smuts, cellular fusion without nuclear fusion has taken place. If only one sporidium falls on a barberry leaf the haploid mycelium produces only pycnia and pycniospores. The pycniospores themselves cannot infect the bar- berry, but if pycniospores of mating type opposite to that of the haploid mycelium already growing in a barberry leaf are carried to that leaf in an infected area, these pycniospores fuse with the hap- loid receptive hyphae protruding from the pycnia and their paired nuclei are thought to migrate through the mycelium. Aecia and bi- nucleate aeciospores (Fig. 8) are known to form as a result of such a union of pycniospores and receptive hyphae. 18 STRUCTURE AND CLASSIFICATION OF FUNGI The aeciospores can infect only wheat, barley, oats, rye, and cer- tain wild grasses. They do not infect barberry. They give rise to a mycelium with paired nuclei, the dikaryon, which eventually forms Fig. 9. Section through a red pusl^le on wheat affected with Puccinia graminis showing the formation of urediospores. Fig. 10. Section through a black pustule on wheat affected with Puccinia graminis showing the formation of teliospores. another kind of spore, the urediospore (uredospore, urediniospore) . These are produced singly on the sporophore, and are binucleate. They are reddish in color and form the red pustules on wheat leaves RUSTS 19 and stems, the red rust. The urediospores can also infect only the wheat, not the barberry, and they spread the disease rapidly. Toward the end of the summer the host plant matures and the infection tends to develop more abundantly on the stems and from the mycelium on the stems there arises a fifth type of spore, the teliospore (teleutospore). The teliospores are surrounded by a thick dark-colored cell wall and are formed in the black pustules on the stems, the black rust. They are two-celled, each spore consisting of two separate protoplasts, each in its own compartment (Fig. 10). Each cell is at first binucleate but, in the teliospore, the nuclei fuse and the uninucleate, diploid stage is found. The teliospores remain dormant over winter. In the process of ger- mination the fusion nucleus under- ^^"- ^\ Germination of a telio- , T • • .1 spore oi ruccinia graminis showing goes reduction division as m the the formation of promycelium and smut spores. The four nuclei be- basidiospores. come separated by crosswalls in the promycelium so that we have a four-celled basidium as in some of the smuts (Fig. 11). From each of these four cells a sporidium, which may be considered a basidiospore, is formed, each with but one haploid nucleus. These spores are capable of infecting the bar- berry but not the wheat. This complex life cycle on two hosts is not a special case but is typical of many of the rusts. Resemblances to the cycle of the mush- rooms and smuts can be seen. The complete cycle is known for a large number of rusts including many of great economic importance. Sometimes the greater economic loss is in the alternate host, that is, the host which corresponds to the barberry in the disease just con- sidered, and in which cell union but not nuclear fusion takes place. Many of the species of rusts complete their life cycle on a single host, and are said to be autoecious. Many, however, like P. graminis do require two different hosts. These are heteroecious. The two host plants required for heteroecious rusts are themselves not closely related, as for instance wheat and barberry, the hosts of the stem rust of wheat, currants and white pine, the hosts of the white pine blister rust, cedars and apple trees, the hosts of the cedar rust of apples. It might be of interest to bacteriologists that the necessity of two different hosts for P. graminis to complete the sexual cycle and the various stages in the life cycle were known in essential de- tails by 1865, largely as a result of the work of the Tulasne brothers 20 STRUCTURE AND CLASSIFICATION OF FUNGI and DeBary. It was not until Theobald Smith, twenty-eight years later, had worked out the cycle of the protozoan parasite of Texas tick fever that such a life cycle was known in animal pathology. The alternate method of fertilization, that is, the pairing of the nuclei from the receptive hyphae and from pycniospores of opposite mating type, was discovered only fairly recently by Craigie in Canada. Physiologic Races. Although the smuts may be cultivated on artificial media, growth in a host plant is usually necessary for the development of the diploid smut spores. The rusts are obligate para- sites, which have not been grown in artificial cultures. In both of these groups, host specificity has developed to a very high degree, most species of smuts being capable of infecting but a single kind of plant, whereas with the rusts, each species of the parasite requires one or two unrelated host plants. But it is known that there occurs a still higher degree of host specificity, for within the species of the parasite there are found different races or strains, morphologically identical, but capable of infecting only certain varieties or "lines" of the host species. These races or strains of parasitic fungi ap- parently arise by mutation and hybridization. Such divisions of a species, capable of infecting only certain lines of the host species, are known as physiologic races, and the phenomenon is referred to as physiologic specialization. Ascomycetes. The Ascomycetes are the largest and perhaps the most important class of the fungi, including such widely different types as the large fleshy morels and the minute one-celled yeasts. Many important plant pathogens are Ascomycetes, and some of the molds important to the bacteriologist also belong to this class. It is characterized by the formation of spores, called ascospores, con- tained within a membrane or sac called an ascus. There are gen- erally eight ascospores in an ascus, but many asci may be formed by one thallus. Although there is a wide diversity in gross characters and external appearances between the various groups of Ascomycetes, the mode of formation of the ascospores is fairly uniform and indi- cates the homogeneity of the class. Ascospores. The formation of the ascospores is a result of sexual fusion of nuclei. The mechanism of their production is simplest in the yeasts. Different stages in the formation of the ascospores of a yeast, Schizosaccharomyces, are shown in Fig. 12. Two contiguous yeasts cells send out minute tube-like processes which meet and fuse ; their nuclei come together and unite, the single nucleus resulting undergoes division three times to form eight daughter nuclei. Each ASCOSPORES 21 of these becomes surrounded by a certain amount of reserve material, and a spore wall is formed. The cell containing these spores (usually four or eight) is the ascus. The ascus is formed less directly in many other yeasts. In these the haploid ascospores fuse in the ascus two by two, or the haploid cells which develop from the ascospores fuse Fig. 12. Various stages in the formation of ascospores in a yeast, Schizosaccha- romyces octosporus. After Guilliermond. with each other or with an unfertilized ascospore. These fused cells then develop into the ordinary yeast cell which is generally supposed to have a single diploid nucleus. After considerable proliferation by budding, some of these cells may undergo reduction division, form- FiG. 13. Various stages in the development of ascospores: A, Eremascus Jertilis; B, Endomycopsis fibuliger. After Guilliermond. ing the ascospores, usually four in number. This process will be elaborated in Chapter X. In the genus Endomycopsis and related fungi, a group of molds closely related to and often classified with the yeasts, the formation of ascospores is equally simple. Two contiguous cells in a filament 22 STRUCTURE AND CLASSIFICATION OF FUNGI of mycelium send out bud-like processes. Sometimes one of these is larger than the other, i.e., there may be a differentiation into an antheridium and oogonium. These unite, their nuclei fuse, and spores are formed immediately as in some of the yeasts. Various stages are shown in Fig. 13. In most of the Ascomycetes, the Euascomycetes, however, the proc- ess is not so simple. After fertilization of the oogonium by the antheridium, the nuclei do not fuse at once and the resulting cell does not at once develop ascospores, but instead gives rise to new filaments of mycelium, the ascogenous hyphae, with paired nuclei which divide conjugately. In many of the Euascomycetes the oogonium is fertilized by an antheridium from the same thallus. They are homothallic and heterogamous. The nuclei do not unite at once but divide separately and occur in pairs, one nucleus of each pair derived from the oogonium, one from the antheridium. These ascogenous hypha is often at first without crosswalls but eventually septa are formed across at least the end cells. These form their sexual spores and in some species proliferate in a manner which has many analogies with the conjugate nuclear division and basidiospore formation of the Basidiomycetes. a O O HJH Fig. 14. Crozier and ascospore production in Ascomycetes. Diagrammatic. Explanation in text. Drawing by Hazel Jean Henrici. ASCOSPORES 23 The paired nuclei come to lie side by side in the terminal cell (Fig. 14a) which bends to form a crozier (Fig. 14a). Each nucleus divides (Fig. 146) and two septa are formed, one isolating a single nucleus in the crozier, now the terminal cell, and one nucleus of opposite sex in the antepenultimate cell (Fig. 14c). The penultimate cell still has two nuclei, one of each sex. The crozier cell and the antepenulti- mate cell now anastamose and simultaneously the penultimate cell either forms another crozier (Fig. 14(i) or else the nuclei fuse (Fig. Fig. 15. Apothecia of Peziza sylvestris. From F. J. Seaver, The North American Cup-Fungi, 1928. 14e). If the former happens we may have several croziers being formed at the same time, for the cell formed by the union of crozier and antepenultimate cells, as well as the penultimate cell, now the new terminal cell, may form a crozier, and thus we may have a com- plicated system of ascogenous hyphae. Eventually, however, the nuclei fuse in the penultimate cells (Fig. 14e) of the ascogenous hyphae. This diploid nucleus undergoes reduction division, and each cell divides twice again (Fig. 14/) and the eight haploid asco- spores are produced in this cell which becomes enlarged to form an ascus. While the ascogenous hyphae are thus proliferating, the mononucleate or multinucleate mycelium arising from the cells which bore the oogonium and antheridium may form a complicated net- work of mycelium to cover the ascogenous hyphae. This becomes the ascocarp. There are many modifications of the above process. By far the greatest number of the species of Ascomycetes have eight ascospores per ascus. Whereas in the lower Ascomycetes the ascospores may be formed on any part of the mycelium, in the higher 24 STRUCTURE AND CLASSIFICATION OF FUNGI forms they are developed in particular areas and become surrounded and protected by a dense mass of tissue, thus forming a special fruit- ing organ (the ascocarp) , This may be arranged as a hollow sphere (the perithecium) or as a cup-like structure (the apothecium). The fruiting bodies of the cup fungi (Fig. 15) are apothecia. Their inner walls are lined by the asci in a continuous layer, the hymenium which appears in section in Fig. 16. In addition to the ascospores, many Ascomycetes also multiply by the production of exogenous asexual spores, the conidia. These are formed in great abun- dance when conditions are favorable for rapid multi- plication of the species, whereas ascospores are often formed only sparsely or under exceptional circum- stances. Life Cycle of Ergot. The parasite {Claviceps purpurea) producing a disease of grains, especially rye, and grasses known as ergot may be taken as an example to illustrate the life history of a typical Ascomycete. This organism is of some importance in medicine, since it produces in the infected grains a substance which produces contractions of involun- tary muscles. It has produced poisoning both in man and domestic animals, producing abortion by inducing contractions of the uterus, and gangrene by causing constriction of blood vessels. It is also used as a drug to produce a firm contraction of the uterus after childbirth. The fungus grows in the seeds, which, as they develop, become much larger than the healthy grains. The tissue of the grain is gradually re- placed by mycelium. During this period the fungus reproduces by the non-sexual conidia (Fig. 17) formed on the surface of the grain. These are accompanied by a secretion of "honey- dew" which attracts insects. They carry the conidia to the flowers of other plants, which thus become infected. As the grain ripens, those seeds which are infected are completely replaced by the mycelium, which now dries out and forms a dense, hard, black mass, the sclerotium. This retains the general form of the seed but is larger and projects considerably from the Fig. 16. Section through an apothecium of Peziza showing asci. Some are undeveloped. Two contain the full com- plement of eight asco- spores. Fig. 17. Formation of conidia by Claviceps pur- purea. Stained section of a grain affected with ergot. Only a portion of the fungus on the surface is shown. SUBCLASSES OF ASCOMYCETES 25 head of grain (Fig. 18). It is these ergot grains which contain the poison. These ergot grains fall to the ground and remain dormant over winter. In the spring, they revive and sprout a number of little stalks which develop rounded masses of tissue at their tips (Fig. 19). These are called stromata. In each stroma a number of pear-shaped areas, the perithecia, are formed (Fig. 20) . One of these is shown in section in Fig. 21. Each perithecium contains a number of asci, and each ascus con- tains eight needle-like ascospores. When mature, these are forcibly discharged into the air and carried by the wind. If they lodge on grain they germinate, forming a mycelium which invades the growing grain and starts the cycle over again. Subclasses of Ascomycetes. The Ascomycetes are subdivided into two main series according to the way in which they are borne in the ascocarp. The first series, the subclass Protoascomycetes, contains the more primitive forms, in which the ascogenous hyphae are lacking. In Endomycopsis and the yeasts like Schizosaccharomyces men- tioned above, the fusion cells give rise to asci at once and hence ascogenous hyphae are lacking. In many of the yeasts, after fusion of two cells, and presumably also of nuclei, the resulting dip- loidal cell proliferates by budding. Here also there are no asco- genous hyphae. In the second series, the Euascomycetes, the fusion Fig. 18. A head of grain affected with ergot showing a sclerotium. Fig. 19. "Germination" of a sclerotium of Claviceps purpurea showing the formation of stromata. cell gives rise to ascogenous hyphae which are now usually thought to have paired nuclei. By far the greatest number of species falls into this group which is often given the rank of subclass. It is some- 26 STRUCTURE AND CLASSIFICATION OF FUNGI times divided into three more or less well defined subclasses. In the first, the Plectomycetes, the asci are scattered on the ascogenous hyphae and are consequently distributed irregularly inside the peri- thecium, like Aspergillus (Eurotium). See Fig. 48. In the second subclass, the Discomycetes, the asci are borne in palisades, a hy- menium, in an open cup or plate-like apothecium, like in Peziza (Fig. 15) . In the third or Pyrenomycetes the asci are contained in a hymenium in perithecia as in Claviceps (Fig. 21). See page 33 for key to subclasses of Ascomycetes. It must be emphasized that there are literally hundreds of species and scores of genera of Ascomycetes the life cycles of which for the most part have not been worked out. The cycles of some of them, how- ever, have been worked out as completely as that of Claviceps purpurea, which has been taken as an example. One may find excellent material for micro- scopic wet mount study of ascospores in the common sap- rophyte Peziza in the spring, or powdery mildews of a num- ber of plants, e.g., lilac, late in the summer or early autumn. Dried material of either will yield very satisfactory preparations when wetted. The ascospores of yeasts, Aspergillus, and other forms of special interest to the bacteriologist will be considered later. Fungi Imperfecti. There are many fungi which possess the char- acteristic mycelium of Ascomycetes, which reproduce by conidia sim- ilar to those formed by known Ascomycetes, yet which do not form ascospores, or whose ascospores have not yet been discovered. These are designated imperfect fungi, or Fungi Imperfecti. It must be con- fessed that probably in many cases the imperfection lies in our knowl- edge of the organism rather than in the organism itself. They must be classified and identified by their conidia. Although the Fungi Im- perfecti have been established as a class equal in rank to the Basid- iomycetes, Ascomycetes, and Phycomycetes, it should be recognized that an attempt is continually being made to classify the latter three Fig. 20. Stained section of a stroma of Claviceps purpurea showing numerous perithecia at the periphery. PHYCOMYCETES 27 classes in a natural system which expresses their phylogenetic rela- tionship, whereas the class Fungi Imperfecti is, to some extent at least, an artificial classification of coniclial stages of natural groups. They consist of "form species," "form genera," "form families," "form orders." From time to time the perfect forms of these are discovered and then the species are removed from the class of Fungi Imperfecti and placed in their proper place in the Ascomycetes. This leads to some confusion. Thus the large genus Aspergillus is placed in the Fungi Imperfecti because most of the knowTi species do not form asco- spores; but such sexual spores have been found in some species, which are therefore included by some au- thors in another genus, Eurotium, of the Ascomycetes. It might be argued that finding ascospores in one species of a genus of imperfect fungi would warrant transferring the whole genus to the Ascoraj'cetes. But this cannot be safely done, since the genera of Fungi Imper- fecti are based upon the mode of formation of their conidia and it is known that diverse types of Asco- mycetes may produce the same sorts of conidia. In a few instances, fungi formerly classified in the Fungi Im- perfecti were found on further study to belong in the Basidiomycetes or Phycomycetes. It is probable, however, that most of the Fungi Im- perfecti are conidial stages of Ascomycetes. See page 94- Unfor- tunately, it is the imperfect fungi which are of least interest to the mycologist and have therefore had, in general, less intensive study, and at the same time they are of most interest to the practical bac- teriologist. Most of the fungi pathogenic for man must be placed in this group, and likewise most industrial molds (not yeasts) be- long to the Fungi Imperfecti. Phycomycetes. The Phycomycetes are the most primitive class of the fungi. Some of them reproduce by both sexual spores and non- sexual spores. There are three subclasses which are so diverse that _ - iMt» m wMm pm ^m i iff ir ^ aBvMirasl^''K9 Fig. 21. Stained section through a peritheciura of Claviceps pur- purea showing the elongated asci. Each of these contains eight needle-like ascospores. 28 STRUCTURE AND CLASSIFICATION OF FUNGI the class can best be described by considering each subclass sepa- rately. Indeed, as stated above, many mycologists consider them so diverse that they divide the Phycomycetes into two or three classes, each equal in rank to the other two classes of perfect fungi. The Archimycetes are primitive forms, mainly aquatic and para- sitic on water plants and animals, though some are important causes of disease of land plants. They are characterized by the absence of mycelium, the thallus being a single cell or an irregular mass, though a rudimentary mycelium is formed by some. They reproduce mainly by motile, flagellated spores called zoospores. In some, sexuality is very primitive. In others, well-defined gametes, oogonia, and antheridia, are formed. There are no forms of importance to the bacteriologist. The Oomycetes are also mostly aquatic forms; some are parasitic on land plants, and some live in soil. They reproduce by sexual spores called oospores and non-sexual zoospores which in many forms are motile. Life Cycle of Saprolegnia. The common water molds of the genus Saprolegnia may be considered to illustrate the life history of an Oomycete. They are found especially on dead animal matter sub- HH wr ^^^^^^^^^^^H ^^^^^^^^^^^^^^B^^^ /' ^^^^^^^^^^^Bw^ ~.^'~' ^ :i^^ R^BaHH^IH^^^H^^Hi^^^ •J^^H . mM Fig. 22. A minnow infected with Saprolegnia. merged in water, as insects and fish. Some are parasitic on fish. The scum which develops frequently on goldfish in aquaria or min- nows kept in bait-pails is due to the growth of this mold. They are especially prone to develop on fish after the latter have been handled or bruised (Fig. 22). The non-sexual spores are formed at the tips of filaments of my- celium projecting into the water. The terminal portion of the fila- ment becomes separated from the rest by a crosswalk The cell so LIFE CYCLE OF SAPROLEGNIA 29 cut off contains many nuclei. Vacuoles appear in various parts of this cell and by growing and coalescing they gradually cut up the enclosed protoplasm into a number of small cells, which develop cell walls and finally two flagella each. Thej^ are the zoospores, and ATM Fig. 23. Sporangia of Saprolegnia with zoospores in various stages of development. the cell in which they are formed is a zoosporangium (Fig. 23). The zoospores are pear-shaped, with the two flagella inserted at the apex. After swimming about for a time they come to rest, lose their flagella, and become rounded. After a resting period, these rounded cells give rise to a new sort of zoospore, bean- shaped, with the two flagella inserted on the side. These then swim about and, if they reach a suitable substrate, come to rest and germinate, forming the coeno- cytic mycelium. Sexual spores are not formed so fre- quently. They are produced by the con- jugation of two morphologically unlike cells, a large oogonium and one or more small tube-like antheridia. These gen- erally develop from neighboring branches of the same filament. Both the oogonium and the antheridium are at first multi- nucleate like the coenocytic mycelium from which they are derived. They become separated from this my- celium by crosswalls. The protoplasm of the oogonium becomes divided into cells containing each a single nucleus. The antheridia penetrate the oogonium and their nuclei fuse two by two with those Fig. 24. An oogonium of Saprolegnia. It has been fertilized by two antheridia and contains six oospores. 30 STRUCTURE AND CLASSIFICATION OF FUNGI of the cells of the oogonium. These fertilized cells then become the oospores, by enlarging and developing a thick membrane. They are capable of remaining dormant for considerable periods of time. Thus in Saprolegnia two types of spores are formed, the endog- enous motile asexual zoospores and the sexual oospores, which are heterogamous and homothallic in origin. In some of the terrestrial ^ forms of Oomycetes parasitic on plants, such as Cystopus, the spores germinate directly by a germ tube and simulate conidia. But these show their relationship by first developing flagella when they lodge in a drop of water and later developing mycelium. Life Cycle of Mucor. In most of the Zygomycetes, the third sub- class of Phycomycetes, the multinucleate character is maintained throughout the life cycle. Mucor may be taken as an example. The aerial mycelium gives rise to non-sexual spores in a sac or membrane, the sporangium. The terminal portion of the filament becomes greatly enlarged, vacuoles appear within the protoplasm, and by en- larging and coalescing these gradually separate the protoplasm into individual cells. Each of the spores so formed contains several nuclei. They mature by developing a spore wall. The inflated tip of the sporangiophore, which projects into the sporangium, is the columella. When the spores are mature the sporangial membrane dissolves away, liberating the spores, which are distributed by the wind. A portion of the sporangial wall remains attached to the base of the columella. (See Fig. 34.) The sexual spores of the Zygomycetes are called zygospores. They are usually isogamous and heterothallic in origin. When two hyphae capable of conjugating come together, their terminal portions become separated by crosswalls. Their cell walls dissolve where they touch, and the two multinucleated masses of protoplasm run together, the nuclei proceeding to unite two by two. The cell develops a thick wall with generally warty or spiny projections from its surface. Various stages in the formation of a zygospore are illustrated in Fig. 25. Zygospores, like oospores, generally remain dormant for consider- able periods. When they revive, they may germinate by putting forth a single filament which at once forms a sporangium and spo- rangiospores or they may produce a more or less extensive mycelium. Further discussion of morphology of Zygomycetes is given in Chap- ter IV. Although there is no morphologically distinguishable differentia- tion into two sexes in most of the Zygomycetes, it can be shown with the heterothallic species that there is a physiological differentiation. ORIGIN AND EVOLUTION OF THE FUNGI 31 By inoculating spores from different thalli on the two halves of a Petri plate culture, it will be found that in some cases where the two thalli come together tlie filaments will fail to fuse and form Fig. 25. Mucor Mucedo. Zygospores: 1, two hyphae in terminal contact; 2, articulation into gametangia a and suspensors b; 3, fu.sion of gametangia; 4, mature zygospore supported by suspensors; 5, germination of zj-gospore. After Bredfeld. zygospores, whereas with other combinations they will fuse. By repeating this experiment with many strains one can demonstrate that two sexes really exist, though they cannot be distinguished by their appearance. These "sexes" are referred to as plus and minus strains. Origin and Evolution of the Fungi. Certain of the Oomycetes, like Saprolegnia, show resemblances to certain of the green algae, 32 STRUCTURE AND CLASSIFICATION OF FUNGI like Vaucheria. This resemblance consists of the coenocytic char- acter of the protoplasm, in the mode or formation of the oospores and the production of motile, endogenous asexual spores. It was there- fore beheved for some time that the Phycomycetes had been de- rived from green algae which had become saprophytic. Similarly a. resemblance was traced between the formation of ascospores in the Ascomycetes and the sexual reproduction in the red algae, and the Basidiomycetes were supposed to have had an independent origin. More recently, however, many mycologists have thought of fungi as a group which has evolved as a separate line from a primitive unicellular form, probably a flagellated protozoan. Some of the Archimycetes show resemblances to the colorless flagellates. It is believed that there has been a continuous evolution from such simple forms to the higher types of Phycomycetes. In addition, the origin of the Ascomycetes has been sought in the Phycomycetes and the origin of the Basidiomycetes in the Ascomycetes. The alternate mononucleate or multinucleate and binucleate mycelium of the Basid- iomycetes and Ascomycetes and the analogies between the clamp connections of the former and the crozier apparatus of the latter point to a close relationship between these two classes. It is thought by some that the fungi thus represent a single independent line of descent from the protozoa and that their resemblance to algae is cir- cumstantial. LITERATURE For the bacteriologist whose interest in the Eumycetes has been aroused by the preceding chapter, or who feels a compulsion out of a sense of duty to make a further study of the true fungi, the following suggestions are made. In the first place, mycology is a science that had advanced further, in many re- spects, by the time that Pasteur and Koch had founded bacteriology, in 1900, than bacteriology has advanced today. Also there are probably at least as many individuals doing research today on pure and applied mycology as are working on problems in bacteriology. Thus there is a larger literature and more facts are known. But this does not make the subject more difficult; rather the contraiy, since monographic treatments have to a great extent combined and systematized what has been discovered and, in spite of the seemingly unneces- sarily large vocabulary, the covering up a lack of knowledge by vocabulary is, in general, a thing of the past as far as mycology is concerned. We bacteri- ologists (including immunologists) can decide for ourselves whether or not this is true in our own science. In general, many of the doubtful points in pure mycology have been confirmed or rejected, whereas in bacteriology, cor- responding features are a subject of investigation, polemics, or conjecture. This does not mean that all is known. Perusal of current botanical journals KEY TO CLASSES AND SUBCLASSES OF PERFECT FUNGI 33 KEY TO THE CLASSES AND SUBCLASSES OF PERFECT FUNGI I. Mycelium coenocytic if present, if absent not rei^roducing by budding or fission. Class PHYCOMYCETES II. Mycelium septate if present, if absent reproducing by budding or fission. A. Sexual spores exogenous. Class BASIDIOMYCETES B. Sexual spores endogenous. Class ASCOMYCETES C. Sexual spores unknown. Class FUNGI IMPERFECTI Subclasses of Phycomycetes I. Mycelium absent or rudimentary. Subclass ARCHIMYCETES II. Mycelium well developed. A. Sexual reproduction heterogamous; non-sexual spores motile or developing motility. Subclass OOMYCETES B. Sexual reproduction in most cases isogamous; non-sexual spores non-motile. Subclass ZYGOMYCETES Subclasses of Ascomycetes I. Asci produced from oogonium directly, or after multiplication by budding of diploid cells. Subclass PROTOASCOMYCETES II. Asci borne on ascogenous hyphae. (Euascomycetes) A. Asci distributed irr.egularly in a closed perithecium. Subclass PLECTOMYCETES B. Asci arranged in a hymenium. 1. Hymenium in an apothecium. Subclass DISCOMYCETES 2. Hymenium in a perithecium. Subclass PYRENOMYCETES Subclasses and Orders of Basidiomycetes I. Basidia alwavs simple; basidiospores on germinating producing mycelium di- rectly. " Subclass HOMOBASIDIOMYCETES (Mushroom and allies) II. Basidia septate or deeply divided or arising from a teliospore or probasidium. Subclass HETEROBASIDIOMYCETES A. Basidiocarp well developed; usually saprophytic. Order TREMALLALES B. Basidiocarp represented by a mass of probasidia, often compound (telio- spore) ; always parasitic on vascular plants. 1. Basidiospores borne on sterigmata, never reproducing by budding. Order UREDINALES (Rusts) 2. Basidiospores sessile on epibasidia, usually capable of reproducing by budding. Order USTILAGINALES (Smuts) 34 STRUCTURE AND CLASSIFICATION OF FUNGI will show how much fundamental work is being done. Either of the following two texts is good for the serious beginner. Bessey, E. a., a Textbook of Mycology, Blakiston, Philadelphia, 1935. Smith, G. M, Cryptogamic Botany, Vol. I, Algae and Fungi, McGraw-Hill, New York, 1938. For a discussion of the morphology and cytology of fungi and the basis of classification, the following two monographs are suggested. The second is difficult I Gwynne-Vaughn, H. C. I., and B. Barnes, The Structure and Development of the Fungi, Cambridge Press, Cambridge, 2nd ed., 1937. Gaumann, E. a.. Comparative Morphology of the Fungi, translated by C. W. Dodge, McGraw-Hill, New York, 1928. For a discussion of the sexuality of fungi, Giiumann's monograph or the following is suggested. Kniep, H., Die Sexualitat cler niederen Pflanzcn, Fischer, Jena, 1928. For a very readable, popular, but truly excellent discussion of an important field of applied mycology, with considerable pure mycology painlessly applied, the following book by an English novelist is highly recommended. L.AEGE, E. C, The Advance of the Fungi, Holt, New York, 1940. A valuable pamphlet giving very full keys through the families, with repre- sentative genera, several sketches, and an excellent glossary, will be found in the following. Martin, G. W., Outline of the Fungi, University of Iowa Studies in Natural History, Vol. 18, Supplement, 1941. CHAPTER II VARIATIONS IN THE LOWER FUNGI * Fungi, like other living things, are subject to variations in form and function. Some of these are but temporary or reversible changes, others are permanent; some occur spontaneously, others result from the action of known environmental factors; some result from changes in the hereditary constitution of a single cell, i.e., they are mutations, whereas others result from an interbreeding of two races or species, i.e., they are hybrids. But it is often difficult or impossible to deter- mine in a given instance whether the observed change is temporary or permanent, spontaneous or induced, mutant or hybrid. These variations are important in explaining the origin and evolution of new races and species, but as they occur in the laboratory they are often very annoying, since they lead to a loss or alteration of specific characters that are being studied, and lead to a great deal of con- fusion in classification and nomenclature. Pleomorphism. If one sends to a type culture collection for cul- tures of several species of dermatophytes, he is likely to receive several tubes bearing different labels, but which contain molds look- ing very much alike — an abundance of white woolly aerial mycelium, with few or no conidia or other diagnostic characters. These molds were quite different when first isolated — some powdery, some velvety, some white, some colored, and wdth different types of spores. But after long-continued cultivation they have gradually lost their dis- tinguishing characters, becoming more and more woolly in char- acter and producing more and more sterile aerial mycelium. Pig- ment production is usually the first character to go. These variations have been described in detail by Grigoraki.^^ Dermatologists, following the usage of Sabouraud, call such changes pleomorphism. But this word was first used by DeBary to designate the series of changes observed in rust fungi as these appear on their different host plants, a phenomenon quite distinct from that which we have under discussion. German mycologists have used a better word, degeneration, and they have designated the change just * This chapter has been used as it was written by Dr. Hcnrici, with the excep- tion of some minor editorial changes. 35 36 VARIATIONS IN THE LOWER FUNGI described as woolly degeneration. This woolly degeneration occurs not only with the ringworm fungi, but also with a variety of molds, such as Aspergillus nidulans, which loses first the ability to form perithecia, and then forms fewer and fewer conidia and more and more sterile mycelium. It is most likely to occur when cultures are frequently transferred on highly favorable media. This fact was recognized long ago by Sabouraud who devised a conservation me- dium, rich in peptone and poor in sugar, on which stock cultures were maintained. These should be transferred only at long intervals. Another type of change observed in cultures of fungi has been called faviform degeneration by Alexander.^ In ringworm fungi it usually follows a stage of woolly degeneration. The mold now grows as a low, fiat, wrinkled, waxy-looking mass of mycelium, without any aerial mycelium or conidia, firmly adherent to the agar. This is the normal appearance of the fungus of favus, hence the name. Although less frequently observed than woolly degeneration, it is seen in a variety of molds, and occurs very constantly with the organism of North American blastomycosis, a fungus which normally grows with an abundance of sterile woolly aerial mycelium. Still another type of change may be observed in certain fungi which normally do not produce any aerial mycelium, but grow as a moist mat on the surface of the agar. Such fungi are Candida Krusei, Sporofrichiwi Schenckii, and Pullularia pidlidans. I have observed with all of these, after long-continued cultivation on Sabouraud agar, the development of dry aerial mycelium, with conidia in the last two. Finally, one may observe changes in yeasts after long-continued cultivation. Normally forming no mycelium, they may develop first elongated cells, then pseudomycelium, and finally some filaments of true, septate, branched mycelium. An extension of this phenomenon is observed in organisms like C. albicans, which on Sabouraud agar normally form an abundance of yeast cells and little mycelium, but which on long-continued cultivation gradually produce more and more mycelium and fewer yeast cells, so that eventually they come to look like C. Krusei. Microbic Dissociation in Fungi. It may be observed that all these variations form a continuous series which may be indicated thus: Yeast -^ Submerged -» Aerial -^ Sterile -> Faviform mycelium mycelium woolly growth with aerial spores mycelium -So far as I know, no single fungus has ever been observed to go through all these changes, unless possibly Blastomyces dermatitidis, SPONTANEOUS VARIATIONS 37 But if the fungus changes on long-continued cultivation, it usually changes in the direction indicated by the arrows. The so-called black yeasts may go through the first three stages, finally appearing as a mold of the genus Cladosporium. See page 111. Ringworm fungi may pass through the last three stages. Although transforma- tions in the direction of the arrows are seen far more frequently than in the reverse direction, these variations are not entirely irreversible. Punkari and Henrici,-^ studying variations in the yeast Crypto- coccus pulchertimus, called attention to the similarities between the variations and those exhibited by bacteria as they change from smooth to rough forms. As the yeast develops into rudimentary mycelium, the colonies become rough and wrinkled. Negroni -* made a similar comparison in the case of Candida albicans. Since the basic mechanisms are unknown in both cases, no definite statement can be made, but one is justified in assuming that the common varia- tions in fungi, as indicated in the diagram above, are of the same general character as the variations commonly observed in colonies of bacteria and usually designated microbic dissociation. Sectors and Secondary Colonies. These variations have been described as appearmg rather gradually in the cultures, but it seems probable that what actually occurs is a rather sudden change in a cell here and there, and then a gradually developing dominance of these new types over the normal types as the strain is continued in further subcultures. Exactly similar transformations may be ob- served to occur rather suddenly in portions of a single culture. As with bacteria, such changes are best seen in colonies, especially giant colonies, i.e., a single large colony allowed to grow for a long time on a Petri plate or in a flask of agar. The occurrence of variation in these giant colonies is usually manifested by the occurrence of sectors, wedge-shaped areas differing in color or texture from the body of the colony. Such sectors represent the growth of the fungus from a cell which has undergone a transformation. As the colony spreads, the growth from this cell differs in appearance from that of other cells at the periphery of the colony. Sometimes little tufts of mycelium or little papillae of yeast cells will occur in an older part of the colony, which are different in character from the rest of the colony. These tufts or papillae correspond to the secondary colonies of bacteria. Spontaneous Variations. Little is known regarding the mechan- isms involved in these variations, but it seems likely that both in- ternal and external factors are involved, and that external agencies merely increase the rate of a change that tends to occur sponta- 38 VARIATIONS IN THE LOWER FUNGI neously. Variations are much more likely to occur in old laboratory strains than in recently isolated cultures. Although it is true that instability is more likely to appear in a strain which has been fre- quently transferred than in one which has been subcultured only at long intervals, it is also true that, once instability occurs, the degree of variation is increased if the culture is allowed to grow for a long time in a single culture, as in giant colonies. Thus staling of the medium and aging of the organism appear to be potent factors. Variants have been described more frequently in parasitic species than in saprophytic ones, but this may be due merely to the fact that mycologists have been more interested in pathogenic species. Induced Variations. Little work has been done on the deliberate production of variants in fungi by the action of external agents. Barnes - reported the development of variants of Thamnidium ele- gans, Botrytis cinerea, and Eurotium herbarium (resulting from the application of heat to the spores). Dickson,^° working with species of Chaetomium, and Nadson and Philippov -^ with Mucorales, ob- tained variants from the action of x-rays. Haenicke " obtained variants of Penicillium species by adding poisons to the medium. Emmons and Hollaender " reported very precise experiments upon the production of mutants of Trichophyton metagrophytes {T. gyp- seum) by the action of ultraviolet light. They found that the mutants observed were similar to those that occur spontaneously, and stated that the ultraviolet light accelerates the rate of mutation. They found that the percentage of mutants among the progeny of the surviving conidia increased with the duration of exposure up to a certain point (at which only about 8 per cent of the spores were still alive) ; with longer exposures and lower survivals, the propor- tion of mutants decreased. Negroni ^* produced rough type colonies of Candida albicans by the use of immune serum, a procedure which has been very fruitful in inducing the S ^ R transformation in bac- teria. Variations in Infection. There is some evidence that variations may occur in the tissues in fungus diseases. Thus Weidmann -^ re- ported a case of trichophytosis of the feet. Cultures were taken over a period of years. At first an organism downy in appearance, and identified as Trichophyton inter digitate, was obtained regularly in cultures. But after five years a fiat powdery type of colony, T. mentagrophytes, appeared. I have been greatly impressed by ob- servations which I have made on organisms isolated from two cases of moniliasis. In one case, an adult, a chronic pulmonary infection was followed by multiple abscesses through the body. From the VARIATIONS IN DIFFERENT CHARACTERS 39 sputum during life, and from the bronchi post-mortem, a typical Candida albicans was isolated. But from abscesses in the kidneys and other viscera, there was obtained a yeast which had the same fermentation reactions, but which failed completely to form my- celium under any conditions. The second case was a child which had suffered for some years from oral and intestinal moniliasis, with repeated attacks of moniliid. From the mouth and feces again C. albicans was isolated repeatedly. At autopsy caseous nodules were found in the peribronchial glands and in the lungs near the hilus. From these were obtained pure cultures of C. Knisei, non-virulent and non-fermentative. It seems very unreasonable to suppose that a simultaneous infection with two closely related species occurred in each of these three cases. It seems much more likely that in all of them the original fungus had undergone a variation within the in- fected tissues. Variations in Different Characters. The variations considered so far have concerned mainly the texture of the colony, which is de- termined largely by the morphology of the fungus. Variations have been observed in a variety of other characters, as pigmentation, virulence, and biochemical activities. It would require too much space adequately to review all the growing literature on variations in fungi. A few papers will be cited, from which further references may be obtained. Chodat ^ made extensive studies of variants of Aspergillus ochra- ceous and Phoma alternariaceum, the latter giving rise to five dis- tinct races. Both morphological and physiological characters varied. Some of the new races were permanent, others reverted. Dodge ^^ obtained an albino race of the red mold, Neurospora sitophila. Emmons ^- described variations in color and texture of the colonies of Microsporum gypseiim. Biltris ^ also described variations of a dermatophyte. Trichophyton mentagrophytes. Wiltshire ^^ found sectors in cultures of Alternaria which gave rise to conidia of the Stemphyllium type. Fabian and IMcCullough " described variations in yeasts, Mackinnon ^^ in Candida albicans. There have been some studies of variations in virulence and host-specificity, but these have been confined to the plant pathogens, especially to smuts and rusts. The intrinsic mechanisms which give rise to variations in fungi are undoubtedly very complex, and far from being fully known. It now seems probable that there occur all the mechanisms known to occur in higher organisms, involving on the one hand the variations caused by a loss or alteration or instability of genes, which are called muta- tions, and on the other hand those due to the combinations and 40 VARIATIONS IN THE LOWER FUNGI segregations of genes involved in sexual reproduction, which result in hybrids. Ever-sporting Races. Once a culture begins to throw variants, it usually becomes continuously unstable, an ever-sporting race. While the parent strain is thus continuously varying, the variants are usu- ally more stable, sometimes apparently permanent. Occasionally variants revert immediately to the parent type. But once a culture of a dermatophyte has become woolly, it is practically impossible to get it to change back to its typical form. Occurring frequently in vegetative mycelium, or in imperfect fungi where sexual reproduc- tion is absent, tending to occur constantly in one direction and often apparently irreversible, such variations seem to show the character- istics of gene mutations, and especially those attributed to unstable genes (see Demerec ^). Reversible Variations. But in many cases variants which seemed for a long time to be permanent have eventually reverted to the parent type. Thus some of the white variants of the red yeast Cryp- tococcus pulcherrimus described by Punkari and Henrici -^ appeared to be permanent but one of these kept by me turned red again after more than a year of continued subcultivation. Negroni -* obtained a partial reversion of the R variant of Candida albicans by growing it in a sterilized culture of the S type to which anti-R type serum was added. Burkholder ^ described a woolly change in the normally slimy fungus, Fusarium Martii var. Phaseoli. When inoculated on beans, the normal host plant, this variant reverted to the normal type. Mutant or Hybrid. The fact that the observed variations are so frequently reversible led Brierley ^ to doubt that they are true muta- tions. He believed that they result rather from a recombination of characters already present in the hereditary constitution of the or- ganism. Such a theory implies that in all cases there occurs some- thing of the nature of sexual reproduction, nuclear^ fusions and segre- gations. Since most of the fungi exhibit sexual reproduction, and many of them complex life cycles in which the various combinations and segregations are often obscure, this seems to be a reasonable theory. There are, however, certain data which indicate that variations may occur without any cell fusions. Thus Punkari and Henrici -^ emphasized the complete absence of spores in the yeast Cryptococ- cus pulcherrimus which they studied, since so far as was known sexual fusion in yeasts always results in the formation of spores. mTHAL FUSIONS 41 This may be questioned in view of the recent demonstration by Winge and Laustsen ^^ that many yeasts are normally diploid, and that sexual fusions actually occur in yeasts formerly believed to be parthenogenetic. ]\Iore convincing as examples of true mutation are the observations of Hanna/^ and of Stakman ^^ and Christensen ^ on the occurrence of mutations in cultures of Ustilago Zeae derived from single spores known to be haploid. Undoubtedly these „exam- ples could be multiplied. Genetics of Fungi. Within the past decade there has grown an extensive literature on the genetics of fungi. This work had its origin in the discovery of heterothallism by Blakeslee. Heterothallism makes it possible to cultivate the sexes of a fungus separately, each in the haploid state, which in turn makes it possible to study the effect of single genes, rather than the complicated condition of pairs of genes which occur in diploid cells. Unisexual cultures may then be crossed at will to produce new combinations. The discovery of heterothallism by Shear and Dodge ^^ in the pink mold Neurospora sitophila led Dodge ^^ to an extensive study of the genetics of this and related fungi. The discovery of an albinistic mutant of this species made possible a series of hybridization experiments. These studies, continued by Lindegren -^ and others, bid fair to make Neurospora as famous an organism as Drosophila in the history of genetics. Similar studies are being made' with other heterothallic fungi, notably the smuts and rusts. More recently "Winge and Laust- sen ^^ have extensively investigated the genetics of yeasts, using the micromanipulator for the isolation of spores from an ascus, and have conducted hybridization experiments with these microorganisms. It would take us too far afield to review this extensive and com- plicated literature, references to which will be found at the end of the chapter. Dodge has recently summarized it as follows: "We know now that new races of fungi are arising through natural hy- bridization. Hybrid structures have been obtained showing domi- nance and Mendelian segregations with crossing-over at reduction which is such an important feature in favoring evolution. We also find in the fungi mutants, lethal factors, deficient chromosomes, sex- chromosomes, sex-linked characters and other genetic features. . . . The fungi in their reproduction and inheritance follow exactly the same laws that govern these activities in higher plants and animals." Hyphal Fusions. When heterothallic fungi conjugate, hyphae de- rived from two spores of different sex fuse. This hyphal fusion re- sults, in the Basidiomycetes. in a new type of mycelium, binucleate. 42 VARIATIONS IN THE LOWER FUNGI with two haploid nuclei in each cell. Such a dikaryophyte presents special problems in genetics peculiar to the fungi. Eventually in some of these binucleate cells the nuclei fuse, and the resulting diploid nucleus divides to give rise to the haploid basidiospores or sporidia. Hyphal fusions are not, however, confined to the Basidiomycetes. They have been observed to occur in a variety of fungi. Vegetative anastomoses occur between neighboring filaments of a single plant, and between neighboring colonies of imperfect fungi. Such fusions do not result in sexual spores. Laibach =^° and Kohler ^^ have called attention to the morphologic and physiologic similarity between these vegetative anastomoses and hyphal fusions in the smuts. The genetic significance of such hyphal fusions is not yet clear. Hansen and Smith ^^ noted the occurrence of hyphal fusions between different single-spore races of Botrytis cinerea, and believed that they resulted in mycelia containing nuclei from both of the parent strains. In this species, as in numerous other imperfect fungi, the conidia as well as the hyphal cells are multinucleate and these authors state that "a multinuclear spore is, therefore, not an individual but, in reality, a colony, and it can, therefore, not give rise to a genetically pure culture unless all of its nuclei are genetically identical." They observed variants in the offspring of cultures derived from hyphal fusions occurring between two different races, and suggested that such strains owe their instability not to mutation but to nuclear heterogeneity. In later work they "ctossed" two species, B. Allii and B. Ricini, and obtained three types which they considered to be new varieties or new species. Davidson, Dowding, and Buller ^ ob- served the occurrence of hyphal fusions in several species of dermato- phytes {Microsporum Audouini, M. Canis, Trichophyton mentagro- phytes). They found that fusions occurred readily between differ- ent strains of the same species, but not between different species, and suggested this as a criterion for species identification. They did not observe variants, and Emmons also failed to observe any evidence of hybridization when different variants of M. gypseum were seeded together. It should be noted here that Spring-^ failed to find any . evidence of heterothallism in dermatophytes. Practical Considerations. What does all this signify to the prac- ticing bacteriologist? It need not disturb his routine work very much. It is not intended to give the impression that all fungi will transform into new types on continued cultivation. This is a tend- ency which has been observed in 'many species of the lower fungi studied by medical and industrial mycologists. But, as with the TAXONOMY 43 bacteria, many species, and some strains of nearly all species, will "stay put" almost indefinitely in artificial cultures. Strains recently isolated from their natural habitat are likely to be very stable, and if old laboratory strains ''go pleomorphic," they can usually be read- ily replaced. Spontaneous variations are of course of importance in explaining the occurrence of new races or physiological types of pathogenic fungi, especially in the plant parasites, and the possibility of developing new industrial yeasts and molds by induced mutations or by hybridization is an attractive field for investigation. But the phenomena of variation in fungi are probably most important from the standpoint of classification and nomenclature. Some of the variants which have been observed differ so much from the parent culture that one might be justified in calling them new species. And some of the variants, particularly those described by Emmons on hi? studies of dermatophytes, are apparently identical with species al- ready known in nature. Taxonomy. Throughout nature individuals exist in infinite vari- ety, and when we group them into species we draw artificial lines which do not actually exist. This has led, throughout the history of biology, to a conflict between the "splitters" who would make a new species of every new individual, and the "lumpers" who recognize only the grossest of differences. "The 'lumper' is the horror of the 'splitter,' the 'splitter' is anathema to the 'lumper'; both are the source of genuine grief and much hardship to conscientious men, who are possessors of normally constituted minds and truly scientific habits." t Conscientious men with normal minds and scientific habits will recognize the modal types about which.individuals fluctuate, and designate these as species. They will recognize the normal limits of variation within these species. Unfortunately in the study of the lower fungi we have suffered from a plethora of splitters and a dearth of lumpers. This is due, in part, to the failure of most workers to take into account the frequent occurrence of slight and often transient variations. Thus we have in the literature of medical mycology probably two or three hundred species names and combinations wdiich differ mainly in slight varia- tions in form, color, or texture of the colonies — characters which are almost never constant. Similar conditions obtain in many other genera of the lower fungi. As our investigations of variation con- tinue, many of these species will necessarily be reduced to synonymy. t W. J. Holland in The Moth Book. 44 VARIATIONS IN THE LOWER, FUNGI Fig 26. Spontaneous variation in a dermatophyte, Trichophyton mentagro- phytes: 1, an original strain; 2, 3, and 4, variants derived from it; 5, another strain; 6, variant derived from it. From C. W. Emmons, Arch. Dermatol. Syphilol. {Chicago), 25, 987 (1932). TAXONOMY 45 Fig. 27. Spontaneous variations in a j^east, Cryptococcus pulcherrimus. Giant colonies derived from a single cell. The original strain was smooth and red. Rough and white variants were obtained. Sectors and secondary colonies may- be seen. From L. Punkari and A. T. Henrici, J. Bad., 26, 125 (1933). 