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Selman a. Waksman tvas born July 2, 1888, in Prihika, a small town in the Ukraine, Russia. His parents were Jacob and Fradia (Lon- don) M'aksman. After f^radiuiting in 1910 from the Fifth Latin School in Odessa, he left for the United States. lie entered the College of Agricidture of Rutgers University in 1911 and received his bachelor of science degree in 1915. He became a natu- ralized citizen the same year. He then was ajypointed research assistant in soil microbiology at the New Jersey Agricultural Experiment Station, and later Research Fellow at the University of California. He obtained a master of science degree from Rutgers University in 1916 and a doctor of philosophy degree from the University of California in 1918. The same year. Dr. Waksman received an appointment as Microbiolo- gist at the New Jersey Agricultural E.xperiment Station at New Brunswick, New Jersey, and as lecturer in soil microbiology at Rutgers University. He became associate professor in 1925, and in 1930 was made professor. He nouy is the head of the Microbiology Department of the College of Agriculture and Experiment Statioji at Rutgers University. In 1931, he teas invited to organize a division of marine bacteriology at the newly established Woods Hole Oceanographic Institution and was appointed marine bacteriologist of that institution. He is a member, honoranj member, or fellow of a number of scien- tific societies in this country and abroad (Brazil, France, Germany, India, Mexico, Russia, Sweden). Among the American societies to tchich he be- longs are the Society of American Bacteriologists, of which he is a former president, the National Academy of Sciences, and the National Research Council. He won the Nitrate of Soda Nitrogen Research Award in 1929, was president of Commission III on Soil Microbiology of the International Society of Soil Science (1927-1935), and was elected a corresponding member of the French Academy of Sciences in 1937. In the summer of 1946. Dr. Waksmax lectured before scientific groups in Europe and was given an honorary degree of doctor of medicine by the University of Liege in Belgium. He holds also honoranj degrees of doctor of sciciwe, awarded to him by Rutgers in 1942 and by Princeton Univer- sitij in 1947, and an honorary degree of doctor of laws from Yeshiva Uni- versity. .Vcti> York, in 194S. Dr. \\'aksman's work in his field has been recognized by several scien- tific societies in recent years. He received the Passano Foundation Award in 1947; the Emil Christian Hansen medal and award from the Carlsberg Laboratories in Denmark the same year; the Netv Jersey Agricultural So- ciety medal; the Albert and Man/ Lasker Award by the American Public Health Association, and the Amonj Award by the American Academy of Sciences, all in 194S. He has published more than SOD scientific papers, and has written, alone or with others, eight books. Among these are Enzymes (1926), Principles of Soil Microbiolosrv (1927, 1932). The Soil and' the Microbe (1932), Humus (1936, 19oS),' Microbial Antagonisms and Antibiotic Sub- stances (1945. 1947), and The Literatvire on Streptomycin. 1944-1948 (I9-IS). Another recent work, edited by Dr. Waksmax, is Streptomycin — Nature and Practical Applications. ANNALES CRYPTOGAMICI et PHYTOPATHOLOGICl Volume 9 The ACTINOMYCETES ANNALES CRYPTOGAMICI et PHYTOPATHOLOGICI {incorpor^ating Annales Bryologici^ edited hy FrANS VeRDOORN, Ph.D. Managing Editor, Chronica Botanica Research Fellow, Arnold Arboretum, Harvard University Botanical Secretary, International Union of Biological Sciences Wij en konncn den Hecr en maker van het geheel Al niet meer verheerlijken, als dat wij in alle zaken, hoe klein die ook in onse bloote oogen mogen zijn, als ze maar leven en wasdom hebben ontfangen, zijn al wijsheit en volmaakthcit. met de uiterste verwondering sien uit steken. Antoni van Lceuwenhoek 1950 WALTHAM, MASS., U.S.A. Published by the Chronica Botanica Company 7k /i The ACTINOMYCETES Their Nature^ Occurrence^ Activities^ and Importance by Selman a. Waksman, Ph.D. Professor of Microbiology, Rutgers Universitij Microbiologist, Neio Jersey Agricultural Experiment Station 1950 WALTHAM, MASS., U.S.A. Published by the Chronica Botanica Company First published MCML By the Chronica Botanica Company of Waltham, Mass., U.S.A. Copyright, 1950. By Selman A. Waksman All riyhts reserved, including the right to reproduce this book or parts thereof in any form Authorized Agents: — New York, N. Y.: Stecher-t-Hafner, Inc., 31 East lOlh Street. San Francisco, Cal.: J. W. Stacey, Inc., 551 Market Street. Ottawa, Ont.: Thorburn and Abbott, Ltd., 104 Sparks Street. Mexico, D. F.: Axel Moriel Sucrs., San Juan de Letran 24-116; Ap. 2762. Lima: Libreria Internacionel del Peru, Casa Matriz. Boza 879; Casilla 1417. Santiago de Chile: Libreria Zamorano y Caperan, Compania 1015 y 1019; CasiUa 362. Rio de Janeiro: Livrarta Kosmos, Rua do Rosario, 135-137; Caixa Postal 3481. Sao Paulo: Livraria Civilizacao Brasileira, Rua 15 de Novembro, 144. Buenos Aires: Acme Agency, Soc. de Resp. Ltda., Suipacha 58; Casilla de Correo 1136. London, W. C. 2: Wm. 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Designed by Frans Verdoorn PREFACE Three and a half decades ago— in the spring of 1914— the u^iter, then a senior at Rutgers College, dug a spade into the earth of the New Jersey Agricidtnral Station experimental plots, to study the distribution of different groups of microorganisvis occurring at different depths in the soil. This operation was repeated monthly, and sterile soil samples were remolded to the laboratory and examined by use of ordinary plat- ing procedures. A relatively simple agar medium was used. Among the soil organisms that attracted the particrdar attention of the yotithful investigator were the actinomycetes. Althotigh he also enumerated the bacteria and the filamentous fungi, he was struck pri- marily by this much-neglected group of soil inhabitants, frequently spoken of as ray fungi and said to belong to the genus Actinomyces or Streptothrix. On December 28, 1915, he presented before the 17th Annual Meeting of the Society of American Bacteriologists, a paper on the subject of "Bacteria, actinomyces and fungi in the soil." In this, his first contribution to the knowledge of the microbiological population of the soil, he said: "The actinomyces grow very slowly; they begin to develop from the bottom of the plate, and to the casual observer the colonies formed look like those of bacteria, even after 5-6 days' incubation; only the some- what mealy or roiigh surface will disclose the fact that they are not bacteria. It requires carefid observation to tell whether those white, pink or grey colonies are bacteria or not. Many cotmts of bacteria might have been confused, when this point was not known, and the fungi and actinomycetes were not taken into consideration." Since this early survey of the occiirrence and abundance of actino- mycetes at different soil depths and in different soil types, the ivriter and his mimerous associates and students have devoted much time to the study of the actinomycetes, their cidtural characteristics, recognition of type species, their classification, their physiological properties and biochemical activities, their importance in the decomposition of pure organic compounds as well as of complex plant atid animal residues in soils, peats, and composts, and finally their ability to produce antibiotic substances. The writer has thus been concerned, d^iring virtually his entire sci- entific lifetime, with the study of the actinomycetes. In summarizing our present knowledge of this interesting and important group of micro- organisms, he has attempted to assemble the work of other investigators, ivith somewhat greater emphasis upon the work done in the laboratories Waksman — x — Actinomycetes of the Department of Microbiology of Rutgers Unix^ersity and the New jersey Agrictdtural Experiment Station. To his many associates, who have helped in making this work pos- sible, the author wnshes to express his sincere appreciation for their nnfailing enthusiasm and continuous interest and collaboration. The writer also Tmshes to express his gratitude to Lt. Col. M. L. LiTTMAN of the Armed Forces Institute of Pathology, for supplying various photographs, to Dr. E. W. Emmons of the National Institute of Health, for reading Chapter XL and to Dr. R. W. Goss of the Uni- versity of Nebraska for reading Chapter X. December 20, 1949 New Brunswick, N. J. %F THE COMPOST how can it he that the grotmd does not sicken? How can you he alive, yon growths of spring? How can you fiirnish health, you hlood of herhs, roots, orchards, grain? Are they not continually puffiijg distemper'd corpses within yoii? Is not every continent work'd over and over with sour dead? Where have yoti disposed of their carcasses? 1 do not see any of it upon you today— or perhaps I am deceiv'd. Behold this compost! behold it well! Perhaps every mite has once form'd part of a sick person—Yet behold! The grass of sprirtg covers the prairies, The summer growth is innocent and disdainfid above all those strata of sour dead. What chemistry! That the winds are really not infectious, That when I recline on the grass I do not catch any disease, Though probably every spear of grass rises otit of what was once a catching disease. Now I am terrified at the Earth! it is that calm and patient. It turns harmless and stainless on its axis, with such endless succes- sions of diseas'd corpses, It distils such exquisite winds out of such infused fetor. It gives such divine materials to men, and accepts such leavings from them at last. Walt Whitman. CONTENTS The Author iv Preface ix The Compost hy Walt Whitman xi Contents xii List of Illustrations xv List of Tables xvii INTRODUCTORY 1 Chapter I TERMINOLOGY, PHYLOGENY, AND TAXONOMY:-Syn ONYMs OF Generic Names of Actinomycetes— Systematic Position AND Classification of Actinomycetes— Identification of Actino- mycetes 3 Chafter 11 IDENTIFICATION AND DESCRIPTIONS OF IMPORTANT TYPES:— Cl,ASSIFICATION OF AcTINOMYCETALES— DESCRIPTION OF SEV- ERAL Important Actinomycetes 22 Chapter 111 MORPHOLOGY AND LIFE CYCLE: -Staining of Actinomy- cetes— General Morphology— Sporulation of Actinomycetes— Types of Growth on Solid and Liquid Media 46 Chapter IV VARIATIONS AND MUTATIONS: -Types of Variation-Cul- ture Consistency 69 Chapter V . METABOLISM OF ACTINOMYCETES— GROWTH AND NUTRITION; PRODUCTION OF ODORS AND PIGMENTS:- NuTRiENT Requirements— Growth and Cell Synthesis of Aerobic Actinomycetes — xiii — Contents AcTiNOMYCETEs— Metabolism of Anaerobic Actinomycetes— Pro- duction OF Odors— Production of Pigments— Thermophilic Ac- tinomycetes 79 Chaffer VI PRODUCTION OF ENZYMES AND OF GROWTH-PRO- MOTING SUBSTANCES:— Production of Enzymes— Production OF Vitamins— Oxidative Mechanisms 100 Chapter Vll ANTAGONISTIC PROPERTIES OF ACTINOMYCETES AND PRODUCTION OF ANTIBIOTICS:-Antagonistic Effects of Ac TiNOMY'CETEs— Production of Antibiotics by Actinomycetes . 107 Chapter VIII DISTRIBUTION OF ACTINOMYCETES IN NATURE:-Oc currence of Actinomycetes in the Soil— Occurrence of Actino- mycetes IN Manures and Composts— Occurrence of Actinomy- cetes IN Water Basins— Occurrence of Actinomycetes in Dust and on Exposed Surfaces of Plants— Occurrence of Actinomy- cetes IN Foodstuffs— Occurrence of Actinomycetes in Animal Systems 133 Chapter IX DECOMPOSITION OF PLANT AND ANIMAL RESIDUES:- Decomposition of Pure Organic Compolinds— Decomposition of Complex Plant Materials— Decomposition of Humus— Ther- mophilic Composts 149 Chapter X ACTINOMYCETES AS CAUSATIVE AGENTS OF PLANT DISEASES:— Potato Scab— Sugar Beet and Mangel Scab— Sweet Potato Pox or Soft Rot— Other Plants Infected by Scab Organ- isms—Other Plant Actinomycoses— Methods of Control . 155 Chapter XI ACTINOMYCETES AS CAUSATIVE AGENTS OF HUMAN AND ANIMAL DISEASES:— Etiology of Infections— Earlier In- vestigations—Recent Studies— Trlie Actinomycosis— Aerobic Ac- tinomyces Infections— Chemotherapy of Actinomycosis . 170 .- ^ m dTl^ . Waksman — xiv — Actinomycetes Chapter XU SUMMARY:— Role of Actinomycetes in Natural Processes— Actinomycetes as Causative Agents of Disease— Actinomycetes AS Agents of Spoilage and Deterioration— Utilization of Actino- mycetes for the Production of Enzymes and Vitamins— Produc- tion OF Antibiotics 187 Afpendix MEDIA USED FOR THE STUDY OF ACTINOMY- CETES 193 Bibliography 199 General Index 223 Index of Organisms 227 Bacteria belong to the most wide-spread of organisms; we may say they are omnipresent; they never fail either in air or water; they at- tach themselves to the surface of all firm bodies, but develop in masses only where decomposition, corruption, fermentation or putrefaction are present. (Ferdinand Cohn, transl. by C. S. Dolley). 0CAI LIST nf ILLUSTRATIONS Figure l.—Streptothrix of Ferdinand Cohn xx Figure 2.— First photograph of a species of Micromonospora (1899) . . 8 Figure 3.— Structure of actinomyces mycehum 11 Figure 4.— Typical growth of aerobic actinomycetes upon agar slants . . 16 Figure 5 a-d.— Different types of branching of aerial mycelium of species of Streptomyces 18/21 Figure 6.—Nocardia asteroides, grown on potato dextrose-beef extract agar 25 Figure 7.—Nocardia asteroides, grown on potato dextrose-beef extract agar 27 Figure 8.— Branching and sporulation of different strains of Nocardia . 29 Figure 9.— Sporulation of straight aerial hyphae of species of Streptomyces 39 Figure 10.— Streptomyces venezuelae, grown on potato dextrose-beef ex- tract agar 44 Figure 11.— Streptomyces sp., grown on potato dextrose-beef agar . . . 47 Figure 12 a-d.— Different forms of sporulation of Micromonospora growing in composts, as shown by contact slide preparations 52/55 Figure 13.— Details of sporulation and of spore germination by S. griseus as shown by electron microscope 56 Figure 14.— Aerial mycelium of a Streptomyces, showing zonation or "fairy ring" formation 58 Figure 15.— Electron micrograph of actinophage 61 Figure 16.— Method of measuring actinophage concentration .... 64 Figure 17.— Variants of Streptomyces griseus growing on yeast extract- glucose agar 70 Figure 18.— Metabolic changes produced by S. lavendulae in aerated and stationary cultures 85 Figure 19.— Metabolic changes produced by S. antibioticus in aerated and stationary cultures 88 Figure 20.— Influence of temperature upon growth and carbon dioxide production by actinomycetes 92 Figure 21.— Decomposition of hemicelluloses by actinomycetes, as meas- ured by CO:; evolution 93 Figure 22.— The use of the agar cross-streak method for testing the ability of actinomycetes to produce antibiotic substances 106 Figure 23 a.— The use of M. tuherctdosis for testing production of anti- biotic substances by actinomycetes 110 Figure 23 b.— Inhibition of streptomycin-sensitive but not of streptomycin- resistant strain Ill Figure 24.— Method of measuring antibacterial or antifungal potency of an antibiotic, by the agar streak method 113 Figure 25. -Streptomycin-producing strain of S. griseus, showing vegeta- tive and aerial mycelium 122 Figure 26.— Method of isolation of streptomycin from metabolite solution 125 Figure 27.— Crystals of the calcium chloride double salt of streptomycin 127 Figure 28.— Metabolic changes produced in the medium by Streptomyces sp. 128 Figure 29.— The course of development of S. alhus and bacteriolytic activ- ity of the culture filtrate, actinomycetin 130 Waksman — xvi — Actinomycetes Figure 30.— Typical growth of soil species of Stre-ptomyces on synthetic media 132 Figure 31.— Relation between density of vegetative mycelium and plate counts of actinomycetes 136 Figure 32.— Different forms of scale produced by S. scabies .... 156 Figure 33.— The relation of soil reaction to the occurrence of potato scab 162 Figure 34.— Influence of the hydrogen-ion concentration on the growth of the potato scab organism 165 Figure 35.— Vegetative mycelium of a pathogenic anaerobic actinomyces 172 Figure 36.— Actinomycosis of lymphatic gland showing granule with my- celial network and peripheral growing edge 174 Figure 37.— An early study of the structure of an actinomyces colony from a bronchopneumonic nodule 178 Figure 38.— The appearance of a Nocardia in the sputum of an infected patient 181 Figure 39.— First use of the generic name Actinomyces C 1877/78) . . 198 Mycology is the Cinderella of Botany and has suffered the dis- advantages of ste-p-sisterhood. The rest of the family at one time or another has received recognition, and occasionally with little warrant except that of importunity. But Cinderella is now ftdly attired for the Ball. Indeed the carriage is waiting. She has all the character- istics which usually attract in that she has developed in a comely manner and has charms of which her devotees are aware, and— she can bring her quiver fidl of rations for the general good. May those who have served her faithftdly benefit for their devotion. . . . (J. Ramsbottom). LIST of TABLES Table 1.— Types of actinomycetes recognized in 1894 9 Table 2.— Comparison of cultural, morphologic and staining reactions of species of Nocardia 30 Table 3.— Comparison of the parasitic and saprophytic actinomycetes . 35 Table 4.— Streptomycin production and streptomycin sensitivity of differ- ent strains of S. griseus and their variants 37 Table 5.— Cultural and physiological characteristics of the streptomycin- producing strain of S. griseiis and its inactive variant 63 Table 6.— Effect of phage upon the growth, phage multiplication, and streptomycin production by different actinomycetes in stationary cul- tures 66 Table 7.— Stability of phage in aqueous suspension upon storage at several temperatures 67 Table 8.— Production of streptothricin by two strains of S. lavendulae and their variants 77 Table 9.— Decomposition of different amino acids by microorganisms . . 81 Table 10.— Decomposition of glycine by different microorganisms in pres- ence of glucose 81 Table 11.— The utilization of carbon and nitrogen sources by S. coelicolor 83 Table 12.— Metabolic changes and efficiency of carbon utilization of S. lavendulae in aerated cultures 84 Table 13.— Acid production by an actinomyces on meat extract-peptone- glucose medium 86 Table 14.— Acid formation from glucose in aerated cultures of S. laven- dulae 87 Table 15.— Influence of reaction on the decomposition of a protein-rich material by actinomycetes 90 Table 16.— Rate of growth of S. griseus and streptomycin production in shaken cultures 90 Table 17.— Metabolic changes produced by S. griseus with different sources of nitrogen 90 Table 18.— Proteolytic activity of actinomycetes in 2 per cent gelatin solution 101 Table 19.— Distribution of bacteriolytic properties among actinomycetes . 102 Table 20.— Occurrence of antagonistic actinomycetes in different soils . 109 Table 21.— Distribution of antagonistic actinomycetes in nature . . . 112 Table 22.— Classification of antibiotics of actinomycetes . . . . 116/117 Table 23.— Inhibition of different actinomycetes by their respective anti- biotics 119 Table 24.— Distribution of antagonistic properties among actinomycetes 120 Table 25.— Inhibition of growth of virulent human tubercle bacilli by different actinomycetes 121 Table 26.— Growth and chemical changes produced by S. griseus under submerged conditions 123 Ta^le 27.— Nitrogen distribution in cultures of S. griseus 123 Table 28.— Antibiotic spectra of streptomycin and grisein 124 Waksman — xviii — Actinomycetes Table 29.— Antibiotic spectra of streptomycin, streptothricin, and anti- biotic 136 129 Table 30.— Numbers of bacteria and actinomycetes in the soil developing on albumen agar 137 Table 31.— Distribution of actinomycetes in various soils 138 Table 32.— Distribution of microorganisms in different soils from Bikini and Rongelap Islands 140 Table 33.— Influence of 1 per cent dried blood upon the microbiological population of the soil 141 Table 34.— Influence of addition of CaCO^ on the numbers of actinomy- cetes in acid soils 141 Table 35.— A list of typical actinomycetes occurring in soils and in com- posts 143 Table 36.— Influence of temperature upon the development of microorgan- isms in manure composts 145 Table 37.— Numbers of microorganisms in an undrained peat bog in Florida 147 Table 38.— Decomposition of xylan by actinomycetes 150 Table 39.— Decomposition of wheat straw by different microorganisms . 152 Table 40.— Decomposition of sedge and reed peat by microorganisms . 152 Table 41.— Decomposition of stable manure by pure cultures of ther- mophilic microorganisms and by a mixed thermophilic population . 153 Table 42.— Certain characters of scab-producing actinomycetes . . . 158 Table 43.— Effect of competition of soil microorganisms upon occurrence of scab 168 Table 44.— Comparative morphological and physiological properties of two common types of pathogenic actinomycetes 171 Les S. chromogena et alba sont des microbes tres repandus dans la terre, surtout abondants dans les racines vegetales et a leur sur- face. ]e les troiivai dans le terreau de jardin jiisqii'd 1 tn. de fro- fondeur; plus has encore le nomhre ahsolu de ces organismes n'est guere considerable, mats depasse neanmoins celui des autres microbes du sol. Cela demontre leur resistance a I'egard des conditions defa- vorahles pour leur nutrition. (M. W. Beijerinck). f k;. \ .'-Stfcplolhyix of Ffiu)inani> C'ohn. The first figures ever to have been published (1875) of an actinomyces (72). w INTRODUCTORY Actinomycetes^ comprise a group of branching unicellular organisms, hich reproduce either by fission or by means of special conidia. They usually form a mycelium which may be of one kind— vegetative or sub- strate—or of two kinds— vegetative and aerial. The actinomycetes are related, on the one hand, to the true fungi or the Hyfhomycetes, with which they have often been classified, and, on the other hand, to the true bacteria or the Schizomycetes, with which they are usually included for purposes of characterization and identification. In one of the early definitions of the actinomycetes (321) they were described as "unicellu- lar microorganisms, liJ, in diameter, filamentous, branching monopodi- ally, seldom dichotomous, producing colonies of radiating structure. They reproduce by fragmentation or oidia-formation; both kinds of spores grow in ordinary media to form filamentous mycelium, never growing into a rod-shaped vegetative state." Frequently, the actinomycetes have been looked upon as a separate group of organisms occupying a position between the filamentous fungi and the bacteria. It has even been suggested that the actinomycetes be considered not only as forming the link between fungi and bacteria, but as representing the original prototypes from which both of these groups of organisms have been derived. Some of the actinomycetes are known to have their counterparts among the bacteria, and others among the fungi. The fact, however, that actinomyces mvcelium and spores are similar in diameter to those of bacteria suggests the advisability of clas- sifying the actinomycetes among the bacteria. A separate order has, therefore, been created, the Actinomycetales, which is distinct from the Euhacteriales, or the true bacteria. Actinomycetes are of universal occurrence in nature. They are found in large numbers in soils, in fresh waters, in lake and river bot- toms, in dust, on plant residues, on food products, in manures, and in composts. They are known to cause various important plant and animal diseases. Occasionally, they induce certain forms of food spoilage, es- pecially because of the peculiar musty odor that they impart. Notwithstanding an extensive literature dealing with the actinomy- cetes, many aspects of their nature and physiolog)^ and even of their role in various natural processes, are still little understood. This is due to ^ The word "actinomycetes" is used in this treatise to designate the organisms under discussion in a plural sense; the words "actinomyces" and "actinomycete" are used in a singular sense, without reference to any specific form, whether it be a member of the genus Actinomyces, or that of any of the other three genera. Waksman — 2 — Actinomycetes certain factors, not the least among which is the confusion regarding their morphology, life cycles, and systematic position; the frequently as- sumed, although totally unjustified, difficulty of their cultivation and identification; and the meagre knowledge of their biochemical activities. Numerous investigators have contributed much valuable information as to the nature and activities of the actinomycetes. This makes possi- ble the recognition of a definite system for characterizing and for classify- ing these organisms. Information has also been accumulated concern- ing their physiology and their importance in natural processes. One particular property of these organisms, namely, their ability to produce a varietv of antibiotic substances, has been utilized for a comprehensive series of investigations in numerous institutional and industrial labora- tories. This has resulted in the isolation of certain agents, which have found application in combating a variety of bacterial infections in man and in animals. Gradually it thus came to be recognized that the actinomycetes are a large and heterogeneous group of microorganisms, comprising several genera and many species. These organisms vary greatly in their physiology and in their role in natural processes. Together with the bacteria and fungi, they contribute to the cycle of life in nature, which results in the liberation, from the complex plant and animal residues, of a continuous stream of available elements, notably carbon and nitro- gen, essential for fresh plant growth. Chapter 1 TERMINOLOGY, PHYLOGENY, AND TAXONOMY Because of their systematic position and their relationships to the bacteria, on the one hand, and to the fungi, on the other, much confu- sion has arisen concerning the taxonomic position of the actinomvcetes This has been further compHcated by the varied terminology used in different countries, and frequently even in the same country, to desig- nate the genera and the species of this group of organisms. The confusion is due to a number of factors, the most important of which may be summarized briefly as follows: 1. In 1875, Ferdinand Cohn (72) designated a culture of a filamen- tous organism found by R. Foerster in the concretions of the lacrymal duct as Streptothrix Foersteri. Cohn emphasized the similarity of this organism to the false-branching Leptothrix, on the one hand, and to the true-branching fungi on the other. The photograph of the organism as prepared by Cohn (Fig. 1) leaves no doubt that this was a true ac-. tinomyces. Soon afterward, in 1877, an infectious agent in cattle dis- covered by Bollinger (42) was named by Harz (166) Actinomyces hovis, because the masses of filaments were arranged radially, which sug- gested the name "actinomyces" or "ray fungus." Neither of these two generic designations has been universally accepted, largely because the first name (^Streptothrix') had been preempted, and the second QActino- myces) has been meeting with much criticism, because the description of the organism was based on its etiology rather than its morpholog)^ and cultural characteristics, and furthermore no pure culture was obtained. 2. Following these two basic contributions to our knowledge of the actinomycetes, numerous investigators, comprising medical workers, plant pathologists, botanists, and bacteriologists, devoted themselves to the study of this group of organisms. This resulted in various overlap- ping descriptions which frequently proved highly confusing, since dif- ferent workers were interested in different aspects of the morphology, physiology, or etiology of the organisms concerned. 3. A large number of generic names were soon added to the first two, without sufficient x:onsideration being given to the fundamental aspects of the morphology and physiology of the organisms themselves. The increasing number of generic designations were then further com- plicated by a large number of species descriptions. These were based either upon the natural substrate from which the organisms were isolated or upon a single physiological property, such as odor or pigment pro- duction when grown in a complex organic medium. Waksman — 4 — Actinomycetes 4. It has now been established that we are dealing here, not with a few species of a highly specialized and limited group of organisms, but with a large and heterogeneous group comprising many thousands of species which occur in numerous natural substrates and which take part in many natural processes. Because of this, it has been generally felt that a more comprehensive study of these organisms and the separation of the group into several genera were justified. One of the main diffi- culties, however, was the problem of digesting a most extensive litera- ture. Until very recentlv, too little was known of the morphology and physiolog}^ of the actinomycetes to justify recognition of basic differences between the different forms in an attempt to establish specific types. Most of the descriptions of the individual species were based largely upon cultural characteristics, usually growth on media highly complex in composition. Production of pigment in the mycelium of the organ- ism and excretion of the pigment into the medium were considered among the most important distinctive characters. The presence or ab- sence of growth on certain media, the liquefaction of gelatin, the diges- tion of milk proteins, and the production of odor were regarded as other distinguishing features. Insufficient recognition was given to the fact that these characteristics varied greatly under different conditions of cul- tivation, such as composition of the medium, oxygen supply, and tem- perature of incubation of the culture. The fact that an organism may undergo various cultural changes when grown for some time on artificial media was also disregarded. Certain aspects of the life cycle of a cul- ture, such as the phenomenon of lysis and the problems of variation and mutation, so common among these organisms, were not recognized at all. In the face of these shortcomings, the difficulty of establishing type cultures is understandable. In most cases, it was much easier to desig- nate any freshly isolated strain by a new name than to identify it with a previouslv established species. Since the comparisons were usually made not with type cultures but with written descriptions, which were frequently -quite inadequate, the resulting confusion is not surprising. Through the years, many new names accumulated, with the resulting difficulty of recognizing the relations of the designated organisms to older or previously described types. With all these limitations, however, information was gradually ac- cumulating concerning proper methods of growing actinomycetes on synthetic media. The specific morpholog)^ of different forms was be- coming established. This helped in recognizing the true systematic position of the group, and pointed to its separation into several distinct and easily recognizable t)^es, which could be raised to the status of genera. No attempt will be made to review in detail the early literature on the actinomvcetes. Such reviews may be found in the monographs of LiESKE (260), Orskov (328), Duch6 (98), Kriss (242), and Kras- Chapter I — 5 — Taxonomy siLNiKov (234, 236), and in many of the earlier (310) and more recent papers (17-20, 106-108, 1 1 1-1 13, 185-192). It is sufiicient here to sum- marize briefly some of the outstanding facts which led to our present knowledge of the terminology and classification of the actinomvcetes. More detailed information concerning the nature, occurrence, and im- portance of certain special groups, notably those that produce animal and plant diseases, the antagonistic forms, and the thermophilic types, will be found in other sections of this monograph. A word must be said here concernina recognition of individual species. At one time it was believed that only very few species of actino- mycetes are found in nature. This belief was based upon observations of the growth of these organisms on complex organic media or upon the appearance presented by the organisms in the substrate from which they were isolated. The presence of a white aerial mycelium was believed to indicate an alhus type; production of a black or brown pigment led to recognition of the chromogenus type; production of a characteristic musty odor gave rise to the odorifer type; when a culture was isolated from an actinomycotic lesion, it was called the hovis type, while an iso- late from a scabby potato was considered as the scabies type. The intro- duction of differential, especially synthetic, media brought out the great variability of the actinomycetes. This frequently led to a multiplicity of names and descriptions based upon minor cultural differences on various media. Thus, we have names after all the colors of the rainbow, such as "albus," "ruber," "roseus," "flavus," "glaucus," "viridis," "laven- dulae," "violaceus," "cyaneus," "niger," and many synonyms of these. Fortunately, sufficient information has now accumulated on the mor- phology of the actinomycetes to justify the separation of this large and highly heterogeneous group of organisms into several genera; cultural, physiological, and often ecological characteristics may be utilized for their separation into species. Synonyms of Generic Names of Actinomycetes:— It is hardly neces- sary to attempt a complete survey of all the generic and specific names that have ever been given to the group of actinomycetes as a whole or to certain constituent forms in particular. In some cases, these names have also been used to designate certain true fungi or true bacteria; in other cases, the names were simple synonyms. Some of the more common designations of the group and their historical significance are listed here: I. Actinomyces Harz (1877).— The most widely used generic name for the actinomycetes is Actinoviyces. It gave rise to the etiological des- ignation of the disease actinomycosis, as well as to the name of the order as a whole, Actinomycetales; the common designation of this group of organisms, an actinoviyces or an actinovtycete, has also been derived from this name. It is made up of two Greek words, actino, meaning ray, and myces, meaning fungus. The specific name of the organism Waksman — 6 — Actinomycetes was given as Actinoviyces hovis. More detailed descriptions were pre- sented in 1890 by Bostroem (44) and by Wolf and Israel (512), the latter having established that actinomycosis in man is caused by an anaerobic form, growing at 37°C., which is also infectious to animals. It has been suggested that this organism be divided into two forms, one causing human diseases, and the other, animal diseases. The reasons for and against the division will be presented later. 