46 VARIATIONS IN THE LOWER FUNGI Fig. 28. H\'brid fungi. Giant colonies from eight different sporidia of Ustilago Zeae obtained from smut spores resulting from a crossing of two strains of corn smut parasites. From J. J. Christensen, Minn. Agr. Exp. Sta. (St. Paul), Tech. Bull. 65 (1929). LITERATURE 1. Alexander, A., Ueber die faviforme Degeneration resp. Unwandlung unserer Dermatophyten, Dermatol. Z., 56, 225 (1929). 2. Barnes, B., Variations in Eurotium herbariorum (Wigg.) Link, induced by the action of high temperatures, Ann. Botany, 42, 783 (1928); Variations in Botrytis cinerea induced by the action of high temperatures, Ann. Botany, 44, 825 (1930); Induced variation. Trans. Brit. Mycol. Soc, 20, 17 (1935). 3. BiLTRis, R., Sur la variabilite des caracteres de I'espece chez les derma- tophytes, Ann. inst. Pasteur, 43, 281 (1929). 4. Brierley, W. B., Variation in fungi and bacteria, Proc. Intern. Congr. Plant Sci., Ithaca, 2, 1629 (1929). 5. BuRKHOLDER, W. H., Variations in a member of the genus Fusarium grown in culture for a period of five years. Am. J. Botany, 12, 245 (1925). 6. Chodat, F., Recherches experimentales sur la mutation chez les champi- gnons. Bull. soc. hot. Geneve, Ser. 2, 18, 41 (1926). 7. Christensen, J. J., Mutation and hybridization in Ustilago zeae, Minn. Agr. Exp. Sta. Tech. Bull. 65 (1929) ; Studies on the genetics of Ustilago zeae, Phytopath. Z., 4, 129 (1931). 8. Davidson, A. M., E. S. Dowding, and A. H. R. Buller, Hyphal fusions in dermatophytes. Can. J. Research, 6, 1 (1932). 9. Demerec, M., Unstable genes, Botan. Rev. 1, 233 (1935). 10. Dickson, H., The effects of x-rays, ultraviolet light and heat in producing saltants in Chaetomium cochliodes and other fungi, Ann. Botany, 46, 389 (1932); Saltation induced by x-rays in seven species of Chaetomium, Aim. Botany, 47, 735 (1933). LITERATURE 47 11. Dodge, B. O., Nuclear phenomena associated with heterothalHsm and homo- thallism in the Ascomycete Neurospora, /. Agr. Research, 35, 289 (1927); Unisexual conidia from bisexual myeelia, Mycologia, 20, 226 (1928); Breeding albinistic strains of the Monilia bread mold, Mycologia, 22, 9, (1930); Reproduction and inheritance in Ascomycetes, Science, 83, 169 (1936); Some problems in the genetics of the fungi, Science, 90, 379 (1939). 12. Emmons, C. W., Pleomorphism and variation in the dermatophytes, Arch. Dermatol. Syphilol. (Chicago), 25, 987 (1932). 13. Emmons, C. W., and A. Hollaender, The influence of monochromatic ultraviolet radiation on the rate of variant production in Trichophyton mentagrophytes. Genetics, 24, 70 (1939) ; The action of ultraviolet radia- tion on dermatophytes, II. Mutations induced in cultures of dermato- phytes by exposure of spores to monochromatic ultraviolet radiation, Am. J. Botany, 26, 467 (1939). 14. Fabian, F. W., and N. B. McCullough, Dissociation in yeasts, J. Bad., 27, 583 (1934). 15. Grigoraki, L., Recherches cytologiques et taxonomiques sur les dermato- phytes, Ann. sci. nat. Botan., 7, 165 (1925). 16. Haenicke, a., Vererbungsphysiologische Untersuchungen an Arten von Penicillium und Aspergillus, Z. Botan., 8, 225 (1916). 17. Hanna, W. F., Studies in the physiology and cytology of Ustilago zeae and Sorosporium reilianum, Phytopathology, 19, 415 (1929). 18. Hansen, H. N., and R. E. Smith, The mechanism of variation in imperfect fungi: Botrytis cinerea. Phytopathology, 22, 953 (1932); The origin of new types of imperfect fimgi from interspecific co-cultures, Zcntr. Bakt., Parisitenk., II, 92, 272 (1935). 19. Kohlee, E., Beitrage zur Kenntnis der vegetativen Anastomosen der Pilze, Planta, 8, 140 (1929). 20. Laibach, F., tJber Zellfusionen bei Pilzen, Planta, 5, 340 (1928). 21. Lindegren, C. C, The genetics of Neurospora, I. The inheritance of re- sponse to heat-treatment, Bull. Torrey Botan. Club, 59, 85 (1932); II. Segregation of the sex factors in the asci of A'', crassa, N. sitophila and N. tetrasperma, Bull. Torrey Botan. Club, 59, 119 (1932) ; III. Pure bred stocks and crossing-over in N. ci'assa. Bull. Torrey Botan. Club, 60, 133 (1933); IV. The inheritance of tan vs. normal, Am. J. Botany, 21, 55 (1934); V. Self-sterile bisexual heterokaryons, J. Genetics, 28, 425 (1934); VI. Bisexual and akaryotic ascospores from A'', crassa. Genetics, 16, 315 (1934); VII. Developmental competition between different genotj'pes within the ascus, Z. Indukt. Abstamm.-u. Vererbungsh., 68, 331 (1935). 22. MACKINNON, J. E., Nuevo sentido de variacion eu Mycotorula albicans, Arch. soc. biol. Montevideo, 7, 162 (1936). 23. N.^DSON, G., and G. Philippov, Influence des rayons x sur la sexualite et la formation des mutantes chez les champignons linferieurs (Mucorinees), Compt. rend. soc. biol., 93, 473 (1925); De la formation de nouvelles races stables chez les champignons inferieurs sous I'influence des rayons X, Compt. rend., Acad. Sci. (Paris), 186, 1566 (1928). 24. Negroni, P., Variacion hacia el tipo R. de Mycotorula albicans, Rev. soc. argentina biol., 11, 449 (1935); Ensayos para obtenir la reversion de la 48 VARIATIONS IN THE LOWER FUNGI forma R a la S de Mycotorula albicans, Rev. inst. bacterioL, dep. nacl. hig. {Buenos Aires), 7, 393 (1936). 25. PuNKARi, L., and A. T. Henrici, A study of variations in a chromogenic asporogenous yeast, J. Bact., 26, 125 (1933) ; Further studies in sponta- neous variation of Tornla pulcherrima, J. Bact., 29, 259 (1935). 26. Shear, C. L., and B. 0. Dodge, Life histories and heterothallism of the red bread mold fungi of the Monilia sitophila group, J. Agr. Research, 34, 1019 (1927). 27. Spring, D., Heterothallism among the dermatophytes, Arch. Dermatol. Syphilol. {Chicago), 24, 22 (1931). 28. Stakman, E. C, and J. J. Christensen, Heterothallism in Ustilago zeae, Phytopathology, 17, 827 (1927). 29. Weidmann, F. D., Morphologic variations in a ringworm species of the toes. Arch. Dermatol, and Syphilol. {Chicago), 13, 374 (1926). 30. Wiltshire, S. P., A Stemphyllium saltant of an Alternaria, Ann. Botany, 43, 653 (1929). 31. WiNGE, O., and O. Laustsen, On two types of spore germination and on genetic segregations in Saccharomyces demonstrated through single spore cultures. Compt. rend. trav. lab. Carlsberg, Sir. physiol., 22, 99 (1937); Artificial species-hybridization in yeast, Compt. rend. trav. lab. Carlsberg, Ser. physiol., 22, 235 (1938); 22, 257 (1939). CHAPTER III METHODS FOR STUDYING MOLDS, YEASTS, AND ACTINOMYCETES Culture Media. Molds may be cultivated on either solid or liquid media in tubes or dishes, as bacteria are. But molds generally grow more slowly than bacteria and, where both are present in the ma- terial to be examined, the latter are apt to overgrow Petri plate cul- tures before the former can develop. For isolation it is therefore desirable to use a medium favorable to the growth of molds and un- favorable for bacteria. Such a medium may be obtained by adding to ordinary nutrient agar a rather large amount of sugar, and by making the medium rather strongly acid, since most molds will tolerate higher degrees of acidity than bacteria will. A medium con- taining 5 per cent glucose and 0.5 per cent tartaric acid in addition to the usual meat extract and peptone is very satisfactory for some fungi but is not suitable for most pathogens of man. If agar is autoclaved in such an acid medium, however, it will be markedly hydrolyzed and will fail to jell on cooling. This difficulty is obviated by sterilizing the glucose and acid separately in concen- trated solution. A solution containing 50 per cent glucose and 5 per cent tartaric acid may be safely autoclaved. Such a sterile solu- tion may be kept on hand and, when need for a medium for isolating molds arises, it can be quickly met by melting a deep tube (about 10 ml.) of ordinary agar (beef extract 0.3 per cent, peptone 1 per cent, pH 7.4) and adding to it 1 ml. of the glucose-tartaric acid solu- tion. This gives a final concentration of nearly 5 per cent glucose and 0.5 per cent tartaric acid and a reaction of about pH 3.8. Less of the glucose-tartaric solution may be used for less acid media. If an agar medium with reduced buffer content and lower pH is used (e.g., A.P.H.A.) less of the glucose-tartaric solution will be used. On these media most molds and yeasts will grow luxuriantly, most bacteria not at all. The growth on the above media is for some purposes, as in count- ing molds in soil, for instance, almost too luxuriant, since some of the rapid growers tend to spread over the plate and crowd out others. Media less rich in nutrients may be preferred for counting molds by 49 50 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES the dilution plate method in soil or other habitats with a large fungus flora. Such a medium has been devised by Waksman *^ especially for soil work, but will prove of value for isolations from other material. It consists of Glucose 10.0 grams Peptone 5.0 grams Monopotassium phosphate 1.0 gram Magnesium sulphate (crystals) 0.5 gram Agar 15.0 grams Water 1 liter Just before use, i.e., after sterilizing and while melted, sulphuric acid is added until the reaction is pH 3.8 to 4.0. About 0.5 to 0.6 ml. of normal acid is sufficient for 100 ml. This medium has less sugar than the preceding. The use of a mineral acid eliminates the danger of the acid being destroyed by the growth of the mold, as might occur with tartaric acid, thus eventually allowing the bacteria to develop. Hydrogen-ion Concentration. Although these acid media serve well for the isolation of most of the common molds and yeasts, they will not permit a growth of all fungi. Many of the pathogenic species in particular may fail to grow. The pH limits of growth have been determined for only a few species of molds and yeasts. Talice *^ published data for a number of fungi pathogenic for animals and for some common saprophytes, when grown on three different agar nu- trient media. Very little difference was found with the different media. A few examples will show the ranges. The organism of North American blastomycosis grew between pH 5 and pH 8, the optimum at pH 7; Sprotrichum Beurmdnni from pH 3.0 to pH 9.6, the optimum at pH 5.0; Microsporum Audouini from pH 5 to pH 7, the optimum at pH 6. Saprophytic species showed a wider range. Rhizopus nigri- cans, for example, grew from pH 2.2 to pH 9.6; Penicillium citrinum showed a similar range, as did also Oospora verticilloides. In many cases fungi which tolerated a wide range of hydrogen-ion concentra- tions failed to show a distinct optimum, the top of the curve form- ing a broad plateau rather than a peak. The parasitic species, Candida albicans, resembled the saprophytic species, growing at all levels from pH 2.2 to pH 9.6, the optimum being pH 7. Some molds may grow in media much more acid than pH 2.2. Starkey and Waksman ^'^ have grown certain molds in poorly buffered media of a normality of as much as 2 and 2.5! These observations have been confirmed. Mr. Owen Sletten (unpublished data) has isolated several molds growing in 2A^ sulphuric acid reagent, pre- sumably deriving their energy from traces of organic materials. NON-REPRODUCIBLE MEDIA 51 These fungi were found to develop in normal and in some cases 2 A^ and 2.5 A'" sulphuric acid to which 1 gram of peptone and 1 gram of glucose per liter were added. Kadisch ^^ studied the influence of the hydrogen-ion concentration upon the growth of various dermatophytes, in the range from pH 6.7 to pH 7.9, and found that all of them grew better in slightly alkaline media. The optimum for most species was pH 7.4. Von Mallinckrodt-Haupt -^ observed that two species of dermatophytes when grown on media nearly neutral in reaction made the media progressively more alkaline (up to pH 8), whereas a Penicillium and a pink yeast produced acid. The alkali-preference of the ringworm fungi was offered by Levin and Silvers ^^ as an explanation for the localization of these fungi in the interdigital spaces and the axillae, where the pR of the skin secretion is higher than on other parts of the body surface. Nearly all actinomycetes prefer an alkaline medium. Soil actino- mycetes grew in the range pH 5 to 9, with the optimum at pH 7 to 8. Jensen, however, found strains in acid peat soils which grew only in the range pH 2.5 to 5.8 with the optimum at pH 3 to 4. See page 362. Types of Media. Culture media used in the study of molds may be divided into three groups: 1. Non-reproducible media, such as pieces or infusions of fruits, vegetables, skin, or hair, which may be valuable for the cultivation of certain species, but which are not reproducible because the sub- strates naturally vary in composition. 2. Reproducible media of unknown composition. In this group would fall the well-known Sabouraud's medium and its various sub- stitutes. These are media containing complex substances such as peptone whose precise chemical constitution is unknown, but which are manufactured by standardized procedures so that the product from one manufacturer is reasonably constant over a period of time, and the same material is available to all laboratories. 3. Synthetic media, i.e., media prepared from pure chemicals of known constitution, which can be reproduced precisely in laboratories throughout the world independently of any manufacturer. Non-reproducible Media. Media of this class are seldom used ex- cept in infusions or for particular purposes, as the cultivation of species of fungi which will not grow upon the other types of media, or which will produce characteristic fruiting bodies only upon a cer- tain substrate. 52 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES Potato plugs may be prepared as for the growth of bacteria, but they are not very useful. Carrot plugs, prepared in the same way, are especially useful in studying spore formation in yeasts. Mois- tened slices of bread are sometimes used, especially in the study of Mucorales. Langeron and Milochevitch " proposed the use of mois- tened grains of barley, w^heat, and rye for the cultivation of derma- tophytes. Conant - found polished rice to be a more favorable me- dium, especially for the development of macroconidia in the genus Microsporum. One part of rice is added to three parts of water in flasks and sterilized in flowing steam on two successive days. The restriction of the dermatophytoses to the skin, nails, and hair has led to a widespread belief that the skin and its appendages con- tain specific nutrients favorable to the dermatophytes. Hair, feathers, leather, and horn have been used for the cultivation of these fungi. Williams*^ has especially studied the growth of dermato- phytes upon hair, using only hair and distilled water as a medium. The various dermatophytes grew, but more slowly and scantily than on Sabouraud's medium. Emmons " grow dermatophytes upon thin shavings of horn in order to study the development of spores. David- son, Gregory, and Birt '^ similarly observed the development of spores of dermatophytes from the continued growth of fungi on the infected hairs after removal from the patient. Memmesheimer ^^ has de- scribed a medium prepared from skin and hair for the cultivation of dermatophytes. The whole skin of guinea pigs is used. To pelts 75 to 80 grams in weight is added 20 ml. of 30 per cent potassium hydroxide and 75 ml. of tap water. This is boiled for 30 to 45 min- utes, almost completely dissolving the skin. The solution is neutral- ized wdth hydrochloric acid and filtered, tap water is added to make 1 liter, and 40 grams of maltose and 18 grams of agar are added. This medium is said to give a more rapid growth of dermatophytes than the usual Sabouraud agar, and also to yield a higher percentage of positive cultures from cases of dermatophytosis. The addition of serum or ascitic fluid to a medium stimulates and improves the growth of some strains of Actinomyces bovis. Other strains grow as well without this addition and it is not necessary for the isolation of most fungi. New or recently isolated strains of Blastomyces and Histoplasma when grown on blood agar at 37° C. grow as budding cells similar to those seen in tissue, but these fungi are probably more easily isolated on Sabouraud's agar medium. It is usually preferable to prepare media from natural materials by making an aqueous solution of the nutritive constituents by in- fusion or decoction. Such liquid media may then be solidified by REPRODUCIBLE MEDIA 53 the addition of agar. A medium widely used by plant pathologists is potato-glucose agar: Peeled sliced potatoes 300 grams Glucose 10 grams Agar 15 grams Water 1 liter The medium is boiled for 20 minutes and strained through cotton. The reaction, slightly acid, is not adjusted. Corn meal agar is also widely used. It is especially valuable in observing the development of chlamydospores and for demonstra- tion of mycelium in Candida albicans. Light-colored corn meal is preferable. If one uses dehydrated media he should make certain that no glucose has been used in the formula. Corn (maize) meal 40 grams Water 1 liter This is heated to 60° C. for 1 hour and stirred frequently. It is filtered through paper and made up to 1 liter; then 20 grams of agar is added. The reaction is not adjusted. If it is sterilized by the fractional method, as is sometimes recommended, trouble may be expected from resistant spores of bacteria in this non-acid medium. Brewery bacteriologists use beer wort and wort agar for the culti- vation of their yeasts, and winery bacteriologists use grape juice or must. Industrial yeasts are said to maintain their special fermenta- tion characteristics better on these natural substrates than on more artificial media. Malt-extract agar may be substituted for beer wort. These media naturally have reactions about pH 4.5 and, if auto- claved, the agar may be softened. Diluted honey has been used for growing both yeast and molds, but it is variable in composition and, save for the special study of honey and nectar yeasts, not very useful. Manure infusion agar has been widely used for the cultivation of various coprophilic fungi. The following method is presented by Gwynne-Vaughan and Barnes.^"^ About 1000 grams of horse, cow, or rabbit dung is soaked in cold water for 3 days ; the liquid is poured off and diluted until it has the color of straw; 2.5 grams of agar is added for every 100 ml. of the diluted fluid. Sterilized soil has been used successfully by Greene and Fred ^^ for maintaining cultures of certain molds with a minimum of physio- logical or morphological degeneration. Reproducible Media. The standard medium used by medical my- cologists for the cultivation of pathogenic fungi is Sabouraud's agar. As originally described, this consisted of 4 per cent of the crude mal- 64 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES tose of Chanut and 1 per cent of the granulated peptone of Chassaing. These were obtainable for many years from the Maison Cogit, 36 Boulevard St. Michel in Paris. Although the ingredients were of un- known composition, they were constant and gave reproducible re- sults. The difficulty in obtaining these ingredients in other parts of the world has led to the development of substitute media which are often also called Sabouraud's agar. Attempts to reproduce the cul- tural characters of dermatophytes as described by Sabouraud on these substitute media indicated that the peptone rather than the sugar was the essential ingredient. For ordinary work a medium containing 2 per cent glucose and 1 per cent Neopeptone (Difco) is a satisfactory substitute for Sabouraud's agar. It may be adjusted to pH 5.6 and is commonly called Sabouraud agar or Sabouraud glucose agar. Pure glucose is more satisfactory than maltose for most fungi; it is more constant in composition, cheaper, and not so likely to change in sterilization. Usually a somewhat better growth is obtained if tap water is used instead of distilled water. There are very few molds, yeasts, or actinomycetes which will not grow abundantly on this medium. It is widely used for saprophytic and industrial fimgi as well as for medical work. The original formula for Sabouraud's agar presented in the pre- ceding paragraph is that of Sabouraud's "proof" agar, originally in- tended primarily for the isolation and identification of dermato- phytes. Species of these fungi have been based largely upon the color and texture of the colonies, which characters may vary mark- edly with slight variations in the medium. In addition to his proof agar, Sabouraud used a conservation agar for the continued cultiva- tion of strains in a collection. He believed that medium was less likely to give rise to pleomorphism in dermatophytes than the proof agar. The conservation medium contained no sugar, only 3 per cent of peptone. Some fungi are more difficult to transfer from this me- dium because they produce fewer aerial hyphae and conidia. The dermatophytes can often be kept safely on the glucose agar if they are placed in the refrigerator (2° to 5° C.) immediately after reaching full development (10 to 15 days) and stored there until the next transfer. The impression that the pathogenic fungi are fastidious in their nutritional requirements and will grow only on Sabouraud's medium is erroneous. Most of them grow well on a variety of media and, on the other hand, Sabouraud's medium is unsuitable for some patho- gens such as Actinomyces bovis. In addition to being a good, easily prepared medium, it is very useful in the identification of dermato- SYNTHETIC MEDIA 55 phytes because it permits comparison with the excellent photographs Sabouraud used to illustrate his taxonomic studies. The ingredients used by Sabouraud are no longer generally available and so many substitute formulas have been proposed that the name Sabouraud 's medium as commonly used has no precise meaning. The substitu- tion of 1 per cent Neopeptone (Difco) and 2 per cent c.p. glucose with 2 per cent agar and adjustment of pH 5.6 gives a medium which produces very nearly the same type of colony as the original formula and makes use of materials readily available and reasonably con- stant in composition. This medium is referred to in this book as Sabouraud or American Sabouraud agar. For most purposes the pH of the medium need not be adjusted. It would of course be highly desirable to obtain a medium of known chemical composition which could be produced in constant form independently of commercial preparations. Unfortunately no medium yet devised will yield a characteristic growth of all of the fungi, especially not of the dermatophytes. Williams ^® found that asparagine was not a satisfactory substitute for peptone. Synthetic Media. Synthetic media are most widely used in bio- chemical studies, since a known substrate is essential in order to study the products of metabolism. With a basic medium of precisely known concentration, one may vary the constituents one by one and so determine the influence of each upon growth, enzyme action, and the like. This aspect of the study of fungi has been extensively re- viewed by Steinberg.^^ The first noteworthy studies of this sort were made by Raulin who developed a formula for the growth of Aspergillus niger containing eleven ingredients, all of known chem- ical composition. Such a medium seems unnecessarily complicated, but has served as the starting point for numerous studies, especially of the effects of small quantities of the metals. Raulin's medium is strongly acid ipH 2.9), and for many species the formula will need modification to provide a more favorable reaction. Synthetic media are further used, where possible, as a standard substrate upon which to observe colony form and especially pigment production. Such a medium which has been widely used is Czapek's medium as modified by Dox and by Thom: Sucrose 30.0 grams Sodium nitrate 2.0 grams Dipotassium phosphate 1.0 gram Magnesium sulphate (crystals) 0.5 gram Potassium chloride 0.5 gram Ferrous sulphate 0.01 gram Water 1 liter 56 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES This medium has been used by Thom for descriptions of species of Penicilhum and Aspergillus, and by Waksman for descriptions of soil actinomycetes. In both cases color of the growth is of great importance in identification. Many fungi cannot, however, readily use sucrose, others cannot utilize nitrates. This medium cannot therefore be used for all fungi. An alternative medium is that of Barnes: Tripotassium phosphate 10 grams Ammonium nitrate 10 grams Potassium nitrate 10 grams Glucose 10 grams Water 1 liter All these synthetic media may, of course, be solidified by the addi- tion of agar unless they are so acid as to hydrolyze agar. Many if not most fungi require minute amounts of other elements, e.g., zinc, but unless purely synthetic media prepared from specially purified chemicals in special glassware are used, sufficient traces of these elements will usually be provided by the ingredients used, the water, or the glassware. Also some fungi require small amounts of growth-promoting substances and in purely synthetic media these may need to be provided for. Dye Media. Various workers have experimented with the addi- tion of dyes to culture media used for the identification of fungi, either to stain the growing mycelium differentially or to serve as pH indicators. Williams ** grew a variety of fungi, mostly pathogenic species, on a 4 per cent peptone, 1 per cent glucose agar to which was added nigrosin, litmus, eosin Y, eosin B, fluorescein, methylene blue and eosin, Wright's stain, neutral red, and Janus green. Von Mal- linckrodt-Haupt ^* grew various dermatophytes on media containing thymol blue, bromophenol blue, bromocresol purple, bromothymol blue, phenol red, and cresol red. Although the various cultures showed differing degrees of dye absorption by the growing fungi, or color changes in the media, no useful differential media have de- veloped from the studies. Negroni and Loizaga ^- grew Candida albicans in a beer wort to which were added basic fuchsin, methylene blue, gentian violet, malachite green, and methyl green in minute quantities (1:50,000-1:10,000). These basic dyes, especially methyl green, induced the production of rough variants. Quantity Production of Mold Mycelium. In many types of re- search, as for obtaining antigens, enzymes, antibiotics, or other in- tracellular products or for large amounts of growth for chemical analyses or animal feeding experiments, it is desirable to obtain a ISOLATION OF PATHOGENIC FUNGI 57 large amount of mycelium. Often oxygen is the limiting factor for growth and the greatest yield is obtained if the molds are grown in a thin layer (about 1 cm. deep) of liquid medium. Flasks take too much incubator space. We have in our laboratories used 1-liter, flat- sided, narrow-mouthed prescription bottles which contain about 100 ml. each of medium. After inoculation, these are placed on their sides and incubated so that the liquid is spread in a broad thin layer. Narrow-mouthed bottles are essential to prevent air contamination. When sufficient growth has occurred, the mycelium may be removed by pulling the mat through the mouth of the tube with a hooked wire, and the excess medium may be removed by straining through gauze or sieve. In our laboratories large amounts of growth for ex- tracting antigens and also for feeding to rats on diets have thus been obtained. In industry special apparatus is used for mass production. Media for Biochemical Studies. In general, one studies the enzyme actions of molds in the same manner as with bacteria, and such media as gelatin, litmus milk, coagulated egg, or blood serum are useful. Sugar fermentations are not important in the identification of molds or actinomycetes, but they are of great importance in identifying yeasts. INIethods for studying sugar fermentations by yeasts will be discussed later. It is frequently desirable to determine the ability of molds and actinomycetes to hydrolyze starch and cellulose. The former may be determined by growing the organism in Petri dishes on agar in which starch has been incorporated. After growth has taken place the agar is flooded with diluted Lugol solution, which will stain the starch blue but will leave a clear zone about the mold if it has a diastatic action. The action on cellulose may be observed by growing the organism in a medium containing various nutrient salts, an inorganic source of nitrogen, but no source of carbon save strips of filter paper. If the mold can digest cellulose, the paper will be softened and eventually dissolved. Isolation of Pathogenic Fungi. In making cultures from derma- tophytosis of the scalp the infected short hair stubs can be collected by means of forceps and placed directly on the surface of Sabouraud's agar slants or, better, placed first in empty sterile Petri dishes for sorting and selection of suitable portions. If long hairs are taken the infected basal portion should be cut off with sterile scissors or scalpel and only this part planted. Two to four plants can be made on one agar slant. In making cultures from dermatophytosis of the skin the lesion should be first cleaned wdth a gauze sponge and 70 per cent alcohol. Care should be taken to avoid lesions recently treated with a fungi- 58 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES cide and to collect material from the active border of the lesion. Scales or the roofs of vesicles can be removed with sterile scalpel or scissors. They can be placed directly upon the surface of agar slants but, especially in dermatophytosis of the foot, isolation of a pure culture is easier if the specimens are placed first in a sterile Petri dish, cut in small pieces with sterile scalpel, and flooded with 70 per cent alcohol. After exposure for 2 minutes, transferring the pieces to agar slants is begun and the procedure is timed so that the last specimens planted have been in the alcohol about 8 minutes. The 70 per cent alcohol, being more bactericidal than fungicidal, exerts a selective action and permits the isolation of a pure culture of the fungus without bacterial contamination. In the isolation of dermatophytes when Staphylococcus and other bacteria may be present on the specimen, the latter should be laid carefully upon the unbroken agar surface at the point where it first touches. The fungus hyphae need no encouragement or aid to pene- trate the agar. Rubbing the specimen across the surface of the agar and breaking the surface of the agar both increase the area over which contaminating bacteria will be deposited and if these bacteria are alive their growth will interfere with the development as well as the isolation of the fungus. If bacteria or saprophytic fungi grow in the primary culture with the pathogen, the latter can be isolated by transferring with a sharp stiff needle some of the conidia or aerial hyphae after they grow beyond the contaminants. Most of the fungi pathogenic for man can be isolated in culture by the simple method of streaking or spreading pus, sputum, blood, macerated tissue, or other pathological material on the surface of Sabouraud agar slants. Usually no preliminary digestion on con- centration is desirable or necessary. A few pathogens require special treatment and these exceptions will be briefly noted here. Actinomyces bovis will not grow on Sabouraud's agar; pus containing this fungus should be planted on veal infusion agar containing 1 per cent glucose. Brewer's thioglycollate glucose medium, or blood agar and incubated anaerobically. Pityrosporum ovale will grow on Sabouraud's agar or similar medium only if it is first covered with a thin layer of lanolin or other fat. A few of the pathogens will grow poorly on any media yet devised and are therefore very difficult to isolate in culture. The separation of pathogenic molds from bacteria is often a diffi- cult problem. In many cases bacteria are much more numerous than the fungi. Here success in isolation is in proportion to the number of cultures made. If a dozen or more slants are inoculated, one has a much better chance of obtaining a pure culture than if he merely ISOLATION OF SAPROPHYTIC ACTINOMYCETES 59 streaks the pus or sputum over a single slant. The use of pour plates may increase the chances of isolating the fungus but is an unwise procedure with such pathogens as Nocardia asteroides and Coccidio- ides immitis in which there is grave danger of inhaling spores from an uncovered Petri dish culture. Often fungi appear in a culture overcrowded with colonies of bac- teria. In such cases the tuft of aerial mycelium may be free of bacteria, or may contain only a few, so that if one carefully touches the very surface of the mold colony he may obtain a relatively or absolutely pure culture. If the fungus produces few or no conidia it may be necessary to postpone subculturing until the fungus has grown well beyond the bacteria or up on the side of the culture tube, when a portion of the mycehum can be removed with a stiff, sharp needle. Isolation of Saprophytic Actinomycetes. In studying actinomy- cetes it must be borne in mind that most of them cannot tolerate an acid medium. The acid-sugar media recommended for yeasts and molds cannot therefore be used. They will for the most part grow on the media used for bacteria, and their growth is generally stim- ulated by the addition of sugar. In the isolation of these organisms the greatest difficulty is experi- enced in separating them from bacteria, since the latter for the most part grow more rapidly and, if they form spreading colonies, will overgrow the plates and suppress the actinomycetes. But most of the actinomycetes can grow with very small amounts of nutrient, and one may use a very weak medium on which many bacteria will not grow. Those that do will not spread much. For general isolation of saprophytic species of actinomycetes, per- haps Czapek's solution agar has been most widely used. On it many bacteria will not grow and those that do are not spreaders. Some difficulty is experienced with spreading molds on this medium. Conn's ^ glycerin asparaginate medium is excellent for isolating non-pathogenic actinomycetes. Glycerol 10 ml. Sodium asparaginate (or asparagine neutralized with NaOH) 1 gram Dipotassium phosphate 1 gram Agar 15 grams Tap water 1 liter Soil extract agar is a "natural" medium which may be used for the isolation of soil actinomycetes. It is made by heating 1000 grams of rich garden soil with 1 liter of water in the autoclave for half an 60 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES hour. The Hquid is decanted and filtered until fairly clear. To 100 ml, of this aqueous soil extract are added 900 ml. of water, 1 gram of glucose, 0.5 gram of dipotassium phosphate, and 15 grams of agar. Isolation of Pathogenic Actinomycetes. Although such weak media serve for the isolation of saprophytic actinomycetes, they are not suitable for the pathogenic species. The strictly anaerobic Actinomyces bovis may be isolated in deep agar shake cultures, 1 per cent of glucose in veal infusion agar being used ; or the method men- tioned previously may be used. Various authorities have recom- mended the addition of blood serum or ascitic fluid, but this has not been found to be advantageous for all strains. Brewer's thioglycol- late medium with glucose is also suitable for A. bovis. The aerobic species may be isolated readily on the same solid media as are used for A. bovis. Single-cell Isolation. Although for most purposes isolation from a mixed culture by plating is readily accomplished, occasions may arise when other procedures are preferable. Where several molds are closely crowded together on a plate, it is sometimes quite feasible to pick off a spore head of a desired species with a sterilized wire under the higher-powered lenses of a binocular dissecting microscope. For certain types of work, as investigations of apparent mutation, cultures known to have developed from a single spore are necessary. Single spores may, of course, be picked up by the use of a micro- manipulator (such as the Chambers instrument) more readily than bacteria, but much simpler procedures are available. Sass ^^ has described such a method which he has used for growing single-spore strains of mushrooms. A spore suspension of proper density is sprayed over the surface of a sterile agar plate, which is then incu- bated for a time. The plate is then examined for well-isolated spores which have germinated, the low-power microscope lens being used. When such a spore is found, a small sterilized spatula with a minute hole in it is placed over the spore and pressed into the agar in such a way as to force a small cylinder of agar (bearing the spore on its upper surface) through the hole. The growing spore may then be easily removed with a sterile needle without danger of touching any other spore. It is simpler and safer to dispense with the perforated spatula, which may touch other spores when it is placed on the agar surface, and to remove the selected spore with a minute spade-shaped needle whose entire cutting edges can be kept in full microscopic view during the entire procedure. Media for Yeast. In general, yeasts may be isolated and cultivated on the same media as are used for molds. Most species tolerate MEDIA FOR YEAST 61 high acidities, and the glucose-tartaric acid medium is the most useful plating medium. Where synthetic media are desired, ammonium salts are preferable to nitrates as a source of nitrogen. Observation of the spores is essential in identifying yeasts. Many species will not form spores readily on ordinary media. A number of methods have been devised to force yeasts to sporulate. Of these the plaster block method. is most commonly used. Plaster of Paris mixed with water is put in small dishes and allowed to harden. When set, it is removed and placed in a larger dish or a large test tube, and sufficient water or dilute (0.1 per cent) peptone solution added to moisten the block thoroughly but not cover its surface. This is then sterilized in the autoclave. A young culture, say not over 24 hours old, is used for inoculation. A generous loopful of the growth is spread over the surface of the plaster. The temperature of spor- ulation is critical, and with unknown species it is advisable to prepare several blocks and incubate them at different temperatures, say 20°, 25°, and 30° C. After 24 hours spores should be searched for by microscopic examination. Other materials, as pieces of clay flower pots or blotting paper, may be used in place of the plaster, the idea being to maintain a limited amount of both nutrition and moisture, though there is some evidence that the calcium sulphate has some specific action in the process. Many yeasts sporulate freely on corn meal agar and sporulation is said to occur quite regularly on Gorodkowa's medium, which has been widely used. The composition is Glucose 2.5 grams Sodium chloride 5 grams Meat extract 10 grams Agar 10 grams Water 1 liter Growth of yeasts on carrot plugs often leads to abundant spore formation. ]\IcKelvey ^^ developed a medium which in our labora- tories has yielded spores more consistently than have others. This consists of a weak carrot infusion (about 150 grams of chopped car- rots to 1 liter of tap water) and 15 grams of agar melted together. Then 3.5 grams of anhydrous calcium sulphate is added and the mixture is thoroughly mixed, tubed, autoclaved, and slanted like any other agar medium. The medium of Mrak and associates ^^ has also given excellent results in our laboratory. Some recent work of Nickerson and Thimann ^* on spore formation in a yeast Zygosaccharomyces may well develop into a method which 62 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES will allow ascospore formation to be much more easily brought about than heretofore. Those workers cultured a strain of Aspergillus niger, subcultures of which may be obtained from the American Type Cul- ture Museum. The medium, after some days of incubation, was filtered and resterilized. When several species of Zygosaccharomyces were inoculated into this medium, conjugation and subsequent "spore formation took place much more rapidly than in ordinary media and a much larger proportion of the cells conjugated. It was determined that two substances responsible for this stimulation of sporulation were also produced by Zygosaccharomyces itself, and it was believed that these substances are ordinarily set free into a medium only as the cells die and autolyze. Nickerson and Thimann furthermore found that the inclusion of riboflavin and sodium glutarate in ordi- nary media for yeasts gave as good results as the Aspergillus filtrate. They did not prove that the sporulation-inducing substances of the mold were the same as the chemicals used, but they did show that the results were the same. This work merits much extension. A new method of Lindegren and Lindegren -^ for inducing spore formation in yeasts is given here. It has not been tried by the authors but in the hands of the Lindegrens it gave excellent results. Presporulation Media Beet leaves extract (1500 ml. H2O, 450 grams beet leaves autoclaved) 100 ml. Beet root extract (1500 ml. H2O, 150 grams beet roots autoclaved) 200 ml. Apricot juice (canned) 350 ml. Grape juice 165 ml. Dried yeast 20 grams Glycerol 25 ml. Agar 30 grams Calcium carbonate 10 grams Water is added to make 1 liter. The mixture is steamed until dis- solved, tubed, and sterilized. Most strains will produce spores di- rectly on the slants in a few weeks. If spores are needed sooner, the mixture is transferred to plaster of Paris. Instead of plaster of Paris blocks, the tubes of Graham and Hastings ^- are used. A mix- ture of equal parts of plaster of Paris (CaS04 anh.) and water are mixed and dispensed into test tubes and solidified in a slanting posi- tion, dried at 50° C. for 24 hours and autoclaved. About 1 ml. of sterilized water is poured over a 3-day growth of yeast on the pre- sporulation medium and allowed to stand 10 minutes. A thick sus- pension is made by stirring. Some of the suspension is poured over UTILIZATION OF SPECIFIC COMPOUNDS 63 the upper portion of the plaster of Paris slant with a sterile pipet. About 3 ml. of water made to pB. 4.0 with acetic acid is pipetted into the lower half of the slant. The inoculated plaster of Paris slants are incubated for 1 to 2 days. Fermentation by Yeasts. With yeasts and yeast-like fungi, if sugars are fermented the fermentation is usually alcoholic, and al- though acid is formed it is not of great significance in identification; an indicator is therefore optional, and greatest attention is given to gas production. In our experience, the observation of fermentation by yeasts requires higher concentrations of sugar and larger amounts of medium than are usually used in studying bacterial fermentation. With 1 per cent sugar in the customary small sugar tubes, results are likely to be variable. A medium containing 3 per cent of the sugar to be tested and 1 per cent of peptone, placed in 30-ml. amounts in 25 by 150 mm. tubes, with inverted 78 by 11 mm. tubes for gas traps, are suitable. Stelling-Dekker ^^ used a 2 per cent solution of the test sugars in yeast, infusion, placed in Einhorn fermentation tubes. To identify yeasts by Stelling-Dekker's key in many in- stances, it is necessary to determine whether the trisaccharide raf- finose is fermented completely or only one third. For this purpose she uses a 4 per cent solution of raffinose in yeast infusion, and the fermentation is studied quantitatively in the van Iterson-Kluyver apparatus. Raffinose is a trisaccharide and each molecule is made up of one molecule of the disaccharide melibiose (%) and one mole- cule of the monosaccharide, fructose (%). If tubes of both raflfinose and melibiose broth are used, it can be determined without the use of special chemical equipment whether all or only one third of the raflfinose is fermented. If both raflfinose and melibiose are fermented, obviously all the raflfinose is fermentable; if raflfinose and not meli- biose, only one third, that is, the fructose portion of the molecule. Wickerham^^ has given details for making fermentation tests with melibiose. Utilization of Specific Compounds. In addition to studying fer- mentation, the identification of yeasts, especially the asporogenous species, requires in many cases a determination of the carbohydrates or nitrogen sources which can be utilized by the organism. A medium, is prepared which contains all the necessary ingredients for growth except a source of carbon, and to this medium the test sugars are added; or a n^edium is prepared which contains a known utilizable sugar (all yeasts can utilize glucose), but with no nitrogen source, to which various test nitrogen compounds may be added. 64 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES Lodder ^^ has presented a simple procedure (originally devised by Beijerinck and called by him an auxanographic method) for deter- mining the utilization of various substrates. For sugars, the basic medium is Ammonium sulphate 5.0 grams Monopotassium phosphate 1.0 gram Magnesium sulphate 0.5 gram Agar 20.0 grams Water 1 liter This medium, melted and cooled, is heavily seeded with the yeast to be tested and poured into Petri dishes. When solidified, the plate cultures are incubated for a few hours to dry the surface. Then small quantities of the dried sugar are deposited on the surface of the agar in labeled areas. Glucose is added to one spot in every case to serve as a control, since all yeasts can utilize this source of carbon. As many as six sugars may be tested on one plate, in addition to the glucose. The sugars dissolve and diffuse into the agar and, if util- izable, the yeasts will grow in that part of the plate culture. Similarly, auxanographs of nitrogen utilization may be made. Here the basic medium is Glucose 20.0 grams Monopotassium phosphate 1.0 gram Magnesium sulphate 0.5 gram Agar 20.0 grams Water 1 liter Peptone, ammonium sulphate, asparagine, urea, and potassium nitrate are the test substances used. Some difficulty may be antici- pated in these highly purified liquid media, for the reason that growth-promoting factors are necessary for some yeasts. Nicker- son ^^ used as criterion a medium including growth-promoting sub- stances, glucose, and nitrates, and tested for nitrate reduction, rather than nitrate utilization. If further work shows that all organisms which are able to utilize nitrates as a sole nitrogen source also reduce nitrates to nitrites, this will be an improvement over previous meth- ods. Nickerson used Dipotassium phosphate 3 grams Magnesium sulphate 0.25 gram Calcium chloride 0.25 gram Potassium nitrate 6 grams Glucose 20 grams Yeast extract 0.1 gram Water 1 liter GIANT COLONIES 65 After growth, the usual color tests for nitrites were made. It is quite possible that the ordinary peptone-glucose solutions such as Sabouraud's with added nitrates would serve as well as the above medium. According to Zimmermann ^^ and Mrak and McClung,^° the aux- anographic method cannot always be relied upon, and no doubt liquid media are to be preferred. The ability to utilize ethyl alcohol is also an important character in the identification of yeasts. This is determined by inoculating the organism into a liquid medium: Ethyl alcohol 30.0 ml. Ammonium sulphate 1.0 gram Monopotassium phosphate 1.0 gram Magnesium sulphate 0.5 gram Water 1 liter The alcohol will have to be added after sterilization. If growth occurs, as manifested by turbidity or sediment, the test is positive. Growth-promoting substances often have to be added to liquid media. In the auxanographic method, the large inoculum provides these substances. Giant Colonies. The form and texture of colonies of yeasts are of diagnostic value, especially those of large isolated colonies (giant colonies) which have been allowed to grow for some time (4 to 6 weeks). See Fig. 114. Since lack of oxygen may modify growth, the containers cannot be sealed, and to prevent too much evapora- tion a large volume of agar is required. For this purpose round narrow-mouthed bottles 5 cm. in diameter and 12 cm. high are suit- able. These are filled with 50 ml. each of the American Sabouraud agar described above and autoclaved. They are inoculated by touch- ing a straight wire to the stock culture, then touching the tip of the wire to the center of the agar surface in the bottle. It is important to hold the bottle in an inverted position while inoculating to prevent air contamination. The agar surface should be allowed to dry a day or two before inoculating. The bottles are incubated at room tem- perature. For clear observation or for photographing, the bottles may be cut at the level of the agar surface by a short cut with a file and pressing the molten end of a glass rod against the file mark. The glass will usually crack neatly around the bottles at this level. The use of Petri dish cultures for photographic surfaces is more con- venient and economical if contamination can be avoided and the plates do not dry too much in the long incubation period. Instead of giant colonies, Stelling-Dekker and Lodder used agar slant growth. 66 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES Temperature of Incubation. The optimum temperatures of molds, yeasts, and aetinomycetes vary rather widely according to the species, and extensive studies are not available for most groups. Fortunately, with many species, the top of the temperature growth curve appears again to be a plateau rather than a peak, so that one has more lati- tude than with most bacteria. It is noteworthy that most of the pathogenic species do not grow best at body temperature but at a point considerably lower. Even the highly parasitic dermatophytes find their optimum temperature at 27° C. according to Kadisch.^^ In general, 30° C. falls near the optimum for most common patho- genic and saprophytic fungi, and the bacteriological laboratory which has frequent occasion to work with these microorganisms should be equipped with a special incubator kept at or near this temperature. Mrak and Bonar ~^ showed that the temperature of incubation has a decided influence on the formation of yeast ascospores as well as on their size. Lower temperatures tend to reduce the size of the asco- spores in relation to the ascus and therefore the individual spores are easier to delineate and study. In some groups, e.g., Rhizopus, the temperature range is of taxonomic significance. Determination of Morphology. The identification of fungi de- pends more upon morphological characters than that of bacteria does. The appearance of the plant mass to the naked eye is fre- quently quite characteristic, and in many cases the organism can be recognized at a glance. It should be borne in mind, however, that both gross morphology and microscopic characters may sometimes vary not only with changes in the composition of the medium but also with the age of the culture. For observing the morphology of molds a much better view can be obtained if the fungus is grown in a Petri dish rather than in a cul- ture tube. Certain precautions must be observed, however. Great care must be exercised in handling plate cultures of pathogenic fungi, and certain agents of pulmonary and generalized mycoses such as Coccidioides immitis and Nocardia asteroides which produce large numbers of easily dissociated air-borne spores and hyphal fragments should never be planted in Petri, dishes. In cases of non-pathogenic fungi it is likewise necessary to avoid dissemination of spores in the laboratory when the Petri dish cover is removed since such spores will be a troublesome source of contamination. Plate cultures can usually be uncovered safely when the colony is young and has just begun to form mature spores. Fortunately many of the morpho- logical features of taxonomic interest ^re rnost easily seen in a young ci DETERMINATION OF MORPHOLOGY 67 The first step in identifying a mold is to determine whether it be- longs to the Phycomycetes or the Ascomycetes (or Fungi Imperfecti). In general, though there are exceptions, this may be readily done by a rather superficial examination. With those species which belong to the Fungi Imperfecti the plant mass is usually relatively compact, the aerial filaments of mycelium are relatively short, and the sur- face is thickly covered with spores which are frequently brightly colored. The surface may be compared to the nap of velvet. In the phycomycetous molds the mycelium is coarser and looser in tex- ture, the aerial hyphae are longer, and the sporangia are less numer- ous. The spore heads and frequently the aerial mycelium are usually dark colored, brown, gray or black. The whole plant mass has a texture comparable to cotton wool. Molds take up considerable water from the medium and give off considerable into the air, which may condense in droplets on the surface of the plant. Large amounts of this transpiration (or gutta- tion) water are characteristic of certain species. The droplets are frequently colored by pigments excreted by the mold. Beginners are frequently apt to jnistake these droplets of moisture on the mycelium, especially if it is colored, for spores or other structures. The general characters of the aerial mycelium and the spore heads may be determined by examining the growth with a magnifying glass or the low-power lens of the microscope from above, after removing the cover of the dish. For this purpose a binocular dissecting micro- scope is ideal. After examining the upper surface of a Petri plate culture, one should reverse the plate and note the under surface of the colony. Frequently very characteristic colors are produced in the submersed mycelium or in the medium itself. These will vary markedly with the medium. Some of the pigments act as indicators, being perhaps yellow on one side of neutrality and red on the other. Occasionally one may find pigment only at the spot where two different molds come together. Particularly one should note if the submersed my- cehum is light or dark in color. This is important as a primary separation of molds belonging to the Fungi Imperfecti. In the examination and culture of sputum, species of Candida are frequently found. The most important of these, Candida albicans, can be readily identified in most cases by its appearance on corn meal agar in a plate culture. The agar is inoculated by placing the inoculum in several parallel streaks across the plate; a stiff needle is used so that some of the inoculum is below the agar surface. After 4 or 5 days of incubation at 20° to 25° C. the species produces char- 68 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES acteristic clusters of blastospores and few to many large spherical chlamydospores. However, strains which have been kept in culture for long periods are not always typical in appearance, and fermenta- tion reactions must be studied for identification. For finer details of morphology it is necessary to prepare slides. Two stiff sharp Nichrome or steel needles are required. A bit of the aerial mycelium is removed with a single thrust of one needle held nearly parallel to the surface of the agar. The mycelium thus re- moved should be touched momentarily to a small drop of 95 per cent alcohol which has been previously placed on a slide, then removed quickly to a drop of 10 per cent sodium hydroxide also previously placed on the slide. It should then be carefully teased apart with two needles and covered with a cover slip. A thin film left on the slide by the rubbing of a finger across it just before the drops of alcohol and sodium hydroxide are deposited will usually prevent the spreading of the former and will facilitate the transfer of the material from one fluid to the other. The alcohol wets the mycelium which otherwise may be nearly opaque owing to the inclusion of air. The sodium hydroxide is a better mounting fluid than -water because it swells the hyphal walls, making certain morphological details more apparent, and the preparation lasts longer. If the preparation is thin the sodium hydroxide crystallizes around the edge, thus sealing the cover and preventing dehydration for several hours. One may thus determine with certainty the presence or absence of septa in the mycelium, the structure of the sporophores and spores, the presence or absence of chlamydospores, and the like. Instead of water some workers may prefer as a mounting fluid Amann's medium, which has the following composition. Phenol, crystals 20 grams Lactic acid, syrup 20 grams Glycerol 40 grams Water 20 ml. These are dissolved together with gentle warming; then the follow- ing is added. Cotton blue 0.05 gram This is the formula given by Linder,-^ save that the amount of dye is greatly reduced. It serves as a combined fixing agent, stain, and mounting fluid. The mycelium and spores will be stained blue; thus much of the dye from the solution will be removed. Such a prepara- tion, however, shows all the parts disarranged and is not permanent. Permanent preparations showing the complete structure of the thallus SLIDE CULTURES 69 may be easily made with most molds by growing them directly on slides or cover glasses. Slide Cultures. Slide cultures may be made either with solid or liquid media. It is best to have the medium not too rich in nutri- ents, since then the mycelium becomes rather densely packed and it is difficult to make out details of structure. The agar should be as clear as possible, because the presence of precipitates obscures the field. Since only small amounts are required, it is quite practical to filter the agar through paper. The slides or cover glasses used must be clean and free from grease, so that the medium will spread in a thin, uniform film. They may be kept in the usual acetic acid- alcohol mixture until used. A convenient culture chamber may be prepared by placing in a Petri dish a piece of glass tubing bent to a V shape. A glass slide is laid across this and the dish, slide, and supporting tubing are sterilized in an autoclave. If, during sterilization, the slide has fallen off its support it can be replaced with sterile forceps. Melted agar is then placed on the slide at a sufficiently high temperature and in sufficient amount to cover with a thin flat layer of agar an area about half the length of the slide and three-fourths its width. When the agar has solidified, the fungus should be planted by streaking spores in two parallel rows extending the length of the agar. Water should then be placed in the bottom of the dish. Distilled water will pro- vide a very humid atmosphere. By the addition of small amounts of salt to the water, various degrees of humidity can be maintained. When the fungus has reached the proper stage of development it can be examined under the microscope. For the study of fine de- tails it must, of course, be covered. If the fungus to be examined is a dry mold it must first be wetted. The slide is held by one end and absolute alcohol is dropped at the upper end of the culture. When it has drained off, the bottom and edges of the slide are wiped with blotting paper and the slide is placed on the table. Then 2 or 3 drops of 10 per cent sodium hydroxide or other mounting fluid is placed upon the culture and a large cover slip is carefully placed over it. The preparation is not permanent but is excellent for exam- ination and photography of the unshrunken mycelium, conidiophores, and spores, and will keep for a few days. Because of the danger of scattering spores this method is not suitable for use in the exam- ination of some fungi pathogenic for man. Cover glasses require less space for incubation than slides, and a number of cover glasses can be incubated readily in a Petri dish. Stained cover glasses can be mounted in balsam to make permanent ^0 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES preparations very readily, but the small amount of nutrient is un- desirable in some cases. A piece of blotting paper or several thick- nesses of filter paper are placed in the bottom of a Petri dish and sterilized in an autoclave. Just before being used, the paper is wetted with sterile water. The cover glasses are cleaned, placed in alcohol, sterilized by flaming, and then placed on the surface of the paper. A tube of the agar is melted in a water bath, cooled to between 43° and 46° C, and inoculated rather heavily with spores of the mold to be studied. With a wire loop one or two loopfuls of the agar are deposited quickly on each cover glass and spread in a rather thin film. The Petri dish may then be incubated. If it is placed in a can, evaporation will be retarded. It is essential to maintain an atmosphere nearly saturated with moisture. For preparations which are to be only temporary, agar is prefer- able to liquid media, since when hardened it will "stay put" on the cover slip. Agar cover slips placed on slides may be examined first, culture side up, with the low- and medium-power lenses, then turned over and mounted in a drop of Amann's fluid for more complete study. Semi-permanent preparations may be made by sealing the edges of the cover slip with asphaltum or other varnishes, first re- moving all excess of the mounting fluid. More permanent mounts may be made if glycerin jelly (1 part gelatin, 6 parts water by weight; 1 per cent phenol added) is used instead of Amann's fluid. This also requires sealing of the mount with asphaltum. Beautiful permanent preparations mounted in balsam may be made from cover slip cultures, but for this liquid media are preferable to agar media because it is almost impossible to obtain a stain which does not color the agar rather deeply. A tube of the liquid medium is inoculated, and then with a sterile pipet small quantities (about 0.01 ml.) are deposited on the cover slips in the Petri dish. The dish must now be handled carefully so that the liquid does not run off on to the filter paper. Czapek's or corn meal solution is suggested for actinomycetes and for those molds which will grow on them. When sufficient growth has taken place the cover slips are removed and thoroughly dried. If drying is not complete the film of mold (or of agar if it has been used) will wash off in the staining opera- tions. It is well to dry the cover slips on some sort of warm plate and to let them continue to dry for about 5 minutes after they appear to be dry. Since the mold is not easily wetted by water, an alcoholic fixing solution is desirable. For ordinary purposes the formalin- alcohol-acetic acid mixture commonly used for plant material is satisfactory. It is made of SLIDE CULTURES 71 Alcohol (50 per cent) 100 mL Formalin (40 per cent) 6.5 ml. Acetic acid (glacial) 2.5 ml. Immersion for a few minutes in this solution will suffice. From this solution the cover slip is washed in several changes of water and then stained. For simple staining a 1 per cent aqueous solution of erythrosin or rose bengal is very satisfactory. These weakly acid dyes may be used with agar preparations, since they do not stain the agar so deeply as they do the mycelium. It may take up to 15 minutes to stain sufficiently. One may also obtain good preparations with agar slide cultures by the use of the acid thionine solution in- troduced by Frost for staining microcolonies of bacteria. The formula is Thionine 1.0 gram Phenol 2.5 grams Acetic acid (glacial) 20.0 ml. Water 400.0 ml. About 1 minute is sufficient for staining. If the staining is pro- longed, the agar will stain too deeply. "With liquid media one may use other stains. Heidenhain's iron hematoxylin gives very clear preparations, but for routine use the simple aqueous erythrosin or rose bengal solution is very satisfactory for molds. For actinomy- cetes these stains are satisfactory but faint. Gram's stain gives a clearer picture if cultures are not too old. One must not rely entirely upon slide cultures for the identification of molds. In some cases growth in the small volume of medium on a cover slip is not altogether typical. Spore heads may be small and imperfectly developed. Some Penicillia, for instance, will form only a single verticil of phialides in slide cultures, whereas they form polyverticillate spore heads in Petri dish cultures. The slide culture supplements but is not a substitute for the examination of the plate culture. Another type of slide culture will prove of great value, particularly in determining details of the arrangement of the aerial mycelium, for example in untangling the branching of the sporangiophores of Mucors. Rather large cover glasses (24 by 40 mm.) are cleaned. With a small hot iron (for instance, the heated end of a small file) a drop of sealing wax or, better, de Khotinsky cement is deposited on each end. With the hot iron this is then spread out to form a layer about 5 mm. wide and 1 mm. or less thick across the ends of the cover glass. A clean slide is now heated in the Bunsen flame and the cover glass is placed upon it with the cement side down. The slide 72 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES should be just hot enough to soften the cement so that it will adhere, not enough to liquefy it so that it will run. One now has a culture chamber arranged as shown in Fig. 29, with a space something less than 1 mm. deep between the cover glass and the slide. A tube of agar is melted, cooled, and inoculated with spores of the mold to be studied. With a sterile capillary pipet some of the agar is transferred to the edge of the cover glass and allowed to run under until part of the space has been filled, as shown in the illustration. If the shdes are prepared just before use, they will have been sufficiently sterilized by the heat used in their preparation. They may then be incubated in a glass staining jar containing moistened blot- ting paper in the bottom. The lid of the jar should be sealed with petroleum jelly or surgeon's plaster. After growth has occurred, the arrangement of both the aerial and sub- merged mycelium may be readily seen. Photomicrographs taken from such slide cultures are shown in Figs. 30 and 46. Paraffin can be used in the preparation and sealing of a culture cell of this type, ( ' — n 1 i^->> v uSukisa, ui^nu,^^ ;'■■-:■.■■ /'■'■ 5-^ t," Fig. 29. Method of grow- ing molds between a slide and cover slip. Fig. 30. Aspergillus sp., shoeing appearance of conidiophores as seen in a slide culture of the type shown in Fig. 29. the sterile melted paraffin being handled in a sterile Pasteur pipet. It is less permanent but more convenient. Microscopic Examination of Yeasts. The vegetative cells of yeasts and the determination of their mode of reproduction (such as type of budding and fission) are best determined in simple wet prepara- tions made by mounting some of the growth from a young, actively growing culture in a drop of water. Smears are not good because of MORPHOLOGY OF ACTINOMYCETES 73 the marked shrinkage and distortion which occurs on drying. The cells may be mounted in a 5 per cent glycerin solution or in Amann's fluid. The vacuoles and "dancing bodies" of yeasts may be stained vitally by suspending the cells in a dilute (1:8000) solution of neutral red. Fraser ^^ has described the differential counting of living and dead yeast cells suspended in solutions of neutral red, Congo red, and methylene blue. Less shrinkage and distortion are observed if a suspension of the cells is made in the fixing fluid and run gradually through the alcohols; it should be centrifuged and resuspended at each change. Finally the sediment should be imbedded in paraffin and sections should be cut as thin as possible. The spores of yeasts may be readily observed in unstained wet preparations after some experience. Beginners are likely to mistake fat droplets and water vacuoles for spores. Spores may be stained differentially in most cases by the use of the same procedures used for staining the spores of bacteria. The following wall give beau- tiful preparations. A dried smear is fixed in 5 per cent chromic acid for 10 minutes, stained in steaming carbol fuchsin for 10 minutes or for several hours in the cold stain, decolorized in 1 per cent sulphuric acid, and counterstained with Loeffler's methylene blue for 2 minutes or more. Spores will be bright red, vegetative cells blue. The cells within which the spores are found are frequently so poorly stained as to be difficult to see. Morphology of Actinomycetes. For studying the morphology of actinomycetes one may use the same methods that serve for the larger molds, but may encounter difficulties due to their extreme minuteness. The very highest magnifications must be used to obtain a clear pic- ture. The slide culture method may be used to good advantage. Here it is particularly important to use a medium poor in nutrients in order to prevent too dense massing of the mycelium. Czapek's medium is satisfactory for many species. For observing the spores and sporophores the method of Drechsler^ has some advantages. It consists in moistening a cover glass with a thin film of albumen. This is then lightly dropped on to the surface of a sporulating colony and gently lifted off again. The spore-bearing filaments will adhere to the cover slip and will be pulled away from the colony, but will retain their normal arrangement. They can then be fixed, stained, and mounted. The anaerobe Actinomyces bovis can be examined by withdrawing young colonies from a glucose veal infusion agar deep culture, crush- 74 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES ing the agar containing the colony under a cover slip, and examin- ing directly. Microscopic Examination of Tissues and Exudates. The diagnosis of fungus diseases of man depends upon direct microscopic examina- tion of tissues and exudates as well as upon cultural methods. In some cases the fungi grow so scantily that, if they are not abundant enough to be found on microscopic examination, it is not likely that they will overcome the competition of contaminants and grow in cultures. In sporotrichosis, however, the small size of Sporotrichum and the difficulties of differentially staining it make its isolation in culture the preferred method of laboratory diagnosis. In all cases, when possible, it is desirable to isolate pathogenic fungi in culture because the final identification of the fungus depends upon deter- ' mination of its characters in artificial culture media. The specific methods of examination and the appearance of the fungus will be discussed in detail in the consideration of the various mycoses. In searching for yeasts, molds, and actinomycetes in pus or other exudates or tissues from infections in man and animals, certain gen- eral facts must be kept in mind. Where it is possible to obtain pus from abscesses which have just been opened surgically before they have formed a fistula communicating with the exterior, it is usually relatively easy to find the organisms. After they have broken open, however, there is always an extensive secondary infection with bac- teria, and it is often almost impossible to find the fungi. In cases where the parasites cannot be obtained in the pus draining from the abscess, they may be found in sections of tissue removed from the wall of the abscess. A biopsy is thus frequently of great diagnostic value. Fungi may be demonstrated in sections of tissue by the Gram- Weigert method or one of its various modifications. Unna *- has described a modification of the Unna-Pappenheim stain which is said to give a very clear picture, especially in skin sections. The sec- tions are removed from water and stained for 5 to 10 seconds in the following solution. Pyronin 0.9 gram Methyl green 0.1 gram Alcohol (96 per cent) 9.0 ml. Glycerol 10.0 grams Aqueous phenol (3^ per cent) to 100.0 ml. These sections are then rinsed in water and quickly dehydrated in absolute alcohol, cleared in xylol, and mounted in balsam. Fungus MICROSCOPIC EXAMINATION OF TISSUES AND EXUDATES 75 elements stain red, nuclei and leucocytes appear bluish green. Sec- tions stained by Giemsa's method show the fungus elements very clearly in some cases. Hematoxylin and eosin will also usually show them, but their coloring is weak. In general, fungus parasites are not so numerous in exudates as bacteria are. One must therefore not be satisfied with a hasty exam- ination but must patiently go over many microscopic fields. Young growing elements of the fungi are all Gram-positive, but old my- celium becomes filled with fat and other materials, and the Gram- staining protoplasm may be but a small portion of the whole. In general, stained smears are not of much use in detecting pathogenic fungi. Some exceptions are: sporotrichosis, where the phagocyted fusiform bodies characteristic of this disease may be best demon- strated in smears of pus; infections with Candida albicans and Cryptococcus neojormans, where the yeast-like bodies may be read- ily demonstrated in smears of pus, sputum, or spinal fluid, stained by methylene blue or by Gram's method; and histoplasmosis, where the small Leishmania-like bodies phagocyted by large macrophages are best demonstrated in smears of bone marrow or splenic pulp, treated like a blood smear, carefully fixed, and stained with Wright's stain or Giemsa's stain. In other fungus diseases the procedure of choice is to examine the pus or sputum or other material wet and usually unstained. If not too thick it can be covered with a cover slip and examined without any preparation. Many specimens need to be diluted or digested in one or another type of mounting fluid. The material most frequently used is a solution of sodium or potassium hydroxide. Such a solution serves to dissolve the leucocytes and other tissue elements, or at least to soften them and make them more translucent, without destroying the fungus elements which for the most part are rather resistant to strong alkali. The strength of the solution varies, according to dif- ferent authorities, from 5 to 40 per cent. The stronger the solution is, the quicker its action and the greater the danger of producing artifacts and of destroying the fungus. In general a 10 per cent solution seems to be most useful. A loop of pus or sputum is stirred up in a drop of this solution on a slide, covered with a cover slip, and allowed to stand 15 to 30 minutes before examination. To pre- vent evaporation the edges of the cover slip may be sealed with petroleum jelly. A number of other mounting fluids may be used. The Amann's fluid with cotton blue described on page 68 will serve to dilute the 76 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES pus, stain both cells and fungus elements, and make them more translucent. Another solution consists of Glycerol 20 parts Ammonia (28 per cent NH3) 10 parts Alcohol 20 parts In examining pus or sputum for fungi, beginners are apt to mistake fat droplets for yeast cells and elastic tissue fibers for mycelium. Fat droplets are highly refractile, and sometimes the edge may ap- pear to be doubly contoured. Adjacent large and small droplets may look like a budding cell. Elastic tissue fibers vary in diameter and branch like mycelium. In both cases, however, there is no internal strycture. Both fat droplets and elastic tissue fibers appear clear and homogeneous, whereas both yeast cells and mycelium will show internal vacuoles and granules. Myelin globules are present in large numbers in some specimens and may be mistaken for yeasts or hyphae. Microscopic Examination of Hairs and Scales. In the diagnosis of dermatophytosis the microscopic examination of hairs and of scales of epidermis is of first importance. Here again the standard procedure is to mount the hairs or scales in a strong alkaline solution to soften the material so that it may be flattened under a cover slip, and to make it translucent so that the fungus elements may be seen. It is important to select hairs which appear to be infected, preferably the stumps of hairs which have broken ofT. The fungi will be found near the root of the hair. The use of ultraviolet light may be help- ful in selecting infected hairs. With both hairs and scales of epi- dermis, it is best to collect material from just back of the advancing border of the lesion, where fungi may be expected to be most abun- dant. The examination of hairs and scales mounted in strong sodium or potassium hydroxide presents all the disadvantages mentioned under the examination of exudates, and additional ones because the alkali may give rise to artifacts that can be mistaken for fungi — crystals of the alkali where evaporation has taken place under the cover slip, and the mosaic fungus. The latter consists of an irregu- lar, branched filamentous structure, without internal granules or vacuoles, that appears in some scales of epidermis when treated with strong alkali. The exact nature of the artifact is unknown. First described by AVeidman,** the mosaic fungus has been further studied by Davidson and Gregory ^ who suggested that it is made up of cholesterin crystals, by Dowding and Orr,^ and by Swartz and USE OF ULTRAVIOLET LIGHT 77 Conant.*'' Most authorities are now agreed that the mosaic fungus is an artifact. Cornbleet * developed an alternative mounting fluid for hairs and scales. Water is added to crystals of sodium sulphide in minimum quantity to obtain complete solution. To this solution an equal volume of alcohol is added. A cloudy precipitate is formed which is redissolved by adding water drop by drop. This alcohol solution makes possible a quicker and more complete wetting of the hair; the sulphide serves to dissolve keratin. Swartz and Conant *° soften scales of epidermis in 5 per cent potassium hydroxide, after which they are washed in several changes of water and then mounted in Amann's fluid with cotton blue (0.5 per cent cotton blue). This method may also be used with hairs. Thus prepared, the mosaic fungus does not appear in scales of epidermis. Clearer preparations with much less (0.05 per cent) cotton blue are obtained but a longer time is required for the stain to be absorbed by the fungi. Lewis and Hopper -° have published numerous clear photographs of fungi in hairs and scales, and of the artifacts which may be confused with them. Use of Ultraviolet Light. The discovery by Margarot and Deveze -" that hairs infected with species of Microsporum exhibit a greenish fluorescence in ultraviolet light has led to a number of studies of this phenomenon both in clinical diagnosis and in the identification of cultures. If the patient is examined in a dark room under filtered ultraviolet light, single infected hairs can be readily seen although there may be no clinically apparent lesion. The method is particularly useful in detecting small satellite lesions in the scalp, infections of the eyebrows, and in checking the results of treatment. Davidson and Gregory ^ showed that the Microsporum species could be distinguished from species of Trichophyton in hairs, the former giving a greenish fluorescence due to a water-soluble sub- stance, the latter a bluish fluorescence due to a substance which could not be extracted with water. Lewis ^^ applied this method to the differentiation of dermatophytes in cultures and found that different species gave fluorescent light of distinguishing color; in particular, Microsporum Canis was said to be distinguished from M. Audouini. Redaelli and Cortese ^^ studied a variety of sapprophytic and para- sitic molds, yeasts, and actinomycetes, and found that, although many of them fluoresced with different colors, the fluorescence varied markedly with the medium and the age of the culture. The medium itself usually gave some fluorescence. Non-pigmented molds were most fluorescent, especially before spores were formed. Lewis and 78 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES . Hopper -° have discussed further the identification of mold cultures by ultraviolet light. Davidson and Gregory ^ described an inex- pensive source of filtered ultraviolet light. It appears that, although the use of fluorescence is a valuable clinical method, the conditions which determine the phenomenon and the specific color of the fluorescence are not yet well enough known for its satisfactory use in identifying cultures in the laboratory. The unreliability of the use of ultraviolet light for identification of cul- tures has very recently been emphasized by Benedek ^ who, however, stressed the great value of this tool in the clinic for distinguishing ringworms of the scalp due to Microsporum from those due to Tri- chophyton. Especially the value of ultraviolet light for detecting carriers was stressed. LITERATURE 1. Benedek, T., Contribution to the epidemiology of tinea capitis. III. Some diagnostic problems in tinea capitis, Mycologia, 36, 598 (1944). 2. CoNANT, N. F., Studies in the genus Microsporum, Arch. Dermatol. Syphilol. {Chicago), 33, 665 (1936). 3. Conn, H. J., The use of various culture media in characterizing actino- mycetes, A^. Y. Agr. Exp. Sta. (Geneva) Tech. Bull. S3 (1921). 4. CoRNBLEET, T., A reagent for demonstrating fungi in skin scrapings and hair, J. Am. Med. Assoc, 95, 1743 (1930). 5. Davidson, A. M., and P. H. Gregory, Convenient source of light for diag- nosis of ringworm of the scalp. Can. Med. Assoc. 7., 27, 176, 485 (1932). 6. Davidson, A. M., and P. H. Gregory, So-called mosaic fungus as an inter- cellular deposit of cholesterol crystals, /. Am. Med. Assoc, 105, 1262 (1935). 7. Davidson, A. M., P. H. Gregory, and A. R. Birt, A clinical and mycological study of suppurative ringworm, Can. Med. Assoc. J., 31, 587 (1934). 8. Dowding, E. S., and H. Orr, Transformation of Trichophyton gypseum into mosaic fungus, Arch. Dermatol. Syphilol. (Chicago), 33, 865 (1936). 9. Drechsler, C, Morphology of the genus Actinomyces, Boian. Gaz., 67, 65, 147 (1919). 10. Emmons, C. W., Dermatophytes. Natural grouping based on the form of the spores and accessory organs, Arch. Dermatol. Syphilol. (Chicago), 30, 337 (1934). 11. Eraser, O. G., The action of methylene blue and certain other dyes on living and dead yeast, J. Phijs. Chem., 24, 741 (1920). 12. Graham, V. E., and E. G. Hastings, Studies on film-forming j'easts. I. Media and Methods, Can. J. Research, C, 19, 251 (1941). 13. Greene, H. C, and E. B. Fred, Maintenance of vigorous mold stock cul- tures, hid. Eng. Chem., 26, 1297 (1934). 14. Gwynne-Vaughan, H. C. I., and B. Barnes, The Structure and Develop- ment of the Fungi, Macmillan, New York, 1939. 15. Kadisch, E., tjber die Bedeutung der Nahrbodenalkalinitat in der Mykol- ogie, Dermatol. Z., 55, 385 (1929). LITERATURE 79 16. Kadisch, E., Der Einfluss der Zuchtungstemperatur auf das Wachstum der pathogenen Hautpilze auf den iiblicken Xahrboden und auf inneren Organen des Meerschweinchen, Arch. Dermatol. Syphilis, 160, 142 (1930). 17. Langeron, M., and S. Milochevitch, Morphologic des dermatophytes sur miheux naturels et miUeux a base de polysaccharides, Ann. parasitol. humainc et comparee, 8, 422, 465 (1930). 18. Levin, O. L., and S. H. Silvers, The possible explanation for the localiza- tion of ringworm infection between the toes, Arch. Dermatol. Syphilol. (Chicago), 26, 466 (1932). 19. Lewis, G. M.. Fluorescence of fungous colonies with filtered ultraviolet radiation (Woods filter), Arch. Dermatol. Syphilol. (Chicago), 31, 329 (1935). 20. Lewis, G. M., and M. E. Hopper, An Introduction to Medical Mycology, Year Book Pub. Co., Chicago, 1939. 21. Lindegren, C. C, and G. Lixdegren, Sporulation in Saccharomyces cere- visiae, Botan. Gaz., 105, 304 (1944). 22. Lixder. D. H., An ideal mounting medium for mycologists, Science, 70, 430 (1929). 23. LoDDER, J., Die Anaskosporogenen Hefen, Iste Halfte, Verhandel. Akad. Wetenschappen, Amsterdam, Adfeel. Natuurkunde, 2nd Sect., 32, 1 (1934). 24. Mallinckrodt-Haupt, A. von, Vitalftirbung mit Indicatorfarben bci Hypho- myzeten, Dermatol. Z., 46, 263 (1926). 25. , P H Messungen bei Pilzkulturen, Dermatol. Z., 55, 374 (1929). 26. McKelvey, C. E., Notes on yeasts in carbonated beverages, J. Bad., 11, 98 (1926). 27. Margarot, J., and P. Deveze, Aspect de quelques dermatoses en lumiere ultraviolette — note preliminaire, Bull. soc. sci. med. biol. Montpellier Languedoc, 6, 375 (1925). 28. Memmesheimer, A. M., Uber eienem neuen Nahrboden fiir Pilzkulturen, Klin. Wochschr., 17, 56 (1938). 29. Mrak, E. M., and L. Bonar, The effect of temperature on asci and asco- spores in the genus Debaryomyces, Mycologia, 30, 182 (1938). 30. Mrak, E. M., and L. S. McClung, Yeasts occurring on grapes and in grape products in CaHfornia, J. Bact., 40, 395 (1940). 31. Mrak, E. M., H. J. Phaff, and H. C. Douglas, k sporulation stock medium for yeasts and other fungi, Science, 96, 432 (1942). 32. Negroni, P., and D. Loizaga, Accion "in vitro" de los colorantes, sobre la morfologia y biologia de Mycotorula albicans, Rev. argentina dermato^ sifiloloyia, 22, 556 (1938). 33. NiCKERSON, W. J., Studies in the genus Zygosaccharomj'ces. Transfer of pellicle-forming yeasts to Zygopichia, Farlowia, 1, 469 (1944). 34. NiCKiERSON, W. J., and K. V. Thimann, The chemical control of conjuga- tion in Zygosaccharomyces. I and II, Am. J. Botany, 28, 617 (1941); 30, 94 (1943). 35. Redaelli, R., and F. Cortese, Sulla fluorescenza degli eumiceti alia luce di Wood, Raggi ultravioletti, 6, 52 (1930); Boll. soc. med.-chirurg. Pavia, 44, 49 (1930). 36. Sass, J. E., The cytological basis for homothallism and heterothallism in the Agaricaceae, Am, J. Botany, 16, 663 (1929). 80 STUDYING MOLDS, YEASTS, AND ACTINOMYCETES 37. Starkey, R. L., and S. A. Waksman, Fungi tolerant to extreme acidity and high concentrations of copper sulfate, /. Bad., 45, 509 (1943). 38. Steinberg, R. A., Growth of fungi in synthetic nutrient solutions, Botan. Rev., 5, 327 (1939). 39. Stelling-Dekker, N. M., Die Sporogenen Hefen, Verhandel. Akad. Weten- schappen, Amsterdam Adjeel. Natuurkunde, 2nd Sect., 28, 1 (1931). 40. SwARTZ, J. H., and N. F. Conant, Direct microscopic examination of the skin. A method for the determination of the presence of fungi, Arch. Dermatol. Syphilol. (Chicago), 33, 291 (1936). 41. Talice, R. v., Le facteur pH en mycologie, son influence sur la culture de certaines especes de champignons parasites de I'homme, Ann. parasitol. hiimaine et comparee, 8, 183 (1930). 42. Unna, p., Jr., tjber Farbung von Fadenpilzen in der Oberhaut, Dermatol. Wochschr., 88, 314 (1929). 43. Waksman, S. A., A method for counting the number of fungi in the soil, J. Bact., 7, 339 (1922). 44. Weidman, F. D., Laboratory aspects of epidermatophytosis. Arch. Dermatol. Syphilol. (Chicago), 15, 415 (1927). 45. WiCKERHAM, L. J., A simple technique for the detection of melibiose fer- menting yeasts, J. Bact., 46, 501 (1943). 46. Williams, J. W., Growth of certain pathogenic fungi on asparagine medium, Proc. Soc. Exptl. Biol. Med., 31, 1176 (1934). 47. ■, Scalp products and hair of men and women as culture media for certain pathogenic fungi, Proc. Soc. Exptl. Biol. Med., 32, 624 (1935). 48. , Effect of dyes on colonies of certain pathogenic fungi, Proc. Soc. Exptl. Biol. Med., 31, 1173 (1934). 49. Zimmermann, J., Sprosspilze im Wein und deren Bestimmung, Zentr. Bakt., Parasitenk., II, 98, 36 (1938). CHAPTER IV MOLDS BELONGING TO THE PHYCOMYCETES No Archimycetes are likely to be of interest to the bacteriologist. Probably the only genus of Oomycetes that he will ever come in contact with is Pythium, some species of which are pathogenic to plants but others of which are soil saprophytes. The mycelium is ordinarily coenocytic, but septa may occasionally be seen in older mycelium. The zoosporangia are usually numerous and rather small, and are mostly borne on the ends of the mycelial strands, although they may be found in the myce- lium, resembling chlamydospores. On germination the contents of the zoosporangia are extruded into a thin-walled vesicle, after which they are differentiated into several motile zoospores (swarm spores) and these are set free. Sexual reproduction has not been seen in some soil species, but most species are heterogamous and homothallic. The Zygomycetes are subdi- vided into two orders, the differ- entiation being based largely upon the structure of the non-sexual spores, the Entomophthorales mul- tiplying by conidia, and the Mu- corales reproducing mostly by sporangiospores. The so-called co- nidia of these phycomycetous fungi are not true exogenous conidia as are found in the Ascomycetes, for in some cases at least careful micro- scopic examination shows that they are really small sporangia con- taining only one or a few sporangiospores. They are sometimes re- ferred to as sporangiola. It is also rather difficult to divide the two orders sharply on the basis described above, as some of the Muco- 81 Fig. 31. Pythium (1, 2, and 5, un- identified species from soil; 3 and 4, from various authors) : 1, mycelium and sporangia; 2, intercalary spo- rangium; 3, zoospore formation; 4, zoospores; 5, oogonium and anther- idia. 82 MOLDS BELONGING TO THE PHYCOMYCETES rales are found to form definite sporangia with numerous sporangio- spores at the ends of the sporangiophores, and conidia (or spo- rangiola) on lateral branches. Other species which resemble the Mucorales in all other respects (and are therefore retained in that order) reproduce only by conidia. Entomophthorales. Members of this order will not be encountered in bacterio- logical work. As their name implies, they are mostly parasitic on insects. One of the best known, Empusa Muscae, causes a disease of the common house fly. The my- celium penetrates the body tissues and forms sporophores on the surface (Fig. 32) . The flies are killed by the fungus, and are frequently found in the fall on walls or windows covered with a whitish powder- like coating. When mature, the conidia are projected forcibly for a distance of several millimeters, and form a white deposit about the dead fly, especially discernible upon window panes (Fig. 33). Mucorales. There are three extensive monographs on the Muco- rales and a very excellent treatise on the Phycomycetes as a whole.^ Fig. 32. Stained section through the body wall of a house fly infected with Empitsa Muscae, showing the development of con- idiophores and conidia. Note the multinucleate character of the my- celium. Fig. 33. A fly dead of Emrmsa Muscae infection. The white powder surround- ing the fly is composed of conidia which have been discharged for some distance. Lendner^ divides the order into two suborders and eight families, Naumov " into three suborders and eight families, and Zycha ' into three suborders and six families. The limits of genera as well as of MUCORACEAE 83 families differ with the different authors. All agree on using the distinction between asexual reproduction by sporangiospores or by conidia (sporangiola) as an important point in subdividing the order. The following key derived from Zycha gives the characteristics of the families. FAMILIES OF MUCORALES A. Sporangia single, multispored sporangia always with columellae. 1. All sporangia multispored. MUCORACEAE 2. Sporangiospores with two kinds of spore-forming apparatus, terminal multi- celled sporangia, and numerous lateral verticillate sporangiola, each with few spores. THAMNIDIACEAE B. Sporangiola or conidia united on special sporophores. 3. Spherical, single, or many-spored sporangiola on specialized enlargements of the fruiting hyphae. CHAENOPHORACEAE 4. Long chains of small sporangiola usually on specialized basal cells. IMostly parasitic on Mucorales. CEPHALIDACEAE C. AU sporangia without columellae, zygotes surrounded by thick covering of hyphae. 5. Sporangia or zygotes single. MORTIERELLACEAE 6. Sporangia or zygotes united in specialized fruiting bodies surrounded by hyphae. ENDOGONACEAE Mucoraceae. The Mucoraceae are a group of molds, frequently referred to as the bread molds, found abundantly in soil, in manure, on fruits, and especially on starchy foodstuffs. They all have the same general structure and appearance and are easily differentiated from other groups of molds by the coarse, non-septate mycelium, by the abundant and loosely meshed aerial mycelium, and by the lack of distinctive colors, the spores being generally black or brown, the mycelium white or gray. In older literature the term mold is fre- quently restricted to fungi of this type, molds of the type of the Fungi Imperfecti being referred to as mildews. The Mucoraceae and Thammidaceae are distinguished from the other families of the order by the presence of a columella in the sporangium. The columella may be looked upon as a septum sepa- rating the sporangium from the sporangiophore, which has become bulged into the sporangium. In addition to sporangiospores some species also reproduce by chlamydospores which appear as round black swellings, like beads, strung on the mycelium. These may be mistaken for zygospores. Several species also characteristically break up into a series of spherical oidia when immersed in liquid where aeration is not abundant. These oidia, yeast-like in appear-' ance, may reproduce for a while by budding, forming new round cells of the same type. Sporangiola are not formed by the Mucoraceae. 84 MOLDS BELONGING TO THE PHYCOMYCETEg Zygospores may be formed by the fusion of neighboring filaments from the same thallus in some cases (as in Zygorrhynchus) or only by fusion of filaments from two neighboring thalli in others (as most of the Mucors and Rhizopus). The first type is said to be homo- thallic, the second heterothallic. As pointed out in Chapter I, in the latter case there is a physiologically distinguishable sex, although the elements are morphologically identical. Since the two sexes can- not be distinguished, they are designated as plus or minus strains rather than as male and female. Sometimes bodies resembling zygo- spores may be formed without fusion of the hypha with another. Such spores are called azygospores. They may be formed by an isolated thallus, or only by hyphae which approach filaments from another thallus of the opposite sign. It is noteworthy that plus and minus strains of different species may exhibit this evidence of sex when grown in contact, even though actual conjugation does not occur. The zygospores are usually large cells, generally black, and with a rough, w^arty exterior. The filaments which form them become expanded near the spore, forming broad supporting bands called sus- pensors. These are sometimes of characteristic form. Unfortunately, zygospores are seldom formed in cultures on artificial media in the laboratory. They are found most frequently on strains freshly iso- lated from their natural habitat. Naturally, with heterothallic varie- ties no zygospores will be seen unless both plus and minus strains are included in the culture. According to Blakeslee, prune extract and moistened slices of bread are media most favorable to the production of zygospores. The following key covers those genera of the Mucoraceae which contain species likely to be encountered by the bacteriologist. A. The fungus spreads over its substrate by stolons or runners. 1. Sporangiophores arise at the nodes of the stolons. RHIZOPUS 2. Sporangiophores arise at the internodes. ABSIDIA B. No stolons or runners are formed. 1. Sporangiophores are simple or branched; sporangia borne apically on the sporangiophore and its branches. a. Zygospores formed from equal gametes, usually heterothallic. aa. Never parasitic on other Mucorales. MUCOR bb. Facultative parasite on certain genera of Mucorales. PARASITELLA h. Zygospores formed from unequal gametes, homothallic. ZYGORRHYNCHUS 2. Sporangia borne only on the lateral circinate branches of the sporangiophore. a. Sporangia globular, columella not constricted. CIRCINELLA b. Sporangia pear-shaped, columella constricted. PIRELLA MUCOR AND RHIZOPUS 85 Mucor and Rhizopus. Of the various genera of the Mucoraceae, Mucor and Rhizopus contain most of the species of Phycomycetes encountered in bacteriological work. Mucor and Rhizopus may be readily differentiated from each other. Because of runners, or sto- lons, Rhizopus tends to cover the surface of the agar plates rapidly, to climb the sides of the Petri dishes and fill the latter with mycelium. The rhizoids, or holdfasts, attach themselves to the under sides of the lids. Although Mucors may fill up the Petri dish with mycelium, they do not thus attach themselves to the lid. Fig. 34. Mucor sp.: (a) young sporulating head; (b) mature sporangium; (c) spores being liberated from sporangium; id) columella after scattering of spores. From S. A. Waksman and R. L. Starkey, The Soil and the Microbe, 1931. The rhizoids or holdfasts of Rhizopus may be seen readily by focusing through the lid of the Petri dish with the low-power lens. The sporangiophores of Rhizopus arise from the nodes of the runners, i.e., at the point where the holdfast or rhizoids are formed. In Mucor the sporangiophores are formed from all parts of the thallus. In Mucor the columella is always either round, cylindrical, or pear- shaped, never hemispherical, and is continuous with the sporangio- phore. In Rhizopus it is hemispherical and rests in a cup-shaped expansion of the sporangiophore called the apophysis (Figs. 34 and 39). In Mucor the spores, though varying in form, are always smooth and regular; in Rhizopus they frequently appear to be angular be- cause they collapse readily when mounted in water. Some species of Mucor show a positive phototropism. Sporangiospores of these species bend toward the source of light. This does not occur with Rhizopus. 86 MOLDS BELONGING TO THE PHYCOMYCETES Fig. 35. Mucor hiemalis: a, sporangiophores ; b, sporan- gium; c, columellae; d, spores. There are many kinds of Mucor that, are worthy of specific rank even to a conservative taxonomist, but owing to the large number of species (Lendner recognizes 51 species, Naumov 93 species and several varieties, and Zycha 42 species) keys for identification are not given here, but reference is made to the original monographs. Lendner's key, in its essential points, is given in the first edition of this book and in Oilman and Abbott's ^ paper on soil fungi. In some ways the keys and description of species by Naumov and particularly by Zycha seem to be pref- erable. Keys based on this system will be found in Oilman,- but those in the original Zycha are more complete. The characters used for identification naturally vary with differ- ent authors. Lendner's system which has heretofore been generally followed uses as important characters for classification the occur- rence of branching of the sporangiophores, their height and thickness, the diameter of the sporangium, the length and thickness of the columella, the dimensions of the spores, the degree of diffluence of the sporangial membrane, the form of the columella, and the shape of the spores. Since zygospores are so rarely formed in cultures, they cannot usually be used in diagnosis. Many of the characters given above are obviously subject to considerable variation even in a single individual and cannot be relied upon too much. The occurrence of branching of the sporangiophore is the most important single character. It cannot be easily determined in many cultures because of the close inter- twining of the hyphae. The develop- ing sporangiophores are frequently un- branched; therefore, one cannot rely upon observations made at the edge of the growing colony where the mycelium is not so dense. Branching is best observed in slide cul- tures of the type shown in Fig. 29. A binocular dissecting micro- scope is of gi-eat value. Three main types of branching are recog- nized: the Monomucors with unbranched sporangiophores, as in Fig. 35; the Racemomucors, with racemosely branched sporangio- FiG. 36. Mucor racemosus: a, sporangiophore; b, sporangium; c, columellae; d, spores; e, chlamydospores on the aerial mycelium ; /, chlamydospores on the submersed mycelium. MUCOR AND RHIZ0P17S 87 phores (i.e., a main stem with lateral branches) as in Fig. 36; and the Cymomucors, with sporangiophores typically branched as in Mucor circinelloidcs (Fig. 38) but frequently rather irregularly branched. Zycha uses in his scheme of classification the presence or the ab- sence of thallospores (Kugelgemmen) and Naumov uses the color of the colony mycelial growth and specialized organs. Neither of these two later workers puts so much stress on the branching of the sporophore. Otherwise, the characters used for identification of species of Mucor are much the same as those used by Lendner. Fig. 37. Mucor racemosus. Production of oidia by submersed mycelium, and multiplication of these by budding. Photomicrograph by dark field illumination. The same authorities are to be consulted for identification of species of Rhizopus. Zycha is much more conservative in his tax- onomy in this genus. He recognizes only 8 species, whereas Lendner describes 22 and Naumov some 30 species. M. Mucedo, being easily obtainable if fresh horse manure is incu- bated in a moist chamber, is perhaps the best-known species of Mucor. It has frequently been chosen as a type of the genus in physiological experiments. It is proteolytic and also capable of splitting fats. It is a cause of spoilage of various foodstuffs at times, is supposed to play a part in the ripening of snuff, may cause a decom- position of leather, and is found in retting flax. It may be recog- nized by the unbranched sporangiophore and cylindrical columella. No oidia or chlamydospores are formed. It is heterothallic. See Fig. 25. 88 MOLDS BELONGING TO THE PHYCOMYCETES M. hiemalis is very similar but the columella is typically spherical (Fig. 35). It is concerned with the retting of flax, secreting an enzyme which dissolves the middle lamella of the intercellular sub- stance. It digests starch and gelatin and ferments dextrose but not sucrose. It also is heterothallic. M. piriformis is recognized by its very large sporangia and pear- shaped columella. It is found on spoiled fruit and is claimed to be a cause of soft rot of pears. M. racemosiis is the most common of the Racemomucors, and though of little practical importance it is of some scientific interest. It is remarkable because it produces an al- coholic fermentation of sugars, and, when it is submerged in sugar solutions, the my- celium breaks up into a series of spherical oidia (Kugelzellen or Kugelgemmen in Ger- man literature) which may continue to grow as single cells by budding, like yeasts (Fig. 37). The association of the yeast-like form with alcoholic fermentation naturally at- tracted great interest. It is curious that these round oidia are formed only by Mucors which also produce alcohol. Their resem- blance to yeasts is only superficial, as they are much larger and each cell contains many nuclei. M. racemosus is easily rec- ognized by the characteristic branching of the sporangiophores. The sporangial wall is frequently covered with tiny crystals of calcium oxalate, and the columella is some- what pear-shaped. Inoculation into glucose broth fermentation tubes will show the pro- duction of gas and yield the spherical budding cells in the sediment. Perhaps the most striking character is the abundant formation of the jet-black chlamydospores in the aerial mycelium. M. racemosus may be distinguished from the closely related M. erectus and M. fragilis by the form of the columella. The latter two species also form slight amounts of alcohol from sugar and give rise to budding oidia in the submerged portions. M. circinelloides is frequently encountered in soil cultures. It ex- hibits true cymose branching (Fig. 38). Budding oidia are formed in mycelium submersed in liquid media. M. plumbeus is also a common soil form. It derives its name from the lead-gray color of Fig. 38. Mucor circi- nelloides: sporangio- phores showing typical cymose branching. MUCOR AND RHIZOPUS 89 the mycelium. It may be identified by the peculiar spiny columella and the prickles on the spores. Although the preceding species form but small amounts of alcohol, another Mucor belonging to the Cymomucor group, M. Rouxii (some- times called M. Rouxianus) has been used industrially for the pro- duction of alcohol. It secretes both diastase and zymase and can therefore produce alcohol directly without any malting process. It is used in the Orient for preparing alcoholic beverages from rice, which are often called wines. The fungus is sometimes marketed under the name Chinese yeast in little balls of rice meal, much as yeast cakes are marketed in the Occident. It has been used commercially for alcoholic production in Europe. More than 5 per cent of alcohol is produced in the fermentation. This species may be recognized by the characteristic Cymomucor branching of the sporan- giophores, the spherical columellae, and large oval spores. In the sub- merged mycelium, large irregular, thick-walled cells may be formed. Like the Racemomucors mentioned above, black chlamydospores appear in the aerial mycelium and budding yeast-like cells in submerged portions. Abundant fat globules may develop in the mycelium, especially on starchy substrates; these take on a deep yellow color. There are several other species related to M. Rouxii which have been isolated from other oriental yeasts, as M, Prainii from India and M. javanicus from Java. Some authors have grouped together those Mucors whiish tend to break up into round oidia, or to produce numerous chlamydospores, in a separate genus, Chlamydomucor, but these structures are found in so many different kinds of molds that they should not be given much weight in classification. Rhizopus nigricans is by far the most common of all the molds be- longing to the Phycomycetes. It is continually encountered in all kinds of bacteriological work as an air contamination, and is par- ticularly annoying because of its ability, by means of its stolons, Fig. 39. Rhizopus nigricans: a, mature sporangium; b, ruptured sporangia, showing the flattened columellae (note the funnel- shaped expansion, apophysis, of the sporangiophore beneath the columella) ; c, spores ; d, a rhi- zoid or holdfast; e, zygospore; /, diagram showing mode of growth by runners. 90 MOLDS BELONGING TO THE PHYCOMYCETES rapidly to overgrow Petri plate cultures (it can cover the entire sur- face of the agar in 24 hours). It is especially important as a cause of spoilage of fruits and stored potatoes, especially sweet potatoes. The small fruits, espe- cially strawberries, are particularly susceptible. The disease in strawberries is known as leak, because of the softening and dripping of the fruit. It is a source of considerable loss in shipment and is prevented by refrigeration. In sweet potatoes, a characteristic soft rot is produced, the main factors determining which are injury to the potatoes and humidity of the storage bins. For a bibliography of the rots caused by R. nigricans, see Heald.* Absidia. The genus Absidia is characterized by the formation of sporangiophores arising from the arched stolons themselves (rather Fig. 40. Absidia sp. from soil: zj^gospore and suspensors with appendages; above, diagram of sporangia and rhizoids. than at the holdfasts, as in Rhizopus). Further differential charac- ters are the rounded or pear-shaped columella, which is quite differ- ent from the flattened hemispherical columella of Rhizopus, and the formation of peculiar curled filaments which surround the zygo- spores, arising from the suspensors. Some species are homothallic. Zygorrhynchus. In Zygorrhynchus the zygospores are formed by the fusion of neighboring branches of the same hypha. This mold is MUCORALES IN FAMILIES OTHER THAN MUCORACEAE 91 therefore homothallic. But there is some apparent differentiation of the conjugating branches, one being larger than the other; only the larger branch develops into a suspensor. This condition then rather approaches the formation of oospores in Saprolegnia (compare Fig. 41 with Fig. 24). Zygorrhynchus is an exception to the isogamy general in the INIucorales. Zygorrhynchus Moelleri is one of the most important of the Phyco- mycetes found in the soil. It is particularly abundant in loose sandy Fig. 41. Zygorrhynchus Moelleri: a, sporangiophore ; h, columellae; c, spo- rangiospores; d, chlamydospores in submerged mycelium; e, zygospore. soils, sometimes forming enough mycelium to bind the loose particles together. In subsoils it is frequently the only fungus present. Soils poor in organic matter seem then to be especially favorable for its growth. It is active in ammonification, but does not decompose cellulose. Circinella. In Circinella the sporangia are formed only on lateral branches, not at the tips of the sporangiophores. These lateral branches frequently arise in whorls, six or eight branches arising from one point at regular intervals along the aerial filaments of mycelium. These lateral branches are also peculiarly curved upon themselves, a condition which is also found in Mucor circinelloides (Fig. 38). Species of Circinella are frequently found on the drop- pings of various animals. Mucorales in Families Other than Mucoraceae. Thamnidium is the only member of the Thamnidiaceae frequently encountered. It has 92 MOLDS BELONGING TO THE PHYCOMYCETES the characteristic of the family already described in the key. See Fig. 42. Cunninghamella (Chaenophoraceae) is a common air con- taminant and is frequently isolated from soils. It has very tenuous TW. 42. Thamnidium : partly diagrammatic, showing terminal sporangium with columella and numerous lateral sporangiola without columellae. rhizoids and forms clusters of unicellular conidia (sporangiola) on the swollen end of a hypha. See Fig. 43. Syncephalastrum (Cepha- lidaceae) is also occasionally encountered. In this several cells in chains are found attached to a sporophore with a swollen tip. Each Fig. 43. Cunninghamella sp.: 1, most of the sporangiola removed from vesicular enlargement of sporophore; 2, small cluster of sporangiola. chain is enclosed within a sporangial wall, but the appearance of the spore head does not always reveal the endogenous character of the spores. See Fig. 44. LITERATURE 93 Mucorales Parasitic on Mucorales. Many spe- cies of the Mucorales are frequently parasitized by other fungi and since both host and parasite are of the same order it may confuse the uninitiated into believing that two kinds of spores are pro- duced from the same hypha. If the parasite be- longs to the Cephalidaceae, and the host to Mucor or Rhizopus, the production of conidia from a sporophore, apparently arising from the same hypha which bears the sporangium, should make one suspect that the culture is parasitized. There are several genera of Cephalidaceae and descrip- tions and discussions of this parasitism will be found in the books of Zycha and of Gaumann. Still more confusing is Parasitella simplex which belongs to the INIucorales and is similar enough to Mucor to be put in that genus by some authorities. An interesting fact in connection with the hetero- thallism of Parasitella is that plus strains of the parasite are said to infect only minus strains of the host and vice versa. It would thus appear that the parasitism may have arisen from an abortive attempt at hybridization. On several occasions in our laboratories many freshly isolated cultures have been parasitized by other fungi when "iso- lated" but for years at a time we have not encountered this phe- nomenon. LITERATURE 1. FiTZPATRiCK, H. M., The Lower Fungi. Phycomycetcs, McGraw-Hill, New York, 1930. 2. Oilman, J. C, A Manual of Soil Fungi, Collegiate Press, Ames, Iowa, 1945. 3. Oilman, J. C, and E. V. Abbott, A summary of the soil fungi, loion State Coll. J. Sci., 1, 225 (1927). 4. Heald, F. D., Manual of Plant Diseases, McOraw-Hill, New York, 2nd ed., 1933. 5. Lendner, a., Les Mucorinees, de la Suisse, Bcitr. Kryptogamenflora Schweiz., 3, 1 (1908). 6. Naumov, N. a., Cles des Mucorinees, translated from the Russian by S. Buchet and I. Mouraviev, Lechevalier, Paris, 1939. 7. Zycha, H., Mucorineae, Kryptogamenflora Mark Brandenburg, 6A, 1 (1935). Fig. 44. Syn- ccphalastrum sp., isolated from soil. Chains of two or three spores surround- ed by a mem- brane are formed over the inflated end of the sporo- phore. These spores are some- times referred to as conidia, though they are really formed in small sporangia. CHAPTER V MOLDS BELONGING TO THE FUNGI IMPERFECTI AND THE ASCOMYCETES Among the molds encountered by the bacteriologist, Ascomycetes are comparatively scarce. The Fungi Imperfecti, however, are abun- dant. Since many of the latter forms show a similarity to or identity with the conidial stages of known Ascomycetes, both classes may conveniently be considered together here. Many species of Fungi Imperfecti, often only after extended study, have been found to be asexual stages of perfect fungi, and un- doubtedly more species in the future 'will be found to have a perfect stage. But all species of Fungi Imperfecti are not necessarily merely conidial stages of perfect fungi ; in the course of evolution they may have permanently lost their ability to form sexual spores at all. Some of the most widespread and vigorous molds have never been shown to possess a perfect stage although they have had almost con- tinuous study for over half a century. The point of view that Fungi Imperfecti are merely conidial stages of sexual fungi has led many mycologists almost to ignore them, or to treat them in their discussions with the perfect fungi which have a similar conidial apparatus and, often, as here treated, to give minor attention to the asexual stage. This in no way helps the worker who wishes to know the identity or to understand the morphology of the mold which he isolates from the lesion of a patient or one which he has found to have industrial or biochemical impor- tance. His mold, as he must study it, does not form sexual spores. Even in those in which the perfect stage has been found, these sexual spores are in many cases produced only rarely and under exceptional conditions. Such a worker must depend upon classifications based on the asexual stage. At best, any classification of the Fungi Imperfecti must be more or less artificial, not necessarily indicating phylogenetic relation- ships. However, it is evident that in many cases species closely re- lated on the basis of their sexual spores had already been classified together on the basis of their conidial stages.