2. Streptothrix Cohn, F. (1875).— Although Streptothrix was the first name proposed for a true actinomyces, it has not recei\'ed wide rec- ognition. This is due largely to the fact that the name had been pre- empted: CoRDA used it in 1839 for a true fungus, which he designated Streptothrix fttsca. Some of the early students of the actinomycetes (32) recognized this and insisted upon the greater justification of the designation Actinoviyces. Another reason why the generic name Strep- tothrix has not been generally accepted is that Cohn himself failed to differentiate sufficientlv between the organism to which he gave this name and the forms designated as Clndothrix, which are now known to be true bacteria. 3. Cladothrix Cohn, F.— The organisms recognized as Cladothrix Cohn represent a group of thread-forming, non-branching bacteria, which produce slimy capsules; they multiply by means of motile conidia ^Cladothrix dichotovm^ and are often found in mouths of animals. The use of this term by many of the early students of actinomvcosis or pseudotuberculosis in man (109, 4) was soon disregarded. 4. Leptothrix Kiitzing, F. T. (1843).— The generic name Leptothrix has often been applied to the actinomvcetes, although it was originally proposed and is now commonly used to designate a group of thread-form- ing, non-branching bacteria. These organisms embrace certain slime- forming iron-bacteria (L. ochraced) and various mouth-inhabiting bacteria (L. huccdis), which later came to be designated as Leptotrichia Trevisan (420). 5. Discomyces Rivolta (1878).— A certain amount of recognition has been accorded the generic name Discomyces. This name had pre- viously been applied to a group of true fungi and has not, therefore, been generally accepted (98). 6. Oospora Wallroth, F. C, (1833).— The name Oospora also was first applied to a group of true fungi. Nevertheless, Sauvageau and Radais (384) attempted, in 1892, to describe the actinomycetes under this genus. Thaxter (415), as well, designated an important group of soil actinomycetes, namely, those that produce potato scab, by this generic name. It was later established that the causative agent of this disease belongs to the true actinomycetes (161). 7. Nocardia Trevisan (1889).— The generic name Nocardia was used to designate an organism belonging to the actinomycetes which was isolated by Nocard from "farcine du boeuf." Wright (519) proposed limitation of this name to a disease condition which is accompanied by Chapter I —7— Taxonomy inflammation and which was, therefore, called nocardiosis, as distinct from actinomycosis. Pinoy (339) included the aerobic forms of actino- mycetes under this generic name, a fact recognized in this treatise. 8. Actinocladothrix Afanassiev (1889).— This name was used to designate the causative agent of actinomycosis in man. Since it had no advantage over the name given by Harz, it has been but litde used. 9. Microuiyces Gruber, M. (1891).— The generic name Micro- niyces was applied to an organism (M. hoffmanni') which apparently belonged to the actinomycetes and which was isolated from the human body. This generic designation was not accepted by other investigators, since it represented no advantage over previous names, nor did it stand for a clearlv recognized tvpe (159). 10. Actinohacterhnn Haas, E. (1906).— To designate organisms that are intermediary between the true actinomycetes and the corynebacteria, the name Actinohacteriwn was suggested. It has not been generally accepted, although existence of these intermediate forms is not denied. 11. ActinohaciJhis Lingieres and Spitz (1904) and Actinohacillus Brumpt (1910) were names applied to nocardia-like organisms, the true nature of which was not sufficiently recognized. The generic name was also used bv Beijerinck, in 1914, for an organism which he had origi- nally described in 1903 as Bacillus oligocarhophihis and for another called Actinomyces QStreftothrix) fmilotro finis. 12. Cohnistreptothrix Pinoy, E. (1911).— In order to diflFerentiate anaerobic actinomycetes from the aerobic forms, Pinoy used this name to designate the former. Castellani and Chalmers (66) as well as Langeron (250) accepted this designation; Orskov (328), however, ap- plied the name to a group of aerobic actinomycetes. 13. Anaeromyces Castellani, A., Douglas, M. and Thompson, T. (1921).— This name was suggested to designate a group of organisms that are intermediary between the genera Mycohacterium and Actino- myces. 14. Aerothrix Wollenweber (1921) was used to designate those actinomycetes which produce aerial mycelium. 15. Pionnothrix Wollenweber (1921) was applied to those forms which do not produce aerial mycelium. This designation and the pre- vious one have received no consideration because of a lack of sufficient characterization of the new genera, or rather subgenera, thus created. The production of aerial mycelium alone is not a sufficiently distinct characteristic to warrant separation of the actinomycetes into generic types, although it is a very important characteristic. 16. Euactinomyces Langeron, M. (1922).— This name was used to designate aerobic actinomycetes, as distinct from the anaerobic Cohni- streptothrix. It has no advantage over those previously suggested. 17. Brevistreptothrix Lignieres (1924).— The generic name Brevi- streptothrix was applied to the actinomycetes of the A. hominis group. 18. Proactinomyces Jensen (1931).— This name was used to desig- Waksman Actinomycetes nate a certain group of actinomycetes, characterized by a special man- ner of sporulation, as will be described later. These forms are some- what related to the genera Corynehacterimn and Mycohacterhwi. The organisms belonging to the genus Proactinomyces were later included by Lehmann and Haag in a separate family Proactinomycetaceae. This group includes such important forms as A. hovjinis Wolf-Israel, Strepto- thrix Israeli Kruse, A. farcinicus, and A. asteroides. The name No- cardia, however, appears to deserve priority in designating this group of actinomycetes. 19. Micromonosfora Orskov (1923).— The generic name Micro- wonos'pora was used to designate those actinomycetes that produce single spores on side«branches. Streftothrix chalcea of Foulerton and A. monosforus of Lehmann and Schutze belong to this group. ^_ >*> Fig. 2.— First photograph of a species of Micromonospora (1899). This organ- ism was a thermophiHc form growing in hot manure compost and called by TsiKLiNSKY Thernioactinomyces vulgaris (429). 20. Thermoactinovtyces Tsiklinsky (1899).— This name was first used to designate thermophilic actinomycetes. Although one of the forms included in this group is definitely a Micromonosfora, as shown in Fig. 2, the fact that forms producing the long-chain type were also in- cluded would preclude the use of TherinoacUnomyces as the generic name. The separation of the thermophilic forms into a separate genus is hardly justified, since organisms which definitely belong to several different genera would be included. The temperature tolerance of cer- tain types of actinomycetes is commonly used only for species separation and not for separation of genera. 21. Mycococcus Bokor (1930).— This name was first used to desig- nate certain nocardia-like organisms (41). It was later applied by Krassilnikov (234) to certain bacteria which appear to be related to the actinomycetes. 22. Asteroides Puntoni and Lconardi (1935).— This name was also Chapter I — 9 — Taxonomy used to designate certain members of the nocardia group of actinomy- cetes. 23. Streptomyces Waksman and Henrici (1943).— In order to sepa- rate those aerobic and nonpathogenic actinomycetes which produce aerial mycelium and which multiply by forming true conidia in chains, from the anaerobic forms, on the one hand, and from the nonconidial types and the single-spore types, on the other, this name was proposed. The generic designation combines the first two names given to the actinomycetes and which ha\'e been most commonly employed in micro- Table 1 : Typjs of actinomycetes recognized in 1894 (132): — Nam2 Observer Synonym Observer A. bovis sulphtireus RiVOLTA A. bovis 0) _ A. foersteri COHN Streptothrix foersteri - A. canis Vachetta A. pleuritic us canis fami/iaris RiVOLTA A. canis Rabe A. bovis farciniats NOCARD Bacillus farcinicus - A. cati RiVOLTA - - A. bovis albus Gasperiot Streptothrix 1,2,3 Almquist S. alba Rossi-Doria A. astenides Eppinger Cladothrix aster, ides _ S. astercides Gasperini S. eppingerii Rossi-Doria A. chromo genus Gasperini S. chr 07720 gen us - S. niger Rossi-Doria Oospora metschnikowi (?) Sauvageau and Radais 0. guignardi (?) Sauvageau and Radais A. bovis luteo-roseus Gasperini - - A. cuniculi SCHMORL S. cuniculi - A. hoffmanni Gruber Micro7)iyces hof7tianni _ A. albido-flavus Rossi-DoRiA S. albido-flava - A. violaceus Rossi-Doria S. violacea _ A. carneus RoSSI-DoRIA S. carnea - A. citreus Gasperini - - A. pluricolor (?) Terni - - A. arborescens Edington - - A. ferrugineus Naunyn - - biological literature, Stre/ptothrix and Actinomyces. This new name obviates the need for using the name Stre-ptothrix, for reasons indicated above, and reserves the designation Actinomyces for the true anaerobic forms to which it was first applied. In addition to the above designations, various other generic names have been used, at one time or another, to designate all the actinomy- cetes or certain constituent groups. These include Actinococcus, Actino- fhyta, Bollingera, Indiella, Indiellopsis, Microsiphonales, Microsforwm, Oidium, and others. Either these names proved to be mere synonyms or they could not be given serious consideration for various other rea- sdns. Waksman — 10 — Actinomycetes As early as 1894, a number of species were already recognized. The names of many of them were considered as synonymous, as shown in Table 1. Systematic Position and Classification of Actinomycetes:— Relation of actinomycetes to bacteria and fungi.— Although they are very often grouped with the fungi, the actinomycetes are related in many respects to the bacteria and are usually classified with the bacteria under the Schizomycetes. The relationship of the actinomycetes to the bacteria is based upon the following properties: 1. The diameters of the filaments and spores of actinomycetes are similar to those of true bacteria and not of fungi. 2. Many actinomycetes reproduce by fragments or oidia that are similar in size and in shape to the rod-shaped and spherical bacteria. 3. Many actinomycetes, especially the pathogens, produce no aerial mycelium; their growth appears similar to that of pleomorphic bacteria, like the members of the genus Corynebacterium. 4. Many actinomycetes are acid-fast, and in their morphology and physiology resemble true bacteria, namely, the members of the genus Mycobacterium. Cer- tain groups among the actinomycetes, especially the genera Actinomyces and Nocardia, show a close resemblance to the mycobacteria. That the actinomycetes show a definite relationship to the fungi, especially the Fungi Imperfecti, is brought out by the following proper- 1. The manner of braliching of the aerial mycelium of many representative groups of actinomycetes, especially the genera Streptomyces and Micramonospora, definitely resembles that of fungi. 2. The production by a large number of actinomycetes of an aerial mycelium and of conidia is definitely typical of many true fungi. 3. The growth of the colonies on the surfaces of liquid and of solid media, as well as their growth in a suspended or submerged condition, is similar to that of fungi and not of true bacteria. Turbidity is not usually produced in the liquid culture. One may, therefore, conclude that the actinomycetes comprise many highly heterogeneous groups of organisms, varying greatly in their mor- phological characteristics, and resembling in some respects true bacteria and in others true fungi. For these reasons, the actinomycetes may ten- tatively be placed in a taxonomical transition group between the Schizo- mycetes and Hyphomycetes, with considerable similarity to, if not actual overlapping of, one or the other. Classification of actinomycetes.— l!^?Lny systems of classifying the actinomycetes have been suggested. These are based upon their activi- ties in a natural environment such as pathogenic and nonpathogenic forms, upon their cultural characteristics such as pigmentation and gela- Chapter I 11 — Taxonomy tin liquefaction, or upon their morphology, especially the manner of sporulation. Buchanan (52) suggested placing the actinomycetes in the order Act'moviycetales with a single family Actinoniycetaceae. The latter was divided into four genera, Actinohacilhts, Leptotrichia, Actinomyces, and Nocardia. Later (53), in the early editions of Bergey's Manual, the Actinoinycetales were di\'idcd into two families, the Actinoniycetaceae with the genera ActiiinhaciUus, Leptotricliia, Actinomyces, and Ery- FiG. 3.— Structure of actinomyces mycelium: (a) S. alhus Gasperini; (hj S. aurantiacus Gasperini Qfrom Krassilnikov, 234). sipelothrix, and the Mycohacteriaceae with eight genera, including My- cobacterium. In later editions of Bergey's Manual, Actinohacilhts was dropped from the first family and Proactinoviyces added; the second family was divided into the genera Corynehacterium and Mycobac- terium. Further changes were made in the final, or sixth edition of the Manual. Lehmann and Neumann (256) divided the order into two families, the Proactinomycetaceae with the genera Corynebacteriiim and Myco- bacterium, and the Actinoniycetaceae with a single genus Actinomyces. K^uYVER and van Niel (224) suggested that the Mycobacteriaceae be removed altogether from the order Actinomycetales. Waksman — 12 — Actinomycetes Several of the systems for classifying the true actinomycetes may be listed as follows: A. Classification of Schahad (1904): I. Non-acid-fast types, liquefying gelatin, producing granules in lesions, with typical swelling of hyphae A. typica. II. Acid-fast types, not producing granules in lesions, without typical swell- ings of lesions A. atypica. 1. Gelatin liquefied A. atypica simplex. a A. alba. b A. flava. 2. Gelatin not liquefied A. atypica psetido-ttiherciilosa. B. Classification of Krainsky (1914): 1. Large colonies (3-5 mm.) produced on solid media; aerial mycelium typically pigmented; oval spores Macroactiriomyces. 2. Small colonies (<3 mm.) produced on solid media; pigmented aerial mycelium; spherical spores Microactinomyces. This system has been applied only to the saprophytic aerobic forms. C. Classification of Chalmers and Christopher son (1916): I. Granules black, noncultivable forms Actinomyces of Babes and Mironescu. II. Granules white, yellow, orange, or red: 1. Cultivated with difficulty, anaerobic types, no arthrospores; granules in masses Cohnistreptothrix. a. Granules yellow C. israeli. b. Granules very small, white C. thihiergi. 2. Cultivated easily, aerobic types, arthrospores produced. . . . Nocardia. a. Clubs present N. hovis. b. No clubs produced: a\ Granules surrounded by a hard shell N. somaliensis. V. Granules without shell: a^. No growth on gelatin N. krattsei. b". Growth on gelatin: a^. Serum coagulated, liquefied: a\ Pathogenic to laboratory animals N. garteni. h\ Nonpathogenic to laboratory animals: a\ Gelatin liquefied N. liquefaciens. V\ Gelatin not liquefied N. convohitus. v. Serum not liquefied: a". Culture yellow orange to red N. astewides. h\ Culture white, then red N. indica. This system was based entirely upon pathogenic forms. D. Classification of Waksman I. (1919): This system, like the previous one, was based largely upon the cultural char- acters of the organisms, embracing, however, mostly soil forms. Whereas the previous system (C) comprised the forms listed here under genera Chapter I — B — Taxonomy Actinomyces and Nocardia, the cultures classitied in this system (D) art' now included under the genus Streptomyces. E. Classification of Wollenxveher (1921): I. Weakly growing strains; aerial mycelium and conidia lacking Subgenus Pionnothrix. This group included A. farcinicus, A. caprae, A. asteroides, A. poly chromogenes, and A. pelletieri. II. More vigorously growing strains, producing aerial mycelium Subgenus Aerothiix. 1. With sclerotial or spirodochial stroma Section Sclerostronni. This group included A. hovis, A. foersteri, A. scabies, and A. aeriiginew^. 2. Sclerotial or spirodochial stroma not significant. a. With brown conidia Section Poliophaerospora. h. With light colored or colorless conidia Section Leucospora. a'. Substrate with variety of colors Subsection Heterochroma. a". With spiral conidial chains Series Helicothrix. b". Without spiral conidial chains Series Ahelicothrix. b'. Substrate with single color Subsection Monochromas. c. With reddish to red conidia Section Erythrinospora. d. With blue conidia Section Glaucospora- F. Classification of Langeron (1923): I. Aerobic forms Euactinoniyces. I. Forms parasitic to man and to animals Section Parasitica. a. Non-acid-fast, thin growth on solid media Majores. b. Acid-fast, tubercle-like growth on solid media Minoies. c. Forms difficult to cultivate, not growing on potato, not liquefying gelatin or serum Breviores. II. Anaerobic forms Cohnistreptothrix. Facultative anaerobic forms, difficult to cultivate; no arthrospores pro- duced. G. Classification of Orskov (1923): I. Typical conidia formation in aerial mycelium Cohnistreptothrix. II. Spore-formation by segmentation Actinomyces. III. Spores produced singly on branches of mycelium. . . . Micromonospora. H. Classification of Lignieres (1924): I. Aerobic, long mycelium, not breaking up into rods Actinomyces. II. Anaerobic, short mycelium, breaking up into long rods Brevistreptothrix. III. No mycelium; cells rod-shaped Actinohacilhis. I. Classification of Jensen (1931): A. No spores formed Proactinomycetaceae. ) I. No mycelium formed: 1. Acid-fast organisms Mycobacterium. Waksman — 14 — Actinomycetes 2. Non-acid-fast organisms Corynehacterium. II. Mycelium formed Proactinomyces. B. Spores formed Actinomycetaceae. I. Spores in aerial mycelium Actinomyces. II. Spores terminally on branches of vegetative mycelium Micronionos'pora. J. Classifcation of Dnche (1934): This classification, like that of C and D, was based largely upon the cultural characteristics of the organisms, and like D, was limited to the conidia-pro- ducing aerobic types, especially of the alhiis group. I. Vigorously growing form.s. 1. Mycelium yellowish, no exopigment. Species included in this group, based on pigmentation of the mycelium, were A. alhus, A. alboviridis, A. roseiis, A. halstedii, A. ■parvus, A. lavendulae. 2. Mycelium yellowish, exopigment not very intense. Descriptions based on soluble pigment, such as A. viridis, A. flavogriseus, etc. 3. Mycelium black, no exopigment, white efflorescence. . . A. reticuli. 4. Mycelium yellow-red, no exopigment, white efflorescence A. alhosforeus. 5. Mycelium yellowish-clear, no exopigment, yellow efflorescence A. citreus. II. Non-vigorously growing forms: 1. Mycelium yellowish, no exopigment, poor yellowish efflorescence.. . A. almquisti. K. Classification of Krassilnikov (1938): I. Actinomycetaceae. 1. Nonseptate mycelium, not breaking into rods Actinom.yces. 2. Unicellular mycelium, later breaking into rods and cocci Proactinomyces. 3. No mycelium, elongated rod-shaped, branching and breaking into coccoid forms Mycobacterium. 4. Cells coccus-like, seldom rod-shaped; resting cells develop in a manner similar to actinomyces spores Mycococcus. II. Micromonosporaceae. Mycelium well developed; conidia produced singly on short conidiophores. Micromonospora. L. Classification of Baldacci (1939): I. Filamentous, often producing two types of mycelium; no conidia formed; cells rod-shaped or coccoid; usually parasitic Mycobacteriaceae. 1. Rod-like organisms, rarely filamentous forms Leptotrichioideae. a. Thin, occasional mycelial hyphae, gram-negative. a\ Cells fusiform Ftisiformis. W. Cells rod-shaped or coccus-like ActinobaciUus. c\ Cells rod-shaped, sometimes filamentous; branched. Pfeifferella. b. Hyphae frequently present, gram-positive. a\ Filaments branched, thickened, showing characteristic granules. Erysipelothrix. Chapter I — 1 5 — Taxonomy V. Filaments unbranched, fragmented into short rods, sometimes with granules and septa Leptotrichia. 2. Filamentous, readily dividing into bacterial segments Proactinomycoideae. a. Filaments with angular growth, dividing into bacteria-like segments. a\ Acid-fast Mycohacterium. h\ Not acid-fast Corynehacterium. b. Long branching hyphae, filaments as in 2a; aerial mycelium may be present but not different from vegetative mycelium: a'. Anaerobic Actinobacterium. V. Microaerobic, sometimes with sclerotial masses. Cohnistre'ptothrix. c\ Aerobic, well-developed mycelium, aerial and vegetative myce- lium undifferentiated Proactinomyces. II. Conidia always produced, with distinct aerial mycelium Actinomycetaceae. 1. Conidia produced singly Micromonos'pora. 2. Conidia seriated and multiple Actinomyces. M. Classification of Waksman II. (1940): I. Mycelium rudimentary or absent Mycohacteriaceae. 1. Nonmotile. a. Acid-fast Mycobacterium. b. Non-acid-fast Corynehacterium. 2. Motile Mycoflana. II. Mycelium produced. 1. Spores formed by segmentation Proactinomycetaceae. a. Anaerobic forms Cohnistreftothrix. b. Aerobes Proactinomyces. III. Vegetative mycelium normally remaining undivided. 1. Conidia formed in chains from aerial hyphae. . . . Actinomycetaceae. Spores produced in chains Actinomyces. 2. Conidia formed terminally and singly on short conidiophores Micro^nonos'poraceae. Spores produced singly Micromonospora. N. Classification of Waksman and Henrici (1943): A. Mycelium rudimentary or absent Mycohacteriaceae Chester. I. Acid-fast organisms Mycohacterium Lehmann and Neumann. B. True mycelium produced. I. Vegetative mycelium fragmenting into bacillary or coccoid elements. Actinomycetaceae Buchanan. a. Anaerobic or microaerophilic, parasitic, not acid-fast Actinomyces Harz. b. Aerobic, partially acid-fast or non-acid-fast. . . . Nocardia Trevisan. II. Vegetative mycelium not fragmenting into bacillary or coccoid ele- ments Streptomycetaceae Waksman and Henrici. a. Multiplication by conidia in chains from aerial hyphae Streptoinyces Waksman and Henrici. b. Multiplication by single terminal spores on short sporophores Micromonospora Orskov. Chapter I _]7_ Taxonomy A detailed discussion of the various species that have so far been described among the actinomycetes, based upon the classification of the organisms into four genera is presented in the latest edition of Bergey's Manual (34). Additional species not included in this Manual are found in Krassilnikov's guide (236). The various principles upon which the recognition of individual species are based are outlined here. A description of the type species within each genus is also presented. Identification of Actinomycetes:— To identify certain individual species of actinomycetes, it is sufficient to give recognition to some of their characteristic properties. These are based upon the occurrence of these organisms in their natural substrates, upon their morphology, upon their cultural characteristics, and upon their biochemical proper- ties. Ecology as a basis of classification.— Although various attempts have been made to classify actinomycetes into several groups on the basis of their natural habitats, no broad system for generalization can ever be developed on this basis alone. It is true that the anaerobic forms, de- scribed here under the genus Actinomyces are largely animal pathogens, and that some of the Nocardia species are also pathogenic. It is also true that the Streptomyces group is characteristic of soils and that the Micro-inonosfora types are found in high-temperature composts, as well as in river and lake waters and bottoms. This alone is hardly sufficient for a separation of the organisms on the basis of their natural occurrence. Such a division would be arbitrary and only approximately true. It has been suggested (250), for example, that the disease-producing actinomycetes be classified on the basis of the specific type of disease; namely, 1. anaerobes, of the Wolf-Israejl type, that attack the abdo- men; 2. aerobes that cause actinomycosis of the lungs, including sapro- phytes occurring in the dust; 3. forms causing swellings in the infectious area, organisms said to be of the so-called Streptothrix type. Actinomycetes are universally present in water basins, in soils, in milk, in or upon other foodstuffs, and in dust. Many attempts have been made to divide these groups upon the basis of their specific habitats. The soil forms, for example, have been separated into plant pathogens and saprophytes; the food-inhabiting types, into odoriferous and non- odoriferous types. These separations, like those based on natural oc- currence, were quite arbitrary. The cosmopolitan nature of many actinomycetes is well established, since species found in one part of the world, are soon discovered also in other parts. Species found in soils may also be found in peats or on foodstuffs. Thus ecology can hardly be considered as a major basis for the classification of actinomy- cetes. Morphology as a hasis for classification.— Ahhough morphological characters are used for the separation of the broader groups of actinomy- cetes, the families and genera, they can also be utilized for the subdivi- Waksman — 18 — Actinomycetes iion of these major groups into smaller units, the species. The nature of the aerial mycelium and the mode of spore formation are the two most distinguishing morphological characteristics. These were em- ployed first by Drechsler, Orskov, and Waksman, and more recently by Jensen, Kriss, and Krassilnikov for the separation of species and even genera. These characters vary, however, and the limits of varia- tion must be established. Krassilnikov came to the conclusion, on the basis of comparative microscopic studies of many cultures freshlv isolated, as well as cultu es <« 1 *%^ ■y Fig. 5 a-d.— Different types of branching of aerial myce- lium of species of Streptomyces: above, long open spirals; Fig. 5b (p. 19), tuft formation of sporulating hyphae; Fig. 5c (p. 20), short compact spirals; Fig. 5d (p. 21), broom shaped structure of sporulating hyphae. grown for 5 years on artificial media, that the form of the sporophores and of the spores is constant for every actinomyces species. Those forms that produce straight, non-spiral-forming sporophores will give rise to straight or slightly bent and wavy, long or short sporophores on all media. Upon reaching maturity, many of the types produce spirals on media favoring the formation of aerial mycelium. Other types vary in this respect, forming spirals on some media and not on others. There may even be variation within the same culture. Synthetic media usually give the most constant morphological characters. Many species that do not form aerial mycelium on organic media will do so on syn- thetic media. Chapter I —19— Taxonomy Drechsler (97) suggested that the nature of the curvature of the spirals can be utilized as a distinguishing character. Krassilnikov, however, observed that most forms turn counter-clockwise (the reverse under the microscope), and only few in the reverse order. The nature of the medium is of great importance in this connection, thus making this character of doubtful taxonomic significance. The fragmentation spores or the true conidia of members of the genus Stre-ptoviyces are spherical, oval, and elongated, whereas the seg- mentation spores or the oidiospores, characteristic of the Nocardia, are i Fig. 5 b (seep. 18). usually cylindrical. The nature of these spores, especially the elongated ones, varies only occasionally. The manner of sporulation is constant. Because of these properties, the morphological characters form the most reliable basis for the separation of these organisms. Cultural characteristics.— The growth and reactions of actinomycetes in culture media have been utilized most extensively for characteriz- ing the individual species. There is, in this respect, however, con- siderable overlapping among the different forms, and one is frequently at a loss to know where to place a freshly isolated culture. Among the most important cultural properties are the following: 1 . Shape and structure of colony, nature of vegetative growth, and appearance of aerial mycelium. 2. Anaerobism vs. aerobism, a very unstable property that cannot be sharply Waksman — 20 — Actinomycetes defined, especially because of the frequent adaptation of anaerobes when freshly isolated to an aerobic form of life upon continued cultivation. 3. Proteolysis vs. non-proteolysis, such as gelatin liquefaction, milk coagulation and proteolysis, serum and egg albumen proteolysis, properties that are quantitative rather than qualitative in nature, with certain few exceptions. 4. Amylolytic v. non-amylolytic action, sucrose inversion vs. non-inversion, lipolysis, etc., properties that also cover phenomena which are largely adaptive in nature but that are valuable as secondary characteristics. 5. Thermophilic vs. mesophilic forms, a phenomenon which is also subject to adaptation and which cannot be very sharply defined because of the many inter- mediary types. 6. Pigment production, one of the most significant properties. Both endopig- ments and exopigments produced on svnthetic and on organic media are given con- FiG. 5 c Qsce p. 18). sideration. Because of this, a number of descriptions have hien based largely upon this property, beginning with the early differentiation between chromogenesis and non-chromogenesis on organic media. On synthetic media, many pigments are produced which resulted in designation of many forms on the basis of the pigment, M'hether present in the vegetative or in the aerial mycelium or whether it is dis- solved in the medium. These pigments vary greatly in nature and intensity with the composition of different media, as well as with conditions of growth and age of the culture. Even with these limitations, however, pigment production is one of the most important and most easily recognizable characteristics, especially when media of known composition and definite conditions of culture are used. 7. Serum diagnosis. This m.ay form a basis for more detailed differentiation of specific types. Aoki (11) established that agglutination reactions can be carried out with actinomycetes as readily as with bacteria; at first he found that the anaerobic forms fall into one group and the aerobic forms into 5 other groups; later, 3 more groups were added establishing in all 9 types. The complement fixation Chapter I — 21 — Taxonomy reaction corresponded well to the agglutination reaction. The agglutinating re- ceptors were present more abundantly in the spores than in the mycelium. V. Magnus (282) has been able to separate various strains of actinomycetes, on a serological basis into acid producers, alkali producers and neutrals; hemoagglutina- tion phenomena were found to occur among 80 per cent of tiie acid producers. 8. Phage specificity. Certain actinomycetes are subject to attack by specific phages; thus, one actinophage attacks only the streptomycin-producing strains of S. grisens, and not others. Biocheuiical characteristics.— This group of properties comprises quantitative rather than quahtative differences. The S. coelicolur group, for example, was found (78) to include forms which differ greatly in type of pigment produced. 1. Fig. 5 d (see p. 18). On the basis of the reduction of nitrate to nitrite, the actinomycetes have been divided (134) into three groups: (a) those that give little or no reduction; (I?) those that give moderate reduction; (c) those that give strong reduction. A similar basis of separation might be suggested for the properties of proteolysis, amvlolvtic action, and sucrose inversion. The ability to utilize specific carbohydrates is another biochemical prop- erty characterizing different types of organisms. On the basis of these various properties, one may feel justified in establishing distinct species within the various genera. Chaffer 11 IDENTIFICATION AND DESCRIPTIONS OF IMPORTANT TYPES Classification of Actinomycetales: — The following classification of the actinomycetes is based entirely upon material included in Bergey's Manual of Determinative Bacteriology (34). For more detailed infor- mation as well as for literature references, the reader is referred to that Manual. Order Actinomycetales Organisms forming elongated, usually filamentous cells, with defi- nite tendency to branching; hyphae not exceeding 1.5[x in diameter, mostly about \\i or less. Usually producing a characteristic' branched mycelium. Multiply by means of special spores, as well as by oidio- spores or by conidia. The special spores are formed by fragmentation of the plasma within the spore-bearing hyphae, the latter being straight or spiral-shaped. The oidiospores are formed by segmentation, or by sim- ple division of hyphae by means of transverse walls, in a manner similar to the formation of oidia among the true fungi. The conidia are pro- duced singly, at the end of special, simple or branching conidiophores. They grow readily on artificial media and form well developed colonies. The surface of the colony may become covered with aerial mycelium. Some of the organisms are colorless or white, whereas others form a variety of pigments. They are either saprophytic or parasitic. In rela- tion to temperature, most are mesophilic, though some are thermophilic. Certain forms are capable of growing at low oxygen tension. Key to the families of order Actinomycetales:— A. Mycelium rudimentary or absent, no spores formed— Family Mycohacteriaceae. I. Acid-fast organisms Mycohacteriwn. B. True mycelium produced: I. Vegetative mycelium divided by segmentation into bacillary or coccoid elements Family Actinomycetaceae. 1. Anaerobic or microaerophilic, usually parasitic, non-acid-fast Actinomyces. Type species— Acfinowyces hovis. 