®- ^^ The problem is complicated by the fact that in many cases it is somewhat difficult to decide just what are the conidia. As pointed 94 VUILLEMIN'S CLASSIFICATION 95 out in the first chapter, many fungi, in* addition to their spores mul- tiply by the formation of oidia or yeast-like cells, by fragmentation of the mycelium, or by budding. These have been differentiated from conidia by the fact that the latter do not grow at once — they must first imbibe water and germinate. But at least some of the growth cells, although they may resemble conidia, are capable of growing at once if separated from the parent mycelium. One may find in some species all transitions between these growth cells and conidia. So it happens that the same organism may be classified in one genus by one authority, and in an entirely different genus by some other writer, owing to different interpretations of the same structure; for instance, the organism of thrush, now generally con- sidered as a species of Candida, has been placed in half a dozen or fnore genera. Moreover, the same fungus may form more than one type of conidium, or may produce its conidia on more than one type of fruiting body. Thus one of the causative agents of chromoblasto- mycosis may produce conidia from flask-shaped conidiophores on some hyphae and show branching chains of conidia from unswollen conidiophores on others. Depending upon which spore-bearing ap- paratus predominated, or which one was seen by or most impressed the worker, the same fungus has been classified in the genus Phialo- phora or in the genus Hormodendrum (Cladosporium). The classification of the Fungi Imperfecti followed by most my- cologists is based upon the system proposed by Saccardo. Another, quite different, classification was introduced by Vuillemin and his system, or modifications of it, is much used in France, Italy, and Latin America. Since so much of the literature on medical mycology has come from those regions, this system cannot be ignored by the bacteriologist. Vuillemin's Classification. Vuillemin divided the Fungi Imper- fecti into three orders on the basis of the characters of the reproduc- tive bodies. Later a fourth order, the Microsiphonales, was added to include the actinomycetes, but more recently this order has been re- duced to a family. Vuillemin's ^~ classification is based upon what were then new interpretations of reproductive bodies. See page 7. CLASSIFICATION OF THE FUNGI IMPERFECTI (VUILLEMIN'S SYSTEM) Order Thallosporales, reproducing by thallospores. Suborder Blastosporineae, reproducing by blastospores. This group includes the non-spore-forming yeasts and fungi with yeast-like forms, as Candida. 96 THE FUNGI IMPERFECTI AND THE ASCOMYCETES Suborder Arthrosporineae, reproducing by arthrospores. This group includes such forms as Geotrichum candidum and, according to the later rearrange- ment, the actinomycetes. It comprises two families. Family Mycodermaceae, with "normal" septate mycelium. Family Nocardiaceae, with "non-septate" mycelium, very fine, of bacterial dimensions (the actinomycetes). Order Hemisporales, reproducing by hemispores. This order contains but one unimportant genus, Hemispora. Order Conidiosporales, reproducing by conidia. Suborder Aleuriosporineae , reproducing by imperfect conidia or aleuriospores. The ringworm fungi would belong here according to Ota and Langeron. Suborder Sporotrichineae, reproducing by true conidia, which are not inter- preted as being borne upon conidiophores, as in Sporotrichum. Suborder Sporophorincae, reproducing by true conidia borne upon true conidiophores. This group would contain most of the common molds of the Fungi Imperfecti, save those of the next suborder. Suborder Phialidineae, reproducing by true conidia borne upon phialides (cfr sterigmata), including Aspergillus and Penicillium. Saccardo's Classification. Saccardo's arrangement makes a pri- mary subdivision into three orders upon the following basis: Certain of the fungi parasitic on plants form conidiophores within a globular mass of protecting mycelium, called a pycnidium, from which conidia are discharged when mature through an opening on the surface of the plant tissue; these forms are placed together in an order known as the Sphaeropsidales. Other plant pathogens produce conidiophores closely packed together, from the surface of a fiat, plate-like mass of pseudoparenchyma on the surface of the host plant, known as an acervulus; such species comprise the order Melanconiales. The third order, the Moniliales (frequently called Hyphomycetales), contains the remaining forms, whose conidiophores are produced neither in pycnidia nor upon acervuli, but are formed from superficial hyphae over the entire surface of the fungus colony. Those molds of interest to the bacteriologist will, of course, all be found in this last order. The Moniliales are further subdivided into groups which have in some works been given the rank of families, in others of suborders. Two of these are based upon a characteristic grouping or bunching of the conidiophores. In the Stilbaceae the conidiophores are clus- tered to form characteristic stalked bodies of cylindrical form, the coremia. In the Tuberculariaceae the clusters of conidiophores form globose bodies witho«t stalks, the sporodochia. Neither of these families contains species important to the bacteriologist, save that coremium-forming Penicillia have sometimes been included with the Stilbaceae and the important genus Fusarium is usually included with the Tuberculariaceae, SACCARDO'S CLASSIFICATION 97 The remaining Moniliales are grouped into two families according to the color of the mycelium. If this is hyaline or brightly colored, the mold is included in the Moniliaceae (Mucidinaceae) ; if it is dark, smoky (black or shades of deep grey, brown, or olive), the mold belongs to the Dematiaceae. This is not a good character for such an important division. Many genera vary considerably in this character and frequently hyaline variants (saltants) are produced in some cultures of the Dematiaceae. These if isolated in nature would be included in a different family from their parent! Perhaps it is fortunate that the Fungi Imperfecti have not been accorded the continual reclassification to which mycologists have (for very good reason) subjected the perfect fungi. Since a natural system at our present stage of knowledge can hardly be attained, any classification has been one for convenience only and such reclassifica- tions as have been proposed are not very great improvements upon the original Saccardo system. The imperfect yeasts and yeast-like fungi are put into four additional families of the Monihales. See page 285. A very large number of genera and species of the Moniliales have been described. A very large proportion of these are rare, many of them of doubtful validity. To present a complete key would defeat the purpose of this book. But experience shows that the molds likely to be encountered by the bacteriologist will fall within a few genera ; fully three-fourths of those encountered in routine work will be species of Aspergillus and Penicillium. We shall, therefore, describe very briefly only a few genera and indicate where further informa- tion may be obtained. The key to the molds belonging to the Fungi Imperfecti published by Lindau in Rabenhorst's Kryptogamenflora has been followed by most mycologists. It has been published in translation in Waksman's Principles of Soil Microbiologij ^^ and in Buchanan's Bacteriology.^ Very complete keys with descriptions of species found in soil will be found in Oilman's Manual of Soil Fungi} Soil forms are naturally likely to appear as laboratory contaminants. The fungi parasitic to man and animals will offer special difficul- ties. A very excellent Manual of Clinical Mycology by Conant * and associates gives descriptions and illustrations of the most com- mon and important species. INIany other forms will be found in Lewis and Hopper's Introduction to Medical Mycology.^" The older books of Castellani and Sartory are no longer of much value. A very large, fairly recent book of Dodge ^ may also be consulted. Jlowever, the inclusion in it of a large number of species for which 98 THE FUNGI IMPERFECTI AND THE ASCOMYCETES pathogenicity has not been proved convincingly, together with an excessive splitting of genera and species, and the inclusion of many fungi. under more than one name with no indication that they refer to the same fungus, makes this book of limited value. Interested bacteriologists are referred to the international journal Mycopath- ologia for recent work on medical mycology. Under an international editorial board, published in Italy and printed in the Netherlands, it is a war casualty. It is to be hoped that this journal will be revived. Aspergillus. The word aspergillus (or aspergillum) means a spe- cial type of brush for sprinkling holy water used in the ceremony the ''Asperges" which Latin word is the first one of Psalm 51:7 that is sung during the ceremony. The re- semblance of the common Aspergillus niger to this brush is striking. The literature on this important genus is enormous but two recent books have so thoroughly covered the field from so many points of view that further references here are neither necessary nor desirable. These books are Thom and Church's monograph The Asper- gilW^^ and Thom and Raper's A Manual of the Aspergilli}^ The genus Aspergillus may be rec- ognized by the very characteristic arrangement of the conidia and conidiophores. The unbranched conidiophore arises from an enlarged cell of the vegetative mycelium, the foot cell, and terminates in a swollen portion, the vesicle. From the latter, there arise a number of little stalks of characteristic bottle shape, the sterigmata or, as they are sometimes called, the phialides. From these primary sterigmata there may arise one, two, or, rarely, more secondary sterigmata. From the tips of the primary sterigmata in some species, or from the tips of secondary sterigmata in those species which have them, chains of conidia are borne. A conidium is formed by a partial abstriction of the sterigma, which has elon- gated lightly, followed by formation of a septum separating the conidium from the sterigma. Further cutting off of the tip of the sterigma thus results in more conidia. Since these spores remain attached together, they occur in chains and the whole arrangement presents the spores arranged on compact masses, "conidial heads," at the tip of the conidiophores. In some species the sterigmata and Fig. 45. Aspergillus niger: a, vesicle and sterigmata; h, foot cells; c, conidia. ASPERGILLUS 99 chains of conidia are borne over the whole vesicle and the spore head is thus spherical ; in other species the sterigmata are found only on the upper part of the vesicle and the sterigmata and chains may spread out, in cross section like a fan, or all sterigmata may point upward, and the head appears like a long cylinder. In some species of Aspergillus, ascospores are found, i.e., these molds are perfect fungi and should be included in the Ascomycetes. A strict interpretation of International Rules would seem to make it obligatory to transfer these species to the genus Eurotium as many authors have done. However, the fact that the whole group of Aspergilli appear to be so obviously homogenous, falling naturally into one genus, together with the fact that so many species do not form ascospores at all, makes it preferable for the sake of con- venience, if for no other reason, to follow the lead of most of the workers who have actually worked extensively with these molds, and put all species in the genus Aspergillus. The ascospores are all more or less of the same basic pattern. The "thickening of the cell-wall of the asco- spore develops in the form of two sym- metrical valves suggesting the arrange- ment found in the shell of a bivalve mollusk {Venus mercenaria) . The ripe ascospore is commonly shaped as a double convex lens with the valves more or less closely in contact at the edges. A series of variations upon this basic pattern occur and characterize particular species." (Thom and Raper.) The ascospores are borne eight per ascus in round to oval asci and the asci are scattered throughout the perithecium in an irregular arrangement. The perithecia are often very abundant and they may determine the color of the colony. In most species of Aspergillus the perithecia are formed on ordinary media with sugar, either regularly and abun- dantly or not at all. They are found throughout the aerial mycelial growth. They are formed after homothallic conjugation. By crush- ing the perithecia under a cover slip, one can usually demonstrate the Fig. 46. A conidiophore of Aspergillus nidulans, showing the foot cell, stalk, vesicle, and chains of conidia. Photomicro- graph from a slide culture. Fig. 47. Aspergillus amstelodami: small conidial head and two small perithecia. X375. From Charles Thorn and Kenneth B. Raper, U. S. Dept. Agr. Misc. Pub. 426 (1941). 100 THE FUNGI IMPERFECTI AND THE ASCOMYCETES asci and ascospores readily. In some cases the asci may be few in number and in some species one may find bodies having the general appearance of perithecia but containing no asci. These are known as sclerotia and are usually looked upon as being incomplete peri- thecia. To identify species of Asper- gillus has been considered an all but impossible task but, as indi- cated in the first edition of this book, the difficulty of identifica- tion is not insurmountable! It would be a simple task for the author to construct some sort of key from Thom and Raper's de- scriptions or to reproduce theirs but, beyond giving the names of the species, little would be gained. Rather the worker is referred di- rectly to their manual.^*' There he will find that by means of a key based primarily upon the color of the conidial heads, the peri- thecia, or the colonies on Czapek's agar, he can readily place his isolate in one of fifteen "species groups." Or he can arrive at the same group by means of another key, based primarily upon morphol- ogy. Then the worker will have to refer to a section devoted to that species group where he will find the fullest kind of de- scription of each separate species. In some cases it is apparent that the identi- fication of species wall be readily made from the keys to species groups and then the diagnosis can be verified from the de- scription itself. In other cases it appears that this will be much more difficult. In all cases the extent of expected variation within a species is indicated, something sadly lacking in many taxonomic treatises. Throughout he will find excellent photo- graphs in abundance, of colonies (many in color) and of morpho- logical details. He will also find exact directions to duplicate the conditions under which the moHs so described and photographed Fig. 48. Section through a perithecium of Asper- gillus sp. showing develop- ment of asci and asco- spores. ASPERGILLUS 101 were made. Identification of a species group only is, for most pur- poses, in effect no identification at all; but it may satisfy the worker, which was often unfortunately, and to Henrici's misgiving, the effect Fig. 49. Aspergillus fimiigaius (Xl^OO) : conidial head. Photograph by Dr. Kenneth R. Raper. of the key to species groups published in the first edition of this book. Hence no key is given here. Aspergillus species are found on a wide variety of substrates. They are numerous in soil and particularly so on dried vegetable matter, as hay and grains. They can apparently tolerate very high osmotic concentrations and extract their necessary water from rela- tively dry substrates. In contrast to Penicillium they can, as a 102 THE FUNGI IMPERFECT! AND THE ASCOMYCETES group, tolerate higher temperatures, many of them growing readily in the 37° C. incubator, a temperature too high for most molds. Among the important species, many of which will be discussed in later chapters, are A. jumigatus and A. nidulans, pathogenic to man and other animals but also occurring widely distributed as sapro- «8 ^» , Fig. 50. Aspergilltts Oryzae (XSOO): conidial head. Kenneth R. Raper. Photograph by Dr. phytes; A. Oryzae which, owing to the abundance of various enzymes, has so many industrial applications; A. niger, and related species, used in citric and gluconic acid production and in assaying available phosphorus and potassium in soil; A. clavatus and other species from which antibiotics may be prepared; A. terreus which produces itaconic acid from sugar; and A. niveus which produces citrinin. Penicillium. The genus Penicillium is characterized by the produc- tion of conidia from sterigmata much like those of Aspergillus, which are produced in clusters or whorls, known as verticils, from short PENICILLIUM 103 branches (metulae) given off, usually also in verticils, from the tip of the conidiophore. The appearance of a low-power view of the spore head is somewhat like that of a brush; and the spore head is called a penicillus, which is Latin for a brush. Some species pro- duce ascospores after homothallic conjugation. These have been extensively studied.*' Our knowledge of the Penicillia has been greatly extended in re- cent years by the extensive work of Biourge ^ and the still more exhaustive treatise of Thom.^^ The latter authority describes (in- cluding three closely related genera) over six hundred species! The differentiation of these is more difficult than that of Aspergilli. In such a condition it is obvious that the precise determination of par- ticular strains must remain a task for specialists. In the earlier literature (and all too frequently even today) papers were published on the ecology, physiology, morphology, etc., of vari- ous "species" of Penicillium without an accurate determination of identity. For instance, any and all green forms were often referred to as Penicillium glaucum. This term has been used so indiscrimi- nately for a variety of species that the name is worthless and should be avoided. The problem of identifying species of Penicillium (and to a less extent other genera as well) is not one that is easily solved by the bacteriologist who works on the biochemistry of molds. If he cannot identify them accurately (and few mycologists even at- tempt to identify species of Penicillium) or if he cannot get them identified by specialists, he had better not give them Latin binomials at all, but deposit them in a culture museum if possible, or at least keep his cultures so that others may verify or extend his work. There are very few of us who have done biochemical work on molds who have failed to make such insufficient identification or to let cul- tures die out. There is thus no way to find the identity of the molds that were studied. One might as well make a physiological study of a "grasshopper," a "wild sunflower," or a "spore-forming bacillus." The Penicillia are subdivided by Thom into four sections, and these are further subdivided into subsections. The basis for the primary subdivision is the nature of the branching of the spore heads, whether this is symmetrical about the axis of the conidio- phore or asymmetrical. The symmetrical types are separated into three groups: the Monoverticillata, with a single whorl of sterigmata at the tip of the conidiophore; the Biverticillata-symmetrica, in which the verticils of sterigmata arise from short branches or metulae, which themselves form a verticil on the end of the conidiophore ; and the Polyverticillata-symmetrica, in which three or more stages of 104 THE FUNGI IMPERFECTI AND THE ASCOMYCETES branching occur. The asymmetrical forms make up the fourth group, the Asymmetrica. This classification can be more readily under- stood from a diagram than from descriptions (Fig. 51). These char- iStcpigmata- Metulae Mono- Bi- Poiy- VGPticillat'a V . - Y ' 5(jmmatTica AscjmmGtnca Fig. 51. Diagram illustrating the different t3^pes of spore heads of Penicillia. acters are subject to some variation, even in a single culture, but a dominant tendency will be manifest, Monoverticillata. Certain molds which formerly were classed as a separate genus, Citromyces, fall in this group. The name was Fig. 52. Penicillia from fruits: a, Penicillium expansum; h, P. italicum; c, P. digitatum. given (by Wehmer) to two species producing citric acid from sugar. These forms were first considered as occupying a position transitional between Aspergillus and Penicillium, since some strains showed a PEKICILLIUM 105 slight swelling of the tip of the conidiophore. But Thom places them with the Penicillia, because numerous other typically penicillate forms have been found subsequently, and because the foot-cell char- acteristic of the Aspergilli is lacking. Species of the Citromyces group were investigated at one time as a possible means of producing citric acid commercially, but without much success. Biverticillata-symmetrica. This section contains a fairly homoge- neous group, of which P. luteum is the commonly known species.- Ascospores are formed regularly by some strains of P. luteum in loose masses of pseudoparenchyma, not in well-defined perithecia. This section is also characterized by the production of pigment, in the form of granules on the mycelium, which varies from yellow through orange to red, the color chang- ing with the species, substrate, and age of the culture. P. pinophilum, produc- ing stains on wood, belongs in this sec- tion, as does also the organism used in gluconic acid production. Poly vert icillata-symmetrica. This is a small group of four species, none of which is of practical importance. Asymmetrica. The great majority of the Penicillia fall in the asymmetric group, including most of those of. any economic impor- tance. This section is divided into several subsections. Velutina. The colonies have a velvety appearance. P. digitatum and P. expansum are important species. Brevi-compacta. Colonies are partly velvety, partly woolly in texture, the penicillus biverticillate and showing characteristically a short compact base with divergent sterigmata and conidial chains. P. stolonijerum, growing on mushrooms and other fungi, and spread- ing by means of aerial runners, belongs here. Lanata-typica. This is characterized by an abundance of aerial mycelium which gives the colony a woolly texture. Conidia appear first in the center of the colony after the felt of aerial mycelium has been established. P. camemberti is an important species. Lanata-divaricata. Here the branches of the penicillus are widely divergent and a looser type of spore head is produced than in the other groups. A series of species characteristically found in soil, of Fig. 53. Penicillia from cheese : a, Penicillium roqueforti; b, P. camemberti. 106 THE FUNGI IMPERFECTI AND THE ASCOMYCETES which P. janthellinum is typical, belongs here. The soil species show yellow to red colors in the mycelium ; the spores are green. Funiculosa. Trailing ropes or bundles of hyphae are found at the edge of the growing colony. Fasciculata. Species forming coremia, or tending to form coremia, are grouped together in this section. P. expansum and P. italicum are important. Fig. 54. Gliocladium : a, conidiophore ; b, branch ; c, metulae ; d, sterigmata; e, clus- ters of conidia. From Conant et al., Manual oj Clinical Mycology, 1944. Fig. 55. Paecilomyces. Single sterigmata (a) bend (b) away from the main axis. Sterigmata (c) are elongated. Conidia (d) are oval. Many conidio- phores resemble those of Penicillium. From Conant et al, Manual of Clinical Mycology, 1944. Although species of Penicillium have been found from time to time in various pathological lesions of man, it is doubtful that any of them is truly pathogenic. Gliocladium. Members of this genus form branched spore heads resembling those of Penicillium, but the conidia become surrounded by a mass of slime which bind together all the spores of one spore head into a rounded mass. But the conidia, in some species at least, are formed in chains, as in Penicillium. Paecilomyces. Paecilomyces is differentiated from Penicillium by its longer tubular sterigmata, the tubular processes being bent away from the axis of the sterigma, and by greater irregularity of the branching, which is only in part verticillate. The perfect stage of this fungus has been found at least once.^- Spicaria is a generic name sometimes used instead of Paecilomyces. SPOROTRICHUM 107 Scopulariopsis. This group contains members which were formerly included with Penicillium, but which differ even more markedly than the above. They may form Penicillium-like branching systems (but frequently the branching is irregular) or the conidiophore may re- main unbranched. The terminal branches which form the spores may not be constricted at their apex to a narrow tubular process, as is characteristic of Penicillium. And finally the large, thick-walled, spiny conidia with their ring at the base (Fig. 56) are quite different from those of Penicillium. Species of this genus are the imperfect stages of one or more genera of perfect fungi. Scopulariopsis brevicaulis (formerly Pen- icilliu7n brevicaule) is a common species of some importance. The spores are yel- lowish brown in color. It is important as a cause of spoilage of various substances. Growing more slowly than many other molds, it takes part in the final disinte- ,• p ,, 1 I Ti • X- Fig. 56. Scopulariovsis gration oi the product. It is very active , . ,. .... 7 . - brevicaulis: comdiophores m proteolysis, producing ammonia abun- ^.^^^ conidia. dantly in gelatin cultures. In the presence of sugars, it produces from arsenical compounds a substance, di- ethylarsine, which has a very characteristic garlic-like odor. This reaction has been used as a test for arsenic, the reaction being said to be more delicate than the usual chemical tests. Only arsenous acid or its salts of the alkaline metals may be detected readily, salts of the heavy metals not so surely, and arsenic sulphide not at all. But disagreeable odors may be produced on other substrates, de- scribed as ammoniacal, or like turnips or cabbage. According to Thom it is an important secondary invader and cause of spoilage of Camembert cheese and other dairy products. It may be found, along with other molds, in corks, and may give rise to very disagreeable odors in bottled products which have been stoppered with such con- taminated corks, without any evidence of mold growth in the product itself. Various strains similar to S. brevicaulis have been isolated from cases of infection about the finger nails (onychomycosis) ; that they are really pathogenic is doubtful. Sporotrichum. The genus Sporotrichum is characterized by the formation of its rather small, round, oval, or pear-shaped conidia sessile on the mycelium or on very small conidiophores, not in chains but in clusters. The conidia arise laterally and at the tips of the conidiophores or hyphal strands from all parts of the mycelium. A 108 THE FUNGI IMPERFECTI AND THE ASCOMYCETES number of species is known, mostly saprophytes, but at least one is known to be a plant pathogen and one, Sporotrichum Schenckii, is the cause of an important mycosis, sporo- trichosis. See page 193. Verticillium. In this genus the erect conidiophores are septate and branched. They are arranged in whorls and these branches rebranch also in whorls. From these numerous tips, whorls of round, elliptical, oval, or short spindle- shaped conidia are borne. Either co- nidia or mycelium or both may be hyaline or they may be slightly pig- mented. Trichoderma. Not infrequently one finds growing on plate cultures a mold which is of a very bright pure green color; the aerial mycelium is very abundant, and frequently little tufts of white (sterile) aerial mycelium project above the conidiophores. Microscopic ex- FiG. 57. Sporotrichum Schenckii from stained slide culture. Fig. 58. Verticillium sp.: a, conidiophores in whorls; b and c, conidia attached to conidio- phores; d, conidia. From Conant et al., Manual of Clinical Mycology, 1944. oo d a Fig. 59. Trichoderma: a, mycelium; b, conidio- phores; c, cluster of co- nidia; d, conidia. From Conant et at., Manual of Clinical Mycology, 1944. amination reveals numerous small clusters of conidia attached di- rectly to the tips of the many-branched conidiophores (Fig. 59). This is Trichoderma viride, one of the most numerous of the soil fungi, and a very common contaminant in bacteriological work. It CiEPHALOTHECIUM 109 is the most active of the soil fungi in ammonification; it liberated 57 per cent of the nitrogen in dried blood as ammonia within 12 days in one experiment. It is also very active in decomposing cellulose. Fig. 60. C ephalosporium Acremontuni. On the other hand, it has no diastatic action at all. This common soil fungus is also a common contaminant and, owing to the abun- dance of the spores and its propensity to grow rapidly and spread over a plate, it may be very troublesome in the laboratory. Cephalosporium. In this genus the conidio- phores are short erect branches of aerial hyphae and are non-septate. The conidia are borne one by one at the tips of the conidiophores but successive conidia push them aside and they form into small balls. These balls of conidia are held together by a secretion of sticky material. Conidia are colorless or nearly so and in most species are elongated or elliptical. C ephalosporium Acremonium is the best known species. Certain species of Cephalosporium are pathogenic to man, being among the organisms capable of causing mycetomas. See page 210. Cephalothecium. Cephalothecium roseum, often called Triothecium roseum, is a fairly common bright pink mold. It may be readily identified by its clusters of two-celled conidia formed in clusters at the ends of short conidiophores. The cell closest to the conidiophore is the smallest. It occurs widely upon a great variety of substrates, fruit, wood, paper, soil, and is weakly patho- genic to some plants. Fig. 61. Conidio- phores and co- nidia of Cephal- othecium roseum. Many of the conidia have be- c o m e scattered. 110 THE FUNGI IMPERFECT! AND THE ASCOMYCETES Fusarium. Fusarium is a large, widespread, and very difficult genus. Forty-odd species and many more varieties have been found in soil and frequently they are encountered as air contaminants. ]\Iany other species are of considerable importance as the cause of diseases of plants. One species, Fusarium oxysponim, has been widely distributed in culture among bacteriologists, bearing the label Trichophyton rosaceum, and this culture, supposedly a dermatophyte, Fig. 62. Fusarium Equiseti: above, macroconidia ; middle, mycelium; below, microconidia. XIOOO. Camera lucida drawing bj^ Dr. Roderick Sprague. but actually a saprophyte," has been used extensively to test sub- stances designed to be used in the treatment or prevention of fungus infections of man. Conidiophores arise in verticillate arrangement from short hyphal branches. On these are borne long fusiform or sickle-shaped multinucleate conidia. The crosswalls of the conidia are frequently somewhat indistinct. In addition to these large multi- nucleate conidia, often designated macroconidia, several species pro- duce also small, one- or few-celled, egg-, pear-, spindle- or kidney- shaped microconidia. See Fig. 62. The perfect stage is known in several species. The genus Fusarium has been monographed by Wollenweber and Reinking.^^ Cladosporium (Hormodendrum). One not infrequently finds as a contaminant on Petri plate cultures a rather small dark olive- "BLACK YEASTS" 111 green colony with a velvety surface. The reverse of the colony is almost black. The same mold is found from platings of soil, espe- cially soil with an abundance of decomposing plant residues. These are found most frequently to be organisms designated as Clado- sporiwn herbarum or H ormodendrum cladosporoides. Cladosporium and Hormodendrum have been separated on the basis of production of two-celled as opposed to one-celled conidia. However, the bi- cellular conidia usually do not develop until late, unicellular spores forming first, and cultures which are regularly one-celled may occa- sionally show a few two-celled spores. It is usually agreed that there is but one genus and species may or may not develop two-celled spores. Since Clado- sporium has priority, this name should be used and Hormodendrum reduced to synonymy.^* One species of Cladosporium is the imperfect form of an Ascomycete, MycosphaereUa Tulas- nei, which is parasitic on various plants. In Cladosporium the conidia are formed dif- ferently than in molds like Aspergillus and Penicillium. In these molds the conidia are -n ^o o u j iiG. 63. Spore head formed by a constriction of the tip of the ste- ^f Cladosporium. rigma, which then forms a second spore that pushes the first one ahead of it, and so on ; thus the terminal spore of a chain is the oldest and frequently the largest. In Cladosporium, however, after the first spores have formed on the conidiophore they bud to form secondary spores and no further conidia are formed directly by the conidiophore. Then only the terminal spores bud and in the chains thus formed the youngest, and frequently the smallest, spores are found at the ends of the chains. Moreover a spore may develop more than one new spore by forming more than one bud and thus we find that the chains of conidia are branched (Fig. 63). These molds are found, as was stated, in soil, and they are also found in large number on decaying leaves, straw, and other vegeta- tion on the surface of the soil. They are said to be of some impor- tance in the spoilage of malt and of stored tobacco. From time to time members of this group are reported as causes of superficial skin lesions in man without clear evidence of their pathogenicity. For relationship of Cladosporium to chromoblastomycosis, see page 199. "Black Yeasts," "Torula nigra," "Monilia nigra." There has been described from time to time a series of yeasts or yeast-like organisms which produce a characteristic black color. They have 112 THE FUNGI IMPERFECT! AND THE ASCOMYCETES been named variously Saccharomyces niger, Torula nigra, Schizo- saccharomyces niger, and Monilia nigra. They have been isolated from a variety of substrates, mostly dairy products. Organisms of this type have been found to produce outbreaks of black spots in Emmenthaler cheese. An apparently identical organism as a cause of spoilage of raw sugar has been reported. Similar forms have been isolated from soil, from air, from commercial yeast cakes, from in- sects, and from certain pathological lesions of man where, however, they may not be in all cases the causative agent of the disease. Fig. 64. "Monilia nigra." There has been some question regarding the identity of the various forms described and their proper classification. Henrici examined a dozen or more strains of so-called black yeasts from various sources, and though each strain might be fitted to the description of one or- ganism or another in certain stages of development, they all showed marked transformations of the same general character on continued cultivation, so that he was not convinced that there is more than one species. These morphologic transformations have been very completely de- scribed ajid illustrated by Maurizio and Staub." When first isolated the organisms grow as soft, pasty, yeast-like cultures, at first a pale yellow color but rapidly becoming a dark greenish black. Examined at this stage, they show only oval budding yeast cells. If pour-plate cultures are made, the deep colonies, at first lenticular, will eventually sprout out numerous radiating filaments of mycelium that develop lateral clustery and chains of budding cells exactly as in cultures of ALTERNARIA 113 Candida species. In later subcultures mycelium also develops under aerobic conditions, the growth becoming tougher in consistency, and eventually producing an olive-green aerial mycelium which gives rise to branched chains of conidia. The aerial conidial apparatus closely resembles that of Clado- sporium, the color also being the same. Hansen was of the opinion that these black yeasts are but yeast-like growth forms of dematia- ceous molds of the type of Cladosporium, and this view was also supported by Lindner. Henrici also concurred in this opinion. Several of the strains which were examined by Henrici produced, at times from the' pasty yeast-like growth, at times from the aerial mycelium, characteristic two- celled conidia exactly like those of Cladosporium. This is probably the explanation for the use of the generic name Schizosaccharomy- ces by Marpmann. The above paragraphs are es- sentially as Henrici wrote them in 1930. Since then Henrici and one of us have isolated several more strains of these organisms. For the most part, the "degeneration" (see Chapter II) was as described. In some cases, for long periods of time, they "degenerated" into the form known as Torula nigra, that is, they formed chains of round black spores directly from the mycelium or from short lateral branches. Unlike in Cladosporium, the chains did not branch. Later, some of these strains appeared wdth branched chains of conidia as in Cladosporium but we have some strains that still show the Torula type of growth. This whole group seems poorly understood but it seems clear that the "black yeasts" are but a unicellular growth phase of certain of the Demat- iaceae including Cladosporium and possibly other genera as well. See Chapter 11. Alternaria. Members of this genus are also frequently encoun- tered by the bacteriologist. They form dark olive-green or brown colonies similar to those of Cladosporium, save that the aerial my- celium is much looser, forming a more woolly type of growth. Molds of this group may be recognized by the peculiar spores of rather large size, multichambered, and composed of a number of Fig. 65. "Torula nigra." When first isolated from soil, it grew as a bud- ding 3'east without mycelium. 114 THE FUNGI IMPERFECT! AND THE ASCOMYCETES cells. These conidia occur in chains, sometimes with short stretches of mycelium between the spores. There are a number of species, many of which are plant pathogens. Stemphyllium is a closely re- lated genus. For a discussion of both genera, consult the articles by Groves and Skolko.^ Species of Alternaria have been found growing in pus of super- ficial wounds, and it has been suggested that they have some patho- FiG. 66. Conidia of Alternaria. genie action. But this is probably not true, because the organisms may be growing merely as saprophytes upon the pus, or more likely upon the cellulose 'of the dressings. Their relation to asthma is mentioned on page 136. Helminthosporium. Next to Cladosporium and Alternaria, prob- ably the most common dematiaceous mold encountered by the bac- teriologist is Helminthosporium. The conidiophores usually arise in groups and are unbranched and septate in most cases. The conidia are large, elongate, and cylindrical or ovate. The ends of the conidia may be rounded or pointed. Many, usually more than four, crosswalls are found in each conidium, which is very dark in color. Curvularia is a related genus. See Fig. 67. NEUROSPORA 115 Neurospora. Under the name of Monilia sitophila, this genus has been known for a long time in its imperfect stage. Several years ago it was found that ascospores were formed readily and more recent studies on cytology and genetics by Shear, Dodge, and Lind- gren (see Chapter II) have made this one of the most completely known of fungi. Because the commonest species of the genus, Neuro- FiG. 67. 1, 2, and 3, H elminthosporium sativum; 5 and 6, Curvularia geniculata. X800. Prepared from outline camera lucida drawings of Dr. Roderick Sprague. spora sitophila, is heterothallic, the ascospores are large and easily isolated mechanically, and growth and sporulation are easily in- duced, this species and mutants of it are excellent "tools" for geneticists. (See Fig. 68.) Since N. sitophila is heterothallic, only the conidial stage will ordinarily be encountered in routine bacteriological work. Conidia are borne from short hyphal branches and the chains are much branched. See Fig. 69. They develop in the same manner as those of Cladosporium. The conidia are cylindrical to ovate and are nu- merous and bright orange-red in color. This together with the fact that the mycelial growth is so copious and floccose, often ascending for several centimeters in the culture tubes, gives the mold a char- acteristic appearance in culture. 116 THE FUNGI IMPERFECTI AND THE ASCOMYCETES N. sitophila is found in soil and on vegetation. It has been espe- cially found in burned-over forest areas. It is most important as a cause of trouble in bakeries because it results in infected bread. Fig. 68. Neurospora sitophila ascospores. The organisms are difficult to eliminate and are said to be extraor- dinarily resistant to heat when they are not wetted. This species may become a serious pest in bacteriological laboratories, and spe- FiG. 69. Neurospora sitophila showing arrangement of conidia. Photograph by- dark field illumination. cial care should be taken not to let the spores be scattered. See Chapter VIII for industrial use of this mold. Monascus. Monascus purpureus is a mold not often isolated by the bacteriologist, but it is one of some importance and interest. Its LITERATURE 117 character of producing a brilliant red soluble pigment is utilized by the Chinese to color various food products. Certain Chinese so- called wines, Chinese red-rice, and soybean cheeses are such products imported into the United States. This organism has been found in silage where it sometimes caused the formation of large "balls" up to one-third meter in diameter.^ The conidia are large and are found singly on the conidiophores, or in short chains. The asci (the fungus is homothallic) are produced in perithecia which are rather small, at least in the strains studied Fig. 70. Monascus purpurcus: 1 and 2, mycelium and conidia; 3, 4, and 5, oogonium and antheridium ; 7, ascogenous and ordinary hyphae ; 6, perithecium. in this laboratory, and the oogonia and antheridia may be easily seen in unstained Petri plate cultures or in stained slide preparations. Although this organism had not been cultured in our laboratories here in Minnesota for over fifteen years, we isolated this species as an air contaminant on one occasion, and years previous to that Henrici had isolated another strain. It is undoubtedly not very common, however. Oilman does not list it as ever being found in soil. LITERATURE 1. BiouRGE, P., Les moissures du groupe Penicillium Link. Etude mono- graphique, La cellule, 33, 1 (1923). 2. Buchanan, E., and R. Buchanan, Bacteriology, Macmillan, New York, 4th ed., 1938. 3. Buchanan, R., Monascus purpureus in silage, Mycologia, 2, 99 (1910). 4. Conant, N. F., D. S. Martin, D. T. Smith, R. D. Baker, and J. L. Calla- way, Manual of Clinical Mycology, Saunders, Philadelphia, 1944. 5. Dodge, C. W., Medical Mycology, Mosby, St. Louis, 1935. 6. Emmons, C. W., The ascocarps in species of PenicilUum, Mycologia, 27, 128 (1935). 7. , Misuse of the name "Trichophyton rosaceum" for a saprophytic Fusarium, J. Bact., 47, 197 (1944). 118 THE FUNGI IMPERFECTI AND THE ASCOMYCETES 8. Oilman, J. C, A Manxwl of Soil Fungi, Collegiate Press, Ames, Iowa, 1945. 9. Groves, J. W., and A. J. Skolko, Notes on seed borne fungi. I. Stem- phylium. II. Alternaria, Can. J. Research, C, 22, 190, 217 (1944). 10. Lewis, G. E., and M. E. Hopper, An Introduction to Medical Mycology, Year Book Pub. Co., Chicago, 1939. 11. Maurizio, a., and W. Staub, Monilia nigra Burri u. Staub. Weitere Unter- suchungen liber Schwarzfleckigkeit bei Emmenthalerkase, Zentr. Bakt., Parasitenk., II, 75, 375 (1928). 12. Ollivier, M., and G. Smith, Byssochlamys fulva, sp. nov., J. Botany, Brit. and For., 71, 196 (1933). 13. Thom, C, The Penicillia, Williams and Wilkins, Baltimore, 1930. 14. , Naming molds, J. Wash. Acad. Sci., 30, 49 (1940). 15. Thom, C, and M. Church, The Aspergilli, Williams and Wilkins, Balti- more, 1926. 16. Thom, C, and K. B. Raper, A Manual of the Aspergilli, Williams and Wilkins, Baltimore, 1945. 17. VuiLLEMiN, P., Classification normale, classement auxiliaire, et groupement pratique des champignons, Compt. rend., Acad. Sci. (Pans), 180, 102 (1925). 18. Waksman, S. a.. Principles of Soil Microbiology, Williams and Wilkins, Baltimore, 2nd ed., 1932. 19. WoLLENWEBER, H. W., and O. A. Reinking, Die Fusarien, Parey, Berlin, 1935. CHAPTER VI FUNGUS DISEASES OF MAN AND ANIMALS— GENERAL CONSIDERATIONS Many different fungi have been described as causing various dis- eases in man and animals. Some of these fungi are known only as pathogens, and the diseases they cause, although sporadic and some- times rare in occurrence, are fairly well known. Other fungi are known both as pathogens associated with lesions in man and as saprophytes in man's environment, and the circumstances under which they sometimes become parasitic are not fully understood. General discussions of the pathogenic fungi are to be found in many review articles and textbooks.^"- "- ^^' ''• ^-' *^' '^ Etiology. In many cases the etiological relationship between mold and disease can be proved by repeated demonstration of the fungus in lesions, its isolation therefrom, production of an infection in ex- perimentally infected animals, and recovery of the fungus from the latter. There are numerous papers in the literature of medical my- cology, however, in which this rigid proof of an etiological relation- ship is lacking. The careless acceptance of an unproved mycotic etiology frequently has led to error. Pinta, a tropical spirochetosis, long thought to be a mycosis from which many different fungi w^ere isolated, is an example of such a mistake. Many of the fungus dis- eases are superficial lesions of the skin or mucous membranes. IMold spores are ubiquitous and it is not at all surprising that they may be cultivated frequently from exposed skin lesions, pus, and sputum. Viable airborne spores of harmless saprophytes are often present in pathological material, and they germinate and grow when the ma- terial is planted upon culture media. The isolation of such a fungus in culture may then be interpreted erroneously as evidence that it was growing in and causing the lesion. One must develop a healthy degree of scepticism with regard to the pathogenicity of fungi isolated in culture from pathological material. In a general consideration of mycoses it is interesting to note that fungi have been more successful as pathogens of plants than of animals. The majority of plant diseases, whether of weeds, trees, or commercially important crop plants, are caused by fungi, and 119 120 FUNGUS DISEASES OF MAN AND ANIMALS the rusts, smuts, mildews, and leaf spots can be mentioned as familiar examples. Comparatively few plant diseases are caused by bacteria. Among the diseases of man and animals, on the other hand, bacterial diseases predominate, and the list of important fungus diseases is short. Furthermore, at least in the case of most of the generalized and frequently fatal mycoses, the fungus appears to be an accidental invader and does not spread from man to man. Fungi therefore ap- pear to be poorly adapted to a parasitic existence in man except in a few instances. Importance of Mycoses. Although fatal fungus diseases in man are less common than bacterial infections they are nevertheless nu- merically important. In Vital Statistics of the United States for 1942 mycoses are reported as causing 359 of 1,385,187 deaths in man. This is less than 0.03 per cent of the total, yet it is more than half the number of deaths caused by either typhoid, tetanus, or polio- myelitis and polioencephalitis; more than the number of deaths due to Rocky Mountain spotted fever and the other typhus-like diseases together ; and nearly twice as many as the sum of all those caused by paratyphoid fever, undulant fever, smallpox, rabies, leprosy, plague, cholera, yellow fever, and relapsing fever. It should be pointed out, of course, that the low mortality rates of some of the well-known diseases just mentioned are due to the enforcement of effective con- trol measures, whereas in the case of the generalized mycoses control measures are not practiced nor, indeed, are they known. Non-fatal mycoses such as the dermatophytoses (ringworm, athlete's foot) are perhaps as common as any bacterial disease. Besides their numerical importance, as compared with some of the better known bacterial diseases, the fungus infections provide useful material for the study of certain biological principles. For example, the phenomena of variability and mutation are more ap- parent in the fungi and can be studied with greater confidence be- cause the larger size and the abundance of measurable morphological characteristics of fungi make it easier to distinguish between a true mutant and a contaminant. Types of Mycoses. Fungus infections are referred to as mycoses, frequently with a prefix or qualifying word to indicate the part affected, as otomycosis (ear), onychomycosis (nail), pulmonary my- cosis. More frequently the type of mycosis is designated according to the etiological agent, as actinomycosis, blastomycosis, coccidioido- mycosis. In any case it is usually necessary to indicate the specific fungus responsible since different fungi may invade the ear or the lungs, and different species of actinomycetes {Actinomyces bovis, HOST SPECIFICITY 121 Nocardia spp.), for example, are etiologically related to different types of actinomycosis. The fungi which cause ringworm have cer- tain peculiarities which set them apart from most of the other patho- genic fungi, and because they are interrelated and the diseases they cause are limited to the skin it is convenient and appropriate to refer to them as the dermatophytes, and to the disease which they cause as dermatophytosis. The term dermatomycosis is sometimes used in the same sense, but the suffix mycosis is generally understood to imply a deeper involvement than is found in dermatophytosis. An- other type of mycosis which is set apart by certain characteristics is the mycetoma. The various mycetomas differ in etiology, but all are characterized by deep invasion of the subcutaneous tissues with the formation of sinuses. In the pus which drains from these sinuses the fungus is found usually in the form of rather firm, well-organized granules which differ in size, shape, consistency, and color accord- ing to the species of fungus involved. Human mycoses can be placed in two groups, those in which there is only a very superficial penetration, represented by the dermato- phytoses, and those involving the subcutaneous tissues and fre- quently causing generalized infections, represented by a considerable number of mycoses of varied types and etiologies. In any attempt to generalize upon the fungus diseases this diversity must be re- membered. The dermatophytoses are caused by a group of closely interrelated fungi, the dermatophytes, which are physiologically adapted to growth upon keratinized structures. They are able to grow saprophytically upon a wide variety of animal and vegetable debris, but their preference for keratinized material, their ability to grow in the epidermis, frequently for long periods without actually causing lesions of clinical importance, and their transmission from one person to another indicate a high degree of adaptability to the parasitic habit. Host Specificity. Some of the dermatophytes show a considerable degree of host-specificity. Microsporum Audouini, for example, is the species usually responsible for epidemics of ringworm of the scalp. It attacks only children, causing in most cases a dry scaling lesion with little host reaction. Because of its good adaptation to the host it is difficult to eradicate and a single epilating dose of x-ray should be given before fungicidal treatment is begun. Although the disease is chronic, with little or no tendency to self-limitation in the child, spontaneous cure usually occurs at puberty. It is thought that the change in endocrine balance occurring at this time makes the host 122 FUNGUS DISEASES OF MAN AND ANIMALS no longer susceptible to parasitism by this fungus. Infection of the adult by M. Audouini is very rare. M. Canis, on the contrary, is a pathogen of cats and dogs and in- vades the human host by accident from an animal source. It does not cause epidemics in man although transmission may occur in instances where there is intimate exposure, as within the family. Both children and adults may be infected. Unlike M. Audouini, M. Canis typically excites considerable reaction on the part of the host, the lesions frequently being edematous and exhibiting pustules. Al- though this difference in the clinical type of lesion may be considered typical, exceptions are too frequent to make it a dependable method of determining which of the two species is involved in a given case. As a result of the host reaction in most cases there is a tendency toward spontaneous shedding of hairs infected by M. Canis and, as Lewis and Hopper ^^ have shown, thorough scrubbing with soap and water may suffice to cure the infection without the use of fungicidal ointments. Similar relationships can be pointed out in the case of several other dermatophytes. Trichophyton violaceum and T. tonsurans are path- ogens of man; they cause chronic dermatophytosis in which the in- fected hair stubs are so firmly anchored that their removal requires special attention, and subsequent fungicidal treatment may be re- quired for a considerable time. By contrast, T. faviforme, which is a pathogen of cattle, and the granulosum and asteroides varieties of T. mentagrophytes, which are pathogens of the horse and other ani- mals as well as man, when they attack man, cause boggy lesions of the type designated kerion. From this type of lesion the infected hairs are spontaneously shed. In rare cases true granulomata are produced. There is occasionally such a severe pustular folliculitis that when the patient is seen in the clinic infected hairs may be difficult to find and several attempts may have to be made before the fungus is isolated in culture. In general, the granular varieties of T. mentagrophytes are more apt to be of animal origin and to evoke a more marked tissue reaction in the human host than are the cottony varieties of that species. There are exceptions, however, io the usual correlations between the type of fungus and the type of lesion, as Dowding and Orr ^^ have pointed out. The species causing dermatophytosis of the foot in man show a high degree of adaptation to this host, in many cases. They may grow for months or years in the epidermis or nails without causing subjective symptoms, becoming clinically important only at rare intervals. A clinical flare-up may be due to altered host resistance; SYSTEMIC MYCOSES 123 a changed environment in the interdigital spaces of the foot due to increased activity on the part of the host, change of season or cli- mate, or to some other unrecognized factor. In any case the fungus is a frequent inhabitant of the epidermis and nails of the feet, and the reactivation of a quiescent lesion is probably a more important factor in the frequent occurrence of dermatophytosis of the foot than is reinfection from an exogenous source. Systemic Mycoses. In spite of the contrasts just cited between the lesions caused by dermatophytes of human origin on the one hand, and of lower animal origin on the other, they become insig- nificant when one contrasts the dermatophytoses with the generalized mycoses. The dermatophytes, despite their adaptation to specific hosts, form a rather closely interrelated group of fungi. They have certain morphological characteristics and physiological adaptations in common, and they appear to be primarily parasites of man and animals. Dermatophytes have been isolated from stable litter and manure, and they undoubtedly grow saprophytically on desquamated epithelium, hairs and similar debris, but their presence in such en- vironments and such substrata is secondary to their parasitism of man and animals. The systemic mycoses, on the other hand, are with few exceptions caused by fungi which are rarely found in man and animals, but apparently have a natural habitat elsewhere and attack man and animals only under exceptional circumstances. In American blasto- mycosis, sporotrichosis, chromoblastomycosis, the type of actinomy- cosis caused by the acidfast Nocardia asteroides, mycetomas of vari- ous types, aspergillosis, and other rarer mycoses, the evidence indi- cates that the fungus has a natural habitat in soil, vegetation, or possibly in some cases in another animal host. The fungi causing these mycoses apparently need to be introduced into the human host by inhalation or subcutaneously by accident on splinters or thorns and under special circumstances in which sensitization may be an important element. These mycoses are not contagious, transmission directly from person to person occurring very rarely if at all. The factors influencing the incidence of these deeper mycoses vary to some extent with the individual mycosis and can best be discussed later in connection with the particular infection. It may be said in general, however, that age may be a factor in some mycoses, occupa- tional exposure appears to be important in others, residence is im- portant in the case of those with an endemic distribution, and re- peated exposure with ensuing sensitization may be important in several. 124 FUNGUS DISEASES OF MAN AND ANIMALS Occupational exposure may be important more because of the op- portunity for repeated exposure than because it provides a single exposure. For example, Aspergillus fumigatus is a common soil fungus and everyone may have inhaled its conidia at times, yet pulmonary aspergillosis in man is rare. It is said to occur most often in France in people engaged in two occupations in which they come into frequent and intimate contact with molded grain, namely, those who feed pigeons in one case, and those who use meal in clean- ing hair in the other. Clinical Course. The theoretical implications involved in the in- itiation of these mycotic infections w^ere discussed by Henrici -^ in a comparison of bacterial infections and certain of the deeper mycoses. He pointed out a fundamental difference between the course of most bacterial diseases and that of most of the deep mycoses. A typical bacterial disease has a sudden onset with rapid resolution. A typical mycosis, on the other hand, has an insidious onset. The develop- ment of the disease is slow and frequently the infection spreads at first only by extension to contiguous tissues. Even in generalized mycoses with fatal termination this early stage of the disease may be most important in respect to time if hematogenous dissemination intervenes late in the disease. The primary lesion may be in the skin or in the lungs, in either case spreading slowly by peripheral exten- sion. When hematogenous or lymphatic dissemination finally occurs the progress becomes rapid and death of the host may ensue. It should be pointed out that there are exceptions to this sequence. Some bacterial diseases follow a slow chronic course and, conversely, in some mycoses there is a rapid extension of the infection. The most common form of coccidioidomycosis is an acute respiratory disease which ordinarily follows a course not unlike that of a com- mon cold or, in more severe cases, influenza. The disseminated form, which is comparatively rare, may follow a rather rapid course to a fatal termination in a few weeks or it may extend over a period of years with very slow progression. Most of the mycetomas, with the exception of the common form of actinomycosis, remain locahzed so that widespread dissemination never occurs. Mycetoma of the foot, for example, may have a duration of 15 or 20 years, yet never ex- tend much above the ankle. Chromoblastomycosis has a similar slow extension without dissemination, but satellite lesions arise by auto- inoculation and influence the rate and extent of the involvement. Sensitization. The importance of trauma in the initiation of some of the mycoses, their slow initial progress, and their final rapid ac- celeration suggest that the fungi causing them grow at first only on SENSITIZATION 125 dead or injured tissue or its products, that they have sHght inherent invasive power, and that they are able to spread only after some change has occurred in the fungi themselves or in their environment. If some change in the fungi has occurred the fungus, when isolated from the disease at a late stage, might be expected to show an in- creased primary virulence when introduced into a new host. Such a change has not been demonstrated. It seems more probable that the host tissues become altered. Increased susceptibility of the host might be explained by the endotoxin theory of Pfeiffer or the allergic theory of von Pirquet. According to the former, as the cells of the parasite die or are killed by the host they liberate toxins which so alter the neighboring host tissue that the surviving cells of the pathogen are able to grow and extend the lesion. According to the second theory it is assumed that the pathogen exerts little toxic action directly upon the host, but that its long-continued presence in the primary lesion sensitizes the neighboring cells so that they become susceptible. Actually both of these mechanisms may be operative. In most of the patho- gens technical difficulties prevent the demonstration of endotoxins and their presence therefore is speculative. There is some circumstantial evidence for the presence of toxins, however, in the case of certain mycoses. In blastomycosis Martin and his associates have shown that the administration of iodides in systemic blastomycosis may cause a rapid extension of the disease unless the patient is first de- sensitized, presumably because of substances liberated by killed cells of the fungus. In the histopathology of coccidioidomycosis the immature cells of the fungus may cause comparatively little response, but upon reaching maturity, when the sporangium ruptures and per- mits the dissemination of the endospores, it incidentally releases a substance, unable to pass the previously intact wall of the fungus cell, which excites an immediate tissue response. At the same time, it is conceivable that this substance may promote the invasion of the neighboring tissues by the newly liberated endospores. As with the bacteria, the pathogenic fungi have various portals of entry to the body which are to some extent specific. Thus there are the dermatophytes mentioned above which grow on the skin and hair; others like the thrush organism are primarily parasites of mucuous membranes, causing lesions of the mouth and vagina. In some cases the primary lesions are in the lungs, the fungi being inhaled. There are fungi which are primarily saprophytes but are capable of growing superficially in the external ear causing otomyco- 126 FUNGUS DISEASES OF MAN AND ANIMALS sis. Other fungi appear to require introduction to the subcutaneous tissues through wounds. Some species of pathogenic fungi have a very low invasive power. In fact, in some of the so-called fungus infections there is no infec- tion in the strict sense, for the parasites do not invade the tissues except to grow in the most superficial epidermal layers. This is true of tinea versicolor; and in many cases of otomycosis the fungus hardly invades the epithelium of the ear but grows as a saprophyte upon the ear wax. In dermatophytosis and in most cases of thrush the fungi invade principally the epithelium and rarely penetrate beyond it. Pathological Anatomy of Mycoses. In the generalized mycoses already mentioned extensive and deep-seated lesions may occur and the disease may spread to other parts of the body either by way of the lymph vessels or the blood stream. The tissue changes brought about in these cases are in part of the nature of abscesses, and in part of the nature of granulomata. Immediately surrounding the parasite there is usually death of the tissues, with softening and the accumulation of leucocytes and the formation of pus. ]\Iany of these abscesses become surrounded by granulation tissue consisting of a dense layer of new fibrous tissue infiltrated with, mononuclear leuco- cytes and sometimes containing giant cells. The degree to which one or the other of these processes predominates varies with the virulence of the strain and the mode of infection. Thus the primary lesions of blastomycosis in the skin are mainly granulomatous, the secondary lesions following a blood-stream distribution are usually pure abscesses. In animals experimentally inoculated with rather large doses, pure abscesses similar to those produced by the pyogenic cocci usually develop. However, in some human infections the lesions may so closely resemble various granulomatous processes, such as those of tuberculosis and syphilis or even cancer, as to make the diagnosis difficult. In experimental infections in laboratory ani- mals the type of lesion may vary widely with the dosage and mode of inoculation. Thus if large doses of Aspergillus fumigatus spores are injected intravenously into pigeons, areas of necrosis with hemor- rhage and very little cellular infiltration will develop about the germinating spores. With very small doses one gets typical tubercles in internal viscera after a much longer time (see Fig. 110). If one blows a mass of spores into the trachea, there develops an acute pneumonia fatal within 24 hours, whereas, if the pigeons are fed on infected grain so that only an occasional spore is inhaled from time TOXINS OF FUNGI 127 to time, there are found cavities in the lungs and a massive growth of mycelium in the air sacs. Toxins of Fungi. It is not clearly understood how the pathogenic fungi injure the tissues. Although fibrosis and giant cell reactions about some lesions bear a resemblance to a foreign body reaction, the extensive necrosis and suppuration which occur in the center of most lesions cannot be readily explained in this way. Moreover, the experimental lesions produced with freshly isolated and highly viru- lent strains of some species, as Aspergillus fumigatus and Candida albicans, are so acute as to suggest that these diseases may be caused by the same mechanisms as those found in bacterial infections. A pigeon inoculated intravenously with a suspension of spores of A. fumigatus may die within 24 hours, showing at autopsy multiple punctate hemorrhages and areas of degeneration or necrosis in par- enchymatous tissues, in short, just such a picture as may be pro- duced by a highly virulent Streptococcus. Such findings naturally suggest that a potent toxin is formed. Considerable investigation has been carried on with regard to the toxins of other pathogenic fungi, but it is largely contradictory and inconclusive. No exotoxins have been definitely established. The occurrence of endotoxins is of considerable theoretical interest. Our conception of endotoxins is based largely upon the demonstra- tion of endoenzymes by Buchner's classical experiment. It is not possible to express the cell sap of bacteria by pressure, as Buchner did with yeasts, because of their minute size. All other attempts to remove toxins from within the cells of bacteria by chemical extrac- tion, autolysis, grinding (after drying or freezing) may be criticized as introducing factors that might readily alter the toxins. How- ever, the cell sap of molds may be expressed readily in a Buchner press. It is remarkable that very few attempts to obtain a toxin from pathogenic fungi by this method are recorded in the literature. Gortner and Blakeslee,^^ during the course of some experiments dealing with antibody production, accidentally discovered that the cell sap of Rhizopus nigricans is highly toxic to rabbits. The toxic substance was obtained both by expressing the juice of the mold and by extraction of dried mycelium with hot water. It gave the reactions of proteins, was non-dialyzable, and was precipitated by alcohol. Intravenous injections in rabbits produced almost imme- diate death, with both symptoms and autopsy findings "practically indistinguishable from anaphylactic intoxication." Subcutaneous in- jections gave rise to abscesses which broke down to form ulcers. The material was quite harmless by mouth. It is quite possible that these 128 FUNGUS DISEASES OF MAN AND ANIMALS results were due to their using rabbits which had somehow become sensitized to the proteins of this mold. Henrici obtained a toxin from R. nigricans which, when injected into rabbits, killed some, but caused anaphylactic reactions only in surviving rabbits given a second injection. A number of investigators have been concerned with the toxins of A. fumigatus because of the high and constant virulence of this fungus for laboratory animals. Renon *^ was unable to find any toxic substance either in the medium or by extracting the mycelium with various solvents. Similar results were obtained by Kotliar,^- Mace,^* and by Martins,^^ although the latter author did observe death with one rabbit inoculated with spores heated to 100° C. On the other hand, Ceni and Besta ^^ found in extracts of A. jumigatus a heat-stable toxic substance affecting mainly the nervous system, and this was confirmed by Bodin and Gautier.'^ Bodin and Lenormand ^ studied the matter further and concluded that there were two toxins, one a substance soluble in fat solvents which pro- duced convulsions and a rapid death in rabbits, the other a volatile substance obtained by distillation which produced paralysis and death in guinea pigs. Henrici ^^ obtained a toxin from the cell sap of a strain of A. jumigatus isolated from aspergillosis in a chick. The toxin was extracted from a glucose-peptone broth culture by squeezing from the mycelial mat as much of the medium as possible, mincing the mycelium with scissors, passing it repeatedly through a food chopper, and expressing the cell sap in a hydraulic press. The toxin resembled the so-called toxalbumins, the exotoxins of bacteria, and the snake venoms in sensitivity to heat, hemolytic action, production of local necrosis and edema, delayed action, and antigenicity. Unlike the toxalbumins, however, its toxicity was augmented rather than de- stroyed by sodium ricinoleate and it appears to be non-protein in nature. In these characteristics and in the type of lesions it produces in animals it resembles the toxin of the poisonous mushroom Amanita phalloides. The cell sap was lethal by subcutaneous, intraabdominal, and intravenous injection (but not by mouth) for rabbits, guinea pigs, mice, and chicks. Injected subcutaneously in rabbits it produced in about 5 hours large boggy lesions in which there was a large amount of gelatinous fluid similar to that in lesions caused by the toxin of Clostridium oedematiens. After a few days the swelling subsided, the center became necrotic and sloughed away leaving an ulcer which required about 10 days to heal. Guinea pigs reacted TOXINS OF MUSHROOMS 129 by developing less gelatinous edema but more necrosis and ulcera- tion. Intravenous inoculation was fatal to rabbits, the survival time decreasing from 10 days to 48 hours with doses varying from 1 cc. to 5 cc. The time of survival varied according to the potency of the particular lot of toxin and the individual susceptibility of the rabbit as well as to the volume of dose. The post mortem findings were hemorrhages in the lungs, serous or serofibrinous effusion into the peritoneal and pleural cavities, sometimes accumulation of fluid around the kidneys, and a pale or mottled liver. There was extensive damage to the secretory tubules of the kidneys. Some rabbits showed necrotic and fatty changes in the liver. Guinea pigs were more uniformly susceptible to the toxin than rabbits, but lesions were similar. In mice and chicks necrosis of renal tubules was the only pathological change found. The toxin is antigenic. Henrici immunized rabbits and demon- strated passive transfer of immunity to guinea pigs and mice. Im- mune serum neutralized the hemolytic action of the toxin in vitro. Toxins of Mushrooms. A word or two may be properly interpo- lated here concerning the toxins of the poisonous mushrooms. These vary with different species. It may be worthwhile to emphasize a fact too often ignored by the amateur mycophagist who seeks to supplement his diet by gathering wild mushrooms. There are many poisonous species of mushrooms and these may closely resemble edible forms. Several genera contain both edible and poisonous species. There is no simple rule or characteristic such as excellent flavor, failure to tarnish a silver coin, or an easily peeled cap which distinguishes an edible from an inedible species. The only safe basis for selecting edible species is a thorough acquaintance with the species collected. A survey of the mushroom poisons has been presented by Ford.^^ In the case of the fly Amanita (Amanita muscaria) one of the poi- sons is an alkaloid, muscarin. The poisons of the most deadly of the mushrooms, A. phalloides (Fig. 71), have been studied by several investigators. The work of Ford has established that there are two poisonous substances present. One is a hemolytic agent called phallin, the other a general toxin known as amanito-toxin. The former is easily inactivated by heat, the latter is not. Laboratory animals may be immunized with extracts of the fungi, their serums developing both antihemolytic and antitoxic properties. The hemo- lytic substance has been isolated and is said to be a pentose-contain- ing glucoside. It is, however, contrary to the opinion of most im- munologists that a non-protein substance may give rise to antibodies. 130 FUNGUS DISEASES OF MAN AND ANIMALS The amanito-toxin alone did not give rise to protective antibodies in Ford's experiments. It was found to be neither a protein, an alka- loid, a glucoside, nor a conjugate sulphate. Contrary to the experience of Ford, de la Riviere ^® found that the toxin affects mainly the nervous system, spasmodic convulsions being ■■ 1 ■ Brl«^' ^ B^^ ^^^B ^^H ^^^^^^^H^^B&;jE ^^^H ^^' B^^^ ii^^l ^^^^B' 'I li 1 ^^^^^^^Bg^x^oci^ "*j^^^^^^^^l Fig. 71. Amanita phalloides, the "Death Cup." The cup-shaped envelope (volva) about the base of the stem and the ring or collar about the stem near the cap are distinguishing signs. (White varieties, of which the above are an example, are sometimes known as A. verna.) the most prominent symptoms. Both authors, agree, however, that the toxin is not bound by nerve tissue. A wide variety of animals, from monkeys to fish, are susceptible. Sheep are apparently im- mune to poisoning by mouth, but succumb to injections. De la Riviere succeeded in obtaining an antitoxin (with the whole extract) by immunizing horses. This not only protected laboratory animals but was shown to have some therapeutic value in cases of human poisoning. Green and Stoesser 2* have studied the effect of sodium ricinoleate upon the toxins of A. phalloides. Whereas this soap destroys the IMMUNITY REACTIONS OF THE FUNGI 131 toxins of diphtheria and tetanus bacilli and scarlet fever streptococci, it augments the toxicity of Amanita and botulinus. Ford proposed a classification of mycetismus (mushroom poison- ing) which recognized five types and listed some of the species of mushrooms associated with each type. In reporting four cases of mushroom poisoning caused by A. phalloides, Vander Veer and Farley, for practical purposes, simplified Ford's classification and recognized a rapid type of poisoning caused by A. muscaria and a delayed type caused by A. 'phalloides ^^ In the rapid type symptoms appear within a few minutes or at most within 3 hours and include "salivation and lacrimation; pupils contracted and not reacting to light or accommodation; nausea and vomiting; abdominal pains with profuse watery evacuations; pulse at times slow and often irregular; dizziness and confusion with convulsions and coma in severe cases; fatal cases, death within a few hours." *^ The prognosis is good even in severe cases if atropine is administered. In the delayed type symptoms appear 6 to 15 hours after ingestion and include sudden onset "with severe abdominal pains; nausea, vomiting, and usually diarrhea; vomitus and stools often showing blood and mucus; extreme thirst; anuria at times; usually jaundice in from two to three days; cyanosis and coldness of extremities; in- creasing prostration with coma and death usually from the fifth to eighth day." *^ The mortality rate is 50 to 70 per cent, often 100 per cent in a given group. If the mushroom meal has contained other poisonous species in addition to A. phalloides the prognosis may be better because the early action of other toxins aids in early evacua- tion. Emetics, gastric lavage, and thorough purging are important even though several hours have elapsed since ingestion. Rest in bed is essential until there is definite recovery. Apparent improvement may be followed by fatal relapse if patients are allowed to resume activity too soon. A high carbohydrate liquid diet is recommended, with forced fluids, intravenous dextrose solution, and physiological salt solution subcutaneously. An antitoxin serum has been used in France and Germany. Immunity Reactions of the Fungi. The available information about immunity reactions in the fungi is somewhat contradictory. It would appear that in general fungi give rise to antibodies only in small amounts and that the antibodies so formed are not highly specific, reacting in some cases with widely divergent types. The difficulty may be due in part to the relatively thick wall of the fungus cell which allows only a slow diffusion of the intracellular proteins into the tissues, This is indicated by the results of Balls ^ 132 FUNGUS DISEASES OF MAN AND ANIMALS who obtained precipitating serums of relatively high titer and specificity with yeasts by inoculating, not the cells, but proteins liberated from them by autolysis. Agglutinins. Most attempts to carry out agglutination reactions with fungi have been unsuccessful. This is due in part to the diffi- culty of obtaining stable suspensions. Generally spores have been used although some workers have used suspensions of ground-up mycelium. Moore and Davis reported that agglutinins were produced in sporotrichosis and considered the agglutination reaction useful in diagnosis and in demonstrating the close relationship between dif- ferent strains of Sporotrichum Schenckii. Cummins and Sanders^* could demonstrate no agglutinins for Coccidioides immitis in experimentally infected animals. Similarly Nicaud^^ found no agglutinins for spores of Aspergillus jumigatus in the sera of human cases of aspergillosis, and Matsumoto ^^ did not obtain any agglutination of spores of A. amstelodami in the sera of intensively immunized rabbits. On the other hand, Widal '° and others found that the sera of sporotrichosis cases would agglutinate the spores of S. Schenckii in rather high dilutions, 1:300 to 1:400 on the average, and this ob- servation has been confirmed by numerous later authors. However, the same Sporotrichum spores are also agglutinated in rather high dilutions by the sera of thrush cases and cases of actinomycosis, according to Widal and his coworkers. This observation, however, was not confirmed by Fineman -' in the cases of thrush studied with American strains of Sporotrichum. Widal and his coworkers found that the sera of thrush cases would agglutinate Sporotrichum spores at a higher titer than they would suspensions of the thrush parasite. Fineman could obtain agglutinins only in a very low titer for Candida albicans in immunized rabbits. According to Epstein ^o this organism agglutinates spontaneously too readily to be used for agglutination reactions. Blakeslee and Gortner ^' ^ obtained rather strong agglu- tinating sera for the spores of Mucor, with some cross reactions. It should be pointed out that the spores of Sporotrichum and of the ■ Mucors have thinner walls than do many mold spores, and they form suspensions fairly readily. Benham demonstrated the usefulness of the agglutination and re- ciprocal absorption of agglutinins tests in determining relationships between species of Candida. Most investigators have found it pos- sible to produce serums of good antigen titer with these yeast-like fungi. ]\Iartin, discussing the application of immunological prin- COMPLEMENT FIXATION 133 ciples to the diagnosis and treatment of mycoses, reported that ag- glutination reactions were generally unsatisfactory for most of the mycoses. Drake ^^ demonstrated "natural agglutinins" against five different yeast-like fungi and concluded that they are normal human serum constituents. Precipitins. Very little study of precipitins has been carried on. Cummins and Sanders ^* recorded negative results with sera of pa- tients and inoculated guinea pigs in coccidioidal granuloma. Michel obtained no reactions with Candida albicans in cases of sprue. Matsumoto obtained precipitates when the sera of rabbits immunized with species of Aspergillus were tested against broth filtrates, but not with extracts of mycelium. The reactions were not definitely specific. Henrici was unable to demonstrate precipitins with sera of rabbits intensively immunized with cell sap of Aspergillus fumi- gatus and C ephalosporium Acremonium. Stone and Garrod *'^ reported that the precipitin and complement fixation reactions of strains of C. albicans they cultivated from thrush were similar. Kesten and Mott ^^ prepared polysaccharides from several yeasts and yeast-like fungi and with them obtained serums which they used to produce precipitin reactions. Complement Fixation. Widal ^° and his coworkers found com- plement fixation positive in sporotrichosis, as with agglutination, and found the same cross reactions with thrush and actinomycosis. Epstein ^° found the complement fixation reaction positive only once in twenty cases of thrush. Cummins and Sanders ^* obtained no com- plement fixation in coccidioidal granuloma. Matsumoto ^'^ obtained more marked results with complement fixation than with precipita- tion in his studies of the Aspergilli, but found no correlation between the grouping of species by complement fixation and their morpho- logical characters. Smith, although much of the details of his work are not yet pub- lished, reported that the complement fixation reaction gives useful information about the progress and probable prognosis in dissem- inated coccidioidomycosis. Martin states that in blastomycosis the titer of complement-fixing antibodies is low even in the presence of extensive skin lesions if the infection is not systemic. AVith increas- ing involvement of the internal organs there is usually a correspond- ing rise in antibody titer. Conversely, a fall in titer indicates in most cases an improvement in the prognosis. Some individuals do not produce antibodies and the complement fixation reaction alone, therefore, is not a reliable diagnostic test. 134 FUNGUS DISEASES OF MAN AND ANIMALS Negroni ^^ isolated a carbohydrate which he beheved came from the capsular material of Candida albicans and was responsible for the agglutinating and complement-fixing properties of the fungus. Martin, using a saline suspension of the yeast-like form of Blasto- myces dermatitidis and otherwise following the usual procedures of the Wassermann tests, demonstrated complement-fixing antibodies in the serum of three of four patients with generalized blastomycosis. There did not appear to be any cross reaction with other pathogenic fungi. He found no relationship between the clinical condition of the patient and the presence of complement-fixing antibodies in the blood. Allergic Reactions. More significant results seem to have been obtained from a study of allergic reactions than from other phases of immunity with the fungi. In 1908 Bloch * established that after an experimental skin infection with Microsporum quinckeanum had been established in guinea pigs, the animals were immune to rein- oculation over the entire skin surface. This immunity is in general not species-specific, the animal remaining also refractory to other distantly related species of dermatophytes. If one does succeed in establishing a second infection, the inflammatory reaction is more acute and the lesion heals more quickly than in a control animal. A similar immunity was demonstrated in human cases where deep- seated lesions occurred. Inoculations of filtrates of old broth cultures of the fungus gave rise to a characteristic papule in human cases if deep-seated lesions were present, but not when the lesions were superficial. This allergic reaction also proved non-specific, infections with different species of dermatophytes giving reactions with the same antigen. Further observations were reported later by Bloch and Massini,^ among them the following interesting experiment. Skin from a pa- tient allergic to ringworm fungi was transplanted to a non-sensitive individual. After the graft was established, it was found that this area of skin was still hypersensitive, although the "native" skin of the recipient did not become sensitized. It was also claimed that deep-seated infections healed more rapidly after establishing an artificial infection with M. quinckeanum, an observation which was confirmed by Plant. The use of trichophytin (extracts of species of Trichophyton) diagnostically and therapeutically has been widely studied by derma- tologists. It seems to have been established that these extracts reg- ularly give reactions in patients in whom deep-seated lesions occur, less regularly in others; that the reaction may persist for some time CUTANEOUS REACTIONS IN OTHER MYCOSES 135 after the lesions have healed; and that some individuals, even those in whom a previous history of ringworm cannot be established, may react; so that, as with tuberculin, a negative reaction is of more diagnostic value than a positive one. Trichophytin is claimed by some to have some therapeutic value in certain deep-seated infec- tions, but the effect is not a specific one. There occurs a focal re- action in the lesion as well as the local one at the site of inoculation, and such therapeutic effect as is noted is to be attributed to this in- creased inflammatory reaction, which is no greater than that ob- tained when irritants are applied locally. The trichophytins have been prepared in various ways. According to Bloch, the best pro- cedure is to evaporate the broth filtrate to one-twelfth its original volume, to which is added the cell sap expressed from the mycelium. The active substance has been chemically investigated by Bloch, Labouchere, and Schaaf,^ who claim that it is a starch-like, nitrogen- containing, levorotatory polysaccharide. Trichophytid. In connection with the allergic reactions of ring- worms, mention should be made of the condition known as tricho- phytid, first described by Jadassohn. This is a generalized skin eruption occurring during the course of a ringworm infection, similar to the generalized eruptions occurring in scrofula or other forms of tuberculosis, and designated tuberculide by Darier. It is as yet un- certain whether this affection is due to a toxic reaction of some sort (perhaps caused by an allergic state of the patient) or to a dis- semination of the fungus by the blood stream. The former hypothesis seems more reasonable, but positive blood cultures have been re- ported. Experimental trichophytid in the guinea pig has been de- scribed."*' Cutaneous Reactions in Other Mycoses. The cutaneous reactions in coccidioidomycosis have been studied more thoroughly than in other mycoses. Dr. C. E. Smith has prepared most of the coccid- ioidin used in skin testing and has had a very extensive experience in its use. He prepares the material by growing strains of Coccid- ioides immitis on a synthetic broth medium like the medium used in the preparation of old tuberculin except that there is a reduction in the amount of glycerin. The composition of the medium and the method of preparation and use are outlined in the section on coccid- ioidomycosis. Coccidioidin appears to be highly specific, but some lots give non- specific reactions and have to be discarded. A small number of per- sons who have had no recognized exposure to Coccidioides react to 136 FUNGUS DISEASES OF MAN AND ANIMALS coccidioidin, and these reactions have so far not been satisfactorily explained. Other mycotic antigens are less specific or have had less extensive use than coccidioidin. Histoplasmin has been prepared by a number of investigators. Palmer/° using histoplasmin prepared from the synthetic broth mentioned above, reported that in student nurses tested there was a very high correlation between positive histoplasmin skin reactions and pulmonary calcification, and suggested that Histo- plasma might be responsible for non-tuberculous calcification. It is known, however, that this histoplasmin gives cross reactions with other mycoses. ^^ A skin-testing antigen prepared from Aspergillus jumigatus at the National Institute of Health and used in testing experimentally in- fected guinea pigs had a primary irritating effect and gave non- specific reactions. The use of a skin-testing antigen in the diagnosis of a mycosis has definite limitations which are not always recognized. A positive re- action may be due to a non-specific factor common to several patho- genic fungi; or, if it is a true specific reaction, the person may have been sensitized by a previous exposure to the fungus quite unrelated to the condition in "^'hich a diagnosis is sought. Skin sensitivity, once it is acquired, persists for many years and perhaps for life. Asthma. Considerable attention has been directed toward mold spores as possible etiologic agents in asthma. It now seems well established that asthma is due to an allergic state toward inhaled substances which may be present in the air as dust. That mold spores may be the exciting agents was suggested by van Leeuven.*^ Cadham ^^ demonstrated that the spores of the wheat rust fungus, Puccinia graminis, excited asthmatic attacks in certain cases he studied. Hansen -^ observed a number of cases apparently due to mold spores. These cases are first detected by cutaneous reactions, but "Hansen applied rather strict criteria for establishing the di'ag- nosis, involving demonstration of the mold in the habitual sur- roundings of the patient, complete relief after removal from these surroundings, an immediate relapse after experimental exposure to the mold in question, and finally a positive Prausnitz-Kustner reac- tion (passive transfer of the skin sensitivity to a normal person). He found various species of Aspergillus most frequently gave reac- tions. Hopkins, Benham, and Kesten "^' ^° reported a case due to a species of Alternaria, and suggested that an eczema from which the same patient suff'ered might be due to a similar cause. TREATMENT OP FUNGUS INFECTIONS 137 Allergists have made extensive surveys of the mold spore content of the air in different geographical areas and at different times of year. It is routine practice to skin-test allergic patients with stock fungus extracts or with extracts of fungi isolated from the particular patient's environment. The role of fungi in allergy has been re- viewed by many allergists, including Sulzberger/^ Peck,'*^ and Brown.^ Treatment of Fungus Infections. The treatment of mycoses is frequently unsatisfactory, the infections persisting for long periods of time in spite of treatment. In actinomycosis, blastomycosis, and histoplasmosis the prognosis is grave and the mortality high. In superficial infections, as the dermatophytoses and thrush, local appli- cations of strong antiseptics may sometimes lead to a rapid cure.^'^ Many of these, however, are also stubborn and persistent. In sporotrichosis the internal administration of iodides causes the rapid disappearance of the lesions. Similar, though less marked, beneficial results are obtained in localized actinomycosis and blasto- mycosis, so that it has come to be generally considered that the iodides are a specific for fungus infections comparable with the arsenicals in protozoan infections. However, Davis ^^ showed for Sporotrichum (as did Reynolds and Henrici ** for an actinomycete) that the iodides have no effect upon the fungi themselves either in vivo or in vitro; growth occurred in media containing as much as 10 per cent of po- tassium iodide. Such therapeutic results as are obtained therefore must be due to an action on the tissues rather than on the parasite. The iodides probably stimulate the formation of fibrous tissue which tends to wall off the organisms. On the other hand Martin and Smith point out that in systemic blastomycosis administration of iodides may cause a rapid extension of the lesions unless the patient is first desensitized. They suggest that the reason for this is that the iodides cause the death of many fungi, and that a toxin is there- upon liberated from the dead fungus cells. Many attempts have been made to find other specific drugs for the treatment of fungus infections, but without much success. Prob- ably because they have been extensively used as fungicides in the treatment of fungus diseases of plants, copper salts and colloidal copper have been used. Neither copper nor sulphur is as useful against the fungus pathogens of man as against those of plants. Tartar emetic and preparations of arsenic and mercury are also used. Although apparent cures and improvements under such treatments have been reported, the results in general are not sufficiently striking to indicate that any specific chemotherapeutic agent has been found. 138 FUNGUS DISEASES OF MAN AND ANIMALS Thymol, instilled into the lesion locally and taken by mouth, has been used with success in some cases of actinomycosis and cocci- dioidomycosis. Sulfadiazine is effective in actinomycosis. Penicillin is also reported useful in this disease, but. has not yet been tried in enough cases to permit a critical evaluation. No effective specific treatment is yet known for most of the sys- temic mycoses. Rest and supportive therapy are recommended to aid the patient in arresting the infection. Many of the mycoses, even of the more severe types, tend to remain localized, spreading by extension only, for long periods. Where medication proves useless in such cases complete surgical excision or even amputation may be the best treatment. LITERATURE 1. 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Lenormand, Recherches sur les poisons produits par Y Aspergillus jumigatus, Ann. inst. Pasteur, 26, 371 (1912). 9. Browx, G. T., Hypersensitiveness to fungi, J. Allergy, 7, 455 (1936). 10. Brumpt, E., Precis de parasitologie, Masson et Cie., Paris, 1936. 11. Cadham, F. T., Asthma due to grain rusts, J. Amer. Med. Assoc, 83, 27 (1924). 12. Cbni, C, and C. Besta, Ueber die Toxine von Aspergillus jumigatus und A. jiavescens und deren Beziehungen zur Pellagra, Centr. Pathol. Anat., 13, 930 (1902). 13. Conant, N. F., D. S. Martin, D. T. Smith, R. D. Baker, and J. L. Call- away, Manual oj Clinical Mycology, Saunders, Philadelphia, 1944. 14. Cummins, W. T., and J. Sanders, The pathology, bacteriology and serology of coccidioidal granuloma, J. Med. Research, -35, 243 (1916). 15. Davis, D. J., The effect of potassium iodide on experimental sporotrichosis, J. Injectious Diseases, 25, 124 (1919). LITERATURE 139 18. DE LA Riviere, R. D., Etude d'une toxine vegetale: la toxine phallinique, Aim. inst. Pasteur, 43, 961 (1929). 17. DowDiNG, E. S., and H. Orr, Three clinical types of ringworm due to Tricho- phyton gypseum, Brit. J. Dermatol. Syphilis, 49, 298 (1937). 18. Drake, C. H., Natural antibodies against yeast-like fungi as measured by slide-agglutination, J. Immunol., 50, 185 (1945). 19. Emmons, C. W., Medical mycology, Botan. Rev., 6, 474 (1940). 20. Epstein, B., Studien zur Soorkrankheit, Jahrb. Kindcrheilk., 104, 129 (1924). 21. FiNEMAN, B. C, A study of the thrush parasite, J. Infectious Diseases, 28, 185 (1921). 22. Ford, W. W., The distribution of haemolysins, agglutinins, and poisons in fungi, especially the Amanitas, the Entolomas, the Lactarius and Inocybes, J. Pharmacol, 2, 285 (1911). 23. Gortner, R. a., and A. F. Blakeslee, Observations on the toxin of Rhizo- pus nigricans, Arn. J. Physiol., 34, 353 (1914). 24. Green, R. G., and A. V. Stoesser, Biological study of mushroom extract and effect of sodium ricinoleate on its toxicity, Proc. Soc. Exptl. Biol. Med., 24, 913 (1927). 25. Hansen, K., tjber Schimmelpilz-Asthma, Verhandl. Deut. Ges. inn. Med., 40, 204-206 (1928). 26. Henrici, a. T., Experimental trichophytid in guinea pigs, Proc. Soc. Exptl. Biol. Med., 41, 349 (1939). 27. , Characteristics of fungous diseases, J. Bad., 39, 113 (1940). 28. , An endotoxin from Aspergillus jumigatus, J. Immunol., 36, 319 (1939). 29. Hopkins, J. G., R. W. Benham, and B. M. Kesten, Asthma due to a fungus — Alternaria, J. Am. Med. Assoc, 94, 6 (1930). 30. Hopkins, J. G., B. M. Kesten, and R. W. Benham, Sensitization to sapro- phytic fungi in a case of eczema, Proc. Soc. Exptl. Biol. Med., 27, 342 (1930). 31. Kesten, H. D., and E. Mott, Soluble specific substances from yeastlike fungi, /. Infectious Disease's, 50, 459 (1932). 32. Kotliar, E., Contribution a Tetude de la pseudotuberculose Aspergillaire, Ann. inst. Pasteur, 8, 479 (1894). 33. Lewis, G. M., and M. E. Hopper, An Introdu^tioii to Medical Mycology, Year Book Pub. Co., Chicago, 1943. 34. Mace, T. C, Etude sur les mycoses experimentales. Arch, parasitol., 7, 313 (1903). 35. Martins, C, Etudes experimentales sur VAspergillu^ fumigatus, Compt. rend. soc. biol, 100, 525 (1928). 36. Matsumoto, T., The investigation of Aspergilli by serological methods. Trans. Brit. Mycol. Soc, 14, 69 (1929). 37. M-s^ERS, H. B., and C. H. Thienes, The fungicidal activity of certain vola- tile oils and stearoptens, J. Am. Med. Assoc, 84, 1985 (1925). 38. Negroni, P., Propiedades antigenicas in vitro de la substancia capsular de Mycotorula albicans, Rev. inst. bacteriol. dept. nacl. hig. {Buenos Aires), 7, 568 (1936). 39. NiCAUD, P., Etude des reactions humorales dans I'Aspergillose, Paris medical, No. 22, p. 531, 1929. 140 FUNGUS DISEASES OF MAN AND ANIMALS 40. Palmer, C. E., Nontuberculous pulmonary calcification and sensitivity to histoplasmin, Public Health Repts., 60, 513 (1945). 41. Peck, S. M., Fungus allergy, J. Allergy, 11, 309 (1940). 42. Pl.\itt, H. C, and 0. Grutz, Die Hyphenpilze oder Eumyceten, Kolle, Kraiis u. Uhlenhuth's Handbuch de Path. Mikroorganismen, Fischer, Jena, 5, 133 (1927). 43. Renon, L., Etude sur rAspergillose chez les animaux et chez I'homme, Masson et Cie., Paris, 1897. 44. Reynolds, G. S., and A. T. Henrici, Potassium iodide does not influence the course of an experimental actinomycosis, Proc. Soc. Exptl. Biol. Med., 19, 255 (1922). 45. Sartory, A., Champignons parasites de I'homme et des animaux, Le Fran- Qois, Paris, 1921. 46. Stone, K., and L. P. Garrod, The Classification of Monilias by serologic methods', J. Path. Bad., 34, 429 (1931). 47. Sulzberger, M. B., Allergy in dermatology, J. Allergy, 7, 385 (1936). 48. VAN Leeuven, W. S., Allergic Diseases, Lippincott, Philadelphia, 1925. 49. Vander Veer, J. B., and D. L. Farley, Mushroom poisoning {mycetismus), Report oj four cases. Arch. Internal Med., 55, 773-791 (1935). 50. WiDAL, F., P. Abrami, E. Joltrain, E. Brissaud, and A. Weill, Serodiag- nostique mycosique, Ann. inst. Pasteur, 24, 1-33 (1910). 51. Dodge, C. W., Medical Mycology, Mosby, St. Louis, 1935. 52. Emmons, C. W., B. J. Olson, and W. W. Eldridge, Studies of the role of fungi in pulmonary disease, I. Cross reactions of histoplasmin, Public Health Repts., 60, 1383 (1945). CHAPTER VII INFECTIONS CAUSED BY MOLDS COCCIDIOIDOMYCOSIS (Valley Fever, Coccidioidal Granuloma) Coccidioidomycosis is an acute, usually mild and self-limited respiratory mycosis, which in exceptional cases becomes chronic and generalized; it then produces granulomatous lesions in almost any organ and has a high fatality rate. History. The first recognized case of coccidioidomycosis was re- ported by Posadas and Wernicke from the Chaco region of Argen- tina. The second and third were seen in 1894 in California by Rix- ford and Gilchrist.^^ * The pathogen was believed to be a protozoan, and Rixford and Gilchrist, seeing a resemblance to Coccidium, fol- lowed a suggestion of Stiles and named it Coccidioides immitis. Four years later Ophuls and IMofiitt,^- studying the third North American case, isolated the organism in culture and showed that it was a fungus. Although the fungus was named under the erroneous im- pression that it was a protozoan the generic name was created for it and actually no protozoa have ever been placed in the genus. The name is therefore valid and the disease is properly designated coc- cidioidomycosis, the name given it by Dickson.^- ^ One frequently hears the disease miscalled coccidiosis which is an unrelated proto- zoan disease common in some animals. The condition was known for 40 years after its discovery as a chronic, generalized, usually fatal granulomatous disease called coc- cidioidal granuloma. After 1936 the investigations of Gifford " and of Dickson revealed that an acute, benign, respiratory disease called Valley fever or desert rheumatism was a mild form of coccidioido- mycosis. The subsequent studies of Dickson, Smith, ^*' ^^' ^*' Aron- son ^ and others have shown that most of the residents of an endemic area react to the intradermal injection of an antigen, coccidioidin, prepared from the fungus. This is interpreted to mean that the * In Chapter VII literature citations will be found at the end of each section. Citations for this section are on page 152. 141 142 • INFECTIONS CAUSED BY MOLDS individual reacting has had a sensitizing contact with the fungus since few non-residents react to the test. This specific skin sensi- tivity is usually acquired early in childhood by the residents of an endemic area, usually without having had a recognized infection. A small percentage (higher in those exposed for the first time as adults) of reacting persons may have had a respiratory infection of some clinical importance. Very few infected individuals (probably a small fraction of 1 per cent of those who become skin sensitized) develop the grave generalized form of the disease. During the period 1893 to 1931 only 254 cases of the generalized form were known from California where it is a reportable disease. There is probably no notable increase in the incidence of coccidioidal granuloma, except as recent mass movements of susceptible adults in the armed forces have enormously increased the number of exposures. However, since 1936 there has been a great increase in the recognition of the acute respiratory type of the disease. Dickson suggested the latter be designated primary coccidioidomycosis and that coccidioidal granu- loma be called progressive or secondary coccidioidomycosis. Clinical. The present concepts of coccidioidomycosis have been well summarized by Smith.^'^ Although primary skin lesions have been reported in a few cases the important portal of entry is the respiratory tract. Cases in which the time of exposure is known or can be estimated accurately show that symptoms may appe&r 8 to 21 days after inhalation of the chlamydospores of the fungus and skin sensitivity to coccidioidin is acquired 10 to 45 days after the exposure. Initial or primary coccidioidomycosis , which follows inhalation of the spores of the fungus, is self-limited and focalized in the lungs. It is usually asymptomatic and is then recognized only by the acqui- sition of skin sensitivity to coccidioidin. However, it may be an acute respiratory condition following an influenzal or pneumonic pattern. There may be pleural, joint, and muscle pains, headache, cough which is usually non-productive, malaise, fever, chills, night- sweats, and anorexia. In some cases there may be formation of pulmonary cavities which close spontaneously and promptly, or per- sist for a considerable time. It is estimated that 2 to 5 per cent of individuals with initial coccidioidomycosis develop erythema no- dosum or erythema multiforme. When this allergic manifestation is present the disease is commonly called San Joaquin fever. Valley fever, desert rheumatism, or desert fever, designations given to the condition as it was seen within the endemic area of the San Joaquin Valley of California long before its etiology was known. Human DIAGNOSIS 143 pathological material of primary coccidioidomycosis has not been available for histological study. It is evident that even when cavita- tion occurs and Coccidioides is present in the sputum the infection is usually effectively controlled and remains well localized.*- ^' ^' ^°' ^^ In a very few primary infections, perhaps one in 500 or 1000, dis- semination of the fungus occurs. This happens if at all usually within a few weeks or months after the primary infection. In some cases this secondary form (originally designated coccidioidal granu- loma) may be the first recognized manifestation of the disease. This was invariably true in the early history of the mycosis, but its rela- tionship to initial coccidioidomycosis is now better understood. This form of the disease is properly designated progressive or dis- seminated coccidioidomycosis. In its clinical manifestations dissemi- nated coccidioidomycosis closely mimics tuberculosis and a differ- ential diagnosis can be made only by demonstration of the fungus, although in certain cases the coccidioidin and tuberculin reactions are helpful. There may be multiple subcutaneous and joint abscesses. Skin lesions may present a verrucous appearance or there may be extensive ulceration. Although in some cases a skin lesion may be the first recognized evidence of infection there may have been, and probably was in most cases, an earlier unrecognized pulmonary in- fection. When dissemination occurs there may be miliary spread to the meninges, bones and joints, lymph nodes, peritoneal cavity, and to any organ, with the notable exception of the digestive tract which is usually spared. The lesions, except for the presence of the fungus, resemble those of tuberculosis to a remarkable degree. Diagnosis. The diagnosis rests finally upon the demonstration of the fungus in pus, sputum, or tissues. Coccidioidin skin and sero- logical tests are, however, helpful, and investigations based on the use of these methods have contributed greatly to knowledge of the disease. Coccidioidin is prepared by growing the fungus for 2 months in a broth medium similar to that used in making old tuber- culin. The formula, as recommended by Dr. C. E. Smith, is as follows. Ammonium chloride (NH4CL) 7.00 grams 1-Asparagine 7.00 grams Dipotassium phosphate c.p. (K2HPO4) 1.31 grams Sodium citrate c. p. (NasCeii^OTd'^AUiO) 0.90 gram Magnesium sulphate U.S. P. (MgS04 -71120) 1.50 grams Ferric citrate U.S.?. VIII (scales) 0.30 gram Glucose of the grade known as cerolose U.S.P. X 10.00 grams Glycerol c.p. (U.S.P.) 25.00 ml. Water to make 1000.00 ml. 144 INFECTIONS CAUSED BY MOLDS "Dissolve asparagine in about 300 cc of hot distilled water, 50 degrees C. Dis- solve each of the organic salts in 25 cc of water, ferric citrate being dissolved in hot water. Add each salt in order, starting with K2HPO4 to the hot aspara- gine solution and mix well each time the salt is added. Then add dextrose and glycerine [glycerol] and finally make up to volume. Fill 1500 cc to each 3 liter Fernbach culture flask. Then sterilize at 115 degrees for 25 minutes. Incu- bate." After the culture has grown for 2 months at room temperature or 30° C. the broth is filtered, made up to the original volume, tested for sterility, a preservative is added, and the product is tested for potency and specificity by skin-testing persons whose degree of sensi- tivity is known. Many lots of coccidioidin fail to meet the latter tests and must be discarded. A suitable lot is stable over ^a pe- riod of several years. The skin test is performed by injecting 0.1 ml. of a dilution of 1:1000 of the filtrate intracutaneously. The test is read after 48 hours as a tuberculin test would be read. Indi- viduals failing to react to the first dose can be retested by a dilution of 1 : 10. Those who have had primary coccidioidomycosis even in an inapparent form and recovered from it retain the skin sensitivity for many years, perhaps for life in many cases. Some individuals appear to lose their skin sensitivity gradually. A coccidioidin skin test may not be informative in the diagnosis of a present condition in an individual who has previously been within the endemic area of coccidioidomycosis for even a few hours since it may merely reflect an early infection from which the patient has recovered. Aronson, Saylor, and Parr ^ in a study of calcified pulmonary nodules in tuberculin negative persons living within endemic areas concluded that some of these nodules may be due to a previous coccidioidomy- cosis. They found a high percentage of reactions to coccidioidin in persons living within the endemic areas of this disease and few or no reactions in other groups. It was shown that there was no cross reaction with tuberculin. Coccidioidin can be used as an antigen in precipitin and comple- ment fixation tests which give some indication of the extent and probable course of the disease. According to Smith, a high antibody titer follows spread of the infection and, conversely, a fall in titer indicates a good prognosis. Skin sensitivity is retained after recov- ery, but in cases of fatal infection it is greatly decreased during the terminal stages. Demonstration of the fungus by direct examination or by culture may be difficult in initial coccidioidomycosis where sputum is often scanty or is not produced. In the disseminated form the fungus can DIAGNOSIS 145 be found more readily. Pus or sputum may be placed on a slide under a cover slip and examined directly. Often the fungus can be demonstrated more easily if the material is placed on the slide and mixed with a drop of 10 per cent sodium hydroxide. This digests the tissue elements while the fungus is relatively resistant. The appearance of the fungus will be described in a later paragraph. Sputum contains so many diverse elements that there may be some difficulty in finding the fungus. In such cases isolation in cultures . , "■ . ' , " ^ "" " • " ' ,«. r-^^ ■/■ ■ W> * •" . \ } » t' • v_^ . . A r '■ ■- ■ ' ^' ; ■ J- '■■ ' • h. ■ - ^ J -■ •;• " ■ ', -- :% .1 i^j^^g^ ■HHH^^K|. Sk. ^wBMH Bl^H^^Er * "^ - ' *• • ■" ■ ' '•'' • i^, ' • • , u^>- ■ W ■-J' ' fJ,-\.:J*-:J ' - . '2i-:- Fig. 72. Coccidioides immitis in sputum after sodium hydroxide digestion: left, an immature spherule and a mature sporangium from which spores have escaped; right, a nearly mature sporangium. may be more productive. This is usually relatively easy because of the rapid growth of the fungus. Sputum. should be streaked out on the surface of agar slants. The digestion and concentration methods used in isolating the tubercle bacillus kill Coccidioides, but sputum may be treated with 0.05 per cent cupric sulphate which destroys most of the bacteria. American Sabouraud agar or a selective cul- ture medium devised at Stanford University may be used. The latter consists of 1 per cent ammonium chloride, 1 per cent sodium acetate, 0.8 per cent tribasic potassium phosphate, and 2 per cent agar. The medium is autoclaved for 10 minutes at 15 pounds pres- sure and 0.04 per cent cupric sulphate is added just before pouring. If plates are used in the isolation of Coccidioides the fungus should be transferred to agar slants soon after it appears and the original plate cultures must be autoclaved promptly. The fungus begins to produce spores usually within 8 to 10 days and there is grave danger of laboratory infection if old plate cultures are opened. It is prob- 146 INFECTIONS CAUSED BY MOLDS able that most laboratory personnel where cultures of Coccidioides immitis are handled sooner or later become infected. Fortunately most of these infections are mild or inapparent, but unnecessary risks should be avoided. If, in making a laboratory diagnosis by culture, there is doubt about the identity of a fungus isolated, 1 ml. of a heavy suspension of the spores can be injected intraperitoneally into a white mouse. Lesions in which the characteristic parasitic growth phase of the fungus can be demonstrated appear in animals killed after 5 or 6 days, and most inoculated mice die in 7 to 14 days. Intratesticular inoculation of guinea pigs has been recommended as a diagnostic procedure but the mouse is a cheaper as well as a more susceptible test animal. Treatment. No specific treatment has been found uniformly suc- cessful in coccidioidomycosis. According to Smith treatment should consist of rest and supportive therapy directed toward assisting the patient in the arrest of the infection. Morphology in Tissue. Coccidioides immitis exhibits in a striking manner the dimorphism found in most pathogenic fungi. In animal tissue the spores of the fungus are small spherical cells 1 to 4 microns ■ in diameter. They are to be found frequently within phagocytes (Fig. 73). These cells never bud but increase in size to upwards of 80 microns in diameter, the usual range of mature cells being 20 to 60 microns in diameter. The structure during this period of develop- ment varies to some extent with the tissue invaded and the strain of fungus. The stainable protoplasm may fill the cell or, especially in experimental infections in mice and guinea pigs, be confined to a comparatively narrow peripheral layer. In the latter case the large central vacuole is eventually filled with an indefinite number of endo- spores (sporangiospores). The process of spore formation was first clearly described in detail by Wolbach and by Ophuls. Their obser- vations have been confirmed by many later students. The proto- plasm becomes cut up by radial and periclinal cleavage planes, the process originating at the periphery and progressing toward the center. The first cleavage planes cut out large multinucleate masses which are usually subdivided by other cleavage planes to form very numerous small spores. However, in some cases the process may be interrupted at an intermediate point and only a few large spores may form. After spore formation is completed the w^all of the parent cell ruptures and the spores pass into the adjacent host tissue where they repeat the cycle. No other cell form is known in the parasitic growth phase. The endospores are infective, as can be demonstrated experi- MORPHOLOGY IN TISSUE 147 Fig. 73. Immature cells of Coccidioidcs imm'tis in experimeutally infected guinea pig. From C. W. Emmons, Mycologia, 34, 454 (1942). Fig. 74. Mature ruptured sporangium of Coccidioidcs immitis and empty spo- rangium invaded by leucocytes. From C. W. Emmons, Mycologia, 34, 456 (1942). 