2. Aerobic, partially acid-fast or non-acid-fast Nocardin. Type species— Nocard/a farcinica. II. Vegetative mycelium normally remaining undivided— Family Stre^tomycetaceae. Chapter II — 23 — Important Types (a) Conidia produced in chains, in aerial hyphae Streptomyces. Type species— Sfrepfowjces alhus. (b) Conidia produced terminally and singly on short conidiophorcs Micromonospora. Type species— MicromoMospora chalcea. Genus I. Actinomyces Harz QStreptothrix Colin; Nocardia Toni and Trevisan; Cladothrix Eppinger, Wolf- Israel fungus; Anaeromyces Castellani; Brevistreptothrix Lignieres; Cohnistrepto- thrix Pinoy). Actinomyces hovis Harz. CDiscomyces hovis Rivolta; Bacterium actinocladothrix Afanasiev; Nocardia actinoviyces Trevisan; Actino- myces hominis Wolf and Israel; Streptothrix actinomyces Rossi-Doria; Cladothrix hovis Mace; Oospora hovis Sauvageau and Radais; Actinomy- ces hovis sidphiireus Gasperini; Nocardia hovis Blanchard; Streptothrix israeli Kruse; Cladothrix actino-myces Mace; Actinomyces israeli Lach- ner-Sandoval; Streptothrix actinomycotica Foulerton; Streptothrix hovis commnnis Foulerton; Streptothrix hovis Chester; Discomyces israeli Gedoelst; Actincnnyces sidphureus Sanfelice; Streptothrix sulphurea Caminiti; Sphaerotilus hovis Engler; Actinohacterium israeli Sampietro; Cohnistreptothrix israeli Pinoy; Proactinomyces israeli Negroni; Actino- myces wolf-israel and Corynehacterium israeli Lentze; Proactinomyces hovis Henrici; Actinomyces israeli Rosebury). According to Baldacci (17), most of the cultures listed as A. hovis comprise forms which have also been designated as A. alhus, A. sid- phureus, etc. These include the four species or varieties of A. hovis created in 1894 by Gasperini, namely, A. hovis sidphureus, A. hovis farcinictis, A. hovis alhus, and A. hovis hiteo-roseus. Waksman's de- scription of A. hovis (443) is said to be equivalent to A. hovis sidphu- reus. Baldacci recommends that this species be considered as A. sid- phureus Gasperini. Baldacci further included among A. hovis, Strep- tothrix hominis Foulerton, Streptothrix luteola Foulerton, Actinomyces hovis Harz fide Waksman, Actinomyces hominis Waksman (sub. A. hominis Bostroem), A. hovis Harz fide Lignieres. Very sparse development of erect aerial hyphae in growths produced in an atmosphere of reduced oxygen tension. These hyphae are oc- casionally septate, but no definite spores are formed; aerial mycelium heavier than vegetative mycelium, one micron or even more in diameter. Arthrospores about 2[jl long. Gram-positive. Acid-fast. The substrate mycelium is initially unicellular, and the branches may extend into long filaments, causing the colony to adhere to the medium, or may give rise more or less quickly to irregular segments and characteristic angular branching. The colonies exhibit a considerable degree of polymor- phism, but no stable variants have been established. Liquid media are usually clear. Compared with the aerobic actinomycetes, the anaerobic organisms Waksman — 24 — Actinomycetes show little biochemical activity. They do not produce soluble pig- ments on protein media or insoluble pigments in their growth; they have no proteolytic action on egg- or serum-containing media; they do not usu- ally clot and do not peptonize milk, and in fact, rarely grow on it at all; they seldom grow on gelatin, and when there is a little flaky growth the tubes when cooled (from the 37°C. necessary for incubation) are found not to have been liquefied; and they have little or no haemolytic action on blood broth or blood agar. Acid is formed from certain sugars: ac- cording to Slack (403) from glucose, maltose, mannitol, sucrose, and lactose; according to Negroni and Bonfiglioli (318) from glucose, galactose, lactose, fructose, maltose, affinose, sucrose, and xylose. Milk also becomes acid. Compared with the human strains, the strains of bovine origin dis- play, according to Erickson (113), cultural and morphological differ- ences. Their colonies are smoother and softer in consistency and are not adherent to the medium. Growth is scantier. The mycelium under- goes fragmentation very rapidly, and extensive ramification is rare. No aerial h}'phae have been found. A much greater degree of uniformity is evident in colony development. Occasional turbidity occurs in liquid media. These strains also show a lesser ability to ferment sugars. Source: Jaw of cattle, udder of swine, and man (dental scum, tonsilar crypts)< Further information on the morpholog)' and phvsiology of this organ- ism is given later (p. 43). In the latest edition of Bergey's Manual, a second species is recog- nized, namely A. israeli, which occurs in human tissues and is said to be responsible for human actinomycotic infections. Genus II. Nocardia Trevisan ^Actinomyces Gasperini, Schottmiiller, Henrici and Gardner; Cohnistrepto- thrix Orskov; Streptotlirix Kruse, Caminiti, Rossi-Doria, Silberschmidt; Cladothrix Eppinger; Brevistreptothrix Lignieres; Actinohacteriuni Haas; Actinocladothrix Afanassiev; Actinohacille Lignieres and Spitz; Actinococcus Beijerinck; Mycococcus Bokor; Asteroides Puntoni and Leonardi; Proactinomyces Jensen.) Slender filaments or rods, frequently swollen and occasionally branched, forming mycelium which after reaching a certain size may give the appearance of bacterial growths. Shorter rods and coccoid forms are found in older cultures. Conidia not formed. The nocardias stain readily, occasionally showing a slight degree of acid-fastness. Aerobic. Gram-positive. The colonies are similar in gross appearance to those of the genus Mycohacterhnn. Paraffin, phenol, and m-cresol are frequently utilized as sources of energy. In their early stages of growth on culture media (liquid or solid), the structure of a nocardia is similar to that of a streptomyces. Both form a typical mycelium: h)q3hae branch abundantly, the branching being true. The hyphae vary in diameter between 2.5[x and 1[k, most of Fig. 6.—Nocardia asteroides, grown on pi^itato glucose-beef extract agar, gram stain, X 100. CPre'pared hy Littman of Armed Forces Institute of Pathology). Waksman — 26 — Actinomycetes them measuring 0.7-0.8[x, according to the species. The mycehum is not septate. The further development of nocardias, however, differs from that of streptomyces cultures: the filaments soon form a transverse w^all and the whole mycelium breaks up into regularly cylindrical short cells, then into coccoid cells. On fresh culture medium the coccoid cells germinate into the mycelium. The whole cycle in the develop- ment of nocardias continues 2 to 7 days. Most frequently the coccoid cells are formed on the third to the fifth day, but those of certain species (Nocardia ruber, for example) can be found as early as the second day. Numerous chlamydospores are sometimes found in older cultures of Nocardia. They are formed in the same way as the chlamydospores in true fungi; the plasma inside the filaments of the mycelium condenses into elongated portions. In older cultures of Nocardia many coccoid cells are changed into "durable" forms. The latter are larger than the vegetative coccoid cells, and the plasma of these cells is thicker than the plasma of vegetative cells. On fresh media the so-called "durable" cells germinate like the spores of Streptomyces. They form 2 to 3 germ tubes. Besides the cells mentioned, numerous involution forms can often be found in older cultures of Nocardia. These cells are thin, regularly cylindrical or coccoid, and are often transformed into a series of spherical or elliptical ampules and a club-like form (2 to more than 3[i). The multiplication of nocardias proceeds by fission, budding, and rarely by special spores. Budding occurs often. The buds are formed on the lateral surfaces of the cells; when they have reached a certain size, they fall off and develop into rod-shaped cells or filaments. The spores are formed by the breaking up of the cell plasm into separate por- tions, usually 3 to 5 in number. Every portion becomes rounded, covered with a membrane, and transforms into a spore; the membrane of the mother cell dissolves and disappears. The spores germinate in the same way as those of Streftomyces; they form germ tubes which develop into a mycelium. The colonies of nocardias have a paste-like or mealy consistency and can be easily taken up with a platinum loop. They spread on the glass and occasionally render the broth turbid. The surface colonies are smooth, folding, or wrinkly. Typical nocardias never form an aerial mycelium, but there are cultures whose colonies are covered with a thin coating of short aerial hyphae, which break up into cylindrical oidio- spores. Many nocardias form pigments. Their colonies are of a blue, violet, red, yellow, and green color. More often the cultures are colorless. The color of the culture serves as a stable character. Krassilnikov (234) divided the genus Nocardia into two groups: ]. Well developed aerial mycelium— substrate mycelium seldom produces cross walls; the threads break up into long thread-like rods; branches of aerial mycelium produce segmentation spores and oidiospores, the latter being cylindrical with sharp '^i ^ -'•♦ Fig. 7.—Nocardia asteroides, grown on potato glucose-beef extract agar, bottom of colony, gram stain, X 975. (,Pre;pared hy Littman of Armed Forces Institute of Pathology). Waksman — 28 — Actinomycetes ends; no spirals of fruiting branches. This group is the same as Group B of Jensen. 2. Typical forms— mycelium develops only at early stages of growth, then breaks up into rod-shaped and coccoid bodies; smooth and rough colonies, dough-like con- sistency; usually do not form aerial mycelium; similar to bacterial colonies; aerial mycelium may form around colonies. The genus Nocardia can also be divided into two groups on the basis of acid-fastness: 1. Partly acid-fast organisms, which are nonproteolytic, nondiastatic, and utilize paraffin; usually yellow, pink, or orange-red in color. 2. Non-acid-fast organisms, which are diastatic, largely proteolytic, and do not utilize paraffin; yellow, orange to black in color. Type Species: Nocardia farcinica Trevisan. QStreftothrix farcinica Rossi-Doria; Oosfora farcinica Sauvageau and Radais; Actinomyces far- cinicus Gasperini; Actinomyces hovis farcinicus Gasperini; Bacillus far- cinicus Gasperini; Cladothrix farcinica Mace; Streptothrix farcini hovis Kitt; Stre-ptothrix nocardii Foulerton; Discoviyces farcinicus Geodoelst; Actinomyces nocardii Buchanan, and many others). Filaments 0.25/j. in thickness, branched. Markedly acid-fast. Gelatin colonies: Small, circular, transparent, glistening. Gelatin stab: No liquefaction. Agar colonies: Yellowish-white, irregular, refractive, filamentous. Agar slant: Grayish to yellowish-white, surface roughened. Broth: Clear, with granular sediment, often with gray pellicle. Litmus milk: Unchanged. Potato: Abundant, dull crumpled, whitish-yellow. Nitrites not produced from nitrates. No soluble pigment formed. Proteolytic action absent. Starch not hydrolyzed. Aerobic, facultative. Optimum temperature 37 °C. CoNANT and RosEBURY (75) recently presented (Table 2) a sum- mary of some of the salient features of different species of Nocardia. Habitat: Associated with disease in cattle, resembling chronic tuberculosis. Transmissible to guinea pigs, cattle, and sheep but not to rabbits, dogs, horses, or monkeys. The last edition of Bergey's Manual contains descriptions of 33 species, with a large nurhber of additional species only incompletely described. Genus III. Streptomyces Waksman and Henrici CStreptothrix Cohn; not Stre-ptothrix Corda; Actinomyces Harz; Discomyces Rivolta; Actinocladothrix Afanassiev; Nocardia Trevisan; Micromyces Gruber; not Micromyces Dangeard; Actinohacterium Haas; Carteria and Carterii Musgrave, Clegg and Polk; Euactinomyces Langeron). ft- 'J { Z *. V'-K - a • 3 \ C V a. : C If \ 1 \ I 6 10 2.0 30 SO u Fig. 8.-Branching and sporulation of different strains of Micromonosporn Qrom Jensen, 186). Waksman 30 Actinoniycetes Table 2: Comparison of cultural, morphologic and .staining reactions of species of Nocardia (75).- Czapek's Fragmen- Color of AciD- Sabouraud's AGAR tation OF Species GRANULE FAST GLUCOSE AGAR (pigment) MYCELIUM Nocardia asteroides Yellowish- + Glabrous, rarely Yellow to + (Eppinger) white, chalky. Moist, orange Blanchard, 1896 with or without clubs soft, folded or wrinkled and granular. Yel- low, orange- ochraceous, red Nocardia Yellowish- + Frequently Yellow to + brasiliensis white, chalky. Folded, orange (Lindenberg) with or cerebriform, ochraceous Cast, and without tenacious and Chalmers, 1913 , clubs dry. Earthy odor. Yellow, orange-ochra- ceous Nocardia madurae Yellowish- - Glabrous. Moist, Cream colored — (Vincent) white, soft, wrinkled. at first; Blanchard, 1896 with or without clubs Cream colored later be- coming pinkish to red Nocardia pellet ieri (Laveran) Pinoy, 1912 Red, with or with- out clubs Small. Glabrous, heaped, wrin- kled. Mucilagi- nous. Coral pink to red Coral red Nocardia paraguayensis (Almeida) Conant, 1947 Black, with clubs Glabrous. Soft, white center. Projecting bor- der adherent, darker Dark cream Organisms growing in the form of a much-branched myceHum with a typical aerial mycelium and spore formation. Aerobic. Sometimes parasitic, with clubbed ends of radiating threads conspicuous in lesions in the animal body. This genus can be divided, on the basis of the structure of sporulat- ing hyphae into five groups: Group 1 : Straight sporulating hyphae, monopodial branching, never produc- ing regular spirals. Group 2: Spore-bearing hyphae arranged in clusters, or broom-shaped arising from compression of the sporophores. Group 3: Spiral formation in aerial mycelium; long, open spirals. Group 4: Spiral formation in aerial mycelium; short, compact spirals. Group 5 : Spore-bearing h)^hae arranged on mycelium in whorls or tufts. Chapter II — 31 — Important Types In group 5, the spore-bearing branches arise from definite knots, in the form of tufts or whorls, on one plane along the mycelium. These tufts consist of 3 to 10 sporophores and are formed more or less equidistant along the mycelium. This type of sporulation is ordinarily produced only on certain media, usualh' synthetic agar, but not on organic media. Tv'pe Species: Streptomyces dims (Rossi-Doria em. Krainsky) Waksman and Henrici. QStreptothrix alha Rossi-Doria; Cladothrix alha Mace; Nocardia alha Chalmers and Christopherson; Cladothrix dichotoma Mace; Streptothrix foersteri Gasperini; Streptothrix 2 and 3 Almquist; Actinomyces saprophyticns Gasperini; Oospora doriae Sauva- geau et Radais; Cladothrix liquefaciens Hesse; Cladothrix invidnerahilis Acosta e Grande Rossi; Actinoviyces chromogemis Gasperini; Strepto- thrix nigra Rossi-Doria; Streptothrix gedanensis I Scheele et Petruschky; Streptothrix graminearxim Berestneff; Actinomyces thermophilus (Berest- neff) Miehe; Cladothrix odorifera Rullmann; Actinomyces chromogenes Gasp. P alha Lehmann and Neumann; Oospora sp. Bodin; Oospora alpha Price-Jones; Streptothrix leucea Foulerton; Streptothrix Candida Petruschky; Streptothrix lathridii Petruschky; Streptothrix dassonvillei Brocq-Rousseau; Streptothrix pyogenes Caminiti; Actinomyces alhus Krainsky; Actinomyces sanninii Ciferri; Actinomyces almqnisti Duche; Actinomyces gotigeroti Duche, and numerous others). This is one of the most widely distributed and most widely described tN'pes in nature. It produces no soluble pigment and abundant white aerial mycelium. Various strains isolated by different investigators have been variouslv described. The most complete recent study was made by Duche (98) and by Baldacci (20). Vegetative hyphae: Branched, l/x in diameter. Aerial mycelium: Abundant white, 1.3 X 1.7^1, with abundant spore formation. Pigment, soluble: None. Aerobic. Odor: Characteristic. Gelatin: Liquefied, no soluble pigment. Bouillon: Flaky growth on bottom with surface pellicle. Milk: Peptonized after having become coagulated. Reaction becomes alkaline. Carrots and other vegetables: Excellent growth. Habitat: Dust, soil, grains, and straw. The last edition of Bergey's Manual contains descriptions of 73 spe- cies of Streptoviyces, and an additional large number of incompletely described species. Genus IV. Micromonospora Orskov (Thermoactinomyces Tsiklinsky). Well developed, fine, nonseptated myceHum, 0.3-0.6[jl in diameter. Grow well into the substrate, not forming a true aerial mycelium at any time. Multiply by means of conidia, produced singly at end of special Waksman — 32 — Actinomycetes conidiophores, on surface of substrate mycelium. Conidiophores short and simple, branched, or produced in clusters. Strongly proteolytic and diastatic. Comprise mostly saprophytic forms. These organisms occur commonly in hot composted manure, in aerial dust, and in soil, in river and lake waters, and in river and lake bottoms. Many are thermophilic and can grow at 65 °C. Key to the species of the genus Micrmnonosfora:— I. Vigorously growing organisms, typically copious spore formation on dextrose- asparagine agar. A. Vegetative mycelium pale pink to deep orange, no typical soluble pigment 1. Micromonospora chalcea. B. Vegetative mycelium orange changing to brownish-black, brown soluble pigment 2. Micromonos'pora ftisca. II. Slowly and feebly growing organisms, with scant spore formation on dextrose-asparagine agar, no soluble pigment. A. Vegetative mycelium pale pink to pale orange 3. Micromonosfora parva. B. Vegetative mycelium yellow to orange-red 4. Micromonospora glohosa. C. Vegetative mycelium blue 5. Micromonospora vulgaris. Type Species: Micromonospora chalcea (Foulerton) Orskov. CStreptothrix chalcea Foulerton; Nocardia chalcea Chalmers and Chris- topherson; Actinomyces chalcea Ford). Formation of a unicellular mycelium which produces distally placed, singly situated spores. No aerial hyphae. No surface growth in liquid medium. The organism absolutely resists desiccation for at least 8 months. Comparison between the power of resistance of the mycelium and the spores, respectively, will no doubt present great difhcultv, be- cause it is almost impossible to ensure that the two constituents are ac- tually detached. Otherwise, the mycelium is but slightly capable of germinating, which may be ascertained by inoculating a water-agar plate liberally with a mixture of mycelial threads and spores. Though virtu- ally all the spores germinate, the mycelial threads have never been found to form new colonies. According to Jensen, vegetative mycelium on dextrose-asparagine- agar is heavy, compact, raised, not spreading much into the medium. Spore layer well developed, moist and glistening, brownish-black to greenish-black, this color sometimes spreading through the whole mass of growth. Liquid media: Growth in form of small, firm orange granules or flakes. Starch: Starch is hydrolyzed. Gelatin: Liquefied. Milk: Digestion of milk with a faintly acid reaction, mostly after a previous coagulation. Chapter II — 33 — Important Types Many strains invert saccharose. Some strains reduce nitrate to nitrite. Most strains decompose cellulose. Proteolytic action seems stronger in this than in the other species of this genus. Optimum temperature for growth, 30-35°C. Thermal death point of mycelium, 70°C. in 2 to 5 minutes. Spores resist 80°C. for 1 to 5 minutes. Habitat: Soil, lake mud, and other substrates. This genus could be subdivided on the basis of the relations of the organisms to temperature, since it includes a number of thermophilic forms which grow readily at 55°-65°C., mesophilic forms having their optimum temperature at 30°C., and organisms growing at low tempera- ture in lakes. Each of these can be divided into diree groups, based on the structure of the spore-bearing hyphae. Among the thermophilic forms, only representatives of the first group have so far been isolated in pure culture, although the existence of the other two groups has defi- nitely been demonstrated in microscopic preparations. These are: Group 1. Simple spore-bearing hyphae. Group 2. Branching spore-bearing hyphae. Group 3. Spore-bearing hyphae in clusters. Description of Several Important Actinomycetes:— In view of the great economic importance of some of the actinomycetes, several species with unusual physiological properties or of great practical value are described in detail here. Actinomyces hovis Harz. A. hovis is an anaerobic pathogen. It's most recent description, under the name of A. israeli, is given by Rosebury (367). This work served as a basis for the following summary. A. hovis is a gram-positive, branching, filamentous organism, non- acid-fast, and not producing spores. The hyphae are usually less than lit. in diameter. In tissue sections made from the lesions of actinomy- cosis, the organism appears in the form of compact granules or colonies which are often visible to the naked eye. The granules are circular or irregular in outline, or may comprise several colonies of different size and shape which have grown together. Each granule consists of a dense mass which stains irregularly in hematoxylin-eosin preparations biit takes the violet dye in sections stained by Gram's method. The ends of individual filaments may be seen around the periphery of the granule, or part of the periphery may be composed of the radially ar- ranged hyaline clubs. These can be stained with eosin. They are several times wider than the filaments, which can sometimes be traced within the structure of the club. In exudates from actinomycosis, certain sulfur granules make their appearance. These are irregularly spherical masses, varying in diame- Waksman — 34 — Actinomycetes ter. They are soft and easily broken under light pressure, but they may occasionally be tough or even calcified. The crushed granule ap- pears as a disorganized mass of irregular, bent and branching filaments, some of which may terminate in the characteristic clubs. In prepara- tions fixed and stained by Gram's method the structure of the granule is lost, the clubs not showing and the picture being that of a mass of irregular bent gram-positive rods. The morpholog)' of the organism has been described as varving from a compact mass of branching mycelium of gram-positive filaments to a mass of short rods which mav be evenly stained or granular, and which show no indication of branching. These differences were found to be associated with roughness or smoothness of the colony. Rough colo- nies, whether grown on an agar surface or in an agar shake culture or in broth, show regular branching; twig-like forms are, however, much more common than long filaments. Intermediate and smooth colonies give a picture resembling that of the diphtheria organism, with granular and polar-stained forms, and with suggestive evidence of branching. Some of the smooth colonies may be derived from rough and clearly branched forms by subculturing; they show evenly stained rods with no distinguishing characteristics. The rough and intermediate forms often show terminal swellings or "clubbed forms" similar to those of the diph- theria organism; but the true clubs do not appear in cultures. White or grayish colonies up to about 1.5 mm. in diameter, are pro- duced in glucose-agar shake cultures at 37°C. within 3 to 6 davs. The rough strains grow in a zone about 5 mm. wide, the upper limit being 0.5 to 2 cm. below the surface of the agar. A few colonies may be pres- ent below or above this zone, but no growth takes place on the surface. Smooth strains show no zone of concentrated growth; the colonies are uniformly distributed from the bottom of the tube to a level 0.5 to 1 cm. from the surface, where growth terminates abruptly. When a colony of a rough strain is transferred with a capillary pipette to a slide, it is usually found to be tough and difficult to break up and emulsify; it shows the characteristic compact branched mycelium. Rough strains grow in glucose broth at 37 °C. as white or grayish masses up to about 5 mm. in diameter at the bottom of the tube, the medium itself remaining perfectly clear. They are often difficult to break up. Intermediate strains tend to grow as smaller particles or gran- ules either at the bottom or along the side of the tube, or as viscid or flocculent masses, with little or no general turbidity. Smooth strains, however, may produce uniform turbidity with or without a viscid or granular sediment. On glucose agar or on brain-heart agar, incubated anaerobically with 5 per cent COo for 4 to 6 days, rough or intermediate strains of A. hovis produce white-gravish to yellowish colonies having a diameter of not more than 1 to 3 mm. These colonies usually adhere to the medium, so that they are hard to remove with an iiwculating needle, often com- Chapter II 35 Important Types ing away all in one piece. The smooth colonies resemble those of white staphylococci or diphtheroids. They are soft and easily broken and emulsified. It was recognized, however, that on anaerobic-COj plates, the colonies are very different from those found on aerobic media; an occasional rough white colony may, on examination, turn out to be a streptococcus. A summary of the morphological and cultural properties of the pathogenic forms as compared to those of the saprophytes is given in Table 3. Table 3: Comparison oj the parasitic and saprophytic actino7nycetes (367): Parasitic actinomycetes Saprophytic actinomycetes Natural habitat- Cellular morphology: Mouth and throat of man and probably of cattle and other animals; obligate parasites; sometimes pathogenic. Branched mycelium, gram-posi- tive, not acid-fast. Marked tendency to fragment into bac- illary forms. Soil, grains and grasses; widely distributed in nature; some pathogenic species, but most forms are non-pathogenic. Branched mycelium, gram-posi- tive; some are acid-fast. Gen- erally little tendency to frag- ment into bacillary forms. Character of Bacteria-like colonies without growth: aerial hyphae; no spores; no pigments. Temperature Optimum, 37°C.; no growth at requirements: 22°C. Relation to Oxygen tolerance limited; gener- oxygen: ally fail to grow or grow poorly under aerobic conditions. Metabolism: Probably never proteolytic. Fer- ment carbohydrates with pro- duction of acid. Species One only: Actinomyces bovis. (Pro- recogni^ed: visional; heterogeneous but not yet satisfactorily subdivided.) Pathogenicity: Causative agent of true actinomy- cosis in man and animals. Colonies more mold-like, often with aerial hyphae and spores (conidia); many produce yel- low, orange or black pigments. Optimum usually 15-20°C. Aerobic; some forms do not grow anaerobically. Many forms actively proteolytic; may utilize carbohydrates with- out acid production. Many, subdivided into several families. Occasional causes of an actinomy- cosis-like disease, very rare in man, and of tropical cutaneous mycetomas, e.g., Madura dis- Streptomyces griseus (Krainsky) Waksman and Henrici S. griseus, as a typical Streptomyces, produces both a vegetative and an aerial mycelium. The former varies in thickness from 0.3 to 2\l (0.5-1.3[x). It is (64) well developed, coenocytic when young, and branched in a typical monopodial form; occasionally two or more branches grow from the same place on the main hyphae; no true septa have been observed in the young vegetative mycelium, but are found in the older mycelium, and especially in the sporulating hyphae. The aerial mycelium is at first whitish, but later changes upon sporulation to yellowish green, with varying shades of cream, gray, buff, and browoiish, depending on strain of organism and culture medium. The sporogen- ous hyphae may be borne directly upon the vegetative mycelium, sev- eral filaments arising from the same vegetative hyphae. Good sporulat- ing strains produce straight, well branched sporogenous hyphae. The spores are produced exogenously in chains on the aerial mycelium, over 200 spores having been counted (64) in a single chain of 3-day old cultures. The aerial sporogenous h)rphae are often clavate and are continuous; transverse septae are laid down simultaneously, di- viding the h^^pha into mononucleate or multinucleate segments. The cells between the septae increase in size, constrictions appearing at the septae, the spores being held in chains and in connection with each other by narrow fragile bridges. The spores vary in shape from spheri- cal to cylindrical and in size from 0.7 to 0.9 X 0.7-1.9[jl, the variations being observed in the same chain, as shown in Fig. 13. The spores germinate at one or both ends, usually at the previous points of attachment to other spores. The germ tubes elongate by apical growth, the spore contents passing into it. The resulting myce- lium branches and later leads to the formation of reproductive mycelium. A nucleus has been demonstrated in the spores, germ tubes, and young mycelium (64). The nuclei move with the cytoplasm. The spores are mononucleate or multinucleate. The cultural characteristics of this organism have been (34) briefly described as follows: Gelatin stab: Greenish-yellow or cream-colored surface growth with brownish tinge. Rapid liquefaction. Synthetic agar: Thin, colorless, spreading, becoming olive-buff. Aerial myce- lium thick, powdery, water-green. Starch agar: Thin, spreading, transparent. Dextrose agar: Elevated in center, radiate, cream-colored to orange, erose margin. Plain agar: Abundant, cream-colored, almost transparent. Dextrose broth : Abundant, yellowish pellicle with greenish tinge, much folded. Chapter II — 37 — Important Types Litmus milk: Cream-colored ring; coagulated with rapid peptonization, be- coming alkaline. Potato: Yellowish, wrinkled. Nitrites produced from nitrates. Proteolytic action in milk and gelatin. The pigment formed is not soluble. Starch is hydrolyzed. Aerobic. Optimum temperature 37°C. The optimum temperature for certain physio- logical reactions is much lower; for example, 25° to 28° for streptomycin produc- tion. Habitat: Peat, soils, river flats, and dust. Numerous strains of this organism have been isolated from habitats which range from the throat of a chicken to that of rich garden soils and cultivated peats. S. griseus represents a most variable group of organisms. This is brought out quite emphatically in an examination of the ability of dif- ferent strains within this species for their ability to produce antibiotic substances. These have been classified into 4 groups. 1. Those strains which produce streptomycin, the amount of antibiotic pro- duced varying greatly with the individual strains under different conditions of culture; they are sensitive to actinophage. 2. Those strains which produce only or predominantly grisein or grisein-like substances; they are resistant to actinophage. 3. Those strains which produce other antibiotics, which are active against gram-positive bacteria only, and the exact nature of which is still unknown. 4. Those strains which produce no antibiotic at all. Another very important characteristic of S. griseus strains is their ability to produce mutants. So far, 2 mutants have been isolated from the streptomycin-producing cultures: (a). A colorless mutant, producing no aerial mvcelium, not producing any streptomycin and sensitive to this antibiotic as brought out in Table 4. (Z?). A pigmented mutant, pro- ducing pink to vinaceous colored vegetative growth, but forming the Table 4: Streptomycin production and streptotnycin sensitivity of different strains of S. griseus and their variants (395) : — Production of Streptomycin Strain or variant Origin streptomycin* sensitivity** Strain No. 4 Sporulating active form 38 >3,125 Strain No. 19 Sporulating active form 128 >3,125 Variant 3 Non-sporulating form 20 Variant 4 Non-sporulating form 16 Variant 6 Non-sporulating form 4 27 Reverted strain Sporulating active form 37 >3,125 • Units of streptomycin in 12-day cultures. •* Units of streptomycin required to inhibit growth of particular strain or variant in 1 ml of medii Waksman — 38 — Actinomycetes typical aerial mycelium; this mutant forms no streptomycin but another antibiotic which is not active against gram-negative bacteria. The life cycle of S. griseiis in relation to the production of strepto- mycin has been described (481) as follows: The growth of S. griseus reaches a maximum in stationary cultures in 10 days and in submerged cultures in 3 to 5 days, followed by the lysis of the mycelium. Growth is accompanied by a gradual rise in the pH value of the culture, and in the ammonia and amino nitrogen contents. The total nitrogen in the mvcelium tends to be higher during the active stages of growth. The production and accumulation of strep- tomvcin parallels the growth of the organism. After maximum activity has been reached, there is a drop in activity, which is rapid in sub- merged cultures. For the production of streptomycin, the presence in the medium of an organic substance is required. This substance may either serve as the precursor of the streptomvcin molecule as a whole or of an important group in the molecule, or it may function as a prosthetic group in the mechanism essential for the svnthesis of the streptomycin. Such a factor can graduallv be synthesized by the organism, when it is provided in the medium in a preformed state, however, as in meat ex- tract or in corn steep liquor, the process of streptomycin synthesis is greatly facilitated. Streptomycin is also formed in purely synthetic media. In addition to the streptomycin complex, S. griseus produces at least 2 other antibiotics one of which, actidione, is active only against fungi, and another, streptocin, which is present in a limited amount in the culture filtrate but more abundantly in the mycelium. Streptocin is soluble in organic solvents and is not active against gram-negative bac- teria. Streptomyces lavendulae (Waksman and Curtis) Waksman and Henrici S. lavendulae also represents a large heterogeneous group of organ- isms which differ greatly in some of their biochemical properties, notably the production of antibiotic substances. The first culture of S. lavendulae was isolated from a New Jersey soil in 1915 (460). Its early description was given (34) as follows: Mycelium and hyphae coarse, branching. Spirals close, 5 to 8/Lt in diameter. Conidia oval, 1.0 to 1.2 by 1.6 to 2.0/li. Gelatin stab: Creamy to brownish surface growth. Liquefied. Synthetic agar: Thin, spreading, colorless. Aerial mycelium cottony, white, becoming vinous-lavender. Starch agar: Restricted, glistening, transparent. Plain agar: Gray, wrinkled. Dextrose broth: Abundant, flaky sediment. Fig. 9.