148 INFECTIONS CAUSED BY MOLDS mentally, but actually are not ef- fective in the direct transmission of coccidioidomycosis in man. Segre- gation of patients is not necessary. The nuclear condition was first described by Emmons '^ who showed that the very numerous nuclei, which frequently lie in a peripheral zone of protoplasm, are typical of those found in other fungi. There is a distinct nuclear membrane en- closing small amounts of baso- philic material and a deeply stain- ing nucleolus. The ultimate spores were described as usually uninu- cleate. Baker, et al.,^ reported that they may be multinucleate. The number of nuclei per spore prob- ably depends upon whether or not progressive cleavage proceeds within the sporangium to the nor- mal final stage of subdivision of the protoplasm. Morphology in Culture. When pus containing Coccidioides is planted upon agar the fungus grows in a very different fashion. The recently liberated spores, large veg- etative cells, and sporangiospores still within the unruptured sporan- gium germinate at once by the de- velopment of hyphae. If, however, the material is incubated under anaerobic conditions on special me- dia there may be a limited devel- opment of the parasitic growth phase. When grown on American Sabouraud agar at 30° C. spores be- gin to form in most strains in about 8 days. Some of the aerial hyphae bear specialized side branches which are about twice the diameter of the hyphae from which they Fig. 75. Cultures of Coccidioides immilis. From C. W. Emmons, Mycologia, 34, 460 (1942). MORPHOLOGY IN CULTURE 149 arise. The protoplasm in these conidiophores condenses or accumu- lates at intervals and septa are formed. A typical conidiophore then resembles a chain of spores separated from each other by spaces devoid of protoplasm. Frequently the original septum can be ob- served midway of the space separating two spores, indicating a con- densation of the protoplasm to form the spore. These spores are usually called chlamydospores and they resemble chlamydospores except in their relationship to spe- .-^yr / \ cialized hyphal branches. As the culture ages similar spores form in many of the aerial hyphae with- out any apparent specialization and the designation of chlamydo- spore seems to be justified. These chlamydospores are highly infec- tious and their accidental inhala- tion by laboratory personnel who handle cultures has resulted in many infections. Spores of this type are formed in large numbers when the fungus grows saprophyti- cally on either natural or artificial substrates and it is supposed that they are present in -^ind-blown dust and initiate infection when contaminated air is inhaled. They have not actually been found in air, and although they have been isolated in culture from soil the actual gi'oui;h of the fungus in soil has not been demonstrated except in the laboratory. When chlamydospores from a culture are injected intraperito- neally into a laboratory animal each spore is capable of rounding up and growing directly into a sporangium as previously described. The spores may remain in short chains during at least the early stages of development in experimentally infected animals, thus gi^'ing rise to a chain of sporangia. Unless one realizes the origin of these chains they may cause confusion, especially when only two sporangia are connected by a short empty cell, thus bearing a superficial resem- blance to conjugating cells. Closely appressed sporangiospores of the parasitic growth phase may also remain adjacent after dissemi- nation from the ruptured sporangium and give a false appearance of budding. Fig. 76. Chlamydospores In a young culture of Coccidioides immitis. From C. W. Emmons, M ycologia, 34, 460 (1942) 150 INFECTIONS CAUSED BY MOLDS Taxonomy. In the method of endospomlation exhibited in the parasitic growth phase of Coccidiodes immitis the resemblance to the Zygomycetes is so great that it seems reasonable to identify the mature Coccidioides cell as a sporangium and its endospores as spo- rangiospores. Baker and his coworkers^ reported that small spo- rangia are produced in culture by some strains, but this phenomenon is not often observed. The hyphae which normally develop in culture are richly septate, a condition which is rare among the Zygomycetes, but in rate of growth and in the general aspect of cultures there is a resemblance. It is true that the sporangium formed in tissue is not associated with a mycelium and that there is no columella, but the sporangia of many of the Zygomycetes (e.g., Mortierella and Syn- cephalis) present so many anomalies that Coccidioides may well find a place within the group. Geographical Distribution. Contrary to the earlier belief that the disease was limited to the Chaco region of Argentina and to the San Joaquin Valley of California, it is now recognized that it occurs throughout the arid Southv/est in southern California, Arizona, and New Mexico, and western Texas. The migration of infected persons from these areas has not established recognized new endemic foci. The cases which have been observed in other parts of the United States, Italy, and elsewhere, are probably in persons who were in- fected in the Southwest and developed the disease after an incubation period, or who were infected from dusty fruit, packing material, or similar sources originating in an endemic focus. Natural Habitat. Coccidioidomycosis is not transmitted from one person directly to another. It is evident therefore that the fungus grows in some habitat outside the human host. The primary lesions, with few exceptions, are in the lungs and they follow inhalation of spores of the fungus. Epidemiological evidence and study of case histories indicate that the spores of the fungus are wind-blown. In- fections follow exposure to dust storms and appear in agricultural workers and others exposed to wind-blown soil. Most primary in- fections occur in the summer and fall and few are seen during the rainy season. It has been assumed, therefore, that the fungus grows in soil during or following the rainy season and that spores mature and are disseminated during the dry season. The fungus has, in fact, been isolated directly from soil, first near Delano, California, at a ranch house occupied by men who had coccidioidomycosis; second in Panoche Valley, California, near a burrow from which a group of university students (who subsequently developed coccidioidomy- cosis) dug a rattlesnake; and third, from five soil samples taken from NATURAL HABITAT 151 various soil types collected on the desert near San Carlos, Arizona, distant from human habitations but near numerous rodent burrows. Hundreds of other attempts to isolate Coccidioides from the upper layers of soil within endemic areas have failed. Recent studies in southern Arizona have suggested the possibility of a rodent reservoir of the disease.- '• ^ Among small rodents trapped in this area the white-footed mouse, grasshopper mouse, and wood rat were never or very rarely infected; but 15 per cent of three species of pocket mice and 17 per cent of one species of kangaroo rat trapped had pulmonary coccidioidomycosis. Surprisingly, a second fungus, Haplosporangium parvum, which resembles Coccidioides in its parasitic phase but is different in culture, was found with even greater frequency (66 per cent of pocket mice) causing a similar pulmonary disease in rodents. No human infections with the latter fungus have yet been recognized, but many individuals who react to the intradermal injection of coccidioidin also react to an antigen (haplosporangin) prepared from this fungus. Coccidioidomycosis in these rodents seemed to be a slowly progres- sive chronic disease which did not exterminate the species and, in fact, appeared to interfere httle with normal development and repro- duction. Further investigations will be required to determine whether these rodents are infected because of their intimate exposure to in- fested soil or whether the fungus is primarily a pathogen of rodents and is present in soil that has been contaminated by infected rodents. Certain circumstances seem to suggest the latter as probable. The disease in rodents is known in species of Perognathus and Dipodomys which are desert species with ranges coinciding generally with a part of the known geographical range of coccidioidomycosis. Species of Peromyscus, however, although more common than Perognathus, and living in adjacent burrows, were rarely found infected under field conditions. Their exposure to the fungus would seem to be equal to that of Perognathus, and they are very susceptible to experimental infections, but they apparently do not serve as hosts under the field conditions investigated. There are no doubt other rodent hosts of the fungus. There appears to be the sort of adjustment between pathogen and the Perognathus host which is compatible with the theory of a rodent reservoir. The difficulty of isolating Coccidioides from desert soil in which it seems to have a spotty distribution not correlated with any recognized differences in vegetation or soil types makes the hypothesis of a rodent reservoir attractive. Coccidioidomycosis occurs also in other animals within the en- demic area. Stiles and Davis ^^ have described the focalized infec- 152 INFECTIONS CAUSED BY MOLDS tion in the mediastinal and bronchial lymph nodes in cattle and sheep, and Farness " has observed the disseminated form of the mycosis in dogs. LITERATURE 1. Aronson, J. D., R. M. Saylor, and E. I. Parr, Relationship of coccidioido- mycosis to calcified pulmonary nodules, Arch. Path., 34, 31 (1942). 2. AsHBURN, L. L., and C. W. Emmons, Experimental Haplosporangium infec- tion, Arch. Path., 39, 3 (1945). 3. Baker, E. E., E. M. Mrak, and C. E. Smith, The morphology, taxonomy, and distribution of Coccidioides immitis Rixford and Gilchrist, 1896, Farlowki, 1, 199 (1943). 4. (jox, A. J., and C. E. Smith, Arrested pulmonary coccidioidal granuloma. Arch. Path., 27, 717 (1939). 5. Dickson, E. C, "Valley fever" of the San Joaquin Valley and fungus Coc- cidioides, Calif, and Western Med., 47, 151 (1937). 6. , Primary coccidioidomycosis, Am. Rev. Tuberc, 38, 722 (1938). 7. Emmons, C. W., Coccidioidomycosis, Mycologia, 34, 452 (1942). 8. , Coccidioidomycosis in wild rodents. A method of determining the extent of endemic areas. Pub. Health Repts., 58, 1 (1943). 9. Emmons, C. W., and L. L. Ashburn, The isolation of Haplosporangium parvum n. sp. and Coccidioides immitis from wild rodents, Pub. Health Repts., 57, 1715 (1942). 10. Farness, O. J., Coccidioidomycosis, J. Am. Med. Assoc, 116, 1749 (1941). 11. Gifford, M. a.. Coccidioidomycosis, Kern County, Aim. Rept. Kern County Dept. Pub. Health, 1938-1939, pp. 73-79. 12. Ophuls, W., and H. C. Moffitt, A new pathogenic mould (formerly de- scribed as a protozoon: Coccidioides immitis pyogenes), Phila. Med. J., 5, 1471 (1900). 13. Rixford, E., and T. C. Gilchrist, Two cases of protozoan (Coccidioidal infection of the skin and other organs, Johns Hopkins Hosp. Repts., 1, 209 (1896). 14. Smith, C. E., Epidemiology of acute coccidioidomycosis with erythema nodosum, Am. J. Pub. Health, 30, 600 (1940). 15. , Parallelism of coccidioidal and tuberculous infections. Radiology, 38, 643 (1942). 16. , Coccidioidomycosis, Med. Clinics N. America, pp. 790-807, 1943. 17. Stiles, G. W., and C. L. Davis, Coccidioidal granuloma (coccidioidomy- cosis), J. Am. Med. Assoc, 119, 765 (1942). 18. Winn, W. A., Pulmonary cavitation associated with coccidioidal infection. Arch. Internal Med., 68, 1179 (1941). THE DERMATOPHYTOSES (Dermatomycosis, Epidermatophytosis, Ringworm, Tinea, "Athlete's Foot") The term dermatophytosis is generally used to indicate a series of diseases caused by a group of fungi which practically never invade THE DERMATOPHYTOSES 153 any other tissue. These fungi for the most part produce only a superficial infection, not tending to involve the deeper tissues or to spread to internal organs, but they are nevertheless important because of their frequent occurrence and, to a certain extent, contagiousness. They are closely related species with numerous intermediary forms which make up a fairly homogeneous group. They are frequently referred to as the dermatophytes. A clear knowledge of this group of fungi can be obtained only by first-hand acquaintance with them. The subject is quite complicated for several reasons. Attempts have been made to classify the causa- tive fungi according to the nature of the disease which they produce; but the same disease may be produced by several different species and, conversely, a single species is capable of producing very different clinical types of disease. On the other hand, attempts at a purely mycological classification based upon morphological characters has been somewhat unsatisfactory because of the pronounced pleomor- phism of the fungi and the incorrect interpretations and evaluation of morphological features.^- ^ * The parasites exhibit a certain amount of geographical specialization, some species isolated from cases in one part of the world being different from those obtained in other parts. Some of the parasites occur only on man, some on cer- tain lower animals. The latter may be transmitted from animals to man. A very large number of species have been described. Many of the species names are synonyms and the number of true species can per- haps be reduced to one tenth the reported number by a critical com- parison of strains and a proper evaluation of variability. For these reasons an extensive knowledge of the group can be attained only after long and intensive study. The subject is one for the specialist. We can do no more here than point out some of the more important general characteristics of the diseases and their etiologic agents, and describe a few of the more common species. Although a very large number of investigators have contributed a voluminous literature to the subject, the work of one man, Sabou- raud,^" overshadows all the rest and dominates the field. To his long-continued, patient, and intensive studies we are indebted for a revival of interest in the subject during the first years of this century. He systematically studied different types of dermatophytosis ; cor- related the clinical features, the microscopic appearance of the fungus in infected hairs, and the colonial and microscopic appearances of * Literature citations for this section will be found on pages 177 and 178. 154 INFECTIONS CAUSED BY MOLDS the fungi in cultures; and made important contributions to the ther- apy of dermatophytosis. His classification of the dermatophytes was based on clinical manifestations for the separation of genera and largely upon the characteristics of the colony for specific separation. The three principal genera which he recognized can be defined in mycological terms and this modification of Sabouraud's classifica- tion will be followed here. His dependence upon colonial appearance to determine species led to an excessive splitting of species and his disregard of earlier specific names makes some of his names invalid. Discussions of the dermatophytes will be found in many other publi- cations.- *'' ^' «' ^°' ^5' ^'> Because of the complexity of the subject it will be necessary to consider the important species of dermatophytes separately. We shall therefore depart from the usual order followed elsewhere in this book and discuss first the morphological and physiological features which relate the dermatophytes to each other and the taxonomic position they occupy. Cultural Characters. The identification of the various species of dermatophytes is made very largely by the appearance of the colonies. Since this is subject to some variation with the composition of the medium, Sabouraud insisted that cultures should be made upon his proof agar. Since the ingredients he used are no longer available or were impure products, substitutes have been required. Plaut and Griitz offered several alternative formulas which they claimed served in place of Sabouraud's medium. Weidman also proposed some satis- factory substitutes. A satisfactory medium which is constant in composition, simple to prepare, and gives satisfactory colony char- acteristics is described on page 55. It contains 1 per cent neopep- tone and 2 per cent glucose. The colonies may be smooth and waxy if no aerial mycelium is formed, or have a chalky surface, or velvety or woolly texture, de- pending upon the abundance and height of the aerial mycelium. They may be yellow or rose or violet in color, but most species are yellowish or white. They may be irregularly folded to form cerebri- form masses, regularly folded in radial patterns, or they may lack folds. These characters are not constant. Pigment production may be lost rapidly on subcultivation, and the texture of the colony is fre- quently altered by the gradual or sudden appearance of an abundance of sterile aerial mycelium, at first as localized tufts on various parts of the colony, but rapidly growing over the whole colony. This is a type of mutation which occurs regularly in some species of derma- CULTURAL CHARACTERS 155 m u • ..^-^^.i^^^^^PS?'*''^-, - ,«^00^^ ^H^^PWS^awi %'i C' "s. Fig. 77. Cultures of dermatophytes: 1, Microsponim Canis; 2, M. Audouini; 3, M. gypseumf A, Trichophyton suljureum; 5, T. rubrum; 6, T. mentagro- phytes. 156 INFECTIONS CAUSED BY MOLDS tophytes and once it has replaced the original type of growth the latter cannot be recovered. Old laboratory cultures are thus fre- quently quite atypical unless they have been properly cared for. If a strain is to be conserved in typical condition a culture should be allowed to grow for 10 days (or longer if required for sporulation by slowly growing species) and then stored at 2° to 5° C. until it is again subcultured. Transfer periods should not exceed 4 months. Strains can be conserved by leaving cultures at room temperature for several months and then, only after the culture is very dry, transferring spores from the upper end of the slant. Under these conditions the sterile overgrowth is usually dead and conidia which have not mutated but have remained viable^ when transferred to new slants, will reproduce the original colony type. This method cannot be depended upon, however, and strains kept in this manner are apt to be lost. From these considerations it will be seen that the precise determination of species is a matter requiring some experience and judgment. Morphology in Culture. In artificial cultures specific and char- acteristic structures of several kinds are produced. Most important of these are the conidia which are produced in large numbers by many strains and species. The conidium of the dermatophytes shows more clearly tban any other single structure the close relationship between the different groups. It varies in size, shape, and abundance to some extent, and in one genus is lacking, but in general it is char- acteristic. The size varies from 2 by 2 microns to 3 by 5 microns. It is spherical to egg-shaped or even clavate, and has a thin smooth wall. The conidium has a broad attachment to the conidiophore and when it breaks loose from this attachment the broad base, fringed by the broken fragments of the end of the conidiophore, can be easily seen if the spore is carefully examined under high magnification. The shape of the spore in some strains of Trichophyton is almost spherical except for this basal facet. In other strains, and in Micro- sporum, the conidium is usually about 3 by 5 microns or larger, and is distinctly clavate, but the basal facet is like that seen in Tricho- phyton. The attachment of the conidium in some species of Trichophyton is rather persistent and in some strains one can observe spores still attached to a phantom hypha in which most of the cells are empty. This characteristic has led the French taxonomists to designate these spores aleuries. In the most common species, however, the name is a misnomer because the conidia are easily detached and these conidia are obviously of the same type in all species. A second definitive MORPHOLOGY IN CULTURE 157 type of spore is the macroconidium. Macroconidia are sometimes called spindle-spores or fuseaux, but the latter terms are descriptive of only the macroconidia of Microsporum. In Alicrosponnn Canis the macroconidia are large, reaching a size of 40 by 150 microns, Fig. 78. Macroconidia of dermatophytes: upper left, Microsporum Canis; upper right, M. gypseum; lower left, Trichophyton mentagrophyles; lower right, Epidermophyton floccosum, 158 INFECTIONS CAUSED BY MOLDS having as many as 12 or 15 crosswalls, and are truly spindle-shaped with thick, rough walls. In M. Audouini a few macroconidia similar to those seen in M. Canis may be found in some strains, but most of Trichophyton Epidermophyton Microsporum Fig. 79. Spore types in the three genera of dermatophytes. them are smaller, have only one or a few crosswalls, and the walls are usually smooth except near the tip. In M. gypseum the macro- conidia are very numerous, broader in proportion to length than in M. Canis, and the walls are nearly smooth. CYTOLOGY 159 In Epiclermophyton no conidia are produced and the macroconidia are clavate or egg-shaped, rounded instead of pointed at the tip, with no septa or only a few, and the walls are thick and smooth. In Trichophyton the macroconidia, when produced, are long clavate spores with rounded end, one or several cells formed by cross septa, and the walls are smooth and thinner than in the other genera. The macroconidia thus serve to identify the genus of dermatophyte. Macroconidia are produced in each of the three genera (although not in every strain of every species), but the type of macroconidium in each genus is distinctive.^ Taxonomy. Some of the dermatophytes occasionally form peculiar knots and twisted masses of hyphae which resemble the ascogonia of some Ascomycetes. These have been interpreted as abortive at- tempts to form asci. They have been referred to by the French authors as nodular organs. Matruchot and Dassonville ^- noted a similarity between certain of the dermatophytes and some species of Gymnoascaceae, and claimed to have produced experimentally a ringworm with a species belonging to this family of the Ascomycetes, Ctenomyces serratus. The Gymnoascaceae are characterized by the formation of asci, not in a compact and well-defined perithecium, but in masses surrounded by a rather loose network of protective mycelium. The clusters of conidia and specialized hyphae produced by some strains of Trichophyton resemble the loose poorly organized ascocarp of the Gymnoascaceae in a superficial way, and several authorities have therefore considered that the dermatophytes are ascomycetes. We do not believe that this point has yet been proved, and prefer to consider the dermatophytes as Fungi Imperfecti, with- out however denying the probability of a relationship to the Asco- mycetes. The variety and variability of the spores formed by the dermatophytes has made it difficult to assign them a position in Sac- cardo's classification. Vuillemin groups them with his Arthrospo- rineae, Ota and Langeron ^^ with the Conidiosporales of Vuillemin's classification. In the hair and skin the dermatophytes sporulate only by a frag- mentation of the mycelium into its component cells. The so-called spores found in the lesions are therefore to be looked upon as oidia or arthrospores. The size and arrangement of these arthrospores give some information about the identity of the fungus, but for specific identification pure cultures must be studied. Cytology. Grigorakis studied the cytology of the ringworm fungi. The cells of the vegetative hyphae and of the macroconidia, chlam- ydospores, and arthrospores are multinucleate. The conidia are uni- 160 INFECTIONS CAUSED BY MOLDS nucleate. Mitotic division of the nuclei was not observed. Mito- chondria and metachromatic granules were also demonstrated. Em- mons confirmed the nuclear findings. Enzymes. The biochemical activities of the skin fungi have been investigated by Tate ^^ who found that all the species which he in- vestigated were proteolytic and secreted lipase, urease, maltase, and diastase. None contained invertase, inulase, lactase, or zymase. The proteolytic enzyme resembled trypsin, acting in an alkaline medium; it did not digest coagulated egg. None of the strains studied could hydrolyze keratin. The fat-splitting activities of ringworm fungi was studied by Mallinckrodt-Haupt, who found that the species studied could grow in mineral solutions with neutral fats as the sole source of carbon, if these were of animal origin, but showed little or no growth with vegetable oils. Main Groups of Dermatophytes. Sabouraud recognized four main groups of dermatophytes, Microsporum, Trichophyton, Epidermo- phyton, and Achorion. These were defined on the basis of the clinical type of lesion produced and the microscopic appearance of the fungus in the lesion, specifically, its relationship to invaded hairs. Thus Microsporum produced on the surface of a parasitized hair a mosaic pattern of small spores, Trichophyton was characterized by a linear arrangement of spores on the hair surface, Epidermophyton did not attack the hair, and Achorion produced peculiar yellow crusts and scutula on the scalp. The first three groups stand as valid genera if defined according to mycological criteria, although a considerable revision of Epidermophyton is required because of its use by some dermatologists for any fungus found growing on the glabrous skin, whether or not it was capable of invading the hair. The fourth genus, Achorion, cannot be defined as a genus of fungi by any valid method, and its species should be distributed to appropriate genera, as we shall see in a later paragraph. Sabouraud divided the genus Trichophyton into subgenera on the basis of the position of the fungus with regard to the hairs: Endothrix species of Trichophyton growing entirely within the hairs; Ectothrix species of Trichophyton growing mainly on the surface of hairs ; and Ectoendothrix or Neoendothrix species occupying an intermediate position between the two other groups. The key to the main groups of dermatophytes presented below is based in part on Sabouraud's clinical classification and in part on mycological criteria.*' The main groups write their own label, so to speak, on the invaded hair, and their identification is often easier KEY TO THE DERMATOPHYTES 161 if one has also seen the patient, but the final identification of species rests upon an examination of a pure culture. In using Sabouraud's system of differentiation, it is obvious that the inspection of hairs is necessary. For this purpose it is of course necessary to select dis- eased hairs. Selection may be facilitated by search under Woods light.^ Too frequently a bunch of perfectly normal hairs is sent to the laboratory for diagnosis ! The fungus is found in the lower part of the hair, that which is within the follicle and extends for a rela- tively short distance above the skin level. Hairs which have broken are of course more likely to show an extensive development of the organism than those which are not. Infections with Trichophyton tonsurans cause the hairs to break off flush with the skin, the stumps appearing as black dots in the fol- licles. These should be extracted with forceps for examination. In all cases the fungi first invade the hairs in the sheath. Whether even- tually it will be found within the shaft or without would appear to depend largely upon the duration of the infection, which in turn is correlated with the degree of in- flammatory reaction. In any case, it is well to remember that the differentiation according to position of the fungus in or on the hair is not an absolute one. In endothrix forms there will be some spores outside the hair, in ectothrix types some within. Fig. 80. Diagram showing the rela- tions of ringworm fungi to the hairs: a, Microsporum; b, Endothrix Tri- chophyton; c, Ectothrix Trichoph- yton. KEY TO THE DERMATOPHYTES Lesions of the Scalp Yellow crusts or scutula present. Clinical favus. 1. Hairs tend to split longitudinally. Colonies waxy, wrinkled, little or no aerial mycelium. Hyphae coarse, distorted, tips swollen and branched. Few conidia, no macroconidia. Trichophyton Schoenleini 2, Colonies white, downy. Conidia and macroconidia formed. Trichophyton quinckearium 162 INFECTIONS CAUSED BY MOLDS 3. Colonies yellowish brown with abundant aerial mycelium and very numer- ous macroconidia. Microsporum' gijpseum 4. Colonies glabrous, wrinkled, reddish violet. Trichophyton violaceum B. No scutula present. Hairs tend to break off square. 1. Suppurative reactions (as follicular abscesses), pustules (of kerion) absent (except Microsporum species of animal origin). a. Hairs broken off at a uniform height, several millimeters above the skin. Much scahng of the epidermis, highly contagious in most cases. Spores on outside of hairs, angular, forming a mosaic. In cultures many spindle- shaped macroconidia with thick, usually rough walls, multicellular (ex- cept M. Audouini). Microsporum b. Hairs mostly broken off flush with the skin, leaving black points. Little scaling of the epidermis. Not so contagious. In cultm-es conidia varia- ble in size, macroconidia very few or lacking. aa. Grows entirely within the hair, both mycelium and spores. Endothrix Trichophyton bb. Grows mainly within the hair, but a few hyphae and spores can be found on the exterior. Neoendothrix Trichophyton 2. Suppurative reactions occur. Lesions of smooth skin also frequently present. Some strains of animal origin. Fungus grows in and on the hair, spores mostly external. a. Spores arranged in rows, not in mosaic. Edoihrix Trichophyton aa. Spores in hair 5 to 8 microns in diameter. Section Megaspores bb. Spores in hair 3 to 4 microns in diameter. Section Microides h. Spores in mosaic pattern. Microsporum Canis Lesions of the Smooth Skin A. Eczema-hke lesions confined to moist parts, as inner surfaces of thighs, axillary regions, between fingers or toes, soles of feet. Not found within hairs. In culture, greenish yellow, no conidia, macroconidia egg-shaped to clavate, thick, smooth walls. Epidermophyton {E. fioccosum) B. Lesions not as above, generally involving hands, arms, general body surface, face, neck. 1. Lesions form intricate patterns of concentric rings with marked scaling. Hairs not invaded. • Trichophyton concentricum 2. Lesions are reddish patches, not raised above the skin level, round to irregu- lar in form, darker at the border, forming rings. Tending to heal in the center, new attacks may occur, forming concentric rings. Generally Microsporum Sometimes Endothrix Trichophyton 3. Lesions are elevated plaques, reddish, round or oval, scaly. Pustules fre- quently present at the border. Ectothrix Trichophyton FAVUS 163 Favus. Favus is caused by Trichophyton Schoenleini in most cases but T. violaceum and Microsporum gypseum occasionally cause clinical favus. The disease occurs not only in man but also in various lower animals, particularly mice, but also cats, dogs, chickens, and some others. The great majority of human cases are contracted by direct or indirect contact with preceding cases, and are due to T. Schoenleini, but some of the species infecting lower animals will also produce the disease in man. The disease occurs particularly in the poor and the unclean. It has almost disappeared in the more advanced countries, but is prevalent in southeastern Europe and northern Africa. It occurs more frequently in children than in adults. The lesions occur most frequently on the scalp, though other parts of the body surface may be affected, but in the latter case the disease has usually been carried from the scalp. The fungus grows be- tween the outer cornified layer of the skin and the inner layer of epithelial cells (]\Ialpighian layer) and forms a very characteristic yellow, cup-shaped mass, the scutulum. The infection begins in a hair follicle, and the hair is usually seen projecting from the scutulum. "When the latter is pulled off, there is left a raw, sometimes suppurating sur- face. By growth and coalescence of the scutula, there may be formed an extensive crusted layer on the scalp. The hairs become opaque and dull, and finally drop out, leaving bald spots. A section through the scutulum shows a radiating feltwork of mycelium, which tends to die off in the center, leaving a granular debris, and to form spores at the periphery. The fungus also invades the shaft of the hair, forming parallel bundles of mycelium through its center. The organism may be demonstrated by a microscopic examination of either the scutulum or a hair. A small portion of the former should be crushed under a cover slip in a 10 per cent sodium hydroxide solu- tion; the hair should also be examined in alkah. In the lesions the mycelium is very irregular, its component cells varying considerably in size and form. The cells tend to break apart, giving the filaments an articulated appearance, and the terminal portions of the filaments Fig. 81. Favus of the scalp showing crusts or scutula. 164 INFECTIONS CAUSED BY MOLDS give rise to a series of round arthrospores. In a preparation from a scutulum as described above, these elements become mixed togetlier and one finds a melange of rounded cells, short oval cells, and longer and shorter fragments of mycelium. The fungus has a similar struc- ture in the hair ; one finds long, articulated filaments of cells of vary- ing size, the terminal portions ending in a series of rounded arthro- spores. A very characteristic feature is the degeneration and disap- pearance of the fungus in the hair, which leaves a series of air bubbles. Cultures are obtained by inoculating minute portions of scutula or hairs on the surface of agar slants. As these cultures are likely to be contaminated by bacteria the specimens should be placed for several minutes in 70 per cent alcohol and a large number should be planted to increase the chance of obtaining some colonies reason- ably pure. The particles of material are transferred to the surface of the agar with a needle, and three or four widely separated inocula- tions are made on a slant. The optimum temperature is 30° C. The various strains vary considerably in rapidity of growth and in colony pattern and several varieties have been named on the basis of this variability. The colonies on agar are at first yellowish and waxy in appearance. As they grow older they become much wrinkled and develop a short whitish aerial mycelium (some strains may fail to do this). In cul- tures the organism is extremely pleomorphic. In slow-growing strains, the mycelium may become articulated and numerous chains of arthrospores appear as in the lesions. In more rapidly growing strains there occurs an extensive development of mycelium which is, however, very irregular; it forms in some places thick, irregular masses having little resemblance to ordinary mycelium. These are called the ameboid forms. Large yellowish thick-walled cells may appear in the course of the mycelium. Finally, filaments at the periphery of the colony end characteristically in a branched cluster of swollen cells. The latter structure is referred to as the favic chandelier. In cultures which form a short aerial mycelium a few conidia can usually be demonstrated. These are typical of the dermatophyte conidia except that they are more variable in size and shape than in most species. A few are so large as to suggest the macroconidia seen in other species of Trichophyton. T. Schoenleini is apparently of variable pathogenicity for lower animals. Typical scutula have been produced by inoculation of the RINGWORM 165 skin in mice. Intravenous and intraperitoneal injections into rabbits produce nodular lesions of the lungs or peritoneum respectively. Cases of favus contracted from lower animals are not nearly so frequent as the purely human form. Of these the mouse type is more frequently seen. It occurs on the smooth skin, rather than the scalp. Multiple scutula may develop, but the disease does not tend to spread and progress as in the infections with T. Schoenleini. The parasite of mouse favus is T. quinckeanum. Ringworm. Ringworm is a parasitic infection of the skin due to various fungi belonging in the genera Trichophyton and Micro- FiG. 82. Ringworm of the smooth skin. sporum. It occurs endemically in some centers of population and at times produces small epidemics. The disease is transmissible from man to man, from lower animals to man, and, rarely, from man to animals.^ It was formerly a very common disease, especially in children of the poorer classes. For a time in Paris special schools were maintained for children with ringworm. Partly as a result of a rapid means of treatment utilizing Roentgen rays, the disease has greatly decreased in prevalence within recent years. The disease is transmitted by means of the spores formed in or on the skin and hairs, either by direct contact or indirectly through combs, brushes, or towels. It may be contracted from animals also 166 INFECTIONS CAUSED BY MOLDS by direct contact, more frequently from the walls of stalls and litter about barns. Children are frequently infected from cats and dogs with which they play. The fungus has been isolated from the litter of animal stalls ; this shows that the fungus is capable of living sapro- phytically upon vegetable matter, as many of the other pathogenic fungi are. The spores can probably remain viable for long periods outside the animal body. The disease may attack all parts of the skin surface. Clinically a division is made between ringworm of the hairy parts and ring- worm of the smooth skin ; ringworm of the nails also occurs and may constitute a reservoir of infection which is difficult to eradicate. The nature of the infection varies considerably according to the degree of inflammatory reaction of the invaded tissues. .Thus a wide variety of clinical types may be recognized, but the differences are more ap- parent than real because fundamentally the pathologic changes are the same in all the types. The infection begins in a hair follicle, from which focus it extends to the surrounding skin and into the hair. In the skin, in addition to redness due to congestion, there occurs scaling caused by an over- production of epithelium in response to the irritation caused by the presence of the fungus, an exudation of fluid, and an accumulation of leucocytes. A very characteristic feature is the formation of con- centric rings of inflammatory reaction. This is, of course, more apparent on the smooth skin. The formation of these rings has not been satisfactorily explained, but may be due to the same mechanism which leads to the formation of concentric rings of growth so fre- quently seen when molds are grown in Petri plate cultures. The infection tends to spread at the periphery and heal in the center. It is the formation of these advancing rings of inflammatory reaction which has given origin to the popular name ringworm (Latin, tinea; French, teigne). Microsporum Ringworm. In Microsporum infection, although the mycelium invades and is found inside the hairs, the spores are formed on the exterior. There is some difference of opinion as to whether these spores are to be considered arthrospores, i.e., produced by frag- mentation of the mycelium, or whether they are conidia. But they occur in irregular clusters not in chains, and are closely packed to- gether on the surface, forming a sort of mosaic. The individual cells are polyhedral in form, not rounded or cylindrical. In Trichophyton infection, for comparison, the mycelium may be within or without the hair, or both, and the spores are formed in both places, but they MICROSPORUM RINGWORM 167 are definitely produced by a fragmentation of the mycelium, and as a result appear in regular parallel rows or chains. Ringworm due to members of the genus Microsporum is some- times referred to as microsporosis, to distinguish it from ringworm caused by Trichophyton. Microsporosis is in the majority of cases a ringworm of the scalp, called tinea capitis by the dermatologists. It occurs most frequently in children; in stubborn and untreated cases caused by Microsporum Audouini it may persist for some years, but usually disappears at puberty. It is the most contagious of the ringworms. During epidemics such as those recently seen in several cities in eastern United States M. Audouini causes a very high per- centage of ringworm of. the scalp. In other parts of the United States M. Canis is of more frequent occurrence and may exceed M. Audouini. In infections caused by M. Audouini the lesion consists of a red- dened scaling area which tends to spread in the characteristic ring form. A number of such patches may form and coalesce to give an irregular area. There is marked scaling of the epidermis. The hairs tend to break off transversely at a height of 2 to 6 mm., leaving the stumps somewhat thickened, whitish, and opaque. Thus there appear a number of very characteristic irregular (moth-eaten) bald patches covered by the short stumps of the diseased hairs, which are fairly uniform in height. Generally inflammatory reactions are not pro- nounced. In infections caused by M. Canis inflammatory reactions are more often present. One can therefore frequently predict, from the clinical appearance, which of the two fungi is the cause of the lesion, but there are so many exceptions to the general rule that it is not a dependable method of determining the etiology. The diagnosis may be established by examining an epilated hair in a drop of sodium hydroxide solution. There will be found on the outside a sheath of spores closely packed together to form a poly- hedral or mosaic pattern, and in the interior of the hair shaft septate- hyphae which tend to break up into arthrospores toward the distal end of the hair stub, and which terminate in growing hyphal tips (frequently dichotomously branched) toward the root of the hair. The spores germinate readily on Sabouraud's medium and give rise to a rather fine septate mycelium. After a few days the mycelium becomes distended here and there, the swollen cells developing into chlamydospores. These are very characteristic of the mycelium of Microsporum. The aerial mycelium is frequently peculiarly twisted, and gives rise to numerous lateral branches of short length and finally, on some of the branches, clavate conidia borne as lateral buds on conidiophores or undifferentiated hyphae. In M. Canis 168 INFECTIONS CAUSED BY MOLDS very numerous large, multicellular, spindle-shaped, rough, and thick- walled macroconidia are also borne on the hyphae. The macro- conidia of M. Audouini are' smaller, have fewer cells, and appear to be depauperate forms of the type. These macroconidia are so dif- ferent from the clavate macroconidia of Trichophyton with smooth, thin walls, rounded tips, and few cells, that there is usually little difficulty in deciding which type is under observation. A number of species of Microsporum have been described. Castel- lani recognized sixteen, of which four were of human origin, the remainder of animal origin. The important species are M. Audouini, seen only in man; M. Canis {M. lanosum, M. Felineum), and M. gypseum, from animal sources. Most of the other described species are only varieties of the first two named. These two species may be differentiated by the characters tabulated below (adapted from Plant and Griitz). Microsporum Audouini Highly contagious, causing school epidemics. Of long duration, resistant to treat- ment. Only the head usually involved, ex- ceptionally areas in the immediate vicinity. Inflammatory reactions mostly lack- ing if vmtreated. Cultures grow slowly. Colonies remain grey or white with a reddish color visible in reverse of culture. Only rarely transmissible to animals from cultures. Microsporum Canis Less contagious, may cause family epidemics. Of shorter duration, about 1 year. Frequently also skin lesions at a dis- tance from the head. Inflammatory reactions often present without any irritation from treat- ment. Cultures grow more rapidly and colo- nies attain a larger size. Colonies become tobacco brown to reddish in center. Rabbits and guinea pigs easily in- fected from cultures. Further differentiation may be made on the basis of colony struc- ture. In M. Audouini there is often a small central knob elevated above the surface of the colony. In some strains the latter is marked radially by four to six deep clefts. The whole surface has a velvety texture. In M. Canis there is a central flat area surrounded by an elevated zone of aerial mycelium with usually no folding. The surface has a woolly texture. Endothrix Species of Trichophyton. The pure endothrix species of Trichophyton are from human sources. Their infections occur mainly in the scalp, the majority of the cases of ringworm of the ENDOTHRIX SPECIES OF TRICHOPHYTON 169 scalp not due to Microsporum being caused by members of this group of molds. They produce persistent infections without much inflammatory reaction. Although most cases occur in younger life, the infection does not, like microsporosis, tend to disappear at pu- berty, but may persist into adult life. Trichophytosis (endothrix) of the scalp is differentiated from microsporosis by the fact that the organism is found almost exclusively within the hair, the spores being arranged in chains. The hairs tend to break off at the skin level rather than a few millimeters above, leaving a smooth bald spot with few hair stumps and little scaling of the skin. In addition the disease almost always involves some part of the smooth skin as well. The disease is sometimes referred to by its French name, peladoide. Many endothrix species of Trichophyton have been described, but most of these names apply to minor variants of two or three species. The important species are Trichophyton tonsurans {T. crateriforme), T. Sabouraudi {T. acuminatum), and T. violaceum. T. tonsurans produces large irregular bald patches, the hairs break- ing off flush with the skin early after infection. The skin of the scalp in these bald patches may appear quite healthy. Some hair stumps may be present. They are dark in color, thicker than the normal hairs, and pull out with some difficulty. Microscopic exam- ination reveals the chains of spores in the interior of the hair. They are round, oval, or cylindrical in form and are produced by simple fragmentation of the mycelium into its component cells. They are much larger than the spores of Microsporum and have thick walls. In addition to the bald patches of the scalp, ring-shaped lesions fre- quently develop on the face, neck and hands. The colonies on Sabouraud's agar are acuminate, i.e., there is a conical peak projecting from the center of the colony. The closely related T. Sabouraudi forms a colony similar in appearance except that there is a central depression. The peripheral portion of the colony is folded into ridges. The colony is creamy white in color, becoming brownish in some strains; the surface is covered with a fine powdery coat of short hyphae and conidia. The conidia are spherical to pear-shaped or clavate in some strains, borne laterally and at the tips of conidiophores or on only slightly differentiated hyphae, Chlamydospores are formed in abundance in the vegetative mycelium. T. violaceum is found in northern Africa, southern Europe, Russia, and America. It produces lesions of the scalp of the same general character as the species just described. In the hair the spores are not so regularly in chains, and they are rounded. The colonies are 170 INFECTIONS CAUSED BY MOLDS of the acuminate type, but when first isolated they are of a deep violet color. Many strains lose the pigment upon continued cultiva- tion. Colorless sectors frequently appear in the pigmented colonies, together with sectors of transitional color. A number of varieties have been named according to these various differences. Few spores are formed in the cultures. Some strains, after cultivation, lose the Fig. 83. Ringworm of the smooth skin showing concentric rings, approaching the imbricate type. typical glabrous wrinkled appearance and produce a short aerial mycelium. On this mycelium a few typical dermatophyte conidia can sometimes be found. Fungi of the endothrix group have a low degree of virulence for laboratory animals producing no lesions or only localized lesions which soon heal. Neoendothrix Species of Trichophyton. Neoendothrix species of Trichophyton are transitional between the ectothrix and endothrix groups as far as the relationship 'of the fungus to the hair is con- cerned. The condition in the early invasion of the hair by an endo- thrix species in which there are hyphae outside the hair shaft as well as within persists in this group. The infections produced are also transitional between those caused by the human and animal types of Trichophytons; they show some tendency to inflammatory reaction, ECTOTHRIX SPECIES OF TRICHOPHYTON 171 but it is not so marked as in the ectothrix species. Infections by these transitional species are seen in subtropical America, Germany, and Austria. There is one main species, Trichophyton epilans [T. cerebriforme, T. plicatile): It produces ringworm of the scalp, the beard, and the smooth skin, with varying degrees of inflammatory reaction. Plant reports isolating it from cats. It may be inoculated on guinea pigs. The colony resembles T. tonsurans but is yellower and the surface is more wrinkled and folded. Ectothrix Species of Trichophyton. Ectothrix species of Tri- chophyton are clinically sharply differentiated from the endothrix Fig. 84. A hair from ringworm of the scalp. This is a small-spored Ectothrix Trichophyton. The spores are seen on the surface of the hair. types by the more pronounced inflammatory reaction which they produce in the skin. This would seem to indicate a greater virulence. These acute types of trichophytosis tend to heal more rapidly than those with a less pronounced inflammation. Whereas with Micro- sporum and Trichophyton infections the skin changes are usually limited to congestion of the advancing border and scaling of the epidermis, with infections from the ectothrix species there is also an exudation of serum and leucocytes into the skin which leads to more or less bogginess and infiltration of the deeper tissues. Not infre- quently pus oozes in small droplets from the mouths of the hair ^ follicles, or distinct pustules may appear. In more acute forms cer- tain areas of skin may become elevated and feel quite boggy when palpated, as though an abscess of some size were developing, and pus oozes from the hair follicles when the skin is squeezed. Such a lesion is called a kerion. Suppurative lesions of the beard are re- ferred to as sycosis. The ectothrix species are divided into two groups, those with small spores in the lesions (about 3 microns in diameter) and those with 172 INFECTIONS CAUSED BY MOLDS large spores (5 to 7 microns in diameter) . Species of the first group grow more rapidly in artificial culture than those of the second. The small-spored species may be confused with Microsporum if the differentiation is made solely on a hasty examination of the hairs, for they present the same general appearance — a few* articulated hyphae within the hair and numerous spores on the surface forming a sheath. However, these spores, unlike those of Microsporum, are Fig. 85. A suppurating ringworm of the beard ("sycosis"). This is due to an Ectothrix Trichophyton. produced by a fragmentation of hyphae and therefore tend to be arranged in chains rather than in an irregular mosaic. The spores of the two fungi are about of a size. In microsporosis one rarely sees as much inflammatory reaction with a tendency to deep infiltra- tion of the skin as is characteristic of the small-spored ectothrix species of Trichophyton. , In adults the small-spored ectothrix species cause infections of the beard region, the smooth skin, especially the extensor surfaces of the forearms, and the feet. In children lesions of the scalp, face and hands occur. The lesions, when on the smooth skin, are usually of the circular type characteristic of ringworms. The small-spored group form white, cream-colored, yellow, or pink colonies which vary from granular, stellate colonies to snow-white, floccose, "powder-puff" colonies. Many species have been described ^nd differentiated on the b^^is of colony type. The most common DERMATOPHYTOSIS OF THE FEET 173 of these are Trichophyton gypseum, T. inter digitale, T. pedis, and T. niveum. This represents a considerable range in colony type, but when many strains are compared intermediate types can be demon- strated readily." The fungi of this group seem to be actually vari- ants of a single species. The oldest name for this species is T. menta- grophytes and this is accepted here as the valid name.^ The members of this group will readily infect guinea pigs. They have been found causing infections of various domestic animals, espe- cially horses but also cats and dogs. The large-spored ectothrix species are also divided into two groups, those with downy colonies, and those with faviform (i.e., resembling T. Schoenleini) colonies. This difference is due to the amount of aerial hyphae, which is formed early and abundantly in the former, later and less abundantly in the latter. The colonies of the faviform group have the waxy appearance of those of the favus parasite. Cultures of the downy type produce conidia as the only fruiting bodies; cultures of the faviform type fail to produce these, forming only mycelium and chlamydospores. All the large-spored ectothrix species may produce ringworm of the smooth skin, scalp, or beard. They occur more commonly in adults than children. They also occur on a variety of domestic ani- mals. The most important species, T. javijorme, is a pathogen of cattle which readily attacks man. Tinea Imbricata. Trichophyton concentricum is the etiological agent of a variety of ringworm occurring in China, the Malay penin- sula, various Pacific islands, and in Central America, known as Tokelau ringworm or tinea imbricata. The concentric rings so char- acteristic of the tineas reach their highest development in this disease. There is much more scaling of the epithelium than in the other ring- worms, and as a result there is produced on the skin a complicated pattern caused by the confluence of concentric rings of scales. The fungus grows within the epithelium between the superficial and deeper laj'ers. It appears in the scales much as does Epi- dermophyton. Cultures are obtained with great difficulty, partly because many of the very numerous hyphae which can be demon- strated in the scale seem to be dead, and partly because the bacterial infection of the scales is usually so high. The scales can be soaked first in 70 per cent alcohol and a large number of plants made. The colonies are at first like those of T. Schoenleini. Few spores are formed in cultures. Dermatophytosis of the Feet. During recent years dermatophy- tosis of the feet (athlete's foot) has attracted increasing atten- 174 INFECTIONS CAUSED BY MOLDS tion.^^' -° The condition was probably overlooked for many years, but there may also be an increase over the occurrence fifty years ago. According to Mitchell/^ this was due in part to the demobiliza- tion of a considerable number of soldiers at the end of World War I who were suffering from this infection and tinea cruris. Perhaps a greater factor has been the increased participation of the population Fig. 86. Dermatophytosis of foot. Hyphae of Trichophyton mentagrophytes in epidermal scales, unstained. in athletics and golf. It is popularly believed that the infection is contracted by going barefoot in dressing rooms and showers where infected desquamated epithelium may be picked up.^ The infection is extraordinarily prevalent among college students. Legge, Bonar, and Templeton " found the disease in 78 per cent of the men and 17 per cent of the women in a survey at the University of California. The much lower incidence in the women was attributed to the use of rubber bathing slippers. The clinical diagnosis should be con- firmed by laboratory examination.^* ^^ The condition begins in the folds between the toes or on the sole. The lesions begin (frequently rather suddenly) as a series of small bhsters, which tend to coalesce. This is followed by scaling and the EPIDERMOPHYTON 175 development of eczema-like lesions. The condition may yield to treatment, but tends to recur, especially during the summer months. Recurrence is probably more important in the incidence of infection than reinfection. The sudden appearance of a lesion after unusual amounts of walking or standing may be due merely to the sudden activation of an old quiescent lesion in the epidermis or on a toe nail. The presence of fungi has been demonstrated in skin showing no evidence of clinical dermatophytosis or in skin at some distance from an apparent lesion. The lesions in the nails are particularly difficult to clear up completely and treatment is often stopped in the belief that cure has been achieved when actually there is still a focus of infection in a toe nail. A variety of fungi have been isolated. Although some of the cases have been definitely associated with tinea cruris and Epidermoph- yton jioccosum has been isolated from a considerable number of cases (20 per cent in Mitchell's series), it is now apparent that the major- ity of cases are caused by various varieties of Trichophyton menta- grophytes {T. gypseum, T. inter digitale, T. pedis) and T. ruhrum. Epidermophyton. The genus Epidermophyton contains a single species, Epidermophyton fioccosum {E. inguinale, E. cruris). The Fig. 87. Tinea cruris, due to Epidermophyton. 176 INFECTIONS CAUSED BY MOLDS use of the name Epidermophyton for other species found on the feet, merely because they are not seen in hairs, is incorrect. E. floccosum is found on the feet as a cause of athlete's foot but it is most im- portant as the cause of tinea cruris or eczema marginatum. It oc- curs on the smooth skin in those parts which are likely to be moist, most frequently the inner surfaces of the thighs, but also in the axillae, the folds of the buttocks, or under the breasts. It is more frequent in warm climates and is especially prevalent in India, where it is apparently one of the conditions known as dhobie itch. It has been responsible for epidemics aboard ship. The afTection is somewhat different from the other ringworms especially in that it does not tend to heal in the center as it spreads at the periphery. The fungus is found in scales of epidermis as septate hyphae break- ing up into chains of oval or round arthrospores. In artificial cul- tures, reproduction takes place entirely by macroconidia, which are not spindle-shaped as in Microsporum, but are clavate or egg-shaped. The walls are thick and smooth. The distal end is rounded. The macroconidium may be one-celled or may have one or more septa. No small conidia are produced. The color of the colony is a char- acteristic greenish yellow. Cultures rapidly become overgrown with the white sterile mutant mentioned earlier. Saprophytic Skin Fungi Pityriasis Versicolor. Pityriasis versicolor is one of the common- est of the dermatophytoses. It is characterized by a brownish dis- coloration of the skin and it occurs most frequently on the trunk. It causes a light branny scaling and, in some cases, slight itching. In non-pigmented areas of the skin the lesion is darker than the surrounding skin. In exposed skin which is tanned the lesion is often lighter than the other skin, apparently because the fungus interferes with the normal sun-tanning. The causative organism, Malassezia furfur, may be found in large numbers in scales of the epidermis mounted in hydroxide solu- tion. It appears as short irregular strands of branched hyphal frag- ments accompanied by large numbers of round spores varying con- siderably in size. When stained with carbol fuchsin the spores are seen to contain several deeply stained bodies of globular form in a less deeply stained protoplasm. The spores tend to be arranged in clusters aud are perhaps produced from the hyphae as spores. LITERATURE 177 Although some investigators have claimed to cultivate the fungus it is evident that most of the fungi isolated in culture have been contaminants. Erythrasma. Erythrasma is an affection of the moist skin areas of the axillae and groin. It is characterized by a brownish to reddish discoloration. The fungus appears in the epidermal scales as very minute (about 1 micron in diameter) branched hyphae and spores. It is generally referred to as Microsporon niinutissimum, but there seems to be little doubt that it is one of the actinomycetes. Piedra. Piedra, or trichosporosis, is an affection of the hairs, not actually a dermatophytosis, occurring in tropical climates. Masses of fungus mycelium grow as little hard knobs on the hairs. Black piedra is caused by an Ascomycete, Piedraia Hortai. White piedra is caused by Trichosporon Beigelii. Both fungi are most common in tropical regions of high humidity. LITERATURE 1. BoxAR, L., and A. D. Dreyer, Studies on ringworm funguses with reference to public health problems, Am. J. Puh. Health, 22, 909 (1932). 2. Catanei, a., Etudes sur les teignes, Arch. inst. Pasteur, 11, 267 (1933). 3. Davidson, A. M., and P. H. Gregory, Note on an investigation into the fluorescence of hairs infected by certain fungi, Can. J. Research, 7, 378 (1932). 4. , The so-called mosaic fungus as an intercellular deposit of cholesterol crystals, J. Ayn. Med. Assoc, 105, 1262 (1935). 5. Emmons, C. W., Pleomorphism and variation in the dermatophytes, Arch. Dermatol. Syphilol. (Chicago), 25, 987 (1932). 6. , Dermatophytes : natural grouping based on the form of the spores and accessory organs, Arch. Dermatol. Syphilol. (Chicago). 30, 337 (1934). 7. Emmons, C. W., and A. Hollaender, The action of ultraviolet radiation on dermatophytes. II. Mutations induced in cultures of dermatophytes by exposure of spores to ultraviolet radiation, Am. J. Botany, 26, 467 (1939). 8. Gregory, P. H., The dermatophytes, Biol. Rev., Cambndge Phil. Soc, 10, 208 (1935). 9. GuiART, J., and L. Grigorakis, La classification botanique des champignons des teignes, Lyo7i med., 141, 369 (1928). 10. Kaufman N-WoLFF, M., tjber Pilzerkrankungen der Hiinde und Fiisse, Der- matol. Z, 21, 385 (1914). 11. Legge, R. T., L. Bonar, and H. J. Templeton, Epidermomycosis at the U^ni- versity of California, Arch. Dermatol. Syphilol. (Chicago), 27, 12 (1933). 12. Matruchot, L., and C. D.^ssonville, Sur le champignon de I'herpes (Tricho- phyton) et les formes voisines, et sur la classification des Ascomycetes, Bull. soc. mycol. France, 15, 240 (1899). 178 INFECTIONS CAUSED BY MOLDS 13. Mitchell, J. H., Further studies on ringworm of the hands and feet, Arch. Dermatol. Syphilol. (Chicago), 5, 174 (1922). 14. Neal, p. a., and C. W. Emmons, Dermatitis and coexisting fungous infec- tions among plate printers, Public Health Bull. 246, 1939. 15. Ota, M., and M. Langeron, Nouvelle classification des dermatophytes, Ann. parasitol., 1, 305 (1923). 16. Peck, S. M., Epidermophytosis of the feet and epidermophytids of the hands, Arch. Dermatol. Syphilol. (Chicago), 22, 40 (1930). 17. Sabouraud, R., Maladies du cuir chevelu. III. Les maladies cryptogamiques. Les teignes, Masson et Cie., Paris, 1910. 18. SwARTZ, J. H., and N. F. Conant, Direct microscopic examination of the skin. Arch. Dermatol. Syphilol. (Chicago), 33, 291 (1936). 19. Tate, P., The dermatophytes or ringworm fungi, Biol. Rev., Cambridge Phil. Soc, 4, 41 (1929). 20. Wise, F., and J. Wolf, Dermatophytosis and dermatophytids, Arch. Der- matol. Syphilol. (Chicago), 34, 1 (1936). BLASTOMYCOSIS (American Blastomycosis, Gilchrist's Disease) Blastomycosis was first described in 1894 by Gilchrist.*' * In a second case reported by Gilchrist and Stokes ' the fungus causing it was isolated in culture. It was thoroughly studied by Ricketts.® Martin and Smith ^ have more recently published a very useful re- view of the disease and reported in detail several cases. Although not common, it has been reported frequently enough from many parts of the United States to establish it as one of the most impor- tant of the systemic mycoses. Clinical. In about half the reported cases of blastomycosis the first complaints were of pulmonary involvement. In a considerable number of the remaining cases the first observed lesions were sub- cutaneous nodules. The distribution of these lesions does not appear to be related to trauma or exposure and it is probable that the pri- mary lesion was actually in the lung and that there was blood stream dissemination of the fungus. Primary blastomycosis of the lungs frequently bears a striking resemblance to pulmonary tuberculosis in its course and symptoms, and is often so mistakenly diagnosed, the first indication of the true nature of the disease being a rather sudden generalization of the infection with the development of sub- cutaneous abscesses. In systemic blastomycosis the lungs are involved in 95 per cent of the cases and it is probable that in most of these instances the primary lesion was in the lungs. Pulmonary blastomycosis may * Literature citations for this section will be found on page 186. CLINICAL 179 resemble miliary tuberculosis or there may be a few large nodules or abscesses. Small cavities are sometimes found. There may be diffuse or focal consolidation. The bones and joints, spleen, kidneys, prostate and central nervous system are frequently involved. Lesions are found in other organs less frequently. In most cases of systemic blastomycosis skin lesions eventually develop.^- ^^ Dissemination by way of the blood stream leads to a development of multiple abscesses throughout the body. These are particularly prone to occur in the subcutaneous tissues, but may also develop in the muscles, under the periosteum of the bones, or in the viscera. The subcutaneous abscesses are quite characteristic and quite differ- ent from the primary skin lesions. They develop painlessly and without much local heat or redness; they are soft and fluctuant, and when opened discharge a considerable amount of pus from which the fungus may be cultivated. The generalized form of the disease is accompanied by a septic type of fever curve and is usually fatal. Finally there are cases of primary cutaneous blastomycosis oc- curring most commonly on the face, hands, wrists, arms, or lower legs, where exposure to trauma or repeated irritation may be im- portant factors in permitting entrance of the fungus through the skin. In many of these cases there has been a definite history of injury preceding the development of the skin lesion. In primary cutaneous blastomycosis lesions frequently begin as pustules which ulcerate and do not heal. There is usually a single lesion although satellite lesions may follow autoinoculation by scratching. The primary skin lesion may be a papule. Around this secondary nodules develop, slowly enlarge, and coalesce. These break down and discharge pus through a number of small fistulae. As the disease progresses there gradually develops a large elevated mass of tissue with an irregular ulcerated surface that resembles somewhat a breaking down cancer, sometimes a tuberculous ulcer. Slight pressure on the mass will cause pus to ooze from a number of minute openings. There is a considerable development of granula- tion tissue which may be covered with a yellowish oozing crust but which frequently becomes verrucous. The border of the typical skin lesion is elevated and slopes sharply to the normal skin. This active border advances slowly, obliterating the normal structures. The older part of the lesion heals, but the resultant scarring is often very disfiguring. The microscopic appearance of the tissue presents some resem- blance to both tuberculosis and cancer. The inflammatory reaction, particularly in the subcutaneous fibrous tissue, is largely gran- 180 INFECTIONS CAUSED BY MOLDS Fig. 88. Blastomycosis of the skin. Fig. 89. Blastomycosis (Gilchrist's disease) of the skin. PARASITIC GROWTH PHASE 181 ulomatous in nature, there being much new-formed connective tissue and considerable infiltration with mononuclear leucocytes. Giant cells may be formed, and the epithelial tissue, in response to irrita- tion, undergoes considerable proliferation and may send long, finger- like processes down into the inflammatory tissue, much as in epitheli- oma. A very constant and characteristic feature of the microscopic pathology is the occurrence of minute abscesses, i.e., spaces filled with polymorphonuclear leucocytes, in the epithelium proper. In these miliary abscesses the parasites are found extracellularly and within giant cells. Baker ^ has recently analyzed the tissue reactions in twenty-three cases. Diagnosis. The diagnosis of blastomycosis is made by the demon- stration of the fungus in tissues or pus. This is necessary because of the similarity of the lesions to those of other granulomatous proc- esses. The size of the fungus makes it relatively easy to find in tissue sections or when pus is mixed with a drop of 10 per cent sodium hydroxide and examined unstained under a cover slip. Martin described a complement-fixation test which appeared to be specific. However, a negative test does not exclude the diagnosis because complement-fixing antibodies are absent from the blood in some cases. Treatment and Prognosis. In the treatment of cutaneous blasto- mycosis sodium iodide intravenously and potassium iodide by mouth have been fairly successful. Tincture of iodine is also applied locally. Currettage of the lesions and x-ray therapy as well as radium have also cured some cases. Occasionally no treatment is effective. In systemic blastomycosis iodides not only fail to arrest the dis- ease but may cause a rapid spread of the lesions. Martin and Smith ^ recommend partially desensitizing the patient by injecting subreact- ing doses of a skin-testing material prepared from the fungus and gradually increasing the dose until little or no reaction is elicited. Iodides can then be safely administered and sometimes they cure the infection. The prognosis in systemic blastomycosis, however, remains extremely bad. Parasitic Growth Phase. Cells of the parasitic growth phase vary from 3 to 24 microns in size, the usual range being 8 to 10 microns. The fungus cell has a thick wall which is sometimes described by the rather ambiguous term, double contoured, because its inner and outer limits can be observed. The cell may bud in a manner re- sembling that of the yeasts or it may elongate or become dumb-bell- shaped and be divided by a crosswall at the point of constriction. When a bud is formed it at first has a thinner wall than the parent 182 INFECTIONS CAUSED BY MOLDS cell. Usually a cell bears a single bud, but one can also find two or more buds arising from one end of a cell or observe a chain of cells representing buds' which have failed to separate. The opening between the parent cell and the young bud (and the plane of attach- ment between two mature cells) has a greater diameter than is seen in the typical budding of yeasts where the bud is typically pinched off and successive buds form at or near the same spot. Fig. 90. Budding yeast-like cells of Blastomyces dermatitidis, in a wet prepara- tion of pus from a case of blastomycosis. The cells of Blastomyces dermatitidis are larger and have thicker walls than those of Candida albicans and the yeasts. They lack the conspicuous capsule produced by Cryptococcus neoformans and are less uniform in shape than the spherical cells of that fungus. B. dermatitidis has sometimes been confused with Coccidioides immitis, but careful search will reveal budding or pseudobudding which is never observed in the latter. No endospores or ascospores are formed by B. dermatitidis. Cultures. The fungus grows slowly, but if primary cultures are not too heavily contaminated it can be isolated in culture readily. Pus can be spread on blood agar plates and incubated at 37° C, wherupon the fungus grows in a manner closely resembling its para- sitic growth phase. Colonies are yellowish white to tan, somewhat mealy or waxy, and of a vermiculate or worm-cast type. A micro- CULTURES 183 Fig. 91. Blastomyces dermatitidis in a young culture, showing transitions from the yeast-like form to mycelium. Hit: -^1. '^'wtBk Fig. 92. Cultures of Blastomyces dermatitidis: left, the mealy type; center, the prickly type; right, the -woolly type. 184 INFECTIONS CAUSED BY MOLDS scopic examination of such a colony sliows many cells which closely resemble those found in tissue, others which are perhaps twice as long as thick and divided into two cells by a central septum, and some which form thick, abortive hyphae. An examination of these types and transitional forms illustrate clearly the morphological dif- ferences between the budding cells of Blastomyces dermatitidis and those of the true yeasts. When pus containing the fungus is streaked on Sabouraud agar and incubated at room temperature or 30° C. the parasitic growth phase produces branching hyphae and the resultant colony is that of a mold. Three types of growth have been described. Colonies may be somewhat friable (the so- called mealy type) , they may con- sist of a glabrous moist growth on the agar surface with coremia (upright composite strands of hy- phae forming spine-like aggregates at the center of the colony), or they may be tangled masses of dry, aerial hyphae. Some colonies are pure white whereas others be- FiG. 93. Blastomyces dermatitidis. p^j^g brown in age. These varia- Conidia in culture. ^-^^^ ^^^ sometimes related to strain differences and may also be observed in a single strain when it is kept in the laboratory over a period of years. Thus the mealy type is most apt to appear in newly isolated strains and microscopic examination will show the presence of pseudobudding forms and abortive hyphae. The presence of contaminating bacteria which may be carried along undetected for many culture generations often modifies the growth habit. Conidia. Gilchrist and Stokes,'^ in their original description of the fungus, and many subsequent observers have described the conidia which develop in cultures. These structures are quite vari- able in size and shape, depending to some extent upon the individual strain. They are spherical to pyriform or oval and range in size from 2 microns in diameter to 3.5 by 5 microns. Some are sessile, budding directly from the hyphae, and others are on lateral stalks of conidiophores 1 to 10 microns long. The walls are smooth. Conidia may be rare, and in strains kept for many years in the laboratory none may be found. TAXONOMY 185 Animal Inoculations. Laboratory animals are somewhat resistant to infection so that animal inoculation is not a useful method of lab- oratory diagnosis. However, when large numbers of spores from a culture are injected typical lesions result, and this is a useful method of confirming the identify of a culture. Spring" found that mice are the most susceptible of the common laboratory animals, guinea pigs being more resistant, and rabbits practically immune. When the animals are inoculated intraperitoneally, small caseous nodules develop on the peritoneal surfaces which contain the budding fungus cells. These are more numerous in mice than in other animals. The type of tissue reaction in experimentally inoculated animals varies with the virulence of the strain and the resistance of the animal, from frank abscesses to lesions like tubercles, including giant cells. Ben- ham 3 stated that dogs and monkeys are most susceptible. DeMon- breun ^ produced in monkeys cutaneous blastomycosis of the type seen in man. Baker ^ discussed experimental blastomycosis in mice. Spontaneous blastomycosis in dogs has been reported. Taxonomy. Both the name of the disease and that of its etiolog- ical agent are. misnomers. Blastomycosis is used in Europe to desig- nate any disease caused by a budding yeast-like fungus. Whether or not this terminology is justified, it has been the source of consider- able confusion. Although Blastomyces dermatitidis produces buds of a sort in its parasitic growth phase, it is unlike the yeasts both in tissue and in culture, as already pointed out. B. dermatitidis, likewise, is an incorrect name for the fungus be- cause that generic name properly belongs to an unrelated group of fungi. Nevertheless, both names are generally recognized, and be- cause of their familiarity we continue to use them here. Misinterpretation of oil droplets and stored food particles within chlamydospores and other structures have led some observers to place B. dermatitidis among the Ascomycetes. It is properly classified among the Fungi Imperfecta Although its relationships are not known, its resemblance in culture to Histoplasma capsulatum should be pointed out. Skin tests indicate an immunological cross reaction between these two fungi. Several species names have been proposed for the fungus causing blastomycosis. A critical examination of the strains named shows that some of them are varieties of B. dermatitidis characterized by minor differences which do not deserve specific differentiation. It is evident that in other cases the multiplication of names has been due to failure to differentiate B. dermatitidis from Coccidioides or other fungi. 186 INFECTIONS CAUSED BY MOLDS Conant and Howell * have recently transferred the fungus causing South American blastomycosis (paracoccidioidal granuloma) to this genus. Aside from the recognized error in the use of Blastomyces it seems reasonable to range the South American fungus, B. brasiliensis, alongside B. dermatitidis. Geographical Distribution. Blastomycosis is an American disease. It appears to be most common in the Mississippi Valley and it has been called the Chicago disease because of the number of cases ob- served in that area. However, it has a wide distribution on this continent; it has extended into Canada. Presumptive cases have been reported from England. Habitat in Nature. The occurrence of the fungus outside the animal body is not known. Circumstantial evidence suggests that it grows as a saprophyte in soil or dead vegetation. LITERATURE L Baker, R. D., Experimental blastomycosis in mice, Amer. J. Path., 18, 463 (1942). 2. , Tissue reactions in human blastomycosis, Amer. J. Path., 18, 479 (1942). 3. Benham, R. W., The fungi of blastomycosis and coccidioidal granuloma, Arch. Dermatol. Syphilol. (Chicago), 30, 385 (1934). 4. CoNANT, N. F., and A. Howell, Jr., Etiological agents of North and South American blastomycosis, Proc. Soc. Exptl. Biol. Med., 46, 426 (1941). 5. DeMonbreun, W. a., Experimental chronic cutaneous blastomycosis in monkeys, Arch. Dermatol. Syphilol. (Chicago), 31, 831 (1935). 6. Gilchrist, T. C, A case of blastomycetic dermatitis in man, Johns Hopkins Hosp. Repts., 1, 269 (1896). 7. Gilchrist, T. C, and W. R. Stokes, A case of pseudo-lupus vulgaris caused by a blastomycete, J. Exptl. Med., 3, 53 (1898). 8. Martin, D. S., and D. T. Smith, Blastomycosis, Arn. Rev. Tuberc., 39, 275, 488 (1939). 9. Ricketts, H. T., Oidiomycosis (blastomycosis) of the skin and its fungi, J. Med. Research, 1, 373 (1901). 10. Spring, D., Comparison of seven strains of organisms causing blastomycosis in man, J. Injections Diseases, 44, 169 (1929). 11. Stober, A. M., Systemic blastomycosis. Arch. Internal Med., 13, 509 (1914). South American Blastomycosis (Paracoccidioidal Granuloma) Historical. Lutz ^ * in 1908 and Splendore ^ in 1909 described a highly fatal disease observed in Brazil and characterized by skin * Literature citations for this section will be found on page 189. APPEARANCE IN CULTURE 187 and mucous membrane lesions and systemic involvement in which budding cells were observed in the pus. Splendore in 1912 named the fungus Zymonema brasiliense. In many of the early studies the disease was confused with coccidioidomycosis; Almeida^ in 1929 summarized the characteristics of the two mycoses and clearly pointed out their clinical differences, the fundamental morphological differ- ences between the fungi, and the lower virulence of this fungus for experimentally infected guinea pigs. Clinical. Unlike coccidioidomycosis, the primary lesions are most often in the mucous membranes of the mouth and nostrils and in- volvement of the gastrointestinal tract is usual. The lesions are ulcers which increase in size by peripheral spread and by the coales- cence of satellite lesions. They cause a rapid and extensive destruc- tion of the tissues. Skin lesions are crusted or nodular and resemble those of American blastomycosis. The regional lymph nodes become enlarged, break down, and drain through sinuses which penetrate the skin. Hematogenous spread of the organism follows. In some cases the first evidence of infection is enlargement of the lymph nodes in the neck. Primary lesions may appear in the gastrointestinal tract in the region of the cecum or appendix, where ulcers develop. The infec- tion spreads by peripheral enlargement of the lesions and by way of the blood stream. The lungs, spleen, liver, and other organs are in- volved when the disease becomes generalized. Diagnosis. The clinical aspects of the disease and the location and appearance of the lesions are fairly distinctive. In the laboratory diagnosis the fungus should be demonstrated in the lesions or a cul- ture should be obtained. Treatment and Prognosis. The disease does not yield to iodides and other antimycotic treatment and is almost invariably fatal in the diagnosed cases. Appearance of the Fungus in Tissues. The fungus is found in pus and in the tissues as a spherical cell 10 to 60 microns in diameter. It reproduces by budding. Some cells produce one or a few buds and resemble Blastomyces dermatitidis. Cells considered typical of this fungus produce many small buds. In optical section these appear as a crown of small (1 to 4 microns) spherical or elongated projections from the cell wall. Appearance in Culture. The fungus grows slowly. When cultures are incubated at 37° C. the fungus reproduces by a process of multiple budding similar to that seen in tissues. At room temperature it pro- duces hyphae which bear conidia similar to those seen in Blastomyces 188 INFECTIONS CAUSED BY MOLDS dermatitidis except that they are less uniform in size and shape and are not so well differentiated from the ehlamydospores which are also present. Strains vary, some producing a glabrous, wrinkled, or vermiculate colony, others extending more widely over the agar sur- face and being covered with short white aerial hyphae. Fig. 94. Blastomyces brasiliensis in liver tissue (XHOO), showing periph- eral small buds from parent cell. Occasionally larger buds are formed. Photomicrograph by Dr. N. F. Conant. Fig. 95. Blastomyces brasiliensis from beef infusion agar, 37°, X^OO. Some- times multiple budding with large peripheral cells is seen, like that sometimes found in tissues. On Sabouraud agar at room tempera- ture, morphology is mycelial, very like that of B. dermatiditis. Photo- micrograph by Dr. N. F. Conant. Taxonomy. Since Splendore named the fungus Zymonema bra- siliense it has been known under a variety of names, most familiar of which is Paracoccidioides brasiliensis Almeida. Conant and Howell - have called attention to the similarities between this fungus and Blastomyces dermatitidis and have transferred it to the genus Blasto- myces. Moore ^ described two additional species, but there is some doubt whether these are more than minor and unstable variations of B. brasiliensis. Geographical Distribution. The mycosis appears to be most com- mon in Brazil and particularly in the state of Sao Paulo, but it is known also in Argentina and other South American countries. Habitat. The habitat in nature of Blastomyces brasiliensis is un- known. There is some evidence of direct transmission of the fungus from man to man. CLINICAL 189 LITERATURE L Almeida, F. P. de, Estudo comparativo do granuloma coccidioidico nos Estados Unidos e no Biasil, Ann. vied. Sao Paulo, 4, 91 (1929). 2. CoNANT, N. r., and A. Howell, The similarity of the fungi causing South American blastomycosis (paracoccidioidal granuloma) and North American blastomycosis (Gilchrist's disease), J. Investigative Dermatol., 5, 353 (1942). 3. LuTZ, A., Uma mj'cose pseudococcidica localisada na bocca e observada no Brasil, Brasil vied., 22, 121, 141 (1908). • 4. MooRE, M., Blastomycosis, coccidioidal granuloma and paracoccidioidal gran- uloma, Arch. Dermatol. Syphilol. (Chicago), 38, 163 (1938). 5. Splendore, a., Sobre um novo caso de blastomycose generalizada, Rev. soc. sci. Sao Paulo, 4, 52 (1909). HISTOPLASMOSIS From 1906 to 1909 Darling, in a series of papers,-' ^ * reported his discovery of a new disease which he had found while searching the pathological material at the Ancon Hospital, Canal Zone, for kala- azar. He recognized in this material features which differentiated the organism from Leishmania, but he believed it to be a protozoan and he erected for it a new genus, Histoplasma, calling the organism Histoplasma capsulatum and the disease histoplasmosis. As addi- tional material was studied it became apparent that the organism was a fungus rather than a protozoan, but not until 1934 when Dodd and Tompkins ^ observed the fungus in blood smears taken before death and DeMonbreun * isolated it in culture was its complete life cycle known. In the same year Hansmann and Schenken ' also isolated the fungus in culture although, because of certain clinical features of their case, they did not identify it with histoplasmosis. Since these important advances in the knowledge of the fungus several cases have been diagnosed before death and the fungus isolated in culture. A study of this material has demonstrated some variability in the vari- ous strains of the fungus, and considerable variation in the clinical aspects of the disease. Clinical. Darling, from his study of preserved material and a review of case records, described the mycosis as one characterized by irregular fever, emaciation, leukopenia, anemia, and splenomegaly. The study of additional cases has demonstrated that some of these features may be lacking in some cases and that there may be other manifestations not at first recognized.^- ^' ^°' ^^' ^- There may be pap- ular or ulcerative skin and mucous membrane lesions, the naso-oral ' * Literature citations for this section will be found on page 193. 190 INFECTIONS CAUSED BY MOLDS cavity commonly presents lesions, and vegetative endocarditis and ulcerative enteritis have been reported. Diagnosis. Histoplasmosis should be suspected in undiagnosed cases presenting the foregoing characteristics. Its diagnosis depends upon the laboratory demonstration of the fungus in cells of the reticuloendothelial system or its isolation in culture. Histoplasma may be present in the circulating blood, and it can be demonstrated Fig. 96. Histoplasmosis: left, Histoplasma capsulatum in blood smear (impres- sion smear of liver of hamster with experimental histoplasmosis, Giemsa stain, X900) ; right, spores ("chlamydospores") of H, capsulatum. during life in smears of blood or of sternal bone marrow stained by Giemsa's or Wright's methods. Mucous membrane lesions of the oral cavity, when present, are probably better sources of material for smears. For its isolation in culture blood, sternal bone marrow, or material from ulcers should be spread on the surface of Sabouraud agar slants which should then be incubated at 30° C. or on blood agar incubated at 37° C. A skin-testing antigen can be prepared by growing the fungus for 3 months on the synthetic broth medium used in the preparation of coccidioidin. When 0.1 ml. of a dilution of 1:1000 of the sterile filtrate from such a culture is injected intra- cutaneously into the patient or into infected guinea pigs the tuber- culin type of delayed reaction is observed. The antigen is known to cross-react with blastomycosis and occasionally with other my- MORPHOLOGY IN CULTURE 191 coses and its usefulness as a diagnostic test remains to be proved. An antigen has also been prepared from glucose broth.«- ^-' " Prognosis and Treatment. With very- few exceptions, recognized cases of histo- plasmosis have terminated in death. There is some evidence indicating that the disease occurs in a mild and unrecog- nized form. Some apparently healthy individuals react strongly to intradermal injections of histoplasmin, and lesions have been found by accident in individuals apparently dying of other causes. How- ever, treatment of diagnosed cases has been ineffective. IMeleney " recommends the use of the organic salts, the trivalent organic preparations, and the pentavalent preparations of antimony. Morphology in Tissue. In tissue the fungus is a small oval budding cell meas- uring about 3 by 5 microns including the capsule. It appears principally within cells of the reticuloendothelial system. Extracellular fungi are also numerous when infection is heavy. The stainable protoplasm of the fungus cell in most pathological preparations is in a cup- shaped mass at one end of the cell. This no doubt represents shrinkage in part and the presence of a normal cell vacuole in part. The minute nucleus is not apparent. Each cell may be surrounded by a narrow capsule. Morphology in Culture. "When Histo- plasma capsulatum is grown in culture on blood agar at 37° C. it produces budding cells like those seen in tissues.^- * Under these conditions of culture newly isolated strains may grow exclusively in the bud- ding form. However, many old strains and all strains when grown on American Sabouraud agar or other common media at 30° C. or at Fig. 97. Histoplasma cap- sulatum. Young and old cultures on modified Sa- bouraud agar medium. 192 INFECTIONS CAUSED BY MOLDS room temperature revert to a hyphal type of growth and appear as white or brownish cottony molds. On the hyphae of such a culture two types of spores are commonly formed. There are small spherical conidia 1 to 3 microns in diameter borne on lateral conidiophores of varying length but usually not exceeding 4 or 5 microns. These conidia may be smooth, in which case they are practically indistin- guishable from the conidia of Blasto7nyces dermatitidis. Other conidia, and particularly the larger ones, have a rough or spiny outer wall. In many cultures and at early stages of sporulation there are conidia intermediate between these rough-walled conidia and a larger type of spore to be described. It is the second type of spore which characterizes H. capsulatum. It is large, varying in size and shape from a spherical cell 10 to 15 microns in diameter to a clavate cell reaching a size of 12 by 20 microns. The outer wall is adorned with warty, finger-like, or occa- sionally spiny excrescences which give it a distinctive appearance. The variation in size and shape of these excrescences and their homo- geneous structure suggest that they are produced by the proliferation of the cell wall itself. They are not asci, as suggested by some in- vestigators. The spores bearing these external structures are usu- ally called chlamydospores, but one can find intermediate forms which seem to relate them closely to the smaller conidia in a single series. No ascomycetous form is known. When these conidia are injected into an experimental animal they give rise to buds which reproduce the budding life cycle characteristic of the parasitic phase of growth. Taxonomy. The fungus was described and named in the mistaken belief that it was a protozoan. However, since the genus was erected for this organism and has never actually included protozoa the name is valid. There are some differences between the various strains of the fungus so far isolated but these are insignificant and do not merit specific separation. A single species, Histoplasma capsulatum, is recognized. Geographical Distribution. The distribution appears to be cir- cumglobal. Reports have come from the United States, Central and South America, Europe, Russia, Java, and Africa. Habitat. The natural habitat of the fungus is unknown. It has been reported in several cases from dogs, but there is not yet suffi- cient evidence to incriminate dogs as a reservoir. SPOROTRICHOSIS 193 LITERATURE 1. CoNANT, N. F., A cultural study of the life-cycle of Histoplasma capsulatum Darling 1906, J. Bad., 41, 563 (1941). 2. Darling, S. T., A protozoan general infection producing pseudotubercles in the lungs and focal necrosis in the liver, spleen and lymph nodes, J. Am. Med. Assoc, 46, 1283 (1906). 3. , The morphology of the parasite {Histoplasma capsulatum) and the lesions of histoplasmosis, a fatal disease of tropical America, Jour. Exptl. Med., 11, 515 (1909). 4. DeMonbreun, W. a., The cultivation and cultural characteristics of Dar- ling's Histoplasma capstdaium. Am. J. Trop. Med., 14, 93 (1934). 5. DoDD, K., and E. H. Tompkins, A case of histoplasmosis of Darling in an infant. Am. J. Trop. Med., 14, 127 (1934). 6. Emmons, C. W., B. J. Olson, and W. W. Eldridge, Studies of the role of fungi in pulmonary disease, I. Cross reactions of histoplasmin. Public Health Repts., 60, 1383 (1945). 7. Hansmann, G. H., and J. R. Schenken, A unique infection in man caused by a new yeast-like organism, a pathogenic member of the genus Sepe- donium, Am. J. Path., 10, 731 (1934). 8. Henderson, R. G., H. Pinkerton, and L. T. Moore, Histoplasma capsulatum as a cause of chronic ulcerative enteritis, /. Am. Med. Assoc, 118, 885 (1942). 9. Humphrey, A. A., Reticuloendothelial cytomycosis (histoplasmosis of Dar- ling), Arch. Internal Med., 65, 902 (1940). 10. Meleney, H. E., Histoplasmosis (reticulo-endothelial cytomycosis) : a re- view. Am. J. Trop. Med., 20, 603 (1940). 11. Moore, M., and L. H. Jorstadt, Histoplasmosis and its importance to oto- rhinolaryngologists. A review with report of a new case, Ann. Otol. Rhinol. Laryn., 52, 779 (1943). 12. Parsons, R. J., and C. J. D. Zarafonetis, Histoplasmosis in man, Arch. Internal Med., 75, 1 (1945). 13. Van Pernis, P. A., M. E. Benson, and P. H. Holinger, Specific cutaneous reactions with histoplasmosis, /. Am. Med. Assoc, 117, 436 (1941). SPOROTRICHOSIS The genus Sporotrichum is characterized by the prockiction of pear- shaped conidia on minute apiculate processes. The first conidia pro- duced by a culture are usually borne at the tips of short simple conidiophores. As the culture ages the conidia are borne laterally on the conidiophores and on the undifferentiated hyphae. Thus, in an old culture there are very numerous dark or smoky-colored conidia borne at the tips of conidiophores and forming sleeve-like masses around conidiophores and hyphae. Clinical. There are two portals of entry, through wounds and through the alimentary tract. The great majority of human cases 194 INFECTIONS CAUSED BY MOLDS have been primarily wound infections, but a few cases presenting a generalized infection without any primary focus have been inter- preted as an invasion through the mucous membrane of the intestinal tract. Davis ^ * was thus able to produce a generalized infection in rats by feeding the organism. A few cases have been reported in which apparently a primary involvement of the lungs occurred. In most of the cases the lesions will be seen in the skin and subcutaneous tissues. Foerster ^ reports that 111 of 146 cases were primary on the hands. The clinical picture of a typical case is so striking that, once seen, the disease will always be readily recognized.^- ^' "• " There will be Fig. 9S. Sporotrichosis. seen extending in a line upon the surface of an extremity, a series of hard, elevated, reddened lumps, the older lesions presenting a fistula from which pus may be expressed. At first glance the lesions look like boils but are not hot and tender, and there is practically no constitutional reaction. The firmness of the nodules suggests a syphilitic gumma. Between the lesions the course of the subcuta- neous lymph vessels can frequently be traced as reddened lines. Al- though in the majority of cases the infection spreads from the pri- mary lesion by way of the lymph vessels, it seldom goes beyond the regional lymph nodes. Cases of generalized infection by way of the blood stream occur but are relatively rare. Metastatic lesions may occur in the lungs, liver, and especially frequently in the testicles. One gains the impression that such generalized cases are more fre- quent in Europe than in America. In some cases the disease may be transmitted from the arm to some other part of the skin surface by contact. Diagnosis. The disease may present so close a resemblance to tertiary syphilis that it has undoubtedly been frequently misdiag- nosed for that disease. An incorrect diagnosis subjects the patient needlessly to a prolonged course of treatment without any benefit ♦Literature citations for this section will be found on page 199, I CULTURES 195 unless iodides are administered. A correct diagnosis with proper treatment (internal administration of iodides) leads to a prompt and permanent cure. Since iodides are commonly used in the treat- ment of tertiary syphilis, one may make an erroneous diagnosis of syphilis in a case of sporotrichosis, and believe that the diagnosis has been confirmed by the results of specific treatment. The diagnosis is established by demonstrating the organism in the pus. In the body tissues and exudates the parasite appears as a small, single-celled, spindle-shaped organism. It is most frequently seen within the polymorphonuclear leucocytes. It reproduces by budding at one end of the cell. In size the cells are comparable to those of some of the larger bacteria, but they are easily recognized by their charac- teristic cigar shape. They have some- times been incorrectly referred to as spores, but they are quite different from ' ^^^ j^^^"^ /^i^^^"^^ the spores formed in cultures. They arc *^- " -'^**^- tS^^-^ the only structures formed in animal Fig. 99. Smear of pus from tissue; no mycelium develops. They sporotrichosis, showing the par- „ . , . . asites withm the leucocytes. are far from bemg numerous m pus from human cases, and may be found only after prolonged search. They are best looked for in smears stained by Gram's method, al- though many of the cells are Gram-negative. Although these bacillus-like cells are never found in ordinary cul- tures, according to Davis ^ they are formed in cultures in blood or blood serum, especially if air is excluded or if sterile tissue is added. Cultures. Cultures are of more value in diagnosis than are smears. Pure cultures may be readily obtained if made from pus aspirated from the younger lesions which have not yet opened; with more dif- ficulty from those which have developed fistulae, for in these latter there is always considerable secondary infection with bacteria. The fungus will not grow readily on dextrose-tartaric agar, and the medium of choice is Sabouraud agar. The character of the growth on agar is strikingly different from that of most molds. At first it is soft and creamy in texture, the surface moist and shiny, whitish in color, and resembles more a cul- ture of bacteria. As the culture grows older, it becomes darker in color, first a light tan which gradually deepens to a coffee brown and may eventually become quite black. The mass becomes firmer in texture, tending to pull off the agar in rather elastic flakes, and the surface becomes more and more wrinkled. Some strains remain 196 INFECTIONS CAUSED BY MOLDS smooth, moist and shiny without developing the cottony masses of aerial mycelimn seen in most molds. Other strains develop a short black aerial mycelium which may be more abundant on some media such as prune agar, and which may be aggregated into coremia-like structures which give the surface of the colony a spiny appearance. If some of this growth is examined under a microscope there are found a tangled mass of branched mycelium and a large number of pear-shaped conidja which are almost all freed from the hyphae when material is mounted for examination in the usual manner. To see their normal relationship it is necessary to prepare slide cul- tures. This was done by de Beurmann and Gougerot ^ by placing sterile slides in wide test tubes with a small amount of dextrose broth into which the organism was inoculated. As it grew, the mold would climb the slide for a short distance, and the slide was then removed and the growth fixed and stained. The methods of slide culture preparation described in Chap- ter I are preferable. In such a preparation some of the conidia will remain in place at the tips and along the sides of simple conidio- phores and on the undifferentiated hyphae. Each conidium is at- tached by a very narrow stalk which, when it breaks, remains in part on the pointed end of the conidium and in part as an apiculate scar on the hypha from which the conidium fell. This stalk may appear almost thread-like in specimens which have been dehydrated and stained. Conidia which have fallen to the surface of the culture fre- quently bud to produce secondary conidia which are borne on narrow sterigmata. Chlamydospores are also produced on the mycelium. Both cultural and morphological characters are subject to consider- able variation. Of the former, the degree of pigmentation is most variable. Some strains may never develop more than a light tan color, others may darken very rapidly. The same strain may form more pigment' on some media than on others, or on the same medium may remain colorless at one time and become pigmented in a later transfer. An inoculation of a pure culture on agar may show pig- mentation in one portion of the colony and none in another. Fig. 100. Sporotrichum Schenckii. Conidia in culture. ANIMAL INOCULATIONS 197 Taxonomy. On the basis of these rather variable cultural char- acters a number of different species of pathogenic Sporotrichum have been described. Buschke and Langer recognized thirteen. It would seem, however, that the differences upon which these species were made are well within the limits of variability of a single strain. Greatest stress has been placed upon the differentiation between the American species, Syorotrichum Schenckii, and the variety described in Europe as S. Beurmanni. These have been separated mainly on the ground of pigment formation, S. Schenckii being light and S. Beurmanni dark; on the degree of spore formation, ;S. Schenckii forming fewer lateral conidia; and on sugar fermentations, S. Schenckii producing acid from lactose, not sucrose, whereas S. Beur- manni is said to ferment sucrose but not lactose. Davis,* however, has shown that both pigment formation and spore formation are too variable to be used for differentiation because it depends more on the nature of the medium and rapidity of transfer than on the strain. Meyer ^ similarly found that sugar fermentations are highly variable. It would seem, therefore, that although different strains may appear somewhat unlike, there is no valid reason for recognizing more than one of these species, S. Schenckii. Animal Inoculations. Although various laboratory animals are susceptible to infection, rats are particularly so. In addition to mak- ing cultures, inoculation into a male white rat is a procedure of con- siderable diagnostic value. The inoculation should be made into the peritoneal cavity. There occurs a generalized peritonitis with minute nodules on all the peritoneal surfaces, and in addition a very pronounced inflammation of the testis, which may be determined without sacrificing the rat. Unlike human lesions, those in the rat contain the typical cigar-shaped cells in great abundance, and these may be found readily in Gram-stained smears. The agglutination reaction with spores of Sporotrichum has been discussed in Chapter VI. This reaction, introduced by Widal and his coworkers, is of considerable diagnostic value; though cross re- actions occur with thrush and actinomycosis, these diseases are not likely to be confused with sporotrichosis. The best diagnostic pro- cedure is the isolation of the organism in culture, or by inoculation of a rat. Davis ^ was unable to differentiate various strains of Sporo- trichum, including some of equine origin, by means of agglutination reactions. Cutaneous reactions are positive but are considered less specific than agglutination. Moore and Davis ^ got no reaction with an extract of the organism of blastomycosis in a case of sporotricho- sis. 198 INFECTIONS CAUSED BY MOLDS Geographic Distribution. The disease, first recognized in this country by Schenck," and shortly afterwards in France by de Beur- mann,^ has since been found in all parts of the world. The great majority of the reported cases have occurred in France, the United States, and South America. There is some evidence of a limited geographical distribution. Thus Ruediger ^° found five sixths of the cases reported in America by 1912 had occurred in the valley of the Missouri River. According to Foerster ^ (1926) 130 out of 148 cases reported in the United States were in the valleys of the Mississippi or its tributaries, and a large proportion of these in the Missouri Valley. Meyer also found that outside of Pennsylvania most of the cases of equine sporotrichosis occurred in the Missouri Valley. This geographical distribution may be due to a greater prevalence of the parasite in this region; to a larger proportion of the popula- tion (agricultural) being engaged in occupations which expose them to infection; or (and this seems the more likely) to the fact that the medical profession in these districts have been on the lookout for such cases. In recent years more and more cases have been re- ported from other parts of the world. This increasing number of cases reported is also probably due to an increased alertness of the medical profession rather than an actually greater prevalence of the disease. Habitat in Nature. The infection may occur in various ways, but in a large proportion of cases it is clear that the fungus has been introduced into the tissues from or on vegetable matter of one sort or another. Thus Foerster ^ noted that 14 of his 18 cases followed wounds of the upper extremities by barberry thorns. The fungus has been found growing free in nature upon a grain by Sartory; it has been isolated at the National Institute of Health from sphagnum moss, which was responsible for several cases among florists; Lurie isolated it from the timbers of a gold mine in South Africa and from miners exposed to that source; and from the histories of numerous cases, we must assume that it is a fairly common saprophyte upon vegetable matter and in soil. Many cases have occurred in farmers, in some cases following wounds caused by agricultural implements. Benham and Kesten ^ demonstrated the saprophytic growth of Sporotrichu7n Schenckii on experimentally inoculated barberry thorns and in the buds of carnations. They refer to the latter as the trans- mission of sporotrichosis to plants, but it is doubtful if that inter- pretation of the results is justified. Spores of S. Schenckii, S. Poae (a pathogen of carnations), S. pruinosum (a saprophyte), S. Gouger- oti, S. Councilmani, and Penicillium brevi-compactum, and sterile HISTORICAL 199 water controls were injected into young buds of carnation. From 23 per cent (sterile water controls) to 87 per cent (*S. Poae) of the buds failed to open normally. S. Schenckii was recovered from 3 of 18 buds inoculated with the fungus, together with 3 other contaminat- ing fungi. The experiment demonstrated the ability of the fungus to grow on dead or damaged plant tissue. The disease also occurs spontaneously in certain of the lower ani- mals, notably horses and rats. A number of human cases have been contracted either directly (by bites) or indirectly from such lower animals. There have been two accidental laboratory infections, one from an equine strain, the other from a culture of human origin. In at least one case there has been direct transmission from man to man. LITERATURE 1. Benham, R. W., and B. Kesten, Sporotrichosis: its transmission to plants and animals, /. Infectious Diseases, 50, 437 (1932). 2. Davis, D. J., Interagglutination experiments with various strains of Sporo- trichum, J. Infectious Diseases, 11, 140 (1913). 3. , Morphology of Sporotrichum Schenckii in tissues and artificial media, J. Infectious Diseases, 12, 452 (1913). 4. , The identity of American and French sporotrichosis, Univ. Wiscon- sin Studies, pp. 105-131, 1917. 5. DB Beurmann, L., and E. Gougerot, Les Sporotrichoses, Alcan, Paris, 1912. 6. FoERSTER, R. H., Sporotrichosis, an occupational dermatosis, J. Am. Med. Assoc, 87, 1605 (1926). 7. Hopkins, J. G., and R. W. Benham, Sporotrichosis in New York State, N. Y. State J. Med., 32, 595 (1932). 8. Meyer, K. F., and J. A. Aird, Various Sporotricha differentiated by the fermentation of carbohydrates, J. Infectious Diseases, 16, 399 (1915). 9. Moore, J. J., and D. J. Davis, Sporotrichosis following a mouse bite, with immunological data, J. Infectious Diseases, 23, 252 (1918). 10. RuEDiGER, G. F., Sporotrichosis in the L^nited States, J. Infectious Diseases, 11, 193 (1912). 11. ScHENCK, B. R., On refractory subcutaneous abscesses caused by a fungus possibly related to the Sporotricha, Bidl. Johns Hopkins Hosp., 9, 286 (1898). CHROMOBLASTOMYCOSIS (Dermatitis Verrucosa, Chromomycosis) Historical. The first reports of chromoblastomycosis were made in 1915 by Lane ^ and Medlar ^° * in Boston. They adopted for the fungus they isolated a name given it by Thaxter, Phialophora ver- rucosa Medlar. In 1920 Pedroso and Gomes,^^ in Brazil, reported * Literature citations for this section will be found on pages 205 and 206. 