— Sporulation of straight aerial hyphae of species of Streptomyces Qroni Krassilnikov, 234). Waksman — 40 — Actinomycetes Litmus milk: Cream-colored ring. No coagulation; peptonized, with strong alkaline reaction. Potato: Thin, wrinkled, cream-colored to yellowish. Nitrites produced from nitrates. Soluble brown pigment formed. Peptonization of milk and gelatin. Starch is hydrolyzed. Aerobic. Optimum temperature 37 °C. Habitat: Widely distributed in soils and other natural substrates. The first antibiotic produced by S. lavendiilae was designated as streptothricin (452). Since then, several other antibiotics have been isolated from members of this group. Some of these antibiotics, notablv, lavendulin, streptolin and streptothricin VI, are similar to streptothricin in their general antimicrobial spectra, but they differ in their quantita- tive effects upon different bacteria, and in their greater or lower toxicity to animals. Some of the antibiotics produced by organisms belonging or closely related to the S. lavendiilae group are distinctly different from streptothricin both chemically and in their antibiotic spectra, as is the case for Chloromycetin. The streptothricin-producing S. lavendidae strains give rise to a number of variants (478). Some of these variants produce on glucose- peptone a blue diffusible pigment; others form a brown pigment. The vegetative mycelium of the blue pigment-forming variants is pale-blue with scattered, small pin-point areas of deep blue. Upon complete sporulation, the vegetative growth is covered with thick lavender-colored aerial mycelium; occasional sunken areas are of a slightly bluish tinge, these areas corresponding to the pin-point regions of the deeper blue. The under surface of the vegetative growth is cream-colored except for the small blue spots. The other variants produce a colorless to cream- colored vegetative growth free of any blue pigment whatsoever; one to two days later, a brown diffusible pigment appears, the growth becom- ing covered with abundant lavender-colored mycelium. On subse- quent transfer on fungus-agar slants, the tw^o types of variants prove to be rather stable. Some of the strains isolated from an actix'C streptothricin-producing culture may lose the property of producing this antibiotic. Other strains may form antibiotics which vary from the typical streptothri- cin either in their antibacterial spectra or in their toxicity to animals. In a comparative study of the relation between growth of the organ- ism and production of antibiotic, it was found (452) that both in sta- tionary and in shaken cultures growth and activity reach a maximum and then decline, the maximum for the first preceding somewhat that of the second. Since the nitrogen in the dry mycelium varies between 7 and 9 per cent, growth may be expressed in terms of the dry weight of the mycelium or in terms of its nitrogen content. It must be con- cluded, therefore, that production of streptothricin is not a result of Chapter II — 4 1 — Important Types autolysis of the mycelium but is due to cell nutrition or to cell synthesis. This renders the mechanism of the production of this substance distinct from that of tyrothricin, for example, which is a result of autolysis of the bacterial cells, or of penicillin, which is produced at a much later stage of growth of the organism, that is, when it reaches an alkaline reaction. The efficiency of utilization of carbon and nitrogen by S. lavendulae is very high. At the maximum growth stage, 65 per cent of the nitro- gen in the glycine added to the medium was found to be converted into actinomyces cell substance. Since as much as 330-350 mg. of mycelial growth was obtained from 1 gm. of raw starch, the efficiency of utiliza- tion of the carbon, considering the carbon content of the starch as well as of the glycine, is about 40 per cent. Strepto-myces veneznelae Ehrlich, Gottlieb, Burkholder, Anderson and Pridham S. veneziielae, the organism that produces Chloromycetin (chloram- phenicol) was isolated from two different soils, one a mulched field near Caracas, Venezuela, and the other a compost soil at Urbana, Illinois (103, 103a). Primary mycelium growing in agar substrates is thin-walled, colorless, hyaline, monopodially branched. Mature vegetative hyphae vary in diameter from 0.9 to 1.8[j, and the branches grow to about iSOji. in length. Sometimes the substratal mycelium forms oval spores by frag- mentation. The aerial mycelium is lavender under the microscope, thick-walled, generally not much branched, straight or slightly and irreg- ularly curved, not forming spirals, having individual filaments that ap- pear stiff, and arising frequently from the primary mycelium at the surface of the substrate. Individual filaments are rarely septate, are 1.0 to 1.8[j- in diameter, and vary in length up to about 350[ji.. In young colonies, the aerial hyphae project outward radially over the surface of the colony and show a lavender color when examined microscopi- cally. The color of colonies when viewed on agar without magnifica- tion is gray to light tan or pink, but not lavender. Distal portions of the aerial hyphae commonly subdivide into unbranched oidial spore chains, which are readily fragmented into small groups or individual spores. The spores are oval to oblong. Mature spores range from about 0.4 to Q.% in diameter and from 0.7 to 1.6[x in length. The spores formed by fragmentation of hyphae in the substrate are generally smaller than those formed from the aerial hyphae. Individual spores arCf colorless at maturity but in mass appear tan to gray when viewed without magnification. They may be stained readily with crystal violet Waksman — 42 — Actinomycetes and other bacteriological dyes. The spores are uninucleate, as deter- mined by Giemsa staining. The two strains of S. venezuelae were similar, in their cultural and physiological properties, to S. lavendulae, although they differed from S. lavendulae in their ability to utilize various carbohydrates. The former utilized arabinose, rhamnose, xylose, lactose and fructose. The utilization of these by S. lavendulae was either negative or ques- tionable. The two sti-ains also differed from S. lavendidae in their sensitivity to actinophage and in serological reactions. Strcptomyces antihioticus (Waksman and Woodruff) Waksman and Henrici A detailed description of S. antihioticus has been given by Waks- man and Woodruff (491). Morphology: Spore-bearing hyphae produced in the form of straight aerial mycelium. The sporophores are arranged in clusters; no spirals formed. The spores are nearly spherical to somewhat elliptical. Gelatin: Dark brown growth on surface, with patches of gray aerial mycelium. Dark pigment produced, which gradually diffuses into the unliquefied part of gelatin. Liquefaction of gelatin at first very slow, later becoming rapid. Potato plug: Folded, brown-colored growth, with a thin black ring on plug, fading into a bluish tinge. No aerial mycelium. Carrot plug: Cream-colored to faint brownish growth. No aerial mycelium. No pigment. Litmus milk: Thick, brownish ring on surface of milk. Mouse-gray aerial mycelium with greenish tinge; growth becomes brown, especially in drier portions adhering to glass. No reaction change, no coagulation of milk, no clearing; whitish sediment at bottom of tube. Old cultures— heavy growth ring on surface of milk, heavy precipitation on bottom; liquid brownish to black in upper portion. Czapek's agar: Thin, whitish growth. Thin, gray aerial mycelium. Peptone media: Production of dark pigment at early stage of growth is very characteristic. Growth brownish, thin, with yellowish-gray to yellowish-green aerial mycelium. Odor production: Very characteristic soil odor. Antagonistic properties: Has a marked antagonistic effect on gram-positive and gram-negative bacteria (much more so on the former than on the latter), as well as on actinomycetes. It is also active against fungi, which vary in degree of sensitivity. Habitat: Found in soil. Isolated on Escherichia coli washed agar plate, using living cells of E. coli as the only source of available nutrients. Stre'ptomyces aureofaciens Duggar S. aureofaciens, the organism that produces aureomycin, was isolated from the soil (99fl). Chapter II —4^— Important Types Pigment production (golden yellow) is well developed in most strains of this organism grown on meat extract-asparagine-glucose agar, or on potato-dextrose agar, and on potato plugs. The substrate my- celium of young colonies is hyaline at first, commonly becoming yellow in 2 to 3 days. The aerial mycelium is white. The first-formed spores are white, but the entire heavily sporing surface of a slanted agar cul- ture gradually changes in 5 to 7 days at 28 °C. through brownish gray to a dark, drab gray. At the same time most of the substrate mycelial color disappears. The reverse color of slants at its best is golden tan, later tawny. Aureomycin is a weakly basic compound which contains both nitro- gen and nonionic chlorine. Aureomycin when treated with alcoholic ferric chloride gives a greenish-brown color by reflected light and red- dish color by transmitted light. The crvstalline free base has the fol- lowing properties: m.p., 168-169°C; solubility in water, 0.5-0.6 mg/ml at 25 °C; soluble in the cellosolves, dioxane, and carbitol; slightly soluble in methanol, ethanol, butanol, acetone, ethyl acetate, and benzene; in- soluble in ether and petroleum ether; very soluble in aqueous solution above pH 8.5, Stre'ptoviyces scabies (Thaxter) Waksman and Henrici Morphology: wavy or slightly curved mycelium, with long branched aerial hvphae, showing a few spirals. Conidia more or less cylindrical; 0.8 to LO by 1.2 to 1.5[x. Gelatin stab: Cream-colored surface growth, becoming brown. Slow lique- faction. Synthetic agar: Abundant, cream-colored, wrinkled, raised. Aerial mycelium white, scarce. Starch agar: Thin, transparent, spreading. Dextrose agar: Restricted, folded, cream-colored, entire. Plain agar: Circular, entire colonies, smooth, becoming raised, lichenoid, wrinkled, white to straw-colored, opalescent to opaque. Dextrose broth: Ring in form of small colonies, settling to the bottom. Litmus milk: Brown ring with greenish tinge; coagulated; peptonized with alkaline reaction. Potato: Gray, opalescent, becoming black, wrinkled. Nitrites produced from nitrates. Brown soluble pigment formed. Peptonization of milk and gelatin. Starch is hydrolyzed. Aerobic. Optimum temperature: 37°C. Habitat: Soil; cause of potato scab. This is a large heterogeneous group of organisms, occurring in nature in the form of many strains. A number of specific organisms, said to be Fig. \0.—Strepto}nyces veneziiclac, grown on potato glucose-beef extract agar, gram stain, X 975. (Prepared hy Littman of Armed Forces Institute of Path- ology). Chapter II — 45 — Important Types causative agents of scab, have been described. Because of lack of ex- perimental demonstration, it is difficult to state how many of these actu- ally cause scab. The ease with which numerous saprophytic actino- mycetes are isolated from the surface of material that has been in contact with the soil justifies these doubts. In a study of the effect of environmental conditions upon the growth of S. scabies, the following conclusions were reached: S. scabies grows within a wide range of temperature (8° to 38° C.)- Good growth and maturity occur between 13° and 32° C, and the opti- mum temperature is about 27° C. Therefore, under average field con- ditions in most potato growing areas, it appears that temperature, as it affects host and pathogen only, cannot be a very important factor in the scab problem. The spores survive temperatures up to 90 °C. (moist heat) for ten minutes. S. scabies is a strong aerobe. The spores will germinate with an extremely small supply of oxygen, but a large amount is required for subsequent development. Maturit)', as indicated by dark aerial hyphae, will not take place in the absence of oxygen. Amount of oxygen, not partial pressure, is the limiting factor for germination and growth. It was found that the germination of spores of S. scabies on nutrient agar was greatly retarded by a lagging film of excess water. The inocu- lum of S. scabies appeared to increase most rapidly at a soil moisture content about optimum for plant growth. The limiting acid reaction for germination of the spores of the strain of S. scabies used was found to be about pH 5.3. Germination oc-' curred most quickly at about pH 8.5, and an optimum development took place at this point. Because of the higher pH of the tuber and a strong tendency of the pathogen to make its habitat (scab pustule) alkaline, severe scab may be expected in soils ranging from a strongly alkaline reaction to at least pH 5.4. Chapter 111 MORPHOLOGY AND LIFE CYCLE Lack of complete understanding of the distinct morphological char- acteristics of the actinomycetes and of their mode of reproduction has been one of the major causes of the existing confusion concerning the nature and systematic position of this group of microorganisms. The fact that some species of actinomycetes resemble the true fungi in many respects whereas other species resemble the true bacteria more closely, and the fact that actinomycetes are characterized by marked variation in morphology and in cultural characteristics, especially yvhen grown on artificial media, have also contributed to the confusion. One of the early students of the actinomycetes, F. Cohn, recorded in 1875 that the "ray fungi," a common designation given to this group of organisms, are fungus-like in nature. This point of view was held by a number of subsequent investigators, notably Thaxter in 1891, Lachner-Sandoval in 1898, Berestnew in 1899, Neukirch in 1902, and more recendy Drechsler, Orskov, Jensen, and others. The pro- duction of a very fine mycelium consisting of unicellular branching hyphae definitely emphasized their similarity to the true fungi. On the other hand, the unicellular nature of the mycelium, its very fine structure, the resemblance in dimensions of the hyphae and of the spores to those of the bacteria, and the appearance of stained prepara- tions prepared in accordance with bacteriological practice— all tend to suggest that one is dealing here either with bacteria or with bacteria-like organisms. Since most of the early and even the more recent investiga- tors cultivated the actinomycetes on complex organic media, the differ- ences in morphological structures and cultural characteristics tended to be obscured. Marked differences became apparent only with the intro- duction of synthetic media for the growth of actinomycetes and with the development of suitable microscopic techniques for examination of these organisms in an undisturbed state. It has become recognized that the actinomycetes possess morphological properties which not only are distinct from those of the bacteria but which are, within certain limits, fairly constant. Staining of Actinomycetes:— In addition to the direct mcdiods of examining the structure of the actinomycete colonies and their growth characteristics, that is, the general methods developed by students of ^s :' - , '^ 1^ ^"'vNM lll^^^> f Fig. \\.—Stycptniiiyces sp., grown on potato glucosc-bcct agar, gram stain, X 975. ^Prepared hy Littman of Armed Forces Institute of Pathology). Waksman — 48 — Actinomycetes fungi and bacteria, certain special methods have also found application. Among these, it is sufficient to mention the following: 1. Method of Henrici.—A drop of melted agar medium is placed on a slide, allowed to cool somewhat, and inoculated with the actinomyces culture. The agar is then spread in a thin film on the slide. The agar may also he allowed to cool first before being inoculated with a sharp needle. The slide is then incubated in a sterile moist chamber. After growth has taken place, the slides are allowed to dry, are fixed in alcohol, and stained. The entire colony, with both vegetative and aerial mycelium can thus be stained and examined in an undis- turbed condition. 2. Method of Drechsler.— "The culture is grown on a synthetic medium, and the fully developed colony is cut from the agar as carefully as possible. A slide smeared with albumin fixative is brought into firm contact with the surface mycelium of the colony, then separated from it, precautions being taken to avoid any sliding of the two surfaces on each other. If the growth is not too young, the upper portions of the aerial mycelium will be left adhering to the slide without much disarrangement. The adhered growth is then killed and fixed at once, and the preparation is stained and mounted in balsam. Preparations in which the spore chains have commenced to disintegrate are impaired by the large masses of free spores. The most convenient fixative agent is 95 per cent alcohol. As a stain, Haidenhain's iron-alum haemotoxylin is good for protoplasmic structures. Delafield's haemotoxylin, allowed to act for 24 hours with the proper degree of decolorization, yields deeply stained, clear preparations showing distinctly the various mycelial structures of the organism. 3. Other methods.— Various special methods have been utilized for preparing actinomyces cultures for staining. It is sufficient to mention the use of a drop of liquid synthetic medium placed on a cover slip, which is then inoculated with a few actinomyces spores and incubated in a sterile moist chamber or in the form of a hanging drop preparation. The liquid medium may also be allowed to flow around an actinomyces colony, which has been removed from an agar plate and placed on a cover slip; the peripheral growth may be stained. All actinomycetes are gram-positive, although certain thermophilic forms, according to Lieske, may be gram-negative at temperatures above 50°C. Most actinomycetes are non-acid-fast. Some of the Nocardia species, however, especially many of the pathogenic forms, are acid-fast (145, 432). The mycelium stains uniformly, except in older cultures. The presence of metachromatic granules has been observed by Brussov (49) and Drechsler (97); this was believed by some investigators to indi- cate that the cultures possess the property of pleomoi-phism. The gran- ules in the mycelium can be readily stained with methylene blue (1:1,- 000) and decolorized by sulfuric acid. Droplets of fat, often pig- mented, can be seen frequendy in the mycelium (242). The forma- tion of vacuoles has also been reported (97). Neukirch (321) dif- ferentiated the ectoplasm of the actinomycetes from the endoplasm, on the basis of staining with dilute methylene blue, the first being dark blue and the second light blue. The presence of nuclei in actinomycetes has aroused considerable Chapter III —49— Morphology discussion. The presence of fine grains in preparations treated with dilute methylene blue was looked upon as substantial evidence of the presence of a nucleus (321). Others considered these granules, how- ever, as merely fatty bodies or of a metachromatic nature. Krassilni- Kov submitted evidence that these granules consist of chromatin sub- stance, which plays the role of a nucleus. Young hyphae contain single grains which are larger in older cultures and could be distin- guished only with difficulty from the rest of the protoplasm. By use of the Feulgen reaction, von Plotho (340) demonstrated that the reacting substance is distributed in the protoplasm of the actino- mycetes in all stages of its development. He reached the conclusion that nuclear substance is present in the cell plasma. This substance can become concentrated into special nuclear bodies, especially in the mature spores. The presence of thymonucleic acid bears evidence of this fact. These results are not in agreement with those obtained by RippEL (361), who believed that the bodies stained by the Feulgen re- action are fats in nature. Jensen described (186) the staining reactions of Micromonosfora as follows: The hyphae and spores stain easily with all the usual bac- terial stains, such as carbol fuchsin, aqueous fuchsin, methylene blue, gentian violet. Delafield's haematoxylin gives fine and clear prepara- tions, especially when material is fixed with sublimate alcohol. The spores stain more intensely than the hyphae. All the strains are gram- positive, but never acid-fast. Whether nuclei are present in the spores and mycelium was difficult to decide because of the minuteness of the objects. Preparations were stained by the method of Schumacher for demonstrating nuclear material. The preparation was dried on a slide and treated for 2 to 4 hours with 25 per cent hydrochloric acid, washed first with water, then for 10 seconds with dilute Na2C03 solution, and finally stained for 30 seconds with carbol thionine. The presence of deeply stained minute granules was demonstrated in old spores, in germinating spores, and in young mycelium. The nuclear method of staining bacteria was applied successfully to the staining of sporulating actinomycetes (222). It consists in fixing the cells with osmic acid in N HCl, and staining with the Giemsa stain. The acid is usually applied for 6 to 20 minutes at 55 °C., and the stain, diluted 1 to 30, 5 to 30 minutes. The preparations are dehydrated with acetone and x)'lose and mounted in Canada balsam or in the weak stain- ing solution. To show the cell boundaries, the osmic acid preparations are placed for 30 minutes in a 5 per cent aqueous solution of tannic acid, rinsed in water, stained for 2 to 4 minutes in crystal violet 1 : 10,000, and mounted in the stain or in water. General Morphology:— ^Colony formation— Growth of an actinomyces on a solid or in a liquid medium results in the formation of a mass of growth usually Waksman — 50 — Actinomycetes designated as a "colony." This is not a true colony in a bacterial sense, since it is not an accumulation of a number of cells originating from a single cell or from several similar cells. It is rather a mass of branching filaments which originated from a spore or from a bit of vegetative my- celium. The actinomyces colony is made up often of two types of mycelium, consisting primarily of vegetative or substrate growth and of secondary aerial or sporogenous growth. These two types of mycelium often show fundamental differences in appearance, composition, and biological ac- tivities. The vegetative mycelium grows into the medium, whereas the aerial mycelium grows on the surface; the well-developed sporulat- ing hyphae and the reproductive spores are produced in the aerial mycelium. Some actinomycetes form only the vegetative mycelium, whereas others produce both types. Vegetative mycelium.— The vegetative growth of the actinomycetes, or the stroma, is usually shiny, gel-like, or lichnoid in appearance and varies in size, shape, and thickness. The color of the growth may be whitish or cream colored, as well as yellow, red, pink, orange, green, or brown. In addition to the insoluble pigments, certain water-soluble pigments are produced. Some of the pigments, notably the brown and the darker or chromogenic pigments are formed upon complex organic media and are a result of the action of certain enzymes of the tyrosinase type, which are able to oxidize some of the organic constituents of the medium to give the particular pigments. The red, yellow, and blue pig- ments are synthetic in nature. When actinomycetes spores are inoculated into a fresh medium, they germinate rapidly, usually within 2 to 6 hours, and give rise to one or more germ tubes, as shown in Fig. 13. These grow into long hyphae or threads which gradually develop into a complex mycelium. The length and diameter of the hyphae differ considerably for the various organisms. Some are straight and long, reaching 600iJ. or more; others are only 50 to lOOix in length, and are much branched and curved. The vegetative mycelium varies in diameter from 0.2 to O.SpL. Occasion- ally, involution forms are produced which have even a greater diam- eter. The structure of the h)rphae also varies with the composition of the medium, the conditions of growth, especially temperature, and the presence of stimulating or injurious substances. On the basis of the length of the hyphae, Lieske (186) divided the actinomycetes into long-hyphal and short-hyphal forms. It is doubtful, however, whether such a sharp line of demarkation can be drawn for all organisms within this group and for all media upon which they are usuallv grown. In older cultures, the vegetative mycelium becomes brittle and readily breaks into fragments of uneven length. The mycelial frag- ments are very small, usually l[x or less in length. Together with the cellular contents they form a granular mass which deposits on the bot- tom of liquid cultures. Some cultures undergo rapid lysis, especially Chapter III —51— Morphology at higher temperatures or when grown under submerged conditions. Others are subject to attack by specific phages. When inoculated into fresh medium, the finer or disintegrated particles give rise to a normal mycelium. Some investigators (158, 226) were lead to consider this phenomenon as symplasm formation, or as a stage in the life cycle of the actinomycetes. Krassilnikov (234) rejected this concept and emphasized the fact that it is not the symplasting mass as a whole but the sporulating bodies present within the lysed material that are respon- sible for the reproductive capacity of the organism. Aerial viyceUmn.—Alany of the actinomycetes, notably members of the genus Streptomyces, are capable of producing an aerial mycelium superimposed upon the vegetative growth. The production of the aerial mycelium by various actinomycetes depends on the culture, com- position of medium, and conditions of incubation. These factors also influence the nature and abundance of the mycelium. The aerial hj^phae vary considerably in length and may have a diameter of \\}. or even 1.4[jl. Usually, they are short and straight or wavy and much branched. Some organisms produce long hyphae that are little- branched, straight, or slightly curved. The aerial mycelium may cover the whole colony either in the form of a cottony mass or as a powdery, chalk-like to almost granular layer. Certain organisms produce an aerial mycelium in the form of tufts or as concentric zones over the vegetative growth; in a few cases, it may be compacted into bodies resembling coremia, the central portion consisting of vegetative growth and the surface of aerial mycelium. These sporulating hyphae represent a well-characterized sporogenous apparatus, consisting of a sterile axial filament bearing branches in an open racemose or dense capitate arrangement. The primary branches may function directly as sporogenous hyphae or may produce branches of the second and higher orders. In the latter case sporogenesis is con- fined to the terminal elements, and the hyphal portions below the points of attachment of branches remain sterile. The morphology of the spore-bearing hyphae of the various acti- nomycetes exhibits distinct individuality and can readily serve as a basis for specific difi^erentiation. The specialized, sporogenous hyphae are distinguished from the sterile hyphae of the aerial mycelium at an early stage of their development. Though the diameter of the sterile mycehum which arises through the elongation of the growing filament tip shows very litde subsequent increase in thickness, the sporogenous hyphae are, in the beginning, thinner than the axial h)q3hae from which they are derived. Increase in thickness of the sporogenous hyphae fol- lows after the final linear extension has been attained. The final di- ameter of the sporogenous hyphae is in most cases appreciably more than that of the vegetative hyphae. (The formation of the aerial from the vegetative mycelium has been ascribed (222) to agglomerations or fusion of filaments which give rise Waksman — 52 — Actinomycetes to "initial cells." These are formed first in the center of the colony, then at the periphery. The "fusion cells" consist of darkly staining nuclear bodies surrounded by protoplasm and later enclosed by cell walls. They grow into the aerial mycelium by a process of sprouting and subdividing. Transverse septa are easily demonstrable in the aerial mycelium. The division of the nuclear cylinders in the cells of this mycelium initiates spore formation. Some actinomycetes produce an aerial mycelium which has the form of "fairy rings." These consist of concentric spore-bearing rings and spore-free rings disposed in zones. It has been suggested that ring formation is a result of diffusion of injurious substances present or formed in the medium or that it is due to the action of light, which produces a change in transpiration and temperature. This phenome- FiG. 12 a-d.— Different forms of sporulation of Micro- vwnospora growing in composts, as shown by contact slide preparations Qwm Waksman, Cordon and Hul- poi, 459).— for b-d, see pp. 53-55. non may be closely related to the autolytic reactions and attack of cer- tain species by phage. The aerial mycelium is variously pigmented, from shades of white or gray, to yellow, orange, red, rose, lavender and green. The dry powderv appearance of the aerial mycelium of actinomycetes and the difficulty of wetting the spores appear to be due to the presence of lipids in their outer walls. These substances are removed bv fat solvents and wetting agents and are destroyed by alkalies. Staining with Sudan IV distinguishes the lipid-containing aerial mycelium from the vegetative mycelium (114). The manner of spore formation depends upon the specific nature of the organism and upon the conditions of cultivation. The conidio- phores or sporophorcs produced on the aerial hyphae comprise several types, as pointed out pre\'ious]y (p. 30). Chapter III —53— Morphology The composition of the medium is oF great importance in influ- encing the manner of sporulation. Synthetic media are best for study- ing this phenomenon. The process of sporulation is favored by dryness, aerobic conditions, and carbohydrate nutrition. Cytology.— The formation of cell walls by actinomycetes has aroused much speculation. Normally, growing mycelium does not show any cell wall; it becomes apparent, however, when the plasma constricts and breaks up into fractions. This can be seen either in old cultures or during the process of sporulation of the aerial mycelium. When the spores thus produced are liberated as a result of the break-up of the sporophore, the empty shells become visible. The cell wall is soluble in 10 per cent KOH solution and in antiformin. When treated with concentrated H0SO4 it is first pigmented dark and is then dissolved. As the great majority of the cultures do not show such septa, it has Fig. 12 b (see p. 52). generally become recognized that an actinomyces colony represents a single-cell type. Drechsler (97) considered the actinomvces myce- lium to be definitely septated, the hyphae being divided into short sections. This phenomenon is particularly striking in cultures belong- ing to the genus Nocardia, but appears only seldom among species of Streptomyces. Orskov (328) also described septa in certain cultures. He believed that this is the first stage in the process of the break-up of the mycelium into fragments. Krassilnikov (234) considered the ob- served formation of septa as merely the beginning of the fragmentation process. In recent studies on the cytology of actinomycetes, using a more refined method of staining, namely, the tannic acid-crystal violet method, septa have been demonstrated (222) conclusively. They are fofmed early in the vegetative mycelium. This mycelium, however, although septated, never breaks up into single cells. Waksman — 54 — Actinomycetes The mycelium of actinomycetes produces true branching of a mono- podial type. Some observers have reported dichotomous branching (98), a phenomenon considered by others as uncertain (97). The formation, by certain species, of nodes from which side branches are produced in the form of whirls has been reported by Waksman for S. reticuU; this was later confirmed by others, notably by Kriss (242), who added another species under the name of S. verti- cillatus. The formation of short side branches which give rise to single spores is characteristic of species belonging to the genus Microvionospora. These spores have often been designated as chlamydospores or mega- spores. Jensen (186) looked upon them, however, not as involution forms but merely as a type of development of rod-shaped cells, often observed among the mycobacteria and the corynebacteria. Fig. 12 c (see p. 52). Among species of Nocardia, Lieske observed the production of swollen cells, which he considered as involution forms. Krassilnikov considered these as normal stages in the life cycle of the organisms. In old cultures, certain swellings of the terminal ends of the hyphae may be observed. These may also be formed under abnormal growth conditions, as in concentrated media or in the presence of substances like caffeine. These swellings may be considered as involution forms, somewhat similar to the clubs produced by pathogenic actinomycetes in the animal body. The separation of actinomycetes on the basis of these formations is open to criticism. Plasmolysis has not been established as yet for the actinomycetes with any degree of certainty. The lytic reactions are due either to auto- lytic enzymes or to specific phages. Sporulation of Actinomycetes:— Spore forviation.— The actinomvces spore has been described as con- taining a spherical, relatively large chromatin body which is surrounded Chapter III —55— Morphology by cytoplasm enclosed in a spore case. When the spore germinates, the chromatin bodies divide, some of the material entering the germ tubes. As the mycelium develops, it becomes filled with granular or rod-shaped chromatin bodies. Lachner-Sandoval (247) was the first to recognize, in 1898, the true manner of sporulation among the actinomycetes. This was be- lieved to be a distinguishing character of the organisms. Two types of spores were found to be produced, both asexually, one by the process of fragmentation and the other by the process of segmentation. The fragmentation spores were looked upon as analogous to spores formed by true fungi. They are formed by the breaking up of the protoplasm within the cell wall into particles or fragments, more or less uniform in size. These fragments are later liberated by the splitting of the cell wall. During the contraction of the fragments, empty and clearer partitions are formed between them, which have been occa- ^po- « ' % »'-» u- ..-. / / 5 Fig. 12 d (see p. 52). sionallv taken for cross walls. When the spores mature, the surface cover becomes less defined and may gradually disappear, as a result of autolysis. The spore-bearing threads thus assume the appearance of chains of cocci, the spores falling apart readily. The surface cover may persist, however, without dissolving, in which case the spores leave through the broken ends of the sporulating hyphae. Sporulation by the fragmentation process begins at the top of the aerial hyphae and proceeds toward the base. This manner of sporulation is characteristic of the genus Streptoviyces. Sporulation by segmentation consists in the simple breaking up of the sporulating hyphae by means of cross walls. At first the hyphae are unicellular. At a certain stage of growth, cross walls are formed anid the hvphae break up into small segments. These are cylindrical in form, with sharp edges and are uniform in size, usually 1-2.5 X 0.7-0.8iJ.. These often have been considered as true oidiospores (321). im Fig. 13.— Details ot spoiulation and of spore germination by S. griseiis as shown by electron microscope: Top, left, aerial sporogenous hypha showing septation prior to spore formation; top, right, more ad\'anced stage in spore formation; bot- tom, left, well matured, four-spored chain; center, spore germinated by a single germ tube; bottom, right, spore germinated by two germ tubes Qfroni Carvajal, 64). Note (top, left) mitotic division taking place in developing spores. Chapter III — 57 — Morphology The cylindrical oidinspores may swell, giving rise to spherical bodies. This manner of sporulation is characteristic of the genus Nocardia and of certain species of Strcptomyces. Drechsler recognized three types of sporulation: (a) by means of true fragmentation, (/?) by means of doubling of the cell wall, (c) by means of contractions similar to segmentation. According to DucHE, the last process alone results in the production of three types of spores: (fi) regular and irregular arthrospores, (I?) microarthrospores, produced in the substrate mycelium, and (c) endospores in the aerial mycelium. The true conidia or fragmentation spores are formed only in the aerial mycelium, whereas the vegetative mycelium gives rise to chlamydospores or arthrospores. These chlamydospores are produced by the concentration of the plasma in the substrate mycelium and are abundant in some species. They are spherical spores (1.5-1.7[j,), v\ath thick plasma, and are sepa- rated from the rest of the hyphae by cross walls. They are distin- guished from involution forms by a thicker, light-reflecting plasma. They are not produced readily on protein media. Spiral formation.— The sporophores in the aerial mycelium are either straight or spiral-forming. The manner of spiral formation is described in detail by Krassilnikov (236). The spirals in the mycelium curve not long before the spores are produced; the branch may be curved completely or only at the end. The number of turns varies in accord- ance with the length of the spiral. There may be as many as 15 or as few as 1 to 3 turns; usually there are 5 to 6. Some species are char- acterized by long spirals and others by short spirals, some by compact and others by extended spirals. The curvature of the branches may be clockwise (dextrorse) or counterclockwise (sinistrorse). Drechs- ler considered the manner of spiral curvature as characteristic of the species. Since certain species show both types of curvature in the same culture, this distinction can hardly be accepted. Not all the aerial hyphae give rise to spores, some of the hyphae being sterile. Nature of spores.— The spores of actinomycetes are spherical (0.8- 03l). in diameter), oval, or cylindrical (0.8-1 X 0.7[ji.). The shape and size of the spores are characteristic of the species, with a certain degree of gradation and variation. Actinomyces spores are reproductive bodies, comparable to fungus spores, rather than resistant bodies like bacterial spores. Actinomyces spores are destroyed by heat at 60° to 65°C. for 10 to 15 minutes. It has been (225) reported that the spores are somewhat more resistant than the mycelium. When brought into a favorable medium, the spores swell and give rise to one to four germ tubes. The different spores vary greatly in this respect. Both the conidia and the oidiospores germinate in a manner sirpilar to that of the corresponding spores of fungi. The germ tubes may appear at one end or at both ends of the cylindrical oidiospore. Waksman 58 Actinomycetes Reproduction can also occur by the vegetative process, namelv, through the growth of pieces of mycehum, and by the formation of buds, which gradually grow into branches, as well as by means of the chlamydospores. The germination of these spores is similar to that of the other reproductive bodies, independent of the hyphae in which they are produced (234). Fig. 14.— Aerial mycelium of a Streptomyces, showing zonation or "fairy ring" formation Qrom Lieske, 260). Sporulation of the Microvionospora is distinct from that of the other genera. The monopodially branched mycelium is similar to that of the other actinomycetes. The conidia are formed on special branches, which are straight and short— 5-1 OiJ. long— and which frequently give rise to other branches, thus producing group-like structures similar to bunches of grapes. Each branch bears at the end a single spore, pro- Chapter III — 59 — Morphology duced by die splitting off of the tip of the hvpha. The conidia are spherical (1.0-1. 3[jl in diameter), oval, or oblong (1.3-1.5 X 1-2ijl). Sporulation occurs most abundantly on synthetic media. Types of Growth on Solid and Liquid Media:— Aerobic organisms.— The colonies of the aerobic actinomycetes, con- sidered by some as pseudo-colonies, differ greatly from the colonies of fungi, on the one hand, and of the bacteria, on the other. They are usually compact, leathery, growing deep into the medium. Only cer- tain few aerobic pathogens produce colonies of a dough-like con- sistency, which makes them similar to the colonies of bacteria. The colonies can be round and smooth, or much-folded, lichnoid to almost barnacle-like in appearance. The edge of the colony, when examined under the microscope, gives a characteristic picture of radiat- ing hyphae. The colony may be produced below or on the surface of the medium. In liquid media, the colonies may be formed individually on the bottom of the container, they may adhere to the surface of the wall of the container, or they may give rise to a ring of growth on the surface of the medium. The surface colonies may coalesce, producing a pellicle of varying degrees of compactness. The colonies may also be flaky in appearance, but they cause no turbidity of the medium. Sometimes the surface growth is similar to that of tubercle bacteria, that is, dough-like and folded, without producing any aerial mycelium. The composition of the medium has an influence upon the nature of the growth. When actinomycetes are grown in a submerged or in a shaken con- dition (452, 517) they produce characteristic small, bead-like colonies, or granules which may completely fill the culture vessel. Probably be- cause of the continuous break-up of the mycelium or the separation of the spores, growth is much more rapid and more abundant in sub- merged culture than in stationary culture. This is particularly true of certain species of Microvionospora. Anaerobic organisms.— The morphology of the anaerobic forms repre- sents a special problem. Erikson (112) made a detailed study of the morphology of 1 5 strains of the microaerophilic types of A. bovis derived from human materials and of 5 strains of bovine origin. A very sparse development of erect aerial hyphae was detected when the human strains were grown in an atmosphere of reduced ox)^gen tension. These hyphae were found to be occasionally septate, but no definite spores were produced; they were of the same diameter as the hyphae of the substratum mycelium. The substratum mycelium is initially unicellular, the branches extending into long filaments, caus- ing the colony to adhere to the medium. This mycelium may give rise to irregular segments, with a characteristic angular branching. The colonies were said to exhibit polymorphism, although no stable variants could be demonstrated. They gave no turbidity in the medium. Waksman — 60 — Actinomycetes The colonies from the bovine strains were smoother and softer in consistency and did not adhere to the medium. Growth was scantier. The mycehum underwent fragmentation very rapidly, giving only traces of extensive ramification. No aerial hyphae were produced. In contrast to the human strains, the bovine strains showed occasional tur- bidity in the medium, and they were less able to ferment sugars, espe- cially salicin and mannitol. No filterable stage could be demonstrated by ultrafiltration experi- ments on either the human or bovine strains, and no evidence could be obtained in favor of any hypothetical life-cycle. Autolysis of actmomycetes.— Only a few actinomycetes are able to show the phenomenon of autolysis. This was reported first for animal pathogens (91) and later also for plant pathogens and for soil sapro- phytes (509). The active agent responsible for the lysis was consid- ered to be either an enzyme or a nontransferrable phage. Of 1,000 or more freshly isolated cultures of actinomycetes studied by Krassilnikov (232) only very few were able to undergo lysis. An organism described as A. albicans at first gave a typical heavy compact growth covered with white aerial mycelium. On continuous transfer, the colonies became flat, smooth, and somewhat moist and lost the property of producing aerial mycelium. The culture was gradually re- duced to a very thin slimy film. It grew more and more poorly, be- coming, on repeated transfer, more rapidly transparent, until it finally ceased to grow altogether. All attempts to keep it alive were unsuc- cessful. Six other strains of a similar nature were isolated later. They belonged to different groups and showed difl^erent degrees of lysis. Among actinomycetes, autolysis may not appear all through the colony, but may affect only certain sectors or spots, the unlysed part of the colony being quite distinct from the lysed part. Frequently lysis begins in the center of the colony and proceeds to the periphery. The mechanism of autolysis among pathogenic forms is similar to that of the saprophytes but proceeds more rapidly (232). Meat-peptone agar is a favorable medium for the study of autolysis. When the cul- ture is grown at 25 °C., plated out and incubated at 30° or 37° (for pathogens), autolysis proceeds very rapidly; in fact, it becomes evident in 4 to 6 hours. Not all the hyphae are lysed uniformly. Some pro- duce chlamydospores, and others, spherical bodies, as well as hyphal fragments. Under favorable conditions, all three types of bodies are able to grow and develop into fresh colonies. The lytic factor is present within the cells of the actinomycetes. It becomes active when growth ceases, although it is possible that there is considerable overlapping of the two processes. When a growing cul- ture is treated by physical or chemical agencies so as to stop growth, lysis begins immediately. If the temperature of the culture is raised to 60° — 70°C., autolysis occurs in a few minutes. The lytic factor is in- Fig. 15— Electron micrograph of actinophage Qrom Woodruff, et ah, 518). Waksman — 62 — Actinomycetes activated when the culture is heated to 100°, but not to 80°, for 5 minutes. The lytic factor of actinomycetes is very specific. It does not act upon other species or even on closely related forms. It is not to be confused with the transmissible or phage factor which affects certain actinomycetes. In contrast to actinophage, the lytic agent acts also upon the dead cells of the organism. A thermophilic actinomyces isolated (207) from composts of horse manure was found to grow well on various media, but it underwent lysis when grown in a synthetic medium containing ammonium sul- fate and starch, after 24 to 48 hours' incubation at 50°C. During growth, the pH of the culture changed from 7.0 to 5.7. The addition of CaCOs to the medium prevented the production of acid as well as of lysis. After maximum growth has been attained, S, griseus, the organism that produces streptomycin, undergoes lysis (395). This takes place more rapidly under submerged than under stationary conditions of cultivation. The whole culture tends to become viscous as a result of formation of the lysed material. Apparently the maximum peak of streptomycin production is associated with the setting in of lysis of the culture. When lysis has progressed too far, production of streptomycin ceases, and even that already produced may be destroyed. Dmitrieff and Souteeff (91) observed that a culture of an or- ganism designated as Actinomyces hovis, and which evidently belonged to the genus Streptomyces, underwent lysis in various media. When the organism was grown on agar media, the production of lysis was found to be associated only with the formation of a certain t)'pe of colony. As a result of lysis, two types of daughter colonies were formed: one was similar to the mother colony and was characterized by capacity for lysis; the other type of colony did not lyse and was morpho- logically different from the first. The cultures that originated from the colonies capable of undergoing lysis were strongly proteolytic and did not form any aerial mycelium. The cultures obtained from nonlysing colonies were less proteolytic and produced a chalky aerial mycelium, which changed the reaction of litmus milk to alkaline. In broth cul- tures, lysis took place in 2 to 3 weeks; it was associated with the living organism and was of the nature of a nonenzymatic and nontransmissi- ble lytic factor. These results are comparable to those obtained later by Schatz and Waksman (395) in the production of inactive strains by S. grisetis. These strains were free of aerial mycelium, produced no streptomycin, underwent much more rapid lysis, and formed much more acid in the medium (Table 5). Effect of actinophage upon actinomycetes.— VJiu^oi.^ and WiE- RiNGA (509) observed that cultures of actinomycetes isolated from in- fected potatoes underwent lysis. This phenomenon was ascribed to the Chapter III 63 — Morphology production of specific transmissible phages. Repeated additions of a filtrate of S. roseus to the culture of the organism resulted in the development of a phage which gave a large number of plaques on solid media inoculated with the actinomyces and inhibited the growth of the organism in liquid media. Phages were also obtained from the patho- Table 5 : Cultural and physiological characteristics of the streptomycin-producing strain of S. griseus and its inactive variant (395) : — Active strain Inactive variant 1. Antibiotic activity . Produces streptomy- cin in both shaken and stationary cul- tures. 2. Growth. Surface growth always heavily sporulated; grayish-green aerial myce- lium. 3- Reaction. Medium always changes to alkaline; pYi 7.5-8.5. 4. Glucose. Glucose completely consumed in 6-8 days in stationary cultures and in 3-4 days in shaken cultures. 5. hy sis in shaken cultures. Shaken cultures produce very fine flocculant growth, tending to lyse slowly after about 15 days. 6. Lysis in stationary cultures. Surface pel- licles stable; any submerged, flocculant growth tends to lyse as the surface pel- licle develops. 7. Viscosity. Culture filtrate not showing any viscosity. 8. Keinoculation . Inoculation of cultures with lysed inactive culture induces no lysis or reduction in activity. 9. Variation. Sporulating strain gives rise to non-sporulating variants. 10. Sensitivity to streptomycin. Very resist- ant to this antibiotic. 1. No streptomycin formed either in shaken or stationary cultures. 2. No sporulating aerial mycelium; scant development of aerial hyphae with slight tendency to form spores in some old cultures. 3- Reaction of medium at first .icid, pH 50-6. 5; later becoming alkaline. 4. Glucose utilized more slowly. 5- Cultures produce at first balls of growth which change into the turbid, floccu- lant type; rapid and complete lysis in 7-10 days. 6. Stationary cultures produce no surface growth but flocculant, submerged my- celial growth which lyses slowly, only after a month or longer. 7. Culture filtrate becomes viscous during or after lysis. 8. Inoculation of cultures with spores of active strain produces growth and anti- biotic activity if some glucose remains. 9. Asporogenous variants may reconvert to active, sporogenous forms. 10. Very sensitive to this antibiotic. genie organisms A. hovis and N. farcinica. A polyvalent phage was obtained from one of the actinomycetes which was also active upon S. scabies, thvs suggesting possible methods of combating potato scab. The phenomena of phage production by actinomycetes was referred to as "microbiophagy." These investigators were thus the first to empha- size the existence of filterable and transmissible agents comparable to bacteriophages, which were active upon actinomycetes. Krassilnikov and Koreniako (237) emphasized the resemblance of the process of autolysis among actinomycetes to the action of phage Waksman 64 Actinomycetes upon bacteria. They reported, however, that the lytic factor of actino- mycetes was highly specific, since it had no efi^ect upon other species or even upon other strains of the same species of actinomyces. When growth of the organism was delayed under the influence of various fac- tors or when the culture became aged, lysis took place. Different cul- tures underwent lysis with varying degrees of rapidity. It was assumed, therefore, that production of the lytic factor or its mode of action dif- fered with the various organisms. At temperatures of 60° to 70°C., lysis occurred in a few minutes. The lytic agent was resistant to a Fig. 16.— Method of measuring actinophage concentration Qfrom Reilly, Harris and Waksman, 355). temperature of 80 °C. for 1 hour but was destroved at 100°C. in 5 minutes. Not only the living but also the dead cells of the organism were affected by the lytic agent. This last suggested that the agent is different in its action from that of true phage. With the discover)^ that the streptomycin-producing strains of S. griseus are subject to attack by a virus or a phage-like agent, the problem of phage action upon actinomycetes entered a new phase. The lysis of the actinomyces produced by the phage appeared to be quite distinct from the lytic phenomena. Saudek and Colingsworth (383) were the first to report that S. griseus is subject to the action of a transmissible lytic agent which had Chapter III —63 — Morphology all the properties of phage. In the presence of young cultures of S. grisetis, the phage de\eloped rapidly and brought about the lysis of the culture. The plaque method was used for measuring the concentration of the phage. Streptomycin production was partly or completely pre- vented by the phage. Cultures of S. griseus resistant to the action of the phage could easily be isolated. Woodruff and Foster (516) exposed to laboratory air for 24 hours a submerged culture of S. grisezis in a stationary condition, with plugs removed from the flask. The freshly formed pellicle showed evidence of plaque formation. The same phenomenon was observed in a factory 500 miles away. Upon transfer of a filtered culture into a fresh culture of S. grisetis, the phage multiplied. It was calculated that after six transfers, each phage particle increased to 75 X lO-*^ particles. The phage was active against all streptomycin-producing strains of S. griseus but not against the non-streptomycin-producing strains. Phage-resist- ant strains developed readily. They retained their capacity to pro- duce streptomycin but were not absolutely free from phage. The phage of S. grisexis had properties similar to those of bacterial phages, as shown both by cultural characteristics and by appearance in photo- graphs made by means of an electron microscope (Fig. 15). The following method can be employed (355) for assaying the activity concentration of phage in a gi\'en preparation. A 3 to 5-day- old shaken culture of a streptomycin-producing strain of S. griseus is filtered aseptically through paper and inoculated on plates. The phage preparation is obtained by inoculating with phage, young cul- tures of S. griseus grown in a shaken condition, allowing the cultures to incubate further for 24 to 72 hours, and filtering them through a Seitz filter. Dilutions of phage, ranging from 1:10'^ to 1:10^-, are added to 10-ml. portions of sterile nutrient agar, previously inoculated with 0. 1-ml. portions of the paper-filtered culture. The agar is poured into plates, which are incubated at 28 °C. for 2 davs. The plaque counts are made, as shown in Fig. 16, and calculated for 1 ml. of the preparation. Some preparations gave 4 X 10^** or more particles per milliliter. The phage preparation is kept in the refrigerator and used as a standard. To illustrate the effect of actinomyces inoculum upon the phage count, three diff"erent concentrations of filtered 7-day-old shaken culture of streptomvcin-producing S. griseus were added to nutrient agar. The plates were inoculated with the same amount of the phage and incubated at 28 °C. for 48 hours. The following results were obtained. Inoculum Plaque counts per cent X 10^ 10.0 391 1.0 698 0.1 756 Waksman 66 Actinomycetes These results show that the plates do not have to be heavily inocu- lated with S. grisetis in order to give uniform growth on the plate of the organism subject to attack by phage, with the resultant formation of plaques. Actinophage of S. griseus was found to attack only the streptomycin- producing strains of this organism. It had no effect on other strains of S. grisezis or on other streptomycin-producing organisms such as S. Table 6: Effect of phage upon the growth, phage multiplication, and streptomycin production by different actinomycetes in stationary cultures (355) : — Phage added* 9d: lys 13 d; lys Organism Phage per ml X 10^ Strepto- mycin Phage per ml X 10^ Strepto- mycin Streptomycin-producing strains of S. griseus gm/ml gm/ml No. 3463 - - 21 + - - 200 5 No. 3475 30 180 -f >50 <5 370 <5 No. 3480 31 189 + 10 <5 30 28 No. 3481 73 174 + 50 <5 260 13 No. 4 43 201 + 30 <5 160 <5 3475-2PR >0.01 40 40 129 + >50 16 370 75 Grisein-producing strain of <5 <5 S. griseus 3478 + <5 <5 Inactive strain of - - <5 S. griseus 3326a -f - - <0.2 <5 Streptomycin-producing S. bikiniensis + 3 <5 30 7 30 33 * Each 60-ml llask of c 1 of M-1 pha^e, i ing to 7 X 10" particles per 1 ml of medium. bikiniensis. In cultures that do not produce streptomycin, the phage did not multiply and in some cases was destroyed or absorbed (Table 6). This actinophage multiplies only at the expense of the living cul- tures of S. griseus but not on the heat-killed organism. Its optimum temperature for multiplication is 28 °C., and it does not grow at 37 °C. or above. However, it can withstand a temperature of 75 °C. for 1 hour but is completely destroyed when heated at 100°C. in 10 minutes. When it is stored at 6''C., there is little loss of activity, but storage at Chapter III _ 67 — Morjjhology 28 °C. or at higher temperatures results in loss of activity, the rate of loss being proportional to the temperature (Table 7). Constancy of actinomyces tyfes.— Each one of the four genera of actinomycetes has clearly defined morphological characters. Although there is a certain amount of overlapping between the species within the different genera, notably between the species of Actinomyces and of Nocardia, or between Nocardia and Stre'ptomyces, the combined morphological and cultural properties well characterize each genus. Very often a species of Streponiyces may lose, by selection or by mutation or natural variation, the property of forming aerial mycelium; it may then appear to become a t)q5ical Nocardia. This was shown to hold true, for example, of the streptomycin-producing strain of S. griseus. When such a change occurs, it is accompanied by a change in the physiology of the organism. Usually, however, the culture re- verts to its original form under proper methods of cultivation. Table 7: Stability of phage in aqueous suspension upon storage at several temperatures (355): — Phage particles X 10^ per ml, after storage* Temperature — OF STORAGE, 3 days 12 days 29 days °C. 6 44 - 60 28 31 20 0.00005 37 37 15 0.0000009 56.5 18 0.001 * At start all preparations contained 36 X 10' particles of phage per ml. This emphasizes the fact that there is a marked interrelation be- tween the morphological and physiological properties of an organism. Ample evidence of this has been established for the rough and the smooth strains of bacteria. Apparendy such interdependence, though of a somewhat different kind, exists also among actinomycetes. The four genera of the actinomycetes have been shown to possess constant morphological properties, with a limited overlapping of the dif- ferent genera. These properties may be summarized as follows: The genus Actinomyces comprises the anaerobic pathogenic forms. It is characterized by a gram-positive, non-acid-fast, branching vegeta- tive mycelium. No aerial hyphae are formed. The mycelium tends to break up into bacillary forms. The genus Nocardia is characterized by the formation of an un- divided substrate mycelium in the early stages of development. Aerial mycelium may be formed among certain members of the group, but it is usually indistinguishable from the substrate mycelium. The non- septated hyphae of both the substrate and the aerial mycelium break Waksman — 68 — Actinomycetes apart into short rods and cocci, by a process of segmentation, compar- able to oidia formation. The spores germinate, giving rise to a true mycehum. Some of the members of this group are characterized by a marked plemorphism, being either acid-fast or non-acid-fast. The angular type of growth described for some of the actinomycetes is also a property of certain members of this group. In recent studies on the nocardias, Erikson (1151?) examined 300 strains, freshly isolated from soil or obtained from culture collections. On immediate isolation, only 9 per cent were partly acid-fast, but on subsequent cultivation on organic matter-rich media, this increased to 31 per cent. These strains ranged from those giving soft mycobacterial type of growth with transient vege- tative mvcelium and very sparse aerial mvcelium to the harder strepto- myces-like varieties. No evidence was obtained of any resting spores or chlamydospores in the vegetative mycelium; the aerial mycelium, if present, does not form any true spores. The nocardias were, there- fore, considered as asporogenous. The genus Stre^ptomyces produces a well-developed nonseptated mycelium. The vegetative mycelium does not divide during its de- velopment but gives rise to a somewhat thicker aerial mycelium, which is formed most readily on synthetic or poor media. The aerial hyphae produce straight or curved sporulating branches. These give rise to conidia, by a process frequently designated as fragmentation. The spores are produced within the sporulating hyphae and are separated from one another by a constriction process. Later they are liberated by constriction of the cell wall and, its subsequent dissolution. The process of segmentation or oidia formation may also occur among the members of this genus. The substrate mycelium may produce chlamy- dospores; the broken bits of mycelium also have the capacity of growing into a fresh mycelium. The genus Microinonospora is characterized by the formation of a well-developed branching mycelium, producing single oval spores on the tip of special sporophores or side branches. These spore-bearing branches may be single or much-branched, the latter giving rise to a mass of spores similar to a bunch of grapes. No surface growth is produced in liquid media, but abundant growth is formed when such media are stirred or shaken at frequent intervals, thus breaking up the spores, which give rise to new clumps or colonies within the media. Micromonosfora may be looked upon as the most highly developed group among the actinomycetes, placing the whole order Actinomv- cetales closest to the fungi. On the other hand, the genus Nocardia is in many respects related to the mycobacteria, and, through them, to the true bacteria. Chapter IV VARIATIONS AND MUTATIONS No other branch of biology offers so rich a field for the study of variations and mutations and for the rapid selection of new varieties, as that of microbiology. This is due to the simple fact that many gen- erations of organisms can be obtained in a very short time. The fact that these organisms can be grown in an absolutely pure culture, free from any other organisms, and that the composition of the medium and the conditions of growth are easily controlled are other contributing factors. Among the various groups of microorganisms that are readilv subject to variations and mutations, the actinomvcetes occupy a prom- inent place. Actinomycetes are greatly influenced, especially in their cultural characteristics, by the composition of the medium and by the conditions of growth. Variations resulting from cultural differences have often led to expressions of doubt concerning the existence of definite types or species among the actinomycetes (261). Because of this doubt, the use of "species-groups" rather than of definite species for the classification of actinomycetes has been suggested (443). The general appearance of the actinomyces colony, the abundance and formation of aerial mycelium, the manner of sporulation, the production and nature of endopigments and exopigments, and the vital- ity of the organism when grown on different media make up the variation complex of actinomycetes. Types of Variation:— General variations.— Early students of actinomycetes recognized the fact that variations among actinomycetes are of several types. Lieske (260) demonstrated that actinomycetes show greater variability in their morphological and physiological properties than do any other group of microorganisms. He classified the types of variations as (a) simple modifications, (Z?) permanent modifications, and (c) mutations, includ- ing the formation of sectors within a colony. Waksman (446) empha- sized that the variations among actinomycetes differ in quantity and in quality, not only under the influence of various environmental condi- tions but even on continued cultivation under the same conditions. Tjhe soluble pigment may be lost or changed in color; the color of the aerial mycelium may change; even the property of forming aerial 1^ ^ r «> .''« ' '"^-^ ^^^^H H^*.-^^^ ^ F ^ ^^^^1 ik-^*ll ^■^ * •'-•^^ • ^ . ^ ^^B '4^Pl^r "^ ^ r^ ^f,^;^ Fig. 17.— Variants of Stre'ptninyces griseus growing on yeast extract glucose agar Qrom Dulanf.y, Ruger, Hlavac, 101a). Chapter IV — 71 — Variations and Mutations mycelium may be lost. The size, shape, and color of colonies, the length and abundance of mycelium, and the manner of spore formation are influenced by the composition of the medium and the age of the culture. In more recent studies (199) the general variations among the actinomycetes have been divided into three classes: (a) adaptive, or amenable to environment; (1?) continuous or fluctuating, as shown by diff^erences in the colonies plated out from the same culture; (c) devel- opmental, resulting in saltations or mutations. The adaptive type is usually characterized by a decrease in the size of the colony, a loss of the capacity to form sporogenous hyphae, a reduction of the ability to utilize certain nutrients, a change in pigment production, and a loss in the capacity to produce antibiotic substances. The continuous type is most clearly marked by the nature and intensity of the pigment pro- duced by the organism, as well as by the capacity to produce a given antibiotic. The developmental variations are expressed in the pres- ence or absence of aerial mycelium, pigmentation of the vegetative or aerial mycelium, and production of antibiotics. Some of these changes can be reversed to the original by growing the organism on special me- dia, such as glycerol nutrient agar, or in some natural medium, such as sterile soil. Other variations or mutations are more permanent or stable in nature, although only on rare occasions. In spite of these many types of variations, the constancy of strains or species of actinomycetes can be maintained if proper care is taken in growing the cultures on suitable media. The recognition of this fact has led some investigators (98, 318) to emphasize the constancy of the characters of actinomycetes, as contrasted to others who denied such constancy. Tempel (414) observed that several actinomyces strains failed to show, under constant conditions of culture, any sudden changes either in morphology or in physiology, which could be considered as muta- tions. The physiological changes due to the effects of temperature, aeration, reaction and composition of medium w^ere confirmed, but these changes were not permanent in nature. Rippel and Witter (361) could not obtain any variabilit)^ among several actinomycetes, either by changing cultural conditions or by irradiation by means of Rontgen rays or ultraviolet rays. Spontaneously occurring sectors gave normal cultures on transfer. Hereditary variations.—Several specific forms of hereditary variation among actinomycetes have received particular consideration. It is suf- ficient to mention the following: (a) transformation of an actinomyces into a mycobacterium-like organism (328, 378), the former being re- generated by cultivation on certain media, such as potato; (I;) trans- formation of an actinomyces into diphtheroid organisms (211); (c) transformation of anaerobic, short hyphal-producing forms into aerobic, long hyphal forms (328); (t?) change of aerial mycelium and strepto- Waksman — 72 — Actinomycetes mycin-producing strains of S. griseus into inactive strains free from aerial mvcelium (395); (e) change of strain of S. griseus from a color- less ^'egetative culture to a pink variant, accompanied by a change in antibiotic-producing capacity. Early students of the actinomycetes (388) observed that the acid-fast organism which causes infection in man gives rise to two subtypes, one simple in nature and liquefying gelatin, and the other producing pseu- dotubercles and not liquefying gelatin. The second form was looked upon as intermediary between actinomycosis and tuberculosis. Numerous references are found in the literature (188) to the trans- formation of actinomycetes, under special conditions of culture, into mycobacterium-like organisms, or into diphtheroid organisms, and vice versa. Of special interest are the transformations of anaerobic, short- hyphaed forms of actinomycetes into aerobic, long-hyphaed forms. Dis- sociation of pathogenic actinomycetes into aerobic and anaerobic strains has frequently been reported (427). It has also been reported (319) that two sorts of anaerobic colonies were isolated from the pus of actino- mycosis, one smooth and composed of gram-negative rods, and the other adherent and composed of gram-positive filaments. These were looked upon as S and R forms of the organism; even a transitional O form was recognized. These variations have often been considered as a part of the life cycle of the organisms. The composition of the medium, that is, whether complex organic or simple inorganic, protein-rich or carbo- hydrate-rich, and its reaction greatly influence the stability of the cul- ture, or the cycle of growth of actinomycetes. This is true also of environmental factors, especially moisture content, aeration, and tem- perature. The presence of other organisms, resulting in antagonistic and associative eff^ects, likewise influences the variation of the culture. Individual variations and group variations may also be distinguished. The size of mycelium fragments, the formation of grains in the disinte- gration of the cells, the formation of conidia and chlamydospores— all influence the cycle of growth of the individual organism, with the re- sulting variations and modifications. The problem of cell polymorphism among actinomycetes has also aroused much attention. This property must be taken into considera- tion in placing any organism in its taxonomic position (191). The formation of new races or strains can be accounted for on the basis of changes, which are expressed by the surface appearance of the colony, whether smooth or rough, by the presence or absence of aerial myce- lium, by the manner of sporulation, by changes in pigmentation, and by other cultural characteristics. Mutations.— The formation of saltants or mutants by actinomycetes must be regarded as in a class by itself, distinct from the variants. The mutations may be said to include the following types: formation of white strains from blue forms; formation of strains free from aerial mv- celium from strains producing such mycelium or vice versa; formation Chapter IV — 73 — Variations and Mutations of sectors pigmented red among orange-yellow strains. These saltations, are accompanied by morphological, cultural, and physiological characters which are quite different from those of the mother cultures. These new strains are so distinct that they might be considered new species, in accordance with the accepted systems of classification. Stable mutants or saltants were obtained and studied in detail by Kriss (242) and Krassilnikov (234). Jensen has shown (188) that under the influence of ultraviolet rays or even spontaneously, two strains of Nocardia isolated from Australian soils gave rise to new forms, some of which resembled t\'pical species of Streptowyces and others of which were closer to the mvcobacteria. Jensen (191) also observed that under the influence of LiCl mycobacteria gave rise to forms that might be con- sidered as species of Nocardia. Recentlv, extensive investigations have been made of the effect of ultraviolet radiations and x-ravs in inducing mutations of various species of Streptomyces. Savage (385) reported that ultraviolet rays were less mutagenic than the x-ravs, the harder rays of 0.710A and 0.210A wave lengths being most efficient. Mutation rates increased with kill- ing rates up to 99.9 per cent of killing. When doses of 1,000,000 roent- gens were used, as high as 50 per cent mutation rates were observed on morphological properties and 40 per cent on streptomvcin production. Bv means of x-rav and ultraviolet light irradiations, Kellner (213) found that most antibiotically inactive cultures gave rise to antibiotic- producing mutants. Of the greatest interest was the fact that a strain of S. griseiis kept for a long time in the culture collection and which was inactive antibioticallv was induced to form a mutant which produced streptomycin. The frequency of active mutants ranged from 0.01 to 1.2 per cent; mutants obtained from the same parent culture varied in their antibiotic spectra. The viability of conidia exposed to ultraviolet ir- radiation could be recovered by illumination with visible light (214). The varations or mutations mav thus influence not only the species characteristics but also the generic characters. Krassilnikov empha- sized that these changes take place from the simpler to the more com- plex forms, as from micrococci to mycobacteria, from mycobacteria to nocardias, and from nocardias to streptomvces; the reverse phenomenon occurs but seldom. This reasoning led Krassilnikov to the conclusion that actinomycetes are present in natural substrates, such as soil, largely in the form of micrococcus stages. Kriss (240, 242) recognized four types of variation— morphological, cultural, physiological, and applied. These may be briefly summarized as follows: Morphological xmriations.— Some of the morphological variations re- ported may be considered here in further detail. Jensen (188) described the production from single-cell cultures of Nocardia foly- chromogenes of two different forms, one a rod-shaped or R-form, and the other a filamentous or F-form. The R-form produces initially a Waksman — 74 — Actinomycetes small unicellular mycelium which soon divides into bacteria-like ele- ments; these multiply by cell division in the manner characteristic of corynebacteria. Two subtypes were recognized for the R-form: the soft or s-t)'pe and the hard or h-type. The s-type, which is the original, pro- duces a soft, pasty growth of red color; the bacteria-like elements are usually short, blunt, little-branched, and partly acid-fast. The h-type produces a dry, crumbly growth, adhering firmly to the medium and consisting of longer and more slender cells, less acid-fast than the s-type and with a marked tendency to form long filaments. The h-type arises spontaneously in, and can also be produced experimentally from, cul- tures of the s-type. Exposure of the h-type to ultraviolet rays gave rise, for example, to a yellow and a white variety of the s-type. The s- and h-types were believed to correspond to the plane and perrugose variants of mycobacteria, and were also comparable to the smooth and rough variants among other bacteria. The F-form represents a stabilization of the initial mycelial stage of the R-form. It is an actinomyces-like organ- ism, consisting of long, delicate, branching hvphae, with a well-devel- oped aerial mycelium, and without any tendency to divide by septa into bacteria-like elements. The F-form was found to arise spontaneously in old cultures of the s-type, but not in the h-type. Its appearance did not seem to be influenced by external factors. Novak and Henrici (326) reported the appearance of a yellow staphylococcus in a Berkefeld filtrate of a broth culture of a saprophytic actinomyces. Under the microscope, the staphylococcus was observed to change first into rods, then into long, branched filaments which could not be distinguished from true actinomyces mycelium. The reverse changes were also observed. The coccus was found to dissociate first into S- and R-forms, then into filterable G-forms. These observations were believed to support the theory that staphylococci are related to the actinomycetes. As pointed out above Krassilnikov described the micrococcus as merely a stage in the normal development of the nocardia rather than as an abnormal mutant. Certain of the characters of actinomycetes appear, however, to be far more constant than those listed above. These include the formation of aerial mycelium on specific media, the formation, nature, and direction of the spirals, the manner of spore formation, and the size and shape of spores. Only seldom do variations occur in such specific characteristics as abundance of the mycelium, lengthening or shortening of hyphae, and size of spores (242). Cultural variations— Among the cultural variations, those of pig- mentation are most striking, since pigments are widely distributed among actinomycetes. This is of particular significance in view of the fact that differentiation of many species is based upon the nature and intensity of the pigment. Even the major subdivisions of some of the groups of actinomycetes have been based upon pigmentation, as was done by Sanfelice, Duche, and others. Evidence of this is found in Chapter IV — 75 — Variations and Mutations the designation of such groups as alhus, flavus, and violaceiis. Waks- MAN also proposed a key for the separation of species of actinomycetes on the basis of the pigment produced on organic and synthetic media, in- cluding soluble and insoluble (or exo- and endo-) pigments. More detailed study has revealed, however, that on continued culti- vation of organisms, the pigment undergoes changes in its nature, or it disappears altogether. Thus an organism designated as A. verne, be- cause of the soluble green pigment produced in the medium, lost that property on continued cultivation. When the characters of an organism are based on pigmentation, it becomes very difficult to make comparisons even if type cultures are available. Thus, one of the most widely used cultures of actinomycetes, the streptomycin-producing strain of S. griseiis, can hardly be recognized either by comparison with the original cultures of Waksman and Curtis or from the original description of Krainsky, since the type culture lost its characteristic pigmentation and Krainsky's description did not quite correspond with the published description of its pigmentation. Among the other cultural variations reported for actinomycetes, the lytic activities of many of the strains deserve consideration, as pointed out previously (p. 60). The phenomenon of Ivsis, whether considered as a part of the life cycle of the organisms or looked upon as stages of de- generation of a culture, has a bearing upon the production of new types. This holds true also for the effect of phage upon the del^elopment of resistant strains. Marked variations in agar-decomposition and pigmentation of S. coeJicoIor have also been observed (408). Erikson (115a) found that the major variations of S. coelicoJor comprise loss of pigmentation, loss of aerial mycelium, and occasionally also loss of agar-liquefaction. Sin- gle spore isolations from aerial mycelium brought out the possibility of inherent differences in the sister spores of the same chain. Spontaneous occurrence of variants may be found more readily in the spores of de- generate colonies, rendered atypical by artificial methods of cultivation, than in the spores of the aerial mycelium of typical colonies. In an agar-liquefying strain, 3 out of 15 spores lost the power to produce the pigrhent and to liquefy agar. A non-agar-liquefying strain, which had lost the power of pigmentation, gave a variant which produced sectored colonies, some of which possessed the blue pigment. Physiological and applied variations.— These can best be described by an analysis of the variation of two important economic groups of actinomycetes, namely, those that cause potato scab and those that pro- duce antibiotics. These physiological variations are usually more quan- titative than qualitative in nature. Potatoes show considerable variation in their resistance to scab. This has been ascribed either to differences in the environment in which the potatoes are growing or to physiological differences of the strains of S. scabies, the causative agent of infection. Waksman — 76 — Actinomycetes ScHAAL (387) has recently shown by means of sectoring of S. scabies strains that as many as nine sectors appeared in a single colony. The sectors varied in the nature of their mycelium, in the rate of growth of the culture, and in pigmentation. Thus the variants showed not only differences in physiological characteristics from that of the parent cul- ture, but even in morphology. The formation of spirals and the direc- tion of turns varied with the culture. There was little variation, how- ever, in the size of the cells. The effects of nutrition were particularly marked. Production of aerial mycelium was inhibited by a high nitrogen content of the medium. The presence of thiamine favored rapid growth of the cultures and pro- duction of sectors. Various cultures of S. scabies isolated from diseased potatoes differed considerably in their pathogenicity. There was no correlation, how- ever, between the pathogenicity and the cultural characteristics of the strain. The variants obtained from a given culture also differed from the mother culture in their pathogenicity to potatoes. Thomas (421) isolated six physiologic races of S. scabies which distinctly differed in pathogenicity on ten different potato varieties or selections. The most favorable sources of carbon for the growth were sucrose, cellulose, inulin, and maltose. Increasing the nitrogen, phos- phorus, and potash content of the medium retarded the production of aerial mycelium. Nitrogen and phosphorus were generally favorable for growth; potash tended to retard it. The different races also showed marked variation in their sensitivity to antiseptics and to extracts of the mycelium of certain fungi. Maximum growth and stability were ob- served on peat soil; mineral soils tended to retard or inhibit growth and increase variability in the races studied. The more pathogenic races were most stable on most media. Some variant types were peculiar to individual races, but certain types were produced frequently by several races, which pointed to a close genetic relationship between those races. These variations make one wonder, therefore, whether the many species described (298) as causative agents of potato scab represent dis- tinct species or only variants of one type of culture. Another important economic group of actinomycetes, namely, the organisms producing antibiotics, show marked variation in culture. Several variants were obtained from S. grisetis. They differed morpho- logically in formation of aerial mycelium, and physiologically in produc- tion of streptomycin, formation of acid, rate of glucose consumption, autolysis, and production of pigment. Intermediary variants were also obtained. The freshly isolated streptomycin-producing strain of S. grisetis formed typical aerial mycelium, characteristic of the species. It changed the reaction of a glucose-containing medium to alkaline, pro- duced characteristic types of surface and submerged growth, underwent only limited lysis, and was markedly resistant to the antibiotic action of Chapter IV — 77 — Variations and Mutations strcptomvcin. On the other hand, the nonsporulating variant produced no aerial mycehum, formed no streptomycin, was sensitive to the antibi- otic action of this substance, was characterized by a tjq^e of growth that in shaken cuhure underwent rapid lysis, and produced acid in the glu- cose-containing medium. Both strains otherwise possessed the various cultural properties which are characteristic of the S. griseus species as a whole, such as lack of pigmentation in organic media and proteolytic and diastatic properties. The nonsporulating strain, when isolated as such, however, would hardly be recognizable as typical S. griseus. In view of these variations, the question was raised: Is it possible that many of the Nocardia species represent degenerate forms of Streptomyees? Another variant of S. griseus produced a red-pigmented vegetative growth. This was accompanied by a loss in capacity to produce strepto- mycin; in its place another antibiotic, pigmented red and active only Table 8 : Production of streptothricin by tivo strains of S. lavendulae and their variants (478) : — Strain or variant Str; EPTOTHRICIN* Strain No. 8 25 Variant 8a <2 Variant 8b 50 Strain No. 14 23 Variant 14a 23 Variant 14b * Units of streptothricin produced in shaken cultures, after 4 days incubation. upon gram-positive bacteria, was formed. This culture if freshly iso- lated from a natural substrate would definitely not be considered as S. griseus. Another antibiotic-producing organism, S. lavendtdae, was also found (478) to vary greatly in culture (Table 8). The variants dif- fered in the amount and nature of soluble pigment in peptone-contain- ing media, in the presence and nature of aerial mycelium and in its pigmentation, and in the production of streptothricin. One strain of the organism gave rise to two variants: one producing bluish colored vegetative growth, initially blue diffusible pigment, and a lavender- colored aerial mycelium with a slightly blue tinge; and the other pro- ducing cream-colored vegetative growth, a soluble brown pigment in peptone media, and a lavender-colored aerial mycelium. Two variants were also isolated from sectors of colonies of another strain of S. laven- dulae: one forming a white aerial mycelium, sometimes showing a faint shade of pink; and the other devoid of aerial mycelium, except for a scant growth of sporulating aerial hyphae on some of the old slants. When the ability of these four variants to produce streptothricin in Waksman — 78 — Actinomycetes shaken cultures was compared with that of the original culture, the first variant of the first strain was almost completely inactive, whereas the second variant of that strain was more active than the parent strain, and the strains free of aerial mycelium were completely inactive. The production of streptothricin by S. lavendulae was thus associ- ated with the ability of the culture to form aerial mycelium, similar to one of the variants of S. griseus. In both cases, the variants which failed to produce aerial mycelium likewise produced culture filtrates which possessed no antibiotic potency. Aerial mycelium is not, of course, a determinant for the formation of antibiotics, since numerous cultures of both S. griseus and S. lavendulae which produce abundant aerial mycelium are unable to form the respective antibiotics. Jones (201) examined 1,298 freshly isolated cultures of Strepto- myces. About 20 per cent of these showed from the start considerable fluctuation in the production of aerial mycelium, 6 per cent producing only vegetative growth in the first transfer. The question was, there- fore, raised: How many representatives of the fluctuating group may be assigned to the genus Streptomyces or Nocardia? Culture Constancy:— In the utilization of actinomycetes for the production of antibiotics, it is highly important to be able to depend upon the constant characters of a culture. In view of the fact that a given organism may be subject to a great many variations due to con- ditions of cultivation and environment, as well as to other conditions, it is essential to be able to come back to the original culture. With the purpose in view, a given culture is usually inoculated into moist sterile soil. After the culture has made some growth, the soil is allowed to dry out. The culture can thus be kept for a considerable time. When required, the soil is plated out and the culture reisolated (200). On comparing cultures kept in soil with similar cultures kept in synthetic media, it was found that the latter tended to lose their capacity for producing aerial mycelium and for abundant sporulation (115). Chapter V METABOLISM OF ACTINOMYCETES-GROWTH AND NUTRITION; PRODUCTION OF ODORS AND PIGMENTS Nutrient Requirements:— Actinomycetes vary greatly in their nu- trient requirements. Some are able to thrive on very simple com- pounds, whereas others grow only on highlv complex organic materials. Moreover, the same organism may be able to adapt itself to a great variety of nutrients, the amount of cell synthesis depending on the availability of the substrate and on the effect upon the growth of the organism of the secondary changes in the medium resulting from the utilization of the particular substrate. Beijerinck (28) was the first to point out that certain organisms are capable of deriving their carbon nutrition and energy needs from the simplest compounds found in the atmosphere. Although he desig- nated one particular organism as a bacillus (B. oJigocarhophilus^ , he himself noted: "We also found another, rarer species belonging to the genus Streptothrix Cohn, with corresponding properties." Lantzsch (251) later demonstrated that even the bacillus of Beijerinck was in reality an actinomyces. Although Beijerinck doubted the need for S, Mn, and Fe in the medium, he emphasized the importance of N, P, K, and Mg. The medium he used consisted of 0.1 to 1.0 gm KNO3, 0.2 gm K0HPO4, 80 mg MgS04.7H,0, 0.5 mg MnS04.4HoO, 0.5 mg FeCU.SHoO per liter of distilled water. When such a medium was inoculated with a small quantity of garden soil, and flasks were plugged with cotton and incubated at 23°-25°C., there appeared "a thin, white or feebly rose- colored, very dr)^ film, difficult to moisten." The medium remained clear. The growth of the film continued for months and resulted in the accumulation of considerable amounts of organic carbon. Beijerinck found that either nitrate or ammonium salt could be used as a source of nitrogen. The carbon was not derived from the COo, but from the volatile carbon compounds of the atmosphere. Beijerinck ascribed to this organism the property of biologic purification of the air. Lantzsch (251) differentiated between the nutrition of two variants of the organism: the filamentous or branched form which assimilated CD; the coccus-like or bacillary form, which assimilated aliphatic hydro- carbons. Waksman — 80 — Actinomycetes In contrast to this simple mode of nutrition, the other extreme may be found in a manure pile. When stable manure consisting of animal excreta mixed with bedding is allowed to lie in an open pile, with air freely admitted, rapid decomposition sets in, as can easily be detected by extensive solution of COo and NH3 and by a rise in temperature. When the temperature reaches 60° to 65 °C., numerous white patches can be seen throughout the pile. When slides are buried in such a pile, then removed and stained, actinomyces growth is found in great abun- dance. Attempts to cultivate these organisms meet with great difficultv, however, largely because the artificial conditions of nutrition do not quite approach the natural nutrients and environment. The actinomycetes as a group obtain their nutrition between the two extremes illustrated. It is no wonder, then, that a great variety of media have been introduced for the growth of actinomycetes. These media are synthetic and organic in nature. For the purpose of cultivation, especially for determining the morphological and cultural properties of the organisms, synthetic media are commonly used. A number of such media are described in the appendix. For certain purposes, however, organic media are required. This is true particularly in the growth of S. griseits for production of streptomycin. In this case, a complex or- ganic substance in the nature of meat extract, yeast extract, corn steep, soybean meal, and others is found necessary for the rapid production of the antibiotic. Although streptomycin and streptothricin can be pro- duced in simple synthetic media, the process is much slower and lower yields are obtained. Carhon sortrces.— Under natural conditions, actinomycetes live on a large number of substrates. They are able to utilize a great variety of simple and complex organic compounds as sources of carbon and of en- ergy. These compounds include organic acids, sugars, starches, hemi- celluloses and cellulose, proteins, polvpeptides and amino acids, nitrogen bases, and many others. Certain actinomycetes can also attack, to a more limited extent, fats, hydrocarbons, benzene ring compounds, and even such resistant substances as lignin, tannin, and rubber. There is considerable selectivity in the utilization of these materials, some sub- tances being consumed far more readily than others. Glucose, maltose, dextrin, starch, glycerol, organic acids, and proteins are the best sources of carbon; these are followed by sucrose and other sugars, by sugar al- cohols, and by sugar acids (309). Cellulose is attacked only by certain organisms. Agar also can be used as a source of carbon and energy by some actinomycetes, notably certain strains of S. coelicolor (363). As a rule, actinomycetes prefer proteins to carbohydrates as sources of carbon. This preference is so pronounced that when a protein or a protein derivative, such as peptone, and glucose or another available carbohydrate are present in the same medium, an actinomyces attacks the protein first, not only as a source of nitrogen but also as a source of Chapter V — 81 — Metabolism carbon, and liberates considerable waste nitrogen in tlie form of ammonia (Tables 9, 10). Most media for actinomycetes contain a certain amount of carbo- hydrate. This is included to enable the organisms to make more ex- tensive growth and to serve as a buffer, since with proteins and protein- derivatives as die onlv source of carbon, ammonia accumulates so Table 9: Decomposition of different amino acids by microorganisms (472): — 100 ml medium containing 1 per cent of amino acid G ROWTH, NH,-N Amino acid Organism DRY WEIGHT PRODUCED Glycine Strep tomjces sp. Trichoderma sp. 59 50 mg 31 24 Alanine Streptomjices sp. Trichoderma sp. 126 80 39 22 Glutamic acid Streptomyces sp. Ps. fltiorescens Trichoderma sp. 169 128 218 28 29 29 rapidly as to make the medium too alkaline for further growth of the organism. That the favorable effect of glucose in increasing the growth of actinomycetes in the presence of peptone or protein is due, partly at least, to the neutralizing effect of the ammonia produced from the pep- tone by the acid produced from the glucose was known to some of the early investigators of this group of organisms (323). In the case of ty- rosin utilization by actinomvcetes, glucose had a favorable effect upon Table 10: Decomposition of glycine by different microorganisms in presence of glucose (472): — 100 ml medium containing 1 per cent glycine and 2 per cent glucose Glycine-N Glucose Growth, NHs-N Organism decomposed decomposed dry w^eight produced Ps. fluorescens mg 26 mg 820 mg 99 mg 3 Streptomyces sp. Trichoderma sp. 36 49 570 1,490 213 804 19 1 the growth of only those organisms that were able to form, from tyrosin, substances that neutralized the acid produced from the sugar. Certain amino acids, like leucine, were utilized by actinomycetes only in the presence of an available carbohydrate. Urea can serve only as a source of nitrogen; not of carbon (125, 307). Actuallv, urea is produced by certain actinomvcetes from pep- tone (160). Waksman — 82 — Actinomycetes Among the organic acids, formic, oxalic, tartaric, and hippuric are unfavorable carbon sources; acetic, citric, and malic are favorable (307). Ethyl alcohol and ethylene glycol (also erythritol and dulcitol) are un- favorable sources; on the other hand, glycerol and mannitol are highly favorable. Starch is an excellent source of carbon for a large number of actinomycetes. Various hemicelluloses, such as mannans, are readily utilized. The ability of actinomycetes to utilize carbon sources has often been used as an aid in species differentiation, especially among species of Streptomyces. Pridham and Gottlieb (348) reported that all species are able to utilize ci-glucose, if-mannose, starch, dextrin and glvcerol, but not erythritol, phenol, cresol and the sodium salts of formic, oxalic and tartaric acids. Certain compounds, however, may be utilized by certain species and not by others. This is true of rhamnose, raffinose, xylose, lactose, mannose, dulcitol, inositol and the sodium salts of acetic and succinic acids. Since the wide interest in the production of antibiotics by actinomy- cetes has arisen, numerous investigations have been carried out on their ability to utilize carbon sources from the point of view of the production of a particular antibiotic. In the case of streptomycin, for example, pentoses were poor carbon sources; among the hexoses, glucose and man- nose were best, the latter being particularly effective when combined with 1 (— ) proline, maltose was the best of the disaccharides; the trisac- charides were inferior; of the polysaccharides, inulin was inferior to starch and dextrin, among the alcohols, mannitol was a promising car- bon source; none of the organic acids proved to be suitable (100). Nitrogen sowrces.— Actinomycetes are unable to fix atmospheric ni- trogen. Proteins, peptones, and amino acids form the best sources of this nutrient for actinomycetes. These are followed by nitrates and ammonium salts. The former is sometimes considered (309) better than the latter, possibly because of the residual effect of the basic ion left from the nitrate as compared with the acid ion left from the ammonium salt, which makes the medium less favorable for the growth of actinomy- cetes. Ammonium sulfate is utilized better than ammonium chloride. Urea and uric acid are readily utilized and converted into complex organic compounds. Some forms of actinomycetes are able also to use nitrites in low concentrations as sources of nitrogen. The capacity for decomposing proteins is widely distributed among actinomycetes. This is shown by the fact that the great majority of actinomycetes are able to liquefy gelatin; the only nonliquefying forms so far known are among the nocardias, notably N. asteroides. Actino- mycetes are able to coagulate milk, and later peptonize it; in fact, pep- tonization commonly occurs without previous coagulation. Coagulation of the milk is a proteolytic effect, due to formation of specific enzymes, rather than an acid effect. Blood serum is liquefied by many of the species. Complex proteins, such as hoof meal and horn meal, are also Chapter V 83 Metabolism attacked (308). Many forms are able to utilize complex humus com- pounds of soil, as will be shown later. Certain organic compounds, such as phospholipids, favor extensive vegetative growth of actinomycetes. When living or heat-killed, washed suspensions of bacteria are used as sources of nitrogen, growth of actinomycetes is not enhanced, even in the case of those forms which have the capacity to bring about lysis of the bacteria. This led Erik- son (115) to conclude that only small quantities of nitrogen result from the lysis of bacteria, in fact, little more than those found in autolyzates of suspensions of insusceptible bacteria. Mineral nutrierits.— Among the mineral nutrients, phosphorus, potas- Table 11: The iitilixation of carbon and nitrogen sources by S. coelicolor (78):- Rela- Rela- tive tive pig- Nitrogen pig- Carbon Rela- ment source! Rela- ment source* tive inten- Final 0.106 tive inten- Final gm/1 growthf sityt /'H gmN/1 growthft sity /-H None 17 8.4 None 30 32 6.7 ^-Glucose, 10 100 100 7.2 1-Asparagine 100 100 6.7 J-Mannose, 10 202 200 7.0 Glycine 83 36 7.0 ^-Galactose, 10 84 97 7.1 /-Leucine 76 36 7.0 ^-Fructose, 10 79 96 7.0 /-Tryptophane 98 82 6.9 ^-Xylose, 10 143 121 7.2 Urea 86 51 7.0 /-Sorbose, 10 26 8.5 NaNO., 18 6.0 /-Arabinose, 10 65 46 5.2 (NH4)2HP04 57 5.7 Starch, 10 107 87 7.0 Ammonium Inulin, 10 32 8.5 acetate 18 5.6 Trehalose, 10 90 38 8.1 Peptone 146 118 6.8 Cellobiose, 10 81 95 6.7 Tryptone 91 106 7.0 Maltose, 10 62 43 7.3 Casitone 116 100 7.0 Lactose, 10 105 64 6.8 Pepticase 175 118 6.4 Sucrose, 10 34 8.4 Cassamino acid s 116 129 6.6 Glycerol,, 10 135 170 6.6 Sodium casseinate 72 53 6.9 Mannitol, 10 82 88 7.1 Gelatin 106 29 6.4 Dulcitol, 10 20 8.6 Egg albumin 44 35 6.0 Sorbitol, 10 27 8.2 Acetic acid, 5 33 33 8.4 Lactic acid, 5 63 8.8 Fumaric acid, 5 69 9.1 Succinic acid, 5 47 8.9 ^/-Malic acid, 10 60 8.9 Tartaric acid, 5 20 8.7 Citric acid, 5 24 8.2 Gluconic acid, 5 82 25 8.2 'Basal medium (g m/l): aspa aginc-0.5, yeast ex ract— 0.5, K2HPOi— 0.5, MgS04 . 7H20- -0.25, and minor ele- . t Dry weight and pigment intensity of glucose control taken as 100. i Basal medium (gm/1): glucose— 10.0, yeast extract— 0.5, K2HPO4— 0.5, MgS04 . 7H2O— 0.25, tt Dry weight and pigment intensity of asparagine control taken as 100. Waksman — 84 — Actinomycetes sium, and magnesium are usually required in varying concentrations; the need for sulfur, calcium, and iron is often questioned, although there is no doubt that certain organisms, such as the grisein-producing types, benefit considerably from the presence of iron. Traces of other elements, such as zinc are frequently found to have a marked effect upon the growth of certain organisms. Although actinomycetes are found only seldom in salt water basins (522), their sensitivity to higher concentrations of different ions may be of interest. KoBER (226) reported that some actinomycetes are able to withstand a high salt concentration, 0.5 to 0.6 molar solutions giving the largest sized colonies, although the smallest number, pointing to the selective action of the salt. He also found, that although MgSOj is not essential Table 12: Metabolic changes and efficiency of carbon utilization of S. lavendulae in aerated cultures (517): — Tryptone Glycine medium medium Mycelium, dry weight, 7ng Glucose consumed, mg NH3-N liberated, mg Nitrogen compounds deaminated, 77ig Lactic acid produced, mg Volatile acid as acetic, mg Conversion of glucose to lactic acid, pr cent Conversion of glycine to acetic acid, pr cent Efficiency of carbon utilization, ^er cent for growth, it has a favorable effect in fairly high concentrations. Cal- cium is not essential for growth, but its lack in the medium has an in- jurious effect unless magnesium is present. Potassium is injurious in concentrations of 0.5 per cent KCl, which can be neutralized, however, by high concentrations of MgS04. Growth and Cell Synthesis of Aerobic Actinomycetes:— In ordi- nary stationary cultures, actinomycetes produce on the surface of the medium a compact pellicle which may be continuous or which may consist of discontinuous masses of growth or even of individual colonies. Occasionally, growth takes the form of a surface ring along the wall of the vessel. Many species give rise to masses of growth only on the bot- tom of the vessel or in the form of flakes or colonies throughout the medium and on the bottom. This type of growth is greatly influenced by the nature of the spores, the amount of air admitted, and the agita- tion of the cultures, which results in breaking up of the mycelium or of the spores, thus initiating fresh growth. In a submerged state, growth of actinomycetes is usually in the form 101 106 488 • 782 4 22 92 162 126 58 4 13 26 8 10 25 14 Chapter V 85 Metabolism of flakes or small colonies. These organisms never produce a diffused type of growth throughout the medium, as do the bacteria. The only reports of causation of turbidity can be traced to some of the so-called bacterial variants. The growth of the actinomycetes can be easily filtered off through paper or with some filter aid, or it can be removed by centrifugation. Those organisms which, like the Microinonosfora, produce only a limited number of spores, grow much better in a sub- merged and aerated state than in stationary culture. The amount of growth produced by actinomycetes depends not only on the nature of the organism but also on the nature of the nutrients, Days Shakzn Stationary Fig. 18.— Metabolic changes produced by S. lavendulae in aerated and stationary cultures Qrom Woodruff and Foster, 517). their availability, their concentration, and on the environmental factors, such as reaction, buffering of medium, aeration, temperature, stage of growth, and lysis. These factors influence not only the total amount of growth but also the mechanisms of transformation of the constituents of the medium, that is, the physiology of the organisms (Table 11). In general, there is a definite relation between the concentration and availability of the carbon and nitrogen sources in the medium and the amount of cell material synthesized by actinomycetes. This is brought out in Table 12 and in Fig. 18 (517). The efficiency of carbon utiliza- tion is greater in stationary than in submerged cultures. The maximum grpwth is attained, however, in submerged and aerated cultures. The carbon efficiency of S. lavendulae attains 35 per cent in stationary cul- Waksman — 86 — Actinomycetes tures, the corresponding efficiency under submerged conditions being only 21 to 23 per cent (517). As growth progresses, the carbon effi- ciency drops. After growth of the organism reaches a maximum, less synthesis takes place. If lysis sets in, the mycelium is destroyed and CO2 and NH3 are liberated. The ratio of consumption of carbohydrate to utilization of nitrogen depends upon conditions of growth, nature of organism, and age of cul- ture. With sugar and tryptone in the medium, the ratio increases to about 300 per cent as growth advances, thus pointing to greater oxida- tion of the carbohydrate in relation to the utilization of tr)'ptone for cell synthesis. This is true especially for submerged cultures, where the abundance of available oxvgen leads to greater oxidation of carbohydrate as compared to the tryptone consumed. Acid froduction hy actinomycetes.— As a result of the growth of ac- tinomycetes in different media, there is always a tendency for the reac- Table 13: Acid production by an actinomyces on meat extract-peptone-glucose medium (340) : — Strain No. Final ;H* Lac tic acid 3 8.4 - 4 8.6 - 5 8.5 - 6 4.9 + 7 4.7 ++ 8 4.8 + 10 5.2 + + * Original pW of medium 5 8, 25 days incubation. tion to become alkaline unless ammonium salts or organic acids are the sole source of nitrogen, with the result that acid ions accumulate in the medium. In the presence of carbohydrates, however, certain organisms are capable of producing certain organic acids, the concentration of the latter depending on the nature of carbohydrate and its concentration. Sooner or later, however, the acid will be decomposed or the organisms will produce neutralizing substances, with the result that the reaction always tends to become alkaline. The tendency is toward the attain- ment of a maximum alkalinity, which is usually 8.6 to 8.8. The alkaline reaction thus produced by actinomycetes is largely due to certain secondary reactions in the medium, such as the accumulation of the basic ion (Na, K) when nitrates are used in the medium as sources of nitrogen, or to the formation of ammonium ions from pro- teins. The fact that certain actinomycetes do not occur in soils having a pH lower than 5.2 was at one time considered to substantiate the asso- ciation of actinomycetes with alkaline reactions. It has now been estab- lished, however, that even fairly acid soils contain a considerable num- Chapter V —87— Metabolism ber of actinomycetes (460). Jensen (185) isolated from a forest soil an actinomyces which even had a definite preference for an acid reac- tion; hence, he named it S. acidofhilns. The fact that various actinomvcetes are able to produce organic acids form carbohydrates has been long recognized (470). Magnus (281) obser\'ed that many of the actinomycetes found in the larynx are able to produce acid of the lactic t)npe (ether-soluble) even in sugar-free media. Plotho (341) confirmed these observations and definitely established the fact that the acid produced by various actinomycetes is of the lactic type, as shown in Table 13. Woodruff and Foster (517) established that S. lavendulae is also capable of producing considerable amounts of lactic acid from carbo- hydrates. The nature of the nitrogen source is of considerable impor- tance in this connection, as shown in Table 13. In the presence of Table 14: Acid formation frofn glucose hi aerated cultures of S. lavendulae (517): — Glucose p\ioi medium* after per cent 3 days 4 days 5 days 6 days 8.2 8.6 8.7 8.8 1 6.8 6.9 7.0 7.4 2 6.5 6.5 6.5 6.5 5 6.2 6.1 5.7 5.7 ♦ Initial ?H was 7.2. glycine, for example, much more sugar was consumed but less lactic acid produced than with tryptone as a source of nitrogen. This is particu- larly true for submerged cultures. In the absence of glucose or with only extremely low concentrations of sugar and in the presence of tryp- tone, ammonia will accumulate in the medium, gradually making it alkaline. In the presence of 1 per cent glucose, however, the pH is lowered appreciably even in buffered media, due to the formation of organic acids. In the presence of 2 per cent glucose, especially in un- buffered media the fW levels may go down as low as 3.2 in 2 days. The changes in reaction are thus parallel to the concentrations of sugar (Table 14). On the basis of the sugar consumed, lactic acid production was found to be equivalent to 25.8 and 7.5 per cent in tr)'ptone and glycine media, respectively. This high conversion took place under conditions of forced aeration. Other actinomycetes, especially S. griseus, also pro- duce lactic acid (395). This was found to hold true particularly for the degenerated strains which have lost the capacity to form aerial my- celium. In addition to lactic acid, S. lavendulae produces a certain amount Waksman Actinomycetes of a volatile acid, apparently acetic. The volatile acid was believed to be formed by the deamination of the glycine. Large amounts of am- monia also were found to accumulate in the medium as a result of proc- esses of deamination. On the basis of 162 mg of glycine deaminated, the volatile acid, calculated as acetic, amounted to 10.3 per cent of the glycine decomposed. Oxygen consumftion.—S. lavendulae oxidizes glucose and glycerol at a very high rate, the oxygen uptake being, respectively, 60 and 45 per cent of the theoretical. The incomplete oxidation is due to assimilation of some of the products for cell svnthesis and to the formation of incom- 4oo- 30O- 200- /oo- ^^ ~rryptone Consumption 7-0- PH [m^^^ y 1 • * -^ / 300- 22S- /so- 7S - y^ Dcxirx)Se Consumtion ^^iX>- AcHnomycin. / / ^" / / zoo- 300- /oo - ^ - Days Sftaken, -Shztianaru Days Fig. 19.— Metabolic changes produced by S. antihioticus in aerated and stationary cultures Qfrani Woodruff and Foster, 517). pletely oxidized products, such as lactic acid. When the organism is allowed to starve for 1 to 2 days in a phosphate buffer solution and un- der aerated conditions, autorespiration will proceed, at a reduced rate, the cells utilizing the reserve cell materials. Deamination.— Washed cell material of S. lavendulae, grown under submerged conditions, was shaken for 18 hours at 30° C. in media con- taining different amino acids and M/30 phosphate buffer at pH 6.8, and the relative deamination measured by ammonia formation. The majority of the amino acids were deaminated under these conditions, arginine and histidine being attacked most readily; (S-alanine was deam- Chapter V — 89 — Metabolism inated only about one-third as readily as J-alanine. Leucine, isoleucine, and certain other amino acids were not deaminated at all or only in mere traces. Utilization of comflex organic coinfounds.—\t has thus been estab- lished that actinomycetes are able to utilize a great variety of organic compounds as sources of both carbon and nitrogen. The nature of the nutrient influences not only growth of the organism, but also its cultural and physiological properties, such as pigmentation, as shown in Table II. Unfortunately, no satisfactory methods have been developed for measuring accurately some of the changes brought about in the decom- position of complex compounds, such as lignins and humus. Actinomycetes are able to attack numerous other complex organic compounds, such as salicylaldehyde (152), paraffin hydrocarbons (113, 521), rubber (405), and chitinous substances (398). Reduction of nitrates.— Niany actinomycetes possess the capacity of reducing nitrates to nitrites. The importance of this process in the nu- trition of the organisms has not been fully established, although a defi- nite parallelism has been observed between growth of the organisms and accumulation of nitrite in the medium. It has also been estab- lished that nitrite can be utilized as a source of nitrogen by many actino- mycetes, provided its concentration in the medium is not high enough to make it toxic (443, 444). Nitrate is never reduced to atmospheric nitrogen or to ammonia. Wherever these products have been reported, their formation was due to secondary reactions rather than to direct reduction of the nitrate. Gaseous nitrogen can be formed by interaction of nitrite and amino acids in an acid medium (2NOo- -f 4NHo -> 3N2 + 4HoO), a combination quite unlikely in cultures of actinomycetes. Ammonia can be produced in a culture containing nitrate when the synthesized cell material undergoes autolysis. Influence of environment on growth of actincnnycetes.— It is com- monly assumed that actinomycetes prefer a neutral or slightly alkaline reaction for their growth, and that they are especially sensitive to a high acidity; many species are not able to grow at pH 4.8 (136, 445), as brought out in Table 15. The inability of most actinomycetes to grow under acid conditions has been used to advantage in the control of cer- tain plant diseases in the soil, especially potato scab. The optimum temperature for growth of most of the actinomycetes usually falls between 23° and 37 °C. Certain actinomycetes are able to grow at temperatures lower than 20 °C. Some organisms prefer temperatures of 20° to 23 °C. Still others are thermophilic in nature and are able to grow at 50° to 65 °C. The more common forms, how- ever, are readily destroved at the higher temperatures, the resistance of the spores being onlv slightly greater than that of the mycelium. When a pulture is kept for 10 minutes at 70° C, not only the mycelium but even the spores lose their viability. Table 15: Influence of reaction on the decomposition of , protein-rich material by actinomycetes (445) : — 100 gm soil + 1 gm dried blood fW OF SOIL NHs-N produced in 28 days zbies S. viridochrotnogenus S . griseus 3.2 36 4.0 5.0 5.8 6.4 7.2 7.7 8.8 9.6 mg 2.2 28.9 68.3 64.3 66.9 62.6 0.4 3.1 0.9 9.4 47.5 65.2 63.3 63.6 mg 1.0 1.1 1.3 40.9 60.0 62.0 530 2.8 0.4 Table 16; Kate of growth of S. griseus and streptcmycin froducticn in shaken cultures (481): — Incubation Growth fW of filtrate Residual glucose Streptomycin days gm mg/ml fig/ml 6.8 10.2 0.048 6.9 93 <5 0.237 8.5 7.6 5 0.394 8,6 56 63 0.370 8.4 0.5 84 0.248 8.7 0.5 62 10 0.140 8.9 0.5 51 Table 17: Metabolic changes produced by S. griseus ivith different sources of nitrogen (458):- Stationary cultures Cell Sugar Ammonia GROWTH, LEFT, NITROGEN, Nitrogen Incubation, MG PER MG PER MG PER Activity SOURCE DAYS 100 ML 100 ML* 100 ML /zg/ml /-H Sodium nitratef 5 208 580 _ 21 7.6 11 264 22 - 58 8.3 Ammonium sulfate 5 228 650 55 13 6.2 11 291 255 53 8 5.6 Peptone 5 212 600 29 38 8.0 11 365 50 45 80 8.5 Glycine 5 236 550 39 43 8.1 11 252 45 72 60 8.7 • Control = 980 mg glucose per liter. t Mineral sources of nitrogen, 3 gm per liter; organic sources, 5 gm pc Chapter V — 91 — Metabolism The optimum temperature for the production of streptothricin by S. Javenduhe (452) Hes between 20° and 28°C.; at 37°C. very Httle of the antibiotic is produced. The course of growth of an actinomyces, consumption of energy and metabohc changes, as influenced by different sources of nitrogen are brought out in Tables 16 and 17. Metabolism of Anaerobic Actinomycetes:— As compared to the aerobic actinomycetes, the anaerobic forms show only limited growth and biochemical activity. According to Erikson, they exert no pro- teolytic action on egg or serum-containing media; they do not clot or hydrolyze milk and, in fact, rarely grow on it; they seldom grow on gelatin, and when there is a little flaky growth the tubes when cooled are found not to have been liquefied; they have little or no hemolytic action on blood broth or blood agar. Certain strains isolated from human infections have been found to show a slight degree of hemolysis on blood-agar plates at different times, but not consistently. They do not produce soluble pigments on protein media or insoluble pigments in their growth. Fermentation of sugars by organisms belonging to the genus Acti- nomyces is not accompanied by gas formation. This reaction is fairly constant. Glucose is the most readily fermentable sugar; maltose, lac- tose, and sucrose come second and are fermented within a comparatively short time by all strains; positive or negative reactions with salicin and mannitol have been found of value in differentiating strains, such as human and bovine (112). A. hovis was reported by Rosebury (367) to have a limited toler- ance for oxygen, which varies, however, among strains. The optimum temperature for this organisim is 37°C., and optimum pH is 7.2 to 7.6. Although A. hovis grows in the absence of a carbohydrate, it is greatly favored by the presence of glucose. It produces acid from carbohy- drates. A. hovis is killed by heating at 62° to 64° C. for 3 to 10 minutes. Like aerobic actinomycetes, it apparently survives drying for a long time, particularly when kept at low temperatures. Lieske, however, reported (260) that anaerobic forms are very sensitive to drying, being unable to survive even for one day. Production of Odors:— Most of the aerobic actinomycetes are characterized by the production of a specific odor, which is typical of freshly plowed soil or of composts. This odor is musty, or earthy, and occasionally fruity, in nature. Rullmann (373) believed that the odor is characteristic only of a single species, which he designated as A. odorifer. According to Lieske, only those aerobic forms that pro- dijce chalky white aerial mycelium with round spores are capable of forming this odor; the nonsporulating forms of the Nocardia type and Waksman — 92 — Actinomycetes those that produce cyhndrical spores do not give rise to any odor. The presence of carbohydrates in the medium favors odor production. The thermophihc actinomycetes are responsible for the more fruity scents, which arise particularly from young cultures. RuLLMANN was the first to make a detailed study of the pungent odor produced by certain species of actinomvcetes. The odoriferous substance is soluble in ether (373, 376). SO Incubation Days Oensii-t^ o/^ MyctUum CO2 EvoLution Fig. 20.— Influence of temperature upon growth and carbon dioxide production by actinomycetes Cf^om Jensen, 192). Thaysen (417) found that this substance is partly soluble in ethyl alcohol, and he considered it to be an organic amine. In high con- centrations, it had a manurial odor, but in high dilutions, especially in slightly alkaline water, it became markedly "earthy." One strain of an actinomyces was grown in broth, the culture distilled at ordinary pressure and the distillate treated with ether. On removal of ether and dilution of the residual substances 2:10,000,000 in water at pH 7.5-8.0, a typical earthy odor was obtained. When the "odor concentrate" was Chapter V 9B Metabolism diluted with water and fish (trout) were placed in it for 1 hour, they absorbed sufficient odor to become markedly tainted and unpalatable (418). IssATCHENKO (182) emphasized the importance of the odor imparted to river waters by the actinomycetes in rendering such water unpalat- able. In view of the fact that actinomycetes are able to develop at a I^QUS o/^ lncu6ation Fig. 21.— Decomposition of hemicelluloses by actinomycetes, as measured by CO2 evolution Qfrom Waksman and Diehm, 462). much more reduced oxygen pressure than that of the atmosphere (1/ 25), the large numbers found in the surface layers of the bottom de- posits are able to grow rapidly and produce an intense odor. If the bottom is sandy, most of the odor will dissolve in the water; if the bot- tom is clay, the odoriferous substance will be retained and will accumu- late. Odoriferous substances may also be produced by actinomycetes on caqao beans (55), milk (120), and other foodstuffs, rendering them inferior in quality or totally unsuitable for human consumption. Waksman — 94 — Actinomycetes Production of Pigments:— Actinomycetes are characterized by the production of a variety of pigments both on organic and on synthetic media. Nearly half of all the species isolated and described produce a pigment of one form or another, on one medium or another. These pigments are usually described in terms of various shades of blue, vio- let, red, rose, yellow, green, brown, black. There are also many grada- tions of these colors. The nature and the intensitv of the pigment are greatly influenced by the composition of the medium and environmen- tal growth factors. The pigment may dissolve into the medium or it may be retained in the mycelium. The pigment is concentrated in the vegetative growth in many cases, and only in the aerial mycelium in others. Certain species produce more than one pigment, as is indi- cated by such names as A. violacenis-niher and A. tricolor. Certain brown shades are often superimposed on the main pigment, especially in organic media. Some of the pigments are synthetic; others are formed as a result of transformation of certain constituents in the medium. This is true especially of the brown and black pigments produced in protein-con- taining media, as first shown by Cohn, in 1875, and later studied ex- tensively by Beijerinck and many others. Production of pigments by actinomycetes has been utilized as an important cultural characteristic in describing the organisms. Never- theless, the ability to form pigments represents one of the most variable properties among the actinomycetes. This variation depends upon many factors, involving not only the nature of the medium, but also the nature and age of the culture and its previous cultivation. The insoluble types of pigments are more constant than the soluble forms. Acids and alkalies exert a marked effect upon the nature and intensity of the pigment. Some of the pigments are soluble in organic solvents, and others are not. The production of water-soluble brown to black pigments on organic media is characteristic of certain actinomycetes, mostlv members of the genus Streptomyces. These organisms have usually been designated as chromogenic forms. The nature and the formation of this pigment were first investigated by Beijerinck (25). The tyrosinase action characteristic of these organisims was believed to explain the mechanism of the production of this pigment. It is insoluble in organic solvents, but soluble in water, in dilute acids, and in alkalies. According to Afanasiev (6), potato scab organisms failed to pro- duce the melanin pigment in the medium when only tyrosine was pres- ent; however, when other nitrogenous compounds were also added, the black pigment was formed abundantly. This was believed to be due to an alkaline reaction that is favorable to the production of the pigment. It was not formed from other amino acids. All plant pathogenic cul- tures were found to be chromogenic. Although Millard and Burr (298) reported that nonchromogenic actinomycetes may also cause Chapter V — 95 — Metabolism scab formation, Afanasiev questioned these reports, since the cuhures did not cause scab under controlled conditions. The pigment produced by S. coelicolor was first studied by Muller in 1908 (306). This pigment is dark blue and diffuses readily into the medium. If the reaction is acid the pigment becomes red; when the reaction of the medium is alkaline, the pigment is blue. Muller observed that this pigment was produced on synthetic media only with starch as a source of carbon. Waksman, however, demonstrated that this pigment or allied pigments are also produced with sucrose and other carbon sources. Chemically, the blue pigment was at first said (240) to belong to the anthocyanins. This was not confirmed, how- ever, (116). In 1914, Beijerinck (26) described, under the name A. cyaneiis, a culture which would now be classified with the Nocardia group and which produced a pigment similar in its properties to the an- thocyanins. This pigment was recently designated as litinocidin and was found to possess antibiotic properties (133). Lieske distinguished two groups of pigments produced by actino- mycetes: (a) the chromophores or pigment which is not excreted from the mycelium into the medium, and (I?) the chromopars or pigment which is readily excreted. The first group comprises various pigments produced in the vegetative mvcclium grown on synthetic media, namely, vellow, orange, red, blue, violet, brown, black, and green; the aerial mvcelium of these cultures may be white, rose, lavender, red, yellow, orange, green, or grey. The soluble pigments are usually yellow, blue, and red; occasionally they are green; and some orange and brown pig- ments are also produced. Kriss (240) could not accept Lieske's separation of actinomyces pigments into the above types or the classification of Duch^ into endo- pigments and exopigments. Even in the case of the chromophore pig- ments, part at least of the pigmented material is dissolved in the me- dium, possibly because of lysis of some of the cells. The solubility of the chromopar pigments in water is due to the greater penetration of the pigment through the cell wall. The chromophore pigments are either insoluble in water and are bound to the proteins or are dissolved in the fats and lipoids of the cell, or they are water-soluble but unable to pass through the living cell plasma; on the death of the cell, the pig- ment may be able to dissolve into the medium. Kriss recognized four types of pigments among the actinomycetes: A. Pigments soluble in water and in 96 per cent alcohol. These pigments are capable of passing through the living cell plasma. They have been subdivided into, (fi) anthocyanins, soluble only in water, and (I?) hydroactinochromes, soluble in water and in alcohol. B. Lipoactinochromes, insoluble in water but soluble in alcohol and in other organic solvents. J C. Pigments insoluble in water and in organic solvents. D. A combination of water-soluble and water-insoluble pigments. Waksman — 96 — Actinomycetes Only a few of the pigments produced by actinomycetes have been studied from a chemical viewpoint. Krainsky (230) examined in detail several actinomyces pigments. S. erythrochromogenus produced a red pigment soluble in water but not in alcohol, ether, or chloroform. The addition of alcohol to an aqueous solution of the pigment brought about its precipitation. Acids and alka- lies had no effect upon it. A yellow pigment was isolated from S. cel- hdosae. It became violet-red in an alkali solution and blue-green in concentrated H2SO4 . This, as well as the red pigment, was considered to be a carotin. The green pigment of S. viridochromogeniis was found to change to red on treatment with concentrated H2SO4 . Waksman demonstrated that the pigment produced by S. violacetis- niher behaved as an indicator, being red in an acid and blue in an alkali; the change in pigmentation took place at pH 6.6 (443). Conn (78) concluded that the two blue pigments produced by two species of Streftomyces, S. coelicolor and S. violacens-rnher, are not identical. The pigment produced by the first is similar but not identical to azo- litmin. On the basis of this differentiation, Conn believed that the two organisms represent distinct species. This concept could not be accepted by Oxford (330), since the pigment contained too little nitro- gen; neither could its phenazine (116) or anthocyanidin nature be ac- cepted. LiESKE studied a carmine-red pigment that became, on boiling in dilute acid, soluble in alcohol and in ether. The brick-red pigment of other strains of actinomycetes becomes soluble only under the action of concentrated HCl; on treatment with H2SO4 it is changed to a blue-green pigment. N. folychroiuogenes produces a red pigment, sol- uble in chloroform, ether, and acid, but not in alcohol, glycerol, water, or dilute alkali; this pigment is also changed to blue-green by H2SO4 . A light yellow pigment produced by certain actinomyces species was found to be insoluble in organic solvents, but soluble in dilute KOH solution; it changed, on treatment with concentrated H2SO4, first to green, then to dark brown. According to Lieske, the green, brown, and violet pigments of the chromophor type are insoluble in common solvents and give a sepia-brown color when treated with concentrated H2SO4. The yellow-red pigment of N. corrallinn was later identified (354) as belonging to the lipochrome group of fat-soluble pigments. Pigment formation by actinomycetes is influenced by the reaction of the medium, aeration, temperature and by the carbon and nitrogen sources, as shown previously in Tarle II. According to Kriss, the composition of the medium has a quantitative rather than a qualitative effect upon pigment production. He measured the adsorption spec- trum of the pigment obtained by extraction with ether and alcohol from S. longis'porus ruber. Although several pigments were thus recog- nized, they were apparently related. The blue pigments of S. coeli- Chapter V — 97^ Metabolism color could be extracted with cold and hot water as well as with alcohol. This pigment became red when treated with acid, and green when treated with 25 per cent alkali solution. The addition of lead acetate to an aqueous solution of the pigment brought about formation of a violet precipitate. As has been pointed out, this pigment was believed to belong to the anthocyanins, a fact not confirmed (116) by further study. Krassilnikov (234) confirmed the observations of Kriss, that an- thocvanins or allied pigments are characteristic of several actinomycetes. The pigment of N. cyanea is soluble in water and in aqueous solutions of alcohol, but not in pure alcohol, acetone, ether, or chloroform. It does not change in color in an acid medium, although in dilute acids the pigment assumes a rose-violet shade; strong acids decolorize the pig- ment. It is produced only on synthetic media with sucrose and glu- cose as carbon sources. Green actinomycetes also produce a water-soluble green pigment, which is the reason for such species names as A. viridis, A. virido- chromogeniis, and A. verne. The pigment is also soluble in glycerol and in alkali solutions, but not in organic solvents. The water-insoluble pigments have been studied only to a limited extent. Among these, the carotenoids produced by the red, orange, and yellow species are of particular interest (234). Reader (354) demonstrated two such pigments among actinomycetes; one of these pigments was designated as corallin, an ether solution of which gives two bands of absorption in the spectrum. The significance of the various pigments, especially the brown and black types, in the nutrition of actinomycetes is still a matter of specula- tion. ScHiBATA (396) suggested that they play a role in the oxygen exchange between the atmosphere and the cells in a manner similar to the role of hemoglobin in animals. Protective mechanisms have been postulated for some of the pigments (234). Thermophilic Actinomycetes:— Among the thermophilic microor- ganisms, or those capable of growing at higher temperatures, such as 50° to 65 °C, the actinomycetes occupy a prominent place. In view of the fact that these organisms occur so abundantly in organic matter-rich materials, one would naturally expect that they should be abundant in high temperature in heaps of hay, composts, and in soils, as will be shown later (p. 144). They are also found in a number of other substrates, such as pasteurized cheese (36). The abundance of thermophilic actinomycetes in nature has been knovm since the work of Globig (138), in 1888. Tsiklinsky (429) was the first to establish, in 1899, that composts contain an abundance of actinomvcetes. The normal temperature for their growth ranges from 50° to 70 °C. Waksman — 98 — Actinomycetes Thermophilic actinomycetes in culture can be isolated by one of several simple procedures. Tsiklinsky inoculated sterile potatoes with compost material and incubated them at 53-55 °C. After 16 hours' incubation, plates were prepared and incubated at the same tempera- ture. Two cultures of actinomycetes were thus obtained, one of which produced chains of spores and may, therefore, be considered as a spe- cies of Streftomyces, and the other produced round or ovoid spores at the end of side branches, caused by the swelling of the tips, thus rep- resenting a true Micromonospora. The second organism was believed to be widely distributed in nature and was designated as Thermoactino- myces vulgaris. It grew at 48-68°C., with an optimum at 57°C. At 37 °C. or at lower temperatures, it remained inert, but became active within 24 hours when incubated at 56-57° C. The spores of this or- ganism were not destroyed at 100°C. even after 20 minutes. The organism also resisted the action of disinfectants and grew readily on most of the ordinary media. It was strongly proteolytic but not amylo- lytic. The Streptomyces form, designated Thermoactinoniyces II, was less proteolytic, and the spores were less resistant to heat. Gilbert (135) isolated several thermophilic actinomycetes from various soils. He included them under one species as A. ihermofhi- lus. The organisms produced a lichnoid growth, with white aerial mycelium which later became gray. The optimum temperature for growth was 55°, with a maximum at 60° C. Most strains ceased to grow even at 45°, although some could be adapted to grow on agar media at 37° and even lower temperatures. Gelatin was slowly lique- fied. MiEHE (295) looked upon the thermophilic actinomycetes as the characteristic organisms inhabiting the decomposing masses of plant material under high-temperature conditions. These hot composts, rather than the soil, were believed to be the natural substrates of the thermophilic organisms. The spores lost their vitality rapidly, espe- cially on agar media, but survived on hay particles. One organism, desig- nated as A. thermophihis Berestneff, grew well at 40°— 50° C, more slowly at 30°, and not at all at 25° and 60°C. The manner of spore formation of this organism suggests that it was also a member of the Micromonospora group. Miehe reported, however, that some of the thermophilic actinomycetes produced spores in a manner similar to that described bv Gilbert. Schutze (399) reported the presence in decomposing clover hay of representatives of two types of thermo- philic actinomycetes, one of which was designated as A. therniophiliis Berestneff and the other as A. monosponis Lehmann and Schiitze. The latter may be definitely considered a member of the Micromono- spora group. Chapter V — 99- Metabolism In a more recent review of the of thermophilic actinomycetes, some Organism: Author: Thermophilic forms Globig Cladothrix thermophile Kedzior Thermomyces lanuginosus Tsiklinsky Thervioactinomyces Tsiklinsky xnilgaris Streptomyces sp. Tsiklinsky Streptothrix thermophile, Tsiklinsky No. 12 Streptothrix thermophile, Tsiklinsky No. 20 Actinomyces sp. Sames Actinomyces thermophiltis Gilbert Actinomyces thermophilus Miehe hterature (36) on the occurrence 20 species were Hsted, namely: Organism: Author: Thermomyces lamioinostis Miehe Actinomyces monosponis ScHtiTZE Streptothrix No. 8 Bruini Streptothrix No. 9 Bruini Streptothrix No. 12 Bruini Actinomyces spinosporiis Velich Actinomyces thermodiasta- Bergey ticits Actinomyces nondiastati- Bergey CilS Streptothrix thermophilus ECKFORD Actinomyces thermophilus Krohn Actinomyces casei Bernstein and Morton Chapter VI PRODUCTION OF ENZYMES AND OF GROWTH- PROMOTING SUBSTANCES Actinomycetes are able to produce a variety of agents which are essential to their own growth or to that of other organisms living in association with them. Some of these substances are of the nature of enzymes, others are vitamin-like substances, and still others are lytic agents. Actinomycetes also produce a varietv of bacteriostatic and bactericidal substances, or antibiotics, which are discussed in Chapter VII. Production of Enzymes:— Actinomycetes produce both extracellu- lar and endocellular enzymes. Only very few of these enzymes have been concentrated and studied in detail. The presence of others has only been demonstrated in the culture medium or in the mycelium of the organism. Proteases.— The wide distribution of proteolytic enzymes among actinomycetes is indicated by the ability of the organisms to liquefy gelatin with different degrees of rapidity and to attack serum protein, coagulated egg-albumin, casein, and vegetable proteins (442), This property has been utilized for species characterization. In nearly half of the species, especially those belonging to the genus Streptomyces, gelatin liquefaction is accompanied by production of a brown pigment. Optimum gelatin liquefaction occurs at a fH of 6.5 to 8.5. Greater acidity is more injurious to the proteolytic process than is a more alkaline reaction. The proteolytic action of actinomycetes, in contrast with that of fungi and bacteria, is not influenced to any great degree bv the presence of glucose or other available carbohydrates. Tlie breakdown of the protein proceeds, through the amino acid stage, to ammonia. This is brought out in Table 18. In some cases it is easy to establish the in- termediary formation of peptides and amino acids; in other cases, it is more difficult. The proteolytic enzymes are fairly resistant to the effect of tempera- ture, since they are able to withstand heating at 70° C. for 30 minutes. According to Lieske, the resistance of the enzvmes to the eff'ect of higher temperature is greater than that of the living cells of the or- ganisms, the latter being killed at 62°-65°C. When the enzymes are Chapter VI — 101 — Enzymes, etc. heated to 80°C., their activity is destroyed. When some of the cul- tures are kept for a long time at 40°-45°C., the reproductive capacity of the cells is destroyed, but their proteolytic functions are not injured (234). The ability to cause proteolysis is more marked for the non- pigmented forms than for the pigmented types. Beijerinck postulated that the pigmented forms are responsible for a solidifying effect pro- duced by the quinone upon the liquid gelatin. To what extent this is responsible for the apparently lower proteolytic action of pigmented cul- tures still remains to be determined. Table 18: Proteolytic activity of actinomycetes in 1 per cent gelatin solution (187): — Results in milligrams of nitrogen per 25 ml of medium 10 days 30 days Spfxies Formol Formol of gelatin NH4-N titration NH4-N titration S. griseus 6.4 16.5 16.2 30.7 Very rapid S. griseoflavus 17.1 22.1 36.6 Very rapid S. cellulosae 16.0 14.5 27.4 Rapid S. olivaceus 13.2 9.2 24.0 Rapid S. fulvissimus 11.8 7.0 29.5 Fairly rapid S. violaceus-ruber 14.5 9.2 39.2 Rapid S. roseus 11.4 10.2 20.2 Slow S. hohiliae 11.2 6.2 26.9 Slow S. viridochromogenes 11.1 10.6 19.8 Slow S. erythrochromogenes 13.0 1.1 20.5 Slow S. phaeochromogenus 9.3 4.4 22.3 Slow S. diastatochromogenes 11.2 11.1 21.5 Slow S . aureus 2.1 8.8 5.8 22.6 Slow Sterile solution - - 0.0 6.8 - In some species, proteolysis occurs only at a late stage in the develop- ment of the organism. This may be due to the formation of endoen- zymes, which are liberated on the death of the cells, as contrasted with the exoenzyme produced at an early stage of the development of the mycelium. This explanation has not been universally accepted, how- ever (234). Bacteriolvsis may often parallel growth inhibition (Table 19). The ability of certain actinomycetes to cause the lysis of various bacteria is characteristic of certain species of Stre^ptamyces. This effect directed upon plant pathogenic bacteria may be of considerable eco- nomic importance. A culture designated as Streftomyces 105 which produced a wine-colored soluble pigment and a white to gray aerial my- celium was found (83) to exert a lysogenic effect upon various species of Phytomonas, Erwinia, and other gram-positive and gram-negative bacteria. Potato extract-glucose agar media were particularly favorable to the production of the Ivtic agent. Cultures of Streftamyces 105 Waksman — 102 — Actinomycetes added to soil infected Phytoinonas tahaci protected the plants against in- fection. The importance of such lytic agents in soil processes and their relation to true antibiotics are matters for further investigation. The ability to coagulate milk and to dissolve the coagulum is also a common property of actinomycetes. Whether this is due to the for- mation of a special enzyme of the nature of rennet or lab or whether it is a property of the proteolytic mechanism of the organisms remains to be determined. Many strains of actinomycetes are able to hemolyze blood cells, as a result of production of hemolysins. These enzyme systems are dis- tinct from the true proteolytic enzymes, and are also fairly resistant to heat. They apparently have no relation to the pathogenic properties of the organisms producing them. Table 19: Distribution of bacteriolytic properties among actinomycetes C503); — Preparatiom Total strains Ac tivity against S. aureus Growth-inhibition B. subtilis lof 164 103 67 ++ + ++ + Number of strains Per cent of strains Number of filtrates 24 14. 6 9 54 32.9 11 86 52.4 47 22 54 1342 32.9 5 4 88 53.7 58 Activ: ity on heat -killed £. coli and S. aureus Number of filtrates 67 23 25 19 - - The species of Nocardia are usually much weaker proteolytic forms than the Streptomyces species. Some of the nocardias, notably the red and green types, do not liquefy gelatin at all. This is true of N. aster- oides, an important pathogen, and of the saprophytic N. riiber, N. viridis, and others. Some of the yellow species (N. (lava) are weak liquefiers. The lemon-yellow forms (N. citrea) and the white types (N. alhd), however, liquefy gelatin energetically. While the proteolytic property of actinomycetes is a constant char- acteristic, it is essential to remember that the occurrence and the rate of proteolysis, as measured by the extent and rapidity of the reaction, are influenced by environment and are variable properties. Although no enzyme preparations comparable to those of certain fungi and bacteria are obtained on a large scale from actinomycetes, there is no doubt that such preparations could easily be obtained. Whether they would have any distinct advantages is hard to tell. Possibly the thermophilic capacity of some of the organisms might yield enzymes which would be more heat-tolerant than those produced by fungi and bacteria. Chapter VI — 10^ — Enzymes, etc. Certain actinomycetes exhibit, for example, marked proteolytic activity upon wool fabrics. The sterile culture filtrate of one organism was found (141) to exert a marked effect not only upon protein de- rived from soybeans, casein, peanut and corn, but also upon wool fiber, to the extent of 5 to 85 per cent. Amylases.— A large number of actinomycetes are capable of bringing about rapid hydrolysis of starch, either to the dextrin stage or directly to maltose and glucose. These reactions are carried out by means of active amylolytic enzymes. This phenomenon was first observed by Beijerinck and Sames C^77^, and later confirmed and extended by Krainsky, Waksman, Lieske, and others. The method of screening a large number of cultures consists in streaking agar media containing starch as the source of carbon, and allowing it to incubate. After 5, 10, 15 and 20 days, the surface of the agar is covered with a solution of I-KI, and the degree of starch hydrol- ysis measured by the width of the clear zone. It is thus possible not only to establish that many species are capable of producing amylolytic enzymes, but the active forms can be selected for further study. For- mation of a zone of 1.0 to 1.5 cm. in 10 days is an index of excellent amylase production. Inorganic sources of nitrogen, especially nitrates, appear to be preferable to organic forms for the production of amylolytic enzymes. Just as in the case of the proteolytic enzymes, the amylases of actino- mycetes are able to withstand the effect of higher temperatures better than are the cells of the organism producing these enzymes. Surovaya (410) obtained a potent diastatic enzyme preparation from a culture of an organism described as S. diastaticus. The culture was grown on a potato medium for the production of the enzyme. The preparation was designated as "superbiolase." It was active at 70° to 100°C. and had an optimum pH at 6.6 to 6.7. The starch was converted first from the insoluble into the soluble form and then to dextrin. Saccharification of the dextrin proceeded much more slowly than starch liquefaction. This points to the possible application of such enzyme preparations to industrial processes where the sugar produced is not essential. Many species of actinomycetes are also able to attack dextrins, gly- cogen, and inulin and to produce the corresponding enzymes. So far, however, no attempt has been made to study these enzyme systems in detail or to utilize them for any practical purposes. Invertase.— The wide distribution of invertase among the actino- mycetes has been pointed out by Krainsky, Caminiti (61), and Waksman. The ability of some species to utilize sucrose as a source of carbon is dependent upon the property of the organisms to produce this enzyme. Although many species of Streptomyces and Nocardia are able to utilize sucrose, the production of invertase has not been established for all forms. It has even been suggested that this property be utilized for Waksman — 104 — Actinomycetes differentiating species; at best, however, this can be only a secondary characteristic, Cellulolytic enzymes.— Although many actinomycetes are able to grow on cellulose as the only source of carbon (231), the production of corresponding enzymes has so far not been demonstrated. Lipase.— The ability of various actinomycetes to produce lipolvtic enzymes has been established (260). The activity of these enzvmes upon natural products is often accompanied by the formation of odor- iferous substances, discussed in Chapter V. The spoilage of cacao by certain species of Streptomyces (55) may possibly be due to the lipolytic effect combined with odor production. Bacteriolytic and antolytic enzymes.— The ability of certain actino- mycetes to dissolve the dead and in many cases also the living cells of many bacteria has been ascribed to the action of specific lytic enzymes or bacteriolysins. This phenomenon was first observed by Lieske, and later studied extensively by Gratia (153, 155), who utilized this process for the preparation of certain bacterial vaccines, such as typhoid vaccine. The bacteriolytic substance produced by S. alhus was designated by Welsch (505) as "actinomycetin." This property is widely distributed among the actinomycetes, as shown in Table 19; as many as 50 per cent of all cultures have been found active against heat-killed cells of E. colt and against living S. aureus. Borodulina (43) and Nakhimovskaia (316) found that among actinomycetes the lytic principle is excreted by the cells into the medium, thereby inhibiting growth of bacteria found in proximity to the lytic principle and, later, dissolving these bacteria. Though re- sistant to heat, this substance was still considered as an enzyme. Krassilnikov believed that this bacteriolytic enzyme is similar to ly- sozyme of animal origin, although marked differences have been estab- lished between the action of this agent and that of the lytic principle of actinomycetes. Among these antibiotic preparations obtained from this group of organisms, two appear to have properties which would place them either with enzvme svstems or with true antibiotics. These are actinomyces lysozvme and actinomycetin. Some of these lytic sys- tems consist of a lipoidal bactericidal substance, a ribonucleinase, and proteolytic enzymes (407). The ability of certain specific phages to attack actinomvcetes has been discussed previously (p. 62). Production of Vitamins:— The favorable effect exerted by certain actinomycetes upon the growth of various fungi was believed (171, 280) to be due to their ability to svnthesize thiamin, which is produced on simple synthetic media. A study has been made of 22 cultures of ac- tinomycetes grown in thiamin-free media; this was followed by the in- oculation of the same cultures with Phycomyces hlakesleeamis. The fact was established that all the cultures produced thiamine or its inter- mediate or its precursor. The production of carotinoids by certain ac- Chapter VI — 105 — Enzymes, etc. tinomycetes has been demonstrated, as mentioned previously (p. 97). The ability of certain strains of S. griseus to produce vitamin B12 has also been established (p. 191). Oxidative Mechanisms:— Actinomycetes possess a number of oxida- tive mechanisms, only few of which are recognized at the present time. Attention may be called, for example, to the ability of "resting cells" of certain species of Streptoviyces to transform aestradiol to oestrone (507). According to Turfitt (430, 431), various species of Nocardia are capable of attacking various steroids, with the possible exception of halogen-substituted derivatives. The oxidation of cholesterol results in the formation of a cholesterone, followed later by molecular fission, the products of which may be utilized by the organisms for their fur- ther growth. Various actinomycetes are also capable of producing penicillinase (506). Ib Fig 22 -The u.e of the agai cross streak method for testing the abUity of actinomycetes to produce antibiotic substancef.-Upper two plates S. l..e„Mae3,W. ^idfle two pla es I U,eniuUe 3440; lower two plates. S- ,«*«„ M96^Tes, bacterta. fern right column, M. ranae B. mycoides, B. suhtilis, (Original.') M. avium E. coli W, M. tuberculosis 607, E. coli R, S. aureus. M. tuherculosis 607R; , (R = forms resistant to streptomycin.) Chafter VIl ANTAGONISTIC PROPERTIES OF ACTINOMY- CETES AND PRODUCTION OF ANTIBIOTICS Antagonistic Effects of Actinomycetes:— Actinomycetes comprise a large number of organisms which have the capacity of inhibiting the growth of and even destroying other microorganisms, namely, bacteria, fungi, and other actinomycetes. Several detailed reviews of this phe- nomenon have been published during the last decade (448, 449, 451, 453). Any student of soil microorganisms who uses the plate method for counting purposes has obser\^ed that some of the colonies of actino- mycetes on the plate are surrounded bv clear zones free from the growth of bacteria and fungi (483). In 1917, Greig-Smith (157) obser\'ed, for example, that when a soil is plated out on a nutritive agar the growth of certain spreading colonies of B. mycoides and B. vnlgatiis may be in- hibited by other colonies on the plate. These will be surrounded by a clear zone 2 to 10 mm. wide where the spreader does not penetrate. Examination of the colonies that produce this toxic effect showed them to consist of actinomycetes. Further study of the various tvpes of colo- nies brought out the fact that the nonchromogenic strains produced the most toxic effect. He postulated, therefore, that the ability of actino- mycetes to antagonize bacteria and fungi may suggest their possible im- portance in the soil as a factor which limits microbial development and thus affects soil fertility processes. Although the soil may thus be considered to be a source of antago- nistic actinomycetes (488), the enrichment or soil with specific patho- genic bacteria, such as M. hiherculosis does not necessarily lead to the development of specific actinomycetes active upon such bacteria (480, 489). The reason for this is that the growth-inhibiting effect of actino- mycetes upon bacteria and fungi is brought about largely through the production of toxic agents, which are now known as "antibiotics." The production of such substances can easily be demonstrated for some or- ganisms by the agar-cross-streak method. In many cases, however, or- ganisms that show inhibition of bacteria on the plate do not produce any antibiotic substance when grown in liquid media. Gasperini (130) was the first to demonstrate the antagonistic action of actinomycetes. He observed that these organisms develop on fungus mycelium, upon which they live to a limited extent in the form of a Waksman — 108 — Actinomycetes parasite, as a result of the faculty that the actinomycetes possess of di- gesting the membrane of these lower fungi. Greig-Smith first demon- strated the ability of actinomycetes to produce antibiotic agents. LiESKE, who tested a large number of actinomycetes for their anti- bacterial action, established that this process is selective in nature, af- fecting only certain bacteria, such as S. anreiis and that different actino- mycetes vary greatly in this respect. Lieske believed, however, that the antagonistic effect of actinomycetes may be due to a specific bacteriolytic enzyme, namely: "Ein bestimmtes bakterienlosendes Enzym konnte aus den Kulturen nicht isoliert werden; dass ein solches in Frage kommt, ist aber bei der grossen biologischen Bedeutung, welche die Vernich- tung von fremden Mikroorganismen in der Natur fiir die Strahlenpilze besitzt, nicht ausgeschlossen." Rosenthal (369) introduced, in 1925, suitable methods for meas- uring bacteriostatic and bacteriolytic activities of actinomycetes. He isolated from the dust an actinomyces culture which he designated the true biological antagonist of the diphtheria organism. The surface of an agar plate was covered with an emulsion of the test bacteria, and the actinomyces culture was inoculated into several spots on the plate. After 2 days the actinomyces colonies were surrounded by large trans- parent zones, whereas the rest of the plate was covered with the growth of the diphtheria organism. In another experiment, the agar was mixed with a heavy emulsion of the diphtheria organism, which had previously been killed by heat, and the mixture poured into the plates. After solidification of the agar, the actinomyces culture was inoculated into several spots on the plates. The actinomyces colonies gradually became surrounded by clear zones, thus establishing the fact that the organism produced a lytic substance which diffused through the agar and dissolved the dead diphtheria cells. Gratia (155) made a careful study of actinomycetes as agents pro- ducing materials (mycophages) that are capable of bringing about the lysis of bacterial cells. These effects were largely exerted upon dead bacteria, although living cells were later found to be affected also (154). The antibiotic substance produced by one of the organisms (A. alhiis') at first considered to be of the nature of an endo- and exo-bacteriolysin (499). It was later designated by Welsch as actinomycetin, as pointed out previously. The lysis of living bacteria was considered to occur in two stages: first, bactericidal effect of the substance upon the living bac- teria; second, bacteriolytic action upon the dead bacteria, this process be- ing helped by cell autolysis (502, 503). The first detailed survey of the distribution of antagonistic actino- mycetes in nature was made by Nakhimovskaia (316). Of 80 cul- tures isolated from a variety of soils, 47 possessed antagonistic properties; however, only 27 of these were found capable of liberating antibiotic substances into the medium (Table 20). These actinomycetes pos- sessed the property of inhibiting the growth of gram-positive bacteria Chapter VII — 109 — Antagonistic Properties but not of gram-negative bacteria or of fungi. These antibacterial properties were manifested, not only in artificial culture media, but also in the soil. Some of the cultures that were antagonistic to bacteria in nutrient media were ineffective, however, in the soil. The effects were more intense in hght, or podzol, soils and much weaker in heavy, or chernozem, soils. The high content of organic matter in the latter types of soil was believed to be one of the factors that resulted in a de- crease in the antagonistic activities of these organisms. When the actinomycetes were allowed to multiply in the soil before inoculation with bacteria, the antagonistic effect was very pronounced even in the presence of a high concentration of organic materials. According to Borodulina (43), actinomycetes are able to antag- onize various spore-forming bacteria and bring about the lysis of the living cells. He found that a thermostable substance was produced on Table 20: Occurrence of antagonistic actinomycetes in different soils (316): — Tota il number of Number of Stra ins producing Nature of soil strains tested antagor ustic strains antibiotics Chernozem 24 10 9 Podzol 11 7 Solonets 4 4 High altitude soil 9 6 Sandy soil 6 5 Dry desert soil 5 4 River bank meadow 14 7 Cultivated soil 7 4 Total 80 47 27 agar media. The activity of this substance was greatly reduced at an alkaline reaction but was favored by an acid reaction. When B. mycoides and an antagonist were inoculated simultaneously into peptone media, no inhibitive effect was produced because the bacterium changed the reaction of the medium to alkaline, thereby making conditions un- favorable for production of the antibiotic substance by the antagonist. When the antagonist was allowed to develop in the medium before the bacterium was inoculated, a strong antibiotic effect became evident in elongation of the vegetative cells of B. mycoides. This was due to a delay in fission and was accompanied by the suppression of spore forma- tion. Krassilnikov and Koreniako (237) also reported that many species of actinomycetes, notably members of the genus Streftomyces, but not of Nocardia, produce a substance that is strongly bacteiicidal to a vari- ety of organisms. This substance was said to be particularly active against nocardias, mycobacteria, and micrococci. It was less active upon spore-forming bacteria and had no action at all on non-spore-forming bac- Waksman — 110 — Actinomycetes teria. Under the influence of this bactericidal factor, the microbial cells were either entirely lysed or were killed without subsequent lysis. The action upon spore-forming bacteria was bacteriostatic rather than bacteri- cidal (238). The antibiotic substance studied by these and other Rus- sian workers was believed to be similar to lysozyme. An attempt to isolate an antibiotic substance from some of the soil actinomycetes was made by Kriss (243). This substance was in- soluble in ether, petroleum ether, benzol, and chloroform, and was re- FiG. 23 a.— The use of M. tnhercidosis for testing production of anti- biotic substances by actinomycetes. Upper horizontal streak H37Rv strain; lower streak fl37RvR (resistant to > 1000 Atg/ml streptomycin): inhibition of streptomycin-sensitive but not of streptomycin-resistant strain Qfrom Williston et al., 510). sistant to the eff^ects of hght, air, and high temperatures. The optimum reaction for its production by Stre-ptoinyces vioJaceus was found to be pH 7.1 to 7.8, the activity not being increased by selective cultivation. Although it was believed that the substance is similar to egg-white lysozyme, the above properties hardly justify this conclusion. The dif- ferences in the antibiotic properties of the various antagonistic actino- mycetes isolated by the Russian investigators definitely point to the fact that more than one antibiotic substance was involved. Chapter VII Ill- Antagonistic Properties Waksman ct al. (468) came to the conclusion that actinomycetes possessing antagonistic properties against bacteria and fungi are widely distributed in nature, especially in soils and in composts. Two hundred and forty-four cultures were isolated at random from different soils. Of these, 106 cultures or 43.4 per cent possessed some antagonistic properties, and 49 cultures or 20 per cent were highly antagonistic. Similar relations were observed in examining a large series of well- identified organisms kept for a number of years in a type culture col- FiG. 23 b.— No inhibition ot stu I'lnnncin-sensitive or of streptomycin- resistant strains Cfroni Williston ct al., 510). lection (503). The antagonistic forms were most abundantly repre- sented by the genus Strepoviyces (Table 21). BuRKHOLDER (56) examined the antagonistic properties of 7,369 cultures of actinomycetes isolated from soil, using various test organisms, namely gram-positive and gram-negative bacteria, acid-fast bacteria, fungi including yeasts and green algae. Of these cultures, 1,869 in- hibited S. mireiis in agar streak plate tests, 261 inhibited E. coli, and 514 showed an antagonistic effect against Candida albicans. J Various other surveys have been conducted dealing with the capacity of large numbers of actinomycetes to inhibit the growth of bacteria as Waksman — 112- Actinomycetes a whole, of certain groups of bacteria (249), of fungi pathogenic to man (391), of viruses (196), and of phages (392, 393). The antagonistic properties of actinomycetes are not limited to mem- bers of the genus Streptomyces. A culture of Nocardia, isolated by Gardner (129) as an air contaminant, was found to produce antag- onistic effects against a variety of gram-positive bacteria. The active substance produced by this organism was designated as 'proactinomycin. A representative of the genus Micromonospora was also reported (503) to be capable of exerting antagonistic effects against certain bacteria. Actinomycetes also reveal antagonistic activities against fungi (424). Table 21 : Distribution of antagonistic actinomycetes in nature (468) :■ Group I* Group II Grou ipIII Group IV Number - of No. of Per No. of Per No. of Per No. of Per Source of cultures cul- cent of cul- cent of cul- cent of cul- cent of ORGANISMS isolated tures total tures total tures total tures total Fertile, manured and limed soil 74 20 27.0 5 6.8 1 1.3 48 64.9 Infertile, unma- nured, limed soil 75 11 14.7 8 10.7 4 5.2 52 69.3 Potted soil 13 1 7.7 1 7.7 11 84.6 Potted soil, en- riched with E. coli 21 1 4.8 4 19.0 4 19.0 12 57.2 Potted soil, en- riched with mixtures of bacteria 15 12 80.0 2 13.3 1 6.7 Lake mud 9 3 33.3 4 44.4 2 22.2 Stable-manure compost 37 1 2.7 20 54.0 4 10.8 12 32.4 Total 244 49 20.1 44 18.0 13 53 138 56.6 • I— Most methods used. -more limited antagonistic properties; IV — no antibacterial effects Alexopoulos (8, 9) made a survey of the antagonistic properties of 80 cultures of actinomycetes, using Colletotrichum gloeosporioides as the test organism. The following results were obtained: 17.5 per cent of the cultures were strong inhibitors, 38.8 per cent were weak inhibitors, and 43.7 per cent had no inhibiting effect upon the fungus. Meredith (291) surveyed the distribution of organisms antagonistic to Fusarium oxysporum ciihense in Jamaica soils. Most of these an- tagonists belonged to the actinomycetes. The antagonists were not evenly distributed in the various soil samples, 10 of the 66 samples yield- ing 44 per cent of the antagonistic organisms. Those actinomycetes Chapter VII — 11^ — Antagonistic Properties that were antagonistic to the Ftisariuni when grown in their own soil- infusion agar were not always antagonistic when tested in soil-infusion agar prepared from other soils. A culture of an actinomyces isolated from a compost produces lysis of Fusarknn. When spores of both or- ganisms were mixed in an agar medium, the fungus at first developed Fig. 24.— Method of measuring antibacterial or antifungal potency of an antibiotic, by the agar streak method Qrom Reilly, Schatz and Waksman, 356). normally, but began to undergo lysis on the fifth day, large sections of the mycelium disappearing. On the seventh day, only chlamydo- spores were observed. After 9 days, the fungus completely disappeared, whereas the actinomyces made a normal growth. The antibiotic active against the Fusarimn was later designated as musarin. It was found to be an optically active acid, of the probable composition of CC35HgoOi4N2)t2, and had an activity of 1:80,000 to 1:100,000 (12). Waksman — 114 — Actinomycetes Leben and Keitt (254) isolated a culture of Streftoviyces which was antagonistic to 29 phytopathogenic fungi, but not to most bacteria. The culture was grown in corn-steep medium in shake flasks. The culture filtrate was acidified to pH 2.5 and the acti\^e substance extracted from the precipitate with ethanol. A preparation was obtained which completely inhibited VenUiria inneqiialis in a 1:8,000,000 dilution and Sclerotinia fructicola in a 1:11,000,000 dilution. The antibiotic is water-insoluble. The above antibiotic was designated as antimvcin. It was purified by extracting the precipitate produced on acidification of medium to pH 2.5 with ethanol. The active material was heat-labile, soluble in various organic solvents and in water at pH 9.3. The active substance inhibited the growth of various fungi and of only very few bacteria (255). Several entities were isolated from antimycin preparations and designated as A, B and C. The A was a nitrogenous phenol (C2S- H40O9N0). The substance inhibits the respiration of Saccharmnyces cerevisiae, of cytochrome oxidase and succinic dehydrogenase. Actinomycetes also exert marked antagonistic effects against species of Fythnini, as in the case of root rot of sugar cane. Of 3,788 cultures isolated from soil and tested against a parasitic strain of Pythimn, 896 or 23.6 per cent showed some antagonistic effect upon the fungus, the effect, in some cases, being marked. The occurrence of such antag- onistic organisms and the extent of their activities were less pronounced in heavy or infested soils than in light soils (79). Certain actinomvcetes were found (511a) to be responsible for the destruction of the mycelium of Ophioholus graminis, the cause of the take-all disease of wheat, in the soil, especially in partly sterilized soils. This parasitizing and antibiotic effect of actinomycetes and of other soil organisms is responsible for the check in the development of Ophiohohts in natural soils. Actinomycetes possess antagonistic properties not only against bac- teria and fungi but also against other actinomycetes (275). The more aerobic species are antagonistic to the less aerobic types. Millard (296) believed that he succeeded in controlling potato scab caused by Streptomyces scabies by the use of green manures such as grass cuttings. The development of scab on potatoes grown in sterilized soil and inocu- lated with S. scahies was reduced by the simultaneous inoculation of the soil with Streptomyces praecox, an obligate saproph\'te (299). By in- creasing the proportion of the latter organism to the pathogen, the degree of scabbing on the test potatoes was reduced from 100 per cent to nil. The sterilized soil provided sufficient nutrients for development of the antagonist, and only a small increase in the control was obtained when grass cuttings were added and sterilized along with the soil. Sandford (379) was unable, however, to control potato scab by in- oculation, with S. scahies and S. praecox, of either steam-sterilized or natural soil containing different amounts of green plant materials. Chapter VII — 115 — Antagonistic Properties These organisms were perfectly compatible on potato dextrose agar, as well as in a steam-sterilized soil medium. The control of scab (299), therefore, was said to have been due, not to the direct action of S. frae- cox, but to certain other undetermined microorganisms favored by the presence of the green manure. S. scabies was found (379) to be very sensitive to various products of fungi and bacteria. When grown in close proximity to various bacteria, the acid production of the latter inhibited S. scabies to a considerable degree. Its complete inhibition was not due to the acid reaction alone, however, since a certain bacte- rium which definitely inhibited the growth of this plant pathogen was also isolated from the soil, thus suggesting the possibility that the bac- terium may have exerted the antagonistic effect. Goss (146, 147) observed that the severity of scab is dependent on the amount of S. scabies present in the soil. This amount was believed to be controlled by the soil microflora. No evidence was obtained as to whether the effect of the soil flora on S. scabies was due to specific or- ganisms. Kjeszling (217, 218) isolated two cultures of bacteria which were antagonistic to S. scabies. When added to the soil, these bacteria prevented the development of scab on potatoes. Among the other antagonistic effects of actinomycetes that may prove to be of great economic importance is their action upon nitrogen- fixing bacteria. Konishi and Fukuchi (229) have shown that cer- tain actinomycetes are able to inhibit the growth, on the plate, of root- nodule bacteria; some of the organisms, like S. flavus, were particularly inhibiting. In association with actinomycetes, none of the nodule cul- tures grew readily. In the soil, however, no effect of the actinomyces cultures was observed upon alfalfa bacteria. The inhibiting effect of actinomycetes upon the growth of Azoto- bacter was first observed by Nikolaieva (323) in 1914. Nickell and BuRKHOLDER (322) found in the soil a large number of actinomycetes that exert a marked inhibiting effect upon the growth of Azotobacter. It was suggested that antibiosis may be responsible for development of these organisms in the soil. The antagonistic effects of actinomycetes upon plant pathogenic bacteria has also been well established. Hino (173) isolated several actinomvcetes active against Ps. solanacearmn. Corynebacteriuvi se- fedonictun, the causative agent of root rot of potato was antagonized by various actinomycetes, some of which produced antibiotic substances and one produced lysis of the bacterium (335). Further studies on this subject were made by McCormack (275). The ability of actinomy- cetes to produce substances active against bacterial viruses or phages has also been established (198). In a natural environment, such as the soil, the development of the antagonistic properties among actinomycetes will occur largely under aerobic conditions. In a less well oxidized environment the actino- mycetes may themselves be antagonized. A bacterium, like B. viega- Waksman — 116 — Actinomycetes Table 22 : Classification of antibiotics of actinomycetes: — A. Soluble in ether and in other organic solvents: I. Pigmented substances: 1. Orange colored; somewhat soluble in neutral aqueous solution; nitrogen- bearing ring compound, highly toxic, C4iH560nN8; largely active against gram-positive bacteria Actinomycin 2. Yellow pigment, active against both gram-positive and gram-negative bac- teria; highly toxic to animals Xanthomycins A and B 3. Compounds related to actinomycin Actinoflavin 4. Red-blue pigment; soluble in aqueous alkaline solution; active against gram- positive bacteria Litmocidin 5. Compounds related to litmocidin Actinorbodin 6. Orange colored; extracted from charcoal adsorbate and from mycelium by ether-alcohol mixture; active largely against M. tuberculosis Nocardin 7. Green pigment, active against gram-positive bacteria Actinomycelin II. Non-pigmented substances: 1. Organic base; soluble in acidified aqueous solution, inhibits mostly gram- positive bacteria Proactino/nycin 2. Largely fungistatic, not bacteriostatic, C27H42N2O7 Actidione 3. Soluble in water; active against various bacteria and fungi Mycomycin 4. Insoluble in water, present in mycelium; active largely against gram-positive bacteria Streptocin 5. Neutral compound; slightly soluble in water, readily soluble in organic sol- vents, contains nitrogen (8.6%) and non-iojiic chlorine (21.7%); active against various bacteria and rickettsiae Chloromycetin 6. Amphoteric; active against bacteria, rickettsiae and certain viruses Terramycin 7. Heat labile, largely active against fungi Antimycin 8. Heat stable, active against fungi Fradicin 9. An acid; active it vivo against the relapsing fever spirochete and enhances the activity of penicillin against the syphilis spirochete Borrelidin B. Insoluble in ether, but soluble in other organic solvents: I. Violet-blue pigmented substance Mycetin II. Colorless, sulphur-containing substance Sulphactin III. Colorless, nitrogenous body, active against parasitic fungi Musurin C. Soluble in water, insoluble in ether and in other organic solvents: I. Bases soluble in aqueous acid solution; removed from charcoal by acid alcohol; active against various gram-positive and gram-negative bacteria. 1. Little activity against Bacillus mycoides, Scrratia marcescens; active against Bodenheimer organism and fungi Streptothricin a. Compounds closely related to streptothricin, but varying in toxicity to animals and showing quantitatively different antibiotic spectra: (a) Streptin (b) Streptolin (c) Lavendulin (d) Actinorubin (e) Antibiotic 136 (f) Streptothricin VI and \'II 2. Active against B. mycoides and S. marcescens, little activity against fungi, no activity against Bodenheimer organism; glycoside (streptidine-streptobiosa- mine) Streptomycin complex Chapter VII — 117 — Antagonistic Properties Table 22 (Cont.) a. Constituents of streptomycin complex: (a) Streptidine-streptobiosamine, C-iiHa^NyOio Strepomycin (b) Mannose derivative of streptomycin Mannosidostrepomycin (c) Reduced streptomycin Dibydrostreptomycin b. Streptomycin-like materials: (a) Antibiotic F, (b) Strepomycin II 3. Active against streptomycin-resistant M. tuberculosis Neomycin 4. Basic compounds active against rickettsiae and larger viruses Aureomycin II. Removed from charcoal by neutral alcohol, soluble in neutral aqueous solutions; narrow antibiotic spectrum against certain gram-positive and gram-negative bacteria Grisein 1. Grisein-like material, still narrower spectrum than grisein, mostly enteric bacteria Antibiotic 5310 D. Proteins and polypeptides: I. Colourless preparation, possessing lytic properties against living gram-positive bacteria and dead gram-negative bacteria Actinomycetin 1. Active fraction of actinomycetin ActinoZ.yme II. Active largely against micrococci; lyses cell membrane Actinomyces lyso^yme III. Combined with orange pigment; largely bacteriostatic against gram-positive bacteria Micromonosporin E. Incompletely described agents: I. Active against the smegma bacillus S/negmatis factor II. Active against bacteriophages Antiphage factors III. Active against viruses Antivirotics F. Agents not produced readily in liquid media; activity obtained only on agar streak; I. Little known substances Insoluble factors theriuvi, may be antagonistic to certain species of actinomycetes but can be antagonized by others. Certain bacteria, Hke Fs. fluorescens, are markedly antagonistic to actinomycetes as a whole, causing their lysis. Numerous fungi are capable of producing antibiotics, such as penicillin and