200 INFECTIONS' CAUSED BY MOLDS a case observed nearly ten years earlier. They assumed that the fungus was the same as that isolated from the Boston case, but further studies showed that this was not true. In 1922 Brumpt - named the South American strain Hormodendrum Pedrosoi. The disease has since been observed in many parts of the world. Clinical, In the first reported case of chromoblastomycosis the lesion was on the buttocks. Lesions have been reported on the hands, arms, face, neck, and shoulders, but the commonest location is on the foot and lower leg. There is frequently a history of trauma such as penetration by a thorn, and the disease is seen most often in bare- footed agricultural laborers in tropical or subtropical countries. The primary traumatic lesion may appear to heal and then ulcerate or, in the absence of known injury, the primary lesion may be a pustule or a papule which slowly increases in size. There may be consider- able infiltration and some serous oozing in the early lesion. In most cases the lesion soon becomes dry and somewhat verrucous, viola- ceous, and sharply limited by a raised margin. In these early stages there is such a close clinical resemblance to American blastomycosis that a differential diagnosis cannot be made without laboratory examination. However, the lesion does not continue to spread periph- erally as in blastomycosis. Its surface becomes more verrucous and raised and in many cases of some years' duration the continued growth produces few or many large cauliflower-like masses on short pedicels. The nodular surfaces of these tumors may be covered by a smooth epidermis, but the skin is thin, and exposed lesions which are bruised or rubbed frequently ulcerate. Other lesions are crusted or rough and scaly. This is the classical appearance of the disease but the character of the lesion is modified by its location. The pedicellate lesions are seen rarely except on the lower leg and foot, and not in the latter location if the pressure of a shoe limits their development. Satellite lesions develop, probably as the result of autoinoculation by scratching. There may be some spread by way of the lym- phatics. In a few cases there appears to have been hematogenous spread. The secondary lesions may be numerous and over a period of several years involve most of the lower leg. Several of the re- ported cases have been of 20 years' duration. The lesions are relatively painless unless there is ulceration and secondary infection, but there may be severe pruritis. Blockage of the lymphatics causes elephantiasis, and many patients complain mostly of the disability caused by this deformity.^- ^' "• ^^' ^- APPEARANCE OF FUNGUS IN TISSUE 201 Fig. 101. Chromoblastomycosis. Chlamy- dospores of Phialophora Pedrosoi in tissue. Diagnosis. The early lesions closely resemble those of North American blastomycosis and must be differentiated by laboratory methods. The older verrucous lesions are more diagnostic. Exam- ination of sections made from the lesion will reveal the brown, thick- walled chlamydospores of the fungus. In some cases one can peel off some of the epidermal scales at the edge of the lesion and mount them in 10 per cent sodium hydroxide under a cover slip as in examining for derma- tophytosis. In such prepara- tions the fungus can sometimes be found growing as brown hy- phae in these superficial scales. The laboratory diagnosis is not complete without the isola- tion of the fungus in culture, because any one of at least three fungi may cause the dis- ease and there is no correlation between a particular clinical type of lesion and one of the fungi causing chromoblastomycosis. Prognosis and Treatment. The prognosis for cure of chromo- blastomycosis is poor but the disease does not become systemic and does not endanger life. Early lesions may be excised or destroyed by electrocoagulation. They sometimes heal under irradiation or after administration of iodides. Some success has been reported in the use of copper sulphate iontophoresis. Appearance of Fungus in Tissue. In the superficial epidermal scales the fungus may be found in the form of septate branching hyphae with thick brown walls. In the subcutaneous tissues how- ever it is present in a more characteristic form as small clusters of chlamydospores. The disease was called chromoblastomycosis be- cause the origin of these clusters was interpreted as a budding proc- ess. Actually, true budding does not occur, although something ap- proaching budding is seen in some cells. In most cases the cell elongates and is divided by a septum. This elongation and septum formation takes place in any plane so that there results a small cluster of cells with th^ck, dark brown walls. If growth of the fungus is rapid these clusters may contain several cells and in such cases they differ only in size from the granules seen in some types 202 INFECTIONS CAUSED BY MOLDS of mycetomas. These clusters of fungus cells may be in giant cells or smTounded by polymorphonuclear leucocytes. Appearance of Fungus in Culture. Examination of sections of the lesion does not indicate that more than a single species of fungus is involved in the etiology of chromoblastomycosis. However, when cultures are made, one of three fungi may be isolated. One is Phialophora verrucosa, first isolated in Boston from the first re- ported case,^' ^° and since isolated from other North American and Fig. 102. Culture of Phialophora Pedrosoi. from South American cases; one is P. Pedrosoi,^^ first isolated in Brazil and since isolated from cases in many parts of the world; and one is P. compactufn isolated in Puerto Rico by Carrion.^ The sec- ond species is the most commonly found. There are some strains of both P. verrucosa and P. Pedrosoi whose individual differences are manifested by colony characteristics but in general these two species are indistinguishable in colony appear- ance. Microscopic examination shows a difference in method of sporulation, but careful study shows that even here there is an evi- dent relationship and the differences are quantitative and not qualita- tive. This point will be discussed further in the paragraph on tax- onomy. P. verrucosa produces short lateral or terminal conidiophores which may be of nearly uniform diameter or may be enlarged midway to form a bottle-shaped or vase-shaped cell (Fig. 103). This conidio- APPEARANCE OF FUNGUS IN CULTURE 203 phore terminates in a flaring cup and the spores are formed by a budding process in the bottom of the cup. As spores are successively formed in this manner they are held together in a spherical mass by Fig. 103. Sporulation in Phialophora: upper left, Phialophora verrucosa; upper right, P. Pedrosoi; lower, both types of sporulation in P. Pedrosoi. In part from Emmons and Carrion, Mycologia, 29, 329 (1937). some adhesive substance so that a ball of spores is often observed at the mouth of the conidiophore. The depth of the cup in an old conidiophore makes this a semi-endogenous type of sporulation. This is the typical manner of sporulation in P. verrucosa, but careful ex- amination of some strains has shown that rarely a few conidiophores bear conidia terminally and laterally in a manner similar to that seen in P. Pedrosoi. 204 INFECTIONS CAUSED BY MOLDS P. Pedrosoi is more variable than P. verrucosa in its manner of sporulation. The eonidiophore is a lateral or terminal branch of nearly uniform diameter which bears at its tip one or more conidia. These arise as buds at the tip and each is capable of proliferation by- budding to produce one or more secondary spores. These in turn can produce tertiary spores, and so on. This results in an arborescent system of branching chains of conidia. In such spore heads the first spores formed are modified as the complex system develops so that they become shield-shaped. This is the type of sporulation typical of Cladosporium and the fungus has, indeed, been commonly classi- fied in the genus Hormodendrum which is a synonym of Clado- sporium. Most spore heads of P. Pedrosoi are small, and the chains of spores are limited in length to two or three. In fact, branching chains of spores are difficult to demonstrate, both because they may be actually very few or lacking in the culture and because when present they almost invariably break up when mounted for examination. Most of the conidiophores bear, instead of branching chains of conidia, a large number of spores which are sessile and clustered about the tip and for some distance below it, forming a sort of sleeve of conidia. Such conidiophores are characteristically crooked and gnarled near the tip where the conidia are borne and, when the latter are shed, show scars where the conidia were attached. Some of the conidia may bear secondary spores. Various combinations of these two types of sporulation are commonly observed in most strains. Carrion * has grouped strains into named varieties characterized by the predomi- nance of one or another type. A third type of sporulation which appears to be identical with that characterizing P. verrucosa was first observed in P. Pedrosoi by Carrion and Emmons,^- * who pointed out the significance of this observation in elucidating the relationship between these two etio- logical agents of one disease. The phialophores are rare in P. Pedrosoi but have been found in practically all strains examined. They are isolated or grouped in clusters on the mycelium, or they are borne as integral parts of the Cladosporium type of spore head. Taxonomy. Three species are recognized, Phialophora verrucosa, P. Pedrosoi, and P. compactuni.'' P. Pedrosoi, because of the vari- ability in its manner of sporulation, has been variously placed in the genera Hormodendrum, Trichosporium, Acrotheca, Fonsecaea, Gomphinaria, Botrytoides, Hormodendroides, and Phialoconidio- phora. Binford and coworkers ^ emended the genus Phialophora to LITERATURE 205 include this species, believing that the similarities between P. ver- rucosa, P. Pedrosoi, and P. compactum, their production of conidia by identical methods, and their common relationship to a single mycosis justified placing them together in one genus. If their ob- viously close relationship is so indicated the valid generic name is Phialophora. Material studied from two widely separated geographical sources, Java f and Canada,^ indicates that a fungus w^iich resembles Pul- lularia pulliilans is sometimes etiologically related to chromoblasto- mycosis. We believe these fungi to be aberrant strains of P. Pedrosoi (see discussion of black yeasts, page 111). Geographical Distribution. Chromoblastomycosis is best known from Brazil, United States (particularly Puerto Rico), and Cuba, but it has also been reported from other areas in South and Central America, the Caribbean, Java, Russia, Africa, and Japan. Habitat in Nature. Conant ^ showed that Phialophora verrucosa occurs in decaying wood where it had been described under the name Cadophora americana. The close resemblance between P. Pedrosoi and saprophytic species of Cladosporium and the story of trauma in many cases of chromoblastomycosis leaves little doubt that P. Pedrosoi is also normally a saprophyte of soil and decaying vegeta- tion. LITERATURE 1. BiNFORD, C. H., G. Hess, and C. W. Emmons, Chromoblastomycosis, Arch. Dermatol. Syphilol. (Chicago), 49, 398 (1944). 2. Brumpt, E., Precis de parasitologle. Masson et Cie., Paris, 3rd ed., p. 1105, 1922. 3. Carri6n, a. L., Chromoblastomycosis. Preliminary report of a new clinical type of the disease caused by H ormodendrum compactum, nov. sp., Puerto Rico J. Pub. Health Trop. Med., 10, 543 (1935). 4. , Chromoblastomycosis, Mycologia, 34, 424 (1942). 5. Carri6n, a. L., and C. W. Emmons, A spore form common to three etiologic agents of chromoblastomycosis, Puerto Rico J. Pub. Health Trop. Med., 11, 114 (1935). 6. CoNANT, N. F., The occurrence of a human pathogenic fungus as a sapro- phyte in nature, Mycologia, 29, 597 (1937). 7. CoNANT, N. F., and D. S. Martin, The morphologic and serologic relation- ships of the various fungi causing dermatitis verrucosa (chromoblasto- mycosis). Am. J. Trop. Med., 17, 553 (1937). 8. Emmons, C. W., and A. L. Carrion, The Phialophora type of sporulation in H ormodendrum Pedrosoi and H ormodendrum compactum, Puerto Rico J. Pub. Health Trop. Med., 11, 703 (1936). t Courtesy of Dr. C. Bonne. t Courtesy of Dr. L. Berger. 206 INFECTIONS CAUSED BY MOLDS 9. Lane, C. G., A cutaneous disease caused by a new fungus {Phialophora verrucosa), J. Cutaneous Dis., 33, 840 (1915). 10. Medlar, E. M., A new fungus, Phialophora verrucosa, pathogenic for man, Mycologia, 7, 200 (1915). 11. Pedroso, a., and J. M. Gomes, Sobre quatro casos de dermatite verrucosa produzida pela Phialophora verrucosa, Ann. paulistas vied, cirurgia, 9, 53 (1920). 12. Weidman, F. D., and L. H. Rosenthal, Chromoblastomycosis ; a new and important blastomycosis in North America, Arch. Dermatol. Syphilol. (Chicago), 43, 62 (1941). ASPERGILLOSIS Aspergillus fumigatus is an important pathogen, especially for birds. One of the earliest definite records of mycotic infection in animals concerns aspergillosis of the air sacs in birds. The disease is common enough in domesticated birds, pigeons, chickens, and ducks to be of some economic importance. Under the name brooder pneu- monia it sometimes occurs in epidemic form in little chicks. Autopsies of wild birds found dead have revealed many cases in all orders of birds, not only seed eaters, but also insect-eating birds and aquatic species. Fox,^ * in autopsies at the Philadelphia Zoological Garden, found cases in practically all the groups of birds, the disease being responsible for death in from 0.6 per cent of the cases (pigeons) to 40 per cent of the cases (penguins). The fungus is also known to invade birds' eggs during incubation and to infect the embryos. Three types of infection occur in birds: infection of the air sacs, a pneumonic form in the lungs, and a nodular form in the lungs. The first type is a superficial infection of the epithelium lining the air sacs, which may pass into the wings or into the abdominal cavity. The wall of the sac becomes much thickened, a thick mat of my- celium covers its surface, and spores usually develop, so that the inner surface of the sac wall has a green color. In the pneumonic form a diffuse infiltrative lesion develops, the lung tissue is con- solidated, and it has a grayish white color. In the nodular form, isolated masses of infiltrated tissues occur, with necrosis in the center, much resembling advanced tubercles in their gross appearance. In those lesions developing in internal tissues not freely exposed to the air no spores develop. The disease in domestic birds, especially when it occurs in epidemic form, can usually be traced to feeding moldy grain. It may be due to damp quarters in which straw or other material has become moldy. * Literature citations for this section will be found on page 210. ASPERGILLOSIS 207 Fig. 104. Section through the lung of a grouse {Bonasa umbellus) dead of spontaneous aspergillosis, showing filaments of mycelium. Gram-Weigert stain. i.^~ "J^;.- m. •'■'..•*.- i - ' ■•' '•k ". N -* i .^*» - "" ' . , s ^ ~ ^ * '*' , '■■ ■ Fig. 105. Histological tubercle in the heart muscle of a pigeon inoculated intravenously with spores of Aspergillus jumigatus. 208 INFECTIONS CAUSED BY MOLDS Infection of the eggs is usually due to moldy nesting material. Ex- periments have shown that if the eggs are carefully cleaned of their fatty coating, infection will not take place. The infected eggs may be detected by candling. Primary lesions of the lungs also occur in various domesticated mammals, though not so frequently as in birds. Cattle, sheep, and especially horses are known to develop aspergillosis. As with the birds, infection comes from contaminated hay or grain, the spores be- ing inhaled. In some cases particles of inhaled vegetable matter have been found in the lesions. The pulmonary lesions may be either nodular or pneumonic as in birds. A. fumigatus is also patho- genic to man. Some of the cases described are infections of the external ear. The extent of the disease may vary from a mere plugging of the ear canal with mycelium, leading to impaired hearing, to ulceration and sup- puration of the walls of the canal, or even to penetration of the drum and invasion of the middle ear. The milder cases are the more numerous. Other species of Aspergillus may also produce this condition, particularly A. niger,' A. nidulans, and A. flavus. According to Siebenmann *' in Germany about 1 per cent of all ear cases are Aspergillus infections. Asper- gillosis of the ear is said to be particularly frequent in India. Aspergillosis of the lung also occurs in man, but it is rare.^- *' ^ The majority of cases have been reported in France, though the disease is also well known in Germany. The disease may be primary, or secondary to some other condition, particularly tuberculosis. Lang and Grubauer reported a case in which bronchiectasis was apparently a predisposing cause. Secondary cases are more common than pri- mary ones. It is quite possible that some of the cases reported as primary were actually secondary to some other disease whose traces were obliterated by the aspergillosis. On the other hand, it is quite probable that some cases of primary pulmonary aspergillosis are overlooked, being mistakenly diagnosed tuberculosis. Clinically the disease resembles tuberculosis very closely, perhaps advancing some- what more rapidly. According to Lapham ^ cases of primary asper- FiG. 106. Aspergillosis. Hyphal frag- ments of Aspergillus fumigatus in human sputum. ASPERGILLOSIS 209 gillosis give positive tuberculin reactions; conversely Nicaud found that an advanced case of tuberculosis gave positive cutaneous reac- tions with an extract of A. fumigatus. Extensive cavity formation occurs in the lungs. The diagnosis is made by finding the mycelium in the sputum, where it occurs as short hyphal fragments, often en- crusted, and by isolating the fungus in culture. See Fig. 106. The latter procedure is easy when sputum is planted on Sabouraud agar slants because the fungus grows fairly rapidly and is not inhibited by bacterial growth. The prognosis is not good. Internal adminis- tration of iodides is said to be of value, even curative, in some cases, but this treatment must be followed with caution since it may result in the rapid spread of the disease as in the case of blastomycosis. A considerable number of cases of primary pulmonary aspergil- losis were reported in France some years ago, especially by Renon."* In these cases the infection was an occupational disease occurring in individuals engaged at that time in occupations which peculiarly subjected them to the possibility of inhaling large numbers of spores. These were the "gaveurs des pigeons" who fattened squabs for the market by filling their mouths with grain, chewing it fine, and then with their tongues forcing the mass into the esophagus of the birds. The second group was the "peigneurs des cheveux" who prepared hair for the manufacture of wigs by mixing it with corn meal to remove oil and then combing it out. In both cases the source of the infection is obvious enough, though in the first it is possible that the infection may have come from the bird rather than the grain, for some of the pigeons were found to have aspergillosis infections of the mouth. Experimentally inoculated into laboratory animals, A. fumigatus produces lesions which vary according to the virulence of the strain and the dosage. Many strains isolated from air or vegetable matter show no pathogenicity. Strains freshly isolated from spontaneous infections may exhibit a surprising degree of virulence, a small dose of spores suspended in salt solution killing a pigeon overnight when inoculated intravenously. No lesions are apparent in such acute infections. With smaller doses or less virulent strain, multiple miliary abscesses occur in various viscera, especially the lungs. Intravenous inoculations into rabbits usually causes death within 3 to 5 days. Multiple minute abscesses in the cortex of the kidneys are the most striking lesions in these rabbits. Subcutaneous or intraperitoneal inoculations produce localized lesions which may not be fatal. Experimental infections may also be produced by causing the spores to be inhaled. If one dusts spores into a tumbler and holds 210 INFECTIONS CAUSED BY MOLDS this over a pigeon's head for a minute or two, rapidly fatal hemor- rhagic pneumonia develops. Henrici - succeeded in producing only very acute infections in this way. On the other hand, by feeding wheat which had been overgrown wdth the mold, he succeeded in two out of four pigeons in obtaining an infection of the air sacs very similar to the natural disease, death occurring in about 6 weeks. Microscopically both the natural and experimental lesions may vary considerably according to the virulence of the strain. Usually there is extensive necrosis in the vicinity of the organisms, with some suppuration. In the nodular lesions of the lungs there may be some production of fibrous tissue. In the lesions branched segmented hyphal fragments may be found (Fig. 106), with conidiophores in various degrees of development where the fungus reaches a surface exposed to the air. In the miliary abscesses produced by intravenous inoculation one finds small masses composed of radiating filaments somewhat resembling a granule of Actinomyces hovis, save that the filaments are fewer and coarser. LITERATURE 1. Fox, H., Disease in Captive Wild Mammals and Birds, Lippincott, Philadel- phia, 1923. 2. Henrici, A. T., An endotoxin from Aspergillus jumigatus, J. Immunol., 36, 319 (1939). 3. Lapham, M. E., Aspergillosis of the lungs and its association with tuber- culosis, /. Am. Med. Assoc., 87, 1031 (1926). 4. Renon, L., Etude sur I'Aspergillose chez les anim,aux et chez I'homme, Masson et Cie., Paris, 1897. 5. Schneider, L. V., Primary aspergillosis of the lungs, Am. Rev. Tuberc, 22, 267 (1930). 6. SiEBENMANN, F., Die Fadenpihe rmd ihre Beziehungen zur otomycosis asper- gillina, Bergmann, Wiesbaden, 1883. t MYCETOMA Carter - * proposed the term mycetoma to designate a type of fungus infection usually localized to the foot and characterized by a conspicuous deformity in which the foot is greatly enlarged. It was frequently seen in India and, according to Carter, Colebrook introduced into medical literature the name Madura foot by which the condition was popularly known near Madura, India. Carter showed that the condition was not an etiological entity and he de- * Literature citations for this section will be found on page 214. DIAGNOSIS 211 scribed the appearance of the fungi he found in two different types of cases, so far as the techniques of that day permitted. Further studies showed that not two but many fungi were capable of causing mycetoma, and Brumpt ^ described several of these and related them to certain clinical types. Pinoy ^ observed that the mycetomas could be separated into two groups on the basis of the size of the fungi, and he proposed a division between the actinomy- coses caused by Actinomyces and Nocardia, and the true mycetomas caused by fungi having larger hyphae than those of the actinomy- cetes. Chalmers and Archibald ^ accepted this division but proposed the name maduromycosis as a substitute for true mycetomas. Their name has been widely used, sometimes in the sense in which it was proposed but more often as a synonym for mycetoma. We prefer the original name, mycetoma, because (1) the name maduromycosis is not specific but designates infections caused by several unrelated species belonging in some eight or ten genera of Hyphomycetes and Ascomycetes, and (2) the name is a source of confusion because of its derivation from a geographical place to which the disease is not limited, and by derivation it suggests both Madurella (a genus of Hyphomycete causing mycetoma) and Nocardia madurae (an actinomycete causing mycetoma of the type not included under maduromycosis) . The name mycetoma properly refers to both types of infection discussed by Pinoy. Clinical. Mycetoma is a fungus infection of the skin and sub- cutaneous tissues characterized by swelling, destruction of tissues (including bone in some cases), formation of fistulae, and production of pus in which there are well-organized mycotic granules. The char- acter of these granules varies according to the species of fungus pro- ducing them. They may be hard or soft; white, yellow, red or black; pin-point or up to 3 or 4 mm. in size; and the fungus hyphae may be the 0.5/a to l^u, hyphae of actinomycetes or the larger hyphae and chlamydospores of Hyphomycetes or Ascomycetes. When the lesion is on the foot the latter is enlarged and there is usually a characteristic convex swelling of the plantar surface. The infection usually follows a wound such as that caused by penetration of a splinter or thorn and this probably accounts for the frequent occurrence on the foot. Lesions may be on the hand, however, and rarely on other parts of the body. Diagnosis. The clinical appearance of a typical case of mycetoma is distinctive, but the diagnosis rests finally upon the laboratory demonstration of the fungus granules. Because of the many fungi 212 INFECTIONS CAUSED BY MOLDS capable of causing mycetoma ^' '' the diagnosis is not complete until the fungus is isolated in culture and identified. Pus should be collected and examined for granules. The shape, size, consistency, and color of these should be noted. A drop of pus containing granules should be placed on a slide under a cover slip. If the pus is thick it can be mixed with a drop of 10 per cent sodium hydroxide. Most of the fungi causing mycetoma grow on Sabouraud agar, although some of them grow very slowly at first. The optimum temperature for most species is near 30° C. Treatment and Diagnosis. Although mycetoma usually remains localized and does not endanger life, a few cases have been observed in which systemic infection has been caused by fungi similar to those found in some rare types of mycetoma. (Actinomycosis caused by Actinomyces bovis is excluded from this discussion.) In the great majority of cases the infection does not extend above the foot even in cases of many years' duration. The prognosis from the view- point of life is therefore good, but no treatment of the infection, except radical surgery, is effective. Appearance of the Fungi in Tissues. In all cases of mycetoma the fungus forms granules or microcolonies in the tissues, but these differ, as noted above. AVhen Nocardia madurae and A^". mexicana are involved the granules are composed of densely packed, radiating, delicate hyphae 0.5/u, to Ijx in diameter. The color is white or yellow. N. somaliensis and A^. Pelletieri also form granules with small hyphae but the color is reddish yellow or red. The granules as well as the hyphae formed by Hyphomycetes and Ascomycetes are somewhat larger than those formed by Nocardia. In some cases chlamydospores are conspicuous elements of the granule. Phialophora Jeanselmei^ and species of Madurella form black granules. The granules formed by Allescheria Boydii [Mono- sporium apiospermum) , Indiella spp., and Aspergillus spp. are white to yellowish. The granule may be composed largely of chlamydo- spores, but usually there is some degree of radial orientation of hyphae. There is sometimes an acidiphilic zone at the periphery of sectioned granules and this staining reaction and the enlarged hyphal tips at the periphery bear a superficial resemblance to the "clubs" which surround the granule of Actinomyces bovis. See Chapter XIII, Appearance in Culture. The large number of species of fungi as- sociated with mycetoma make it impossible to generalize about their characteristics in culture. A few representative fungi will be de- scribed briefly. GEOGRAPHICAL DISTRIBUTION 213 Species of Nocardia retain in culture the small size which char- acterizes their hyphae in granules in pus. Nocardin madurae grows on agar as a glabrous, wrinkled, grey colony which is in some in- stances covered with short, white, aerial hyphae. No conidia are formed but in old cultures the cells formed by fragmentation of the hyphae serve as reproductive structures. N. mexicana grows slowly and forms heaped, folded colonies rather than spreading widely over the agar surface. The color varies from white (in strains or cultures with aerial hyphae) to yellow or orange. Most strains have a strong musty odor. See page 379 for further discussion of myce- tomas caused by Nocardia spp. Phialophora Jeansehnei grows on Sabouraud agar as a mouse- grey to olive-colored colony closely resembling that of P. verrucosa which causes chromoblastomyco- sis." There is also a close micro- scopical resemblance between the two fungi. Species of IMadurella are grey to black and most of them grow slowly, forming dome- shaped colonies in which conidia are very few or entirely lacking. Perhaps the most frequent cause of mycetoma in the United States K\9 ,: ■at ' 7^^- 4 ' Fig. 107. Allescheria Boydii. Co- nidial stage {Monosporium apiosper- viuvi) on Sabouraud agar. From Conant et al., Manual of Clinical Mycology, Saunders, 1944. is Monosporium apiospermum. It has been shown recently that this fungus is the imperfect form of Allescheria Boydii.^ The fungus produces on Sabouraud agar a rapidly spreading, floccose, mouse- grey colony. The conidia are borne singly or in small groups at the tips and sides of simple or branched conidiophores. They are el- liptical, egg-shaped, or clavate, with a truncate base, and, under the microscope, they are brown. They are 3.5 to 7.5 by 5 to 15/x. The ascocarp of A. Boydii is globose, 50 to 200/a in diameter, with- out ostiole (cleistocarpous) and the wall is thin and dark brown. The asci are subglobose, 8 to 20;a in diameter, evanescent, and each contains eight ovoid ascospores 4 to 4.5 by 6 to 7.5/1, in size with slightly thickened brown walls. Geographical Distribution. Mycetoma occurs throughout the world but is more common in tropical and subtropical countries in persons who do not wear shoes and so are more often exposed to 214 INFECTIONS CAUSED BY MOLDS injury. The specific agents of mycetoma are to some extent geo- graphically limited. Thus, Nocardia madurae is more common in southeastern Asia and the Pacific islands. N. mexicana is seen in southern United States and Mexico. The other etiological fungi, except for a few rare or poorly known species, seem to have a wider distribution. Habitat. The fungi of mycetoma are not transmitted from per- son to person but are obviously related to trauma in most cases. It is assumed that the fungi ordinarily grow in soil or on dead vegeta- tion and become pathogenic only when introduced by accident into the subcutaneous tissues. LITERATURE 1. Brumpt, E., Les mycetomes, Arch. Parasitol., 10, 489 (1906). 2. Carter, H. V., On "Mycetoma" or the fungus-like disease of India, Trans. Med. Phys. Soc. Bombay, 7, 206 (1862). 3. Carrion, A. L., and J. Knott, Mycetoma by M onosporium apiospermum in St. Croix, Virgin Islands, Puerto Rico J. Pub. Health Trop. Med., 20, 84 (1944). 4. Chalmers, A. J., and R. G. Archibald, A Sudanese maduromycosis, Ann. Trop. Med. Paras., 10, 169 (1916). 5. Emmons, C. W., Allescheria Boydii and Monosporium apiospermum, My- cologia, 36, 188 (1944). 6. , Phialophora Jeanselmei comb. n. from mycetoma of the hand. Arch. Path., 39, 364 (1945). 7. Gammel, J. A., The etiology of maduromycosis. Arch. Dermatol. Syphilol. (Chicago), 15, 2il (1927). 8. PiNOY, E., Actinomycoses et mycetomes. Bull. inst. Pasteur, 11, 929, 977 (1913). 9. Symmers, D., and A. Sporer, Maduromycosis of the hand. Arch. Path., 37, 309 (1944). CHAPTER VIII BIOLOGICAL ACTIVITIES OF MOLDS ECOLOGY OF MOLDS The molds in many habitats grow more slowly than do the bacteria. Consequently we do not generally find them growing to any great extent in environments where they have to compete with the latter. However, where conditions are unfavorable for bacteria, there will nearly always be found one or more species of mold capable of de- veloping. Thus we find them growing abundantly on starchy foods which are not favorable to most bacteria, in foods containing a high percentage of sugar which inhibit bacteria because of their high osmotic pressure, in habitats too dry for bacterial development, and in acid materials, as fruit juices or sour milk. Although they grow slowly, they may develop on materials which we would ordinarily consider as supplying but very small amounts of nutrients, like tanned leather, linen, or cotton cloth, if these are damp enough. In fact, molds may develop in the most surprising situations, and may grow on very unusual substrates. They not in- frequently appear in laboratory reagents of various kinds, where small traces of organic matter may be present, sometimes in solutions in which one would think life would be impossible. Thus molds are quite versatile in their ability to adapt themselves to particular en- vironments, and it is difficult to make generalized statements without noting numerous exceptions. . Moisture is of course requisite for growth, since molds like all other biological forms must absorb food in solution. They can get along, however, wuth much smaller amounts of water than are required by bacteria, and in particular can grow in solutions of much higher osmotic pressure. Thus they may be found forming scums on the brine solutions of pickling vats, or on the surfaces of hams or other salt meats; it is well known that they wdll grow on syrups and jellies which will not permit the growth of bacteria. Relative humidity of the atmosphere was found by Thom and Shaw"® to be a critical factor for the growth of molds on butter, very little growth taking place if the humidity was below 70 per cent. 215 216 BIOLOGICAL ACTIVITIES OF MOLDS On the other hand, Lewis and Yesair ^- found humidity had little effect on the growth of mold on Frankfurter sausages, probably be- cause the substrate contained sufficient moisture. There are probably very few, if any, strictly anaerobic molds, and the great majority are strictly aerobic. However, a few species, notably some of the Mucors and certain strains of the Penicillia may grow to some extent under reduced oxygen tension. The organism used in ripening Roquefort cheese, Penicillium roqueforti, can de- velop with less oxygen than is required by most molds. Williams, Cameron, and Williams " have reported the isolation of two strains of "facultatively anaerobic mold of unusual heat resistance." These organisms were identified as strains of an undescribed species of Penicillium by Thom. They proved to be capable of growing in high vacuum. The different species of molds vary markedly in their temperature requirements. See Chapter III. In general it may be stated that the optimum temperature for most species lies somewhere near 30° C. Some growth will take place at temperatures considerably below this, and most forms may grow somewhat at temperatures up to 37° C. or even higher. Most species of Penicillium have their opti- mum temperature between 20° and 25° C. and may fail to grow at temperatures above 30° C. On the other hand, w^th many species of Aspergillus the optimum temperature will be around 35° C. One species pathogenic for birds {Aspergillus fumigatus) finds its opti- mum at 40° C. The thermal death points also vary markedly with the species. Some types of spores are of course much more resistant than vegetative mycelium but not nearly so resistant as the spores of bacteria. Thom and Ayres ^* have studied the heat resistance of mold spores with regard to pasteurization of milk. A temperature of 62.8° C. for 30 minutes was sufficient to destroy practically all. Macy, Coulter, and Combs ^^ likewise found molds easily destroyed by pasteurization processes. Flashing for 30 seconds at a tempera- ture of 73.9° to 79.4° C. was necessary to obtain an equal degree of sterilization. Lewis and Yesair found that 60° C. for 5 minutes was sufficient to kill all the molds from meat products which they studied. With dry heat, of course, higher temperatures are required. One of the strains of the facultatively anaerobic penicillia referred to previ- ously, described by Williams and coworkers, produces sclerotia of unusually high resistance to heat. NUTRITIONAL REQUIREMENTS OF MOLDS 217 NUTRITIONAL REQUIREMENTS OF MOLDS Adequate supplies of elements such as carbon, nitrogen, hydrogen, oxygen, phosphorus, sulphur, and magnesium must be furnished the molds. The sugars, sucrose, glucose, or fructose, serve as excellent sources of carbon for most fungi. Other sugars including pentoses, alcohols, organic acids, oils, higher paraffins, and polysaccharides have also proved capable of satisfying, at least partially, the carbon require- ments of certain molds. Tamiya,*'^ who has carried out one of the more elaborate studies on the relation between chemical structure and assimilability, has made the interesting observation that some compounds can serve satisfactorily for respiration but not for growth. He further noted that no constant relation exists between respiration and growth since the former was found to vary with the source of carbon. Steinberg **" has noted that practically all tests of assimil- ability of a carbon source are based on experiments with pure com- pounds. Since fungi under normal conditions grow on mixtures of carbon compounds, he has suggested that the results of tests with single carbon sources cannot serve as final tests of assimilability. In general, the molds are capable of utilizing a large number of nitrogen compounds. Robbins ^^ has suggested that fungi fall into four groups when classified on the basis of their nitrogen require- ments. The groups may be referred to as the nitrogen-fixing, the nitrate, the ammonium, or the organic nitrogen compound users. The first group, according to Robbins, is capable of utilizing nitrate, ammonium, or organic nitrogen in addition to being able to use atmospheric nitrogen. Those organisms in the second group, in- capable of using gaseous nitrogen but able to use nitrate nitrogen, can grow also with ammonium or organic nitrogen sources. The organisms of the third group are capable of developing only in the presence of ammonium or organic nitrogen compounds. The last group consists of those organisms which can satisfy their nitrogen requirements only with organic nitrogen sources. However, it should be noted and emphasized here in connection with the first group of molds that the belief in the general ability of the fungi to fix gaseous nitrogen is no longer held. Therefore, there would be very few, if any, molds which would be placed in the first group. It should also be pointed out that the nitrogen requirements of an organism are not fixed but vary with the source of carbon. Robbins' scheme 218 BIOLOGICAL ACTIVITIES OF MOLDS of classification need not be altogether discarded, however, because the nitrogen requirements of the fungi could be based on comparative responses with an identical carbon source. That heavy metals play an essential role in the nutrition of molds has been recognized since the earliest investigations dealing with the cultivation of these fungi in synthetic media. In addition to phos- phorus, sulphur, magnesium, and potassium, certain other elements are not only desirable but also often necessary to obtain the maxi- mum yield of fungi from synthetic media. Originally it was held that elements added to the media in minute quantities were beneficial because these substances acted as chemical stimulants. It was postu- lated that the accelerated and increased growth of fungi in media containing these elements was due to the physiological response of the organism to the toxic properties of these elements, now known to be essential. This concept was based on the notion that poisons when added in minute quantities act as stimulants. It was assumed, of course, that the control media were free of trace elements. Hence growths on such media were considered normal and increases in growth were thought to be due to the "stimulating effect" of the added heavy metals. Steinberg ^^ has presented evidence to disprove this chemical stimulation theory. By using extremely efficient meth- ods of purification, this investigator was able to prepare media free of traces of heavy metals. The growth of Aspergillus niger was so scanty in such media and such large increases in yield were obtained when zinc and iron were added that he regarded the chemical stim- ulation theory as untenable. He considered these two elements just as essential to the nutrition of the mold as carbon and nitrogen. In addition to zinc and iron, elements such as copper, manganese, molybdenum, and gallium are now considered not only requisite for maximum growth but also absolutely essential for growth in general of the filamentous fungi. It is generally safe to assume that these elements, which are required only in very minute quantities, are normally present as impurities in sufficiently large amounts (in the chemicals, in most samples of distilled water prepared by the usual methods and glassware used for cultural purposes) despite the fact that these elements are not purposely included in most media used for culturing these organisms. In addition to the above-mentioned elements which must be fur- nished the molds, some fungi require certain organic substances for growth. Thus thiamin has been found essential or beneficial for the growth of most molds. Robbins and Kavanagh ^-' ^^ have shown that some fungi require the intact thiamin molecule; others may re- NUTRITIONAL REQUIREMENTS OF MOLDS 219 quire only the pyrimidine and thiazole portions; and still others may require only the pyrimidine or thiazole fractions, capable of syn- thesizing whichever portion is lacking. Biotin, pyridoxine, p-amino- benzoic acid, choline, and inositol are also required by some fungi. Advantage has been taken of the essentiality of these substances for the growth of certain fungi by various investigators to devise assay methods because the growth is often proportional to the amount of these substances in the culture medium. To a set of cultural vessels containing a medium nutritionally complete in every respect, ex- cept for the test substance for which the assay is being made, are added varying quantities of a material with an unknown content of the test substance. The degree of growth, being dependent on the amount of the test substance, indicates the amount of the test substance in the material. Similar assay methods have also been devised for certain amino acids using molds as test organisms. The principle of using molds for such assays has been employed to meas- ure the available phosphorus and potassium in soil samples. Some very interesting and fundamental studies have been carried out using mutants of Neurospora. Horowitz and Srb ^^ obtained seven mutants of Neurospora crassa incapable of synthesizing arginine, i.e., arginine had to be furnished these mutants for them to grow. These mutants were obtained by exposing the parent or- ganism to ultraviolet and x-radiations; presumably a certain gene or set of genes was thus destroyed. Each of these mutants differed from the normal by a different gene. One strain grew only when arginine was added. Others grew on either arginine or citrulline, thus showing that the latter could be converted to arginine. Others proved capable of using arginine, citrulline, or ornithine. Thus it was deduced that ornithine, too, could be converted to arginine. Each of the mutants capable of utilizing ornothine was also able to use citrulline, whereas the reverse was not always true. Because no strain was found capable of utilizing ornithine and arginine but not citrulline, it was concluded that citrulline was an essential intermediate in the syn- thesis of arginine from ornithine. The importance of this type of investigation with molds should not be underestimated. By such studies, metabolic cycles as the above-described ornithine cycle of Krebs and Hensleit occurring in higher biological forms can be read- ily studied with the lower biological forms such as molds. The ap- plications of such types of investigations are not limited to the fungi themselves but are of use in studying the nutritional and metabolic activities of higher biological forms. 220 BIOLOGICAL ACTIVITIES OF MOLDS MOLD FERMENTATION Citric Acid Fermentation. Various molds produce citric acid from sugars but strains of Aspergillus niger are apparently more active than other species, and have been used more extensively for both experimental and commercial purposes. Wehmer,^^ who was study- ing the fermentations of molds, first noted that some produce citric acid. He named these molds Citromyces, but they were later classi- fied with the monoverticillate Penicillia. Currie and Thom ^^ found that with some strains there was a lag between the curves for total acidity and oxalic acid, an acid then known to be produced by some molds, during the fermentation. This led to a search for another acid, which was subsequently identified by Currie ^* as citric acid. This was considered an intermediary product in the fermentation. According to this worker, the oxida- tion of sugar by A. niger proceeded as follows. Carbohydrate -^ Citric acid — » Oxalic acid -^ CO2 Although this view is no longer held, Currie made other contributions to our knowledge of the citric acid fermentation. He showed that the proportion of oxalic and citric acids could be controlled by pH and the addition of inorganic salts. Low pH was found to favor the production of citric acid and suppress the formation of oxalic acid. Furthermore, it minimized the danger of contamination. A large number of fungi have since been found capable of produc- ing citric acid. Strains of Aspergillus, Penicillium, Mucor have been found to produce this acid, but strains of the A. niger group have proved the most satisfactory in the production of citric acid. The most desirable strains are those which efficiently convert sugar to the acid, are easily cultivated, retain their biochemical characteristics, and produce the least amount of other metabolic products. Doelger and Prescott,^*^ among others, corroborated the findings of Currie and carried out further extensive studies on the techniques of this fermentation. They found that the successive transfer of spores in the same medium stimulates the mold to give high yields of citric acid. They also observed that it was best to seed only one fourth to one half of the surface area of the medium. Where high yields were obtained, the molds produced very little if any spores. Thus sporulation, or lack of it, could be used as an index of the efficiency of a fermentation, according to these men. CITRIC ACID FERMENTATION 221 Although many organic substances may be fermented to citric acid, sucrose and fructose have generally given the best results. Doelger and Prescott found that in batches which were allowed to ferment for 9 to 12 days, mashes containing 14 per cent sucrose were found to give the highest yields. They recommended the use of sucrose or technical glucose for industrial fermentations. These sugars were preferable to maltose or molasses. Fructose was also found to give high yields but its use would not be commercially feasible. Increasing the sugar content, or replacing part of the su- crose with glucose or fructose, or partially hydrolyzing sucrose dur- ing the sterilization process lowered the yields. However, molasses is used in present-day commercial practice. Currie and Doelger and Prescott have shown that the molds gen- erally produce more citric acid when inorganic salts containing potas- sium, phosphorus, magnesium, sulphur, and nitrogen are added to the fermentation liquor. Conflicting reports are found in the litera- ture concerning the addition of iron and zinc. There is a distinct possibility that the strains of molds used react differently to the additions of these metals. Doelger and Prescott noted that the source of water used made a difference in the yields obtained. This observation may be linked with the mineral requirements of the molds. Where the water is deficient in trace amounts of these ele- ments the addition of metals may help, and where there is already an abundance they may exert a toxic effect. These men also found that the pH range of 1.6 to 2.2 was the most suitable for carrying out the fermentation with their organism. They recommend the use of hydrochloric acid in adjusting the pH to this range. Sulphuric, nitric, acetic, and formic acids were found to be inferior to this mineral acid. Whereas Wehmer and others have advocated the use of calcium carbonate to neutralize the acid formed during the fermentation, Prescott and Dunn ^* advise against its use. They maintain that its absence favors higher yields, shortens the fermentation periods, and decreases the possibilities of contamina- tion. Doelger and Prescott also studied the influence of the ratio of the surface area to the volume of the fermentation mash. They advocated the use of shallow pans of aluminum of high grade of purity for growing the mold and carrying out the fermentation. In such pans, there would be large surface areas of mycehum exposed to relatively shallow layers of medium. Agitation of the medium was found to be undesirable. 222 BIOLOGICAL ACTIVITIES OF MOLDS The optimum temperature range was found to be from 26° to 28° C. and the fermentation was generally completed in 7 to 10 days. The optimum amount of air passed over the mold mycelial mats varies with each installation of equipment, too low or too high supplies decreasing the yields of citric acid. While 60 per cent of the sugar may usually be recovered as citric acid, yields as high as 87, 90.7, and even 100 per cent have been reported. After the fermentation is completed, the liquor is drained off and the mat pressed to remove any residual citric acid. Calcium car- bonate may be used to adjust the pH of the liquor to approximate neutrality and calcium citrate is precipitated from a hot solution. The addition of sulphuric acid removes the calcium, which settles out as calcium sulphate, and citric acid is then recovered. • Cahn ' has recommended that cane or beet pulp impregnated with molasses or sucrose be fermented at 20° to 35° C. He claims that the production of citric acid with the use of solid material shortens the fermentation period to 3 or 4 days and, because the fermentation proceeds so rapidly, the deleterious effects of bacterial contamina- tion are obviated. Yields of 45 per cent on the basis of sugar in the molasses or 55 per cent on the basis of sucrose were claimed. The exact details employed in the commercial production of citric acid have not been made available to the public as yet. The reader is referred to the publications of Currie, Doelger and Prescott, and to Industrial Microbiology by Prescott and Dunn for further gen- erally known details on the techniques employed in this tricar- boxylic acid fermentation. Various theories have been suggested for the mechanism of the production of citric acid. Because the theory must be such as to explain its formation from two up to seven and even twelve carbon compounds, and because it must account for the high yields men- tioned above, those which have been proposed up to now have been considered untenable. In general there seem to be two schools of thought. One school maintains that the hexose chain is not broken but becomes trans- formed to citric acid with its forked chain. The other proposes that the hexose is initially split to shorter carbon chain compounds and subsequently built up to citric acid. Challenger and his associates ^° and Franzen and Schmitt ^^ pre- sent evidence in support of the hypothesis that glucose is not broken down but is converted to the forked tricarboxylic acid. They sug- gest the following series of reactions. C6H1206 Glucose GLUCONIC ACID FERMENTATION COOH -^ COOH -^ COOH -^ COOH • • • • CHOH CHOH CH2 CH2 CHOH CHOH C:0 HOC -COOH 223 CHOH CHOH CHOH C:0 • • CHOH CH2 CH2OH COOH COOH Gluconic Saccharic /S,-,-Diketo- acid acid adipic acid CH2 COOH Citric acid Bernhauer/ however, feels that the gluconic and saccharic acids found in the mold cultures (the presence of which is advanced as supporting evidence by the proponents of the first school of thought) originate from a side reaction. Various hypotheses have been ad- vanced by the advocates of the second group of workers who main- tain that sugar is first broken down and the intermediary substances subsequently condensed to form citric acid. The mechanism pro- posed by Bernhauer and Bockl - is typical of the various ones which have been suggested in that all or most believe that a condensation of a dicarboxylic acid and acetic acid occurs. (See reactions on the following page.) As yet, even among the workers who claim that there is a con- densation of intermediate carbon compounds, there seems to be a divergence of opinions. For greater details, consult the publications of Bernhauer and Iglauer,^ Ciusa and Briill,^^ Chrzaszcz and Janicki,^^ and Wells and his associates.'^^ The latter group of workers has carried out careful carbon bal- ance experiments which demonstrated that some of the theories which have been advanced are quite untenable. They obtained yields of citric acid which could not be explained by many of the fermentation mechanisms which have been proposed, i.e., the actual yields proved to be higher than the theoretical. Furthermore the citric acid-carbon dioxide ratios found by them experimentally were higher than the theoretical. Gluconic Acid Fermentation. That bacteria and molds are capable of transforming glucose to gluconic acid by a simple oxidation has been known for some time. As early as 1878, Boutroux noted that a bacterium, "Mycoderma aceti" {Acetobacter aceti) , could produce this acid. In 1922, IMolliard *° observed that "Sterigmatocystis nigra" {Aspergillus niger) grown on sucrose mashes produced gluconic acid 224 BIOLOGICAL ACTIVITIES OF MOLDS COOH CH2 HCCOOH CH2 COOH C6H1206 - Glucose -^ 2CH3C00H Acetic acid -H2O +CH3COOH > COOH > -H2 CH2 CH2 COOH Succinic acid -Hajf +H2 COOH CH CH COOH Fumaric acid +H2OJI' -H2O • COOH CHOH CH2 COOH Malic acid -H2 — > +H20 COOH ; ^ COOH -H2O CH CH2 CCOOH HOC -COOH CH2 CH2 COOH Aconitic acid COOH Citric acid ,,-C02 ' CH2 CCOOH CH2 COOH Itaconic acid ' in addition to citric and oxalic acids. Bernhauer ^ in 1924 noted that a strain of A. niger produced gluconic acid in the presence of calcium carbonate. He found that contrary to what was found in the citric acid fermentation (where relatively high temperatures, abundant supplies of nitrogen, and heavy mats are desirable) gluconic acid fermentation was favored by low temperatures, low supplies of nitro- gen, and thin mat productions. Men of the Northern Regional Re- GLUCONIC ACID FERMENTATION 225 search Laboratory of the U. S. Department of Agriculture, May, Herrick, Wells, Moyer, and their associates, have carried out exten- sive studies on the methods and apparatus which are best suited for the production of this acid. In the original shallow-pan method investigated by Herrick and May," the organism used was Penicilliwn purpurogenum var. rubrisclerotium (Thorn No. 2670). With this method of production, the gluconic acid yield was the best with a high concentration of sugar, 20 to 25 per cent solution of glucose, and a temperature of 25° C. The efficiency in the conversion of sugar to gluconic acid was found to be affected by the ratio of surface to volume of liquid, a ratio between 0.25 and 0.30 being the most feasible. Although agitation of the nutrient solution proved favorable when the con- centration of the sugaif was low, it did not affect the production of the acid when the sugar-concentration was high. The fermentation could be carried out successfully over a wide pH range, 3 to 6.4. Yields of 55 to 65 per cent were obtained in about 11 days. The mold was grown on a glucose-salt solution in aluminum pans placed in a sterilizable chamber. Since Schreyer," working with A. jumaricus, first demonstrated in 1928 that the yield of this acid could be increased by agitation, aeration, and the use of calcium carbonate, a number of investigators have studied this technique of fermentation. With submerged growths of P. chnjsogenum, aerated with filtered and humidified air. May, Herrick, Moyer and Wells ^^ were able to obtain 80 to 87 per cent yields in about 8 days. The temperature used was 30° C. and calcium carbonate was added at the rate of 1 gram for every 4 grams of glucose. In addition to glucose, salts were added to the medium and nitrogen was supplied in the form of ammonium nitrate. Herrick, Hellbach, and May 2« have\also developed a rotary drum, submerged growth method using a strain of A. niger. The advantage of the rotating drum, submerged growth method (which they have developed to a pilot plant scale) over the aerated and agitated, sub- merged growth method was that the fermentation time could thus be cut down considerably, 80 per cent yields being obtained in a little over 2 days. The rotary drum apparatus is essentially a horizontally mounted, hollow, aluminum cylinder closed at both ends and equipped with buckets and baffles placed on the inside walls. The drum is slowly rotated to keep the fermenting culture aerated and mixed. A serai-continuous process has also been developed.*^ For specific 226 BIOLOGICAL ACTIVITIES OF MOLDS details of the processes, the reader is referred to the publications of the Northern Regional Research Laboratory investigators. Miscellaneous Minor Fermentations. Gallic Acid. The produc- tion of citric and gluconic acids depends on fermentative changes brought about by molds. However, the production of gallic acid de- pends on a hydrolytic change, the hydrolysis of tannin. Scheele in 1787 first discovered gallic acid in an infusion of gallnuts which had been acted upon by a mold, but Van Tieghem *'^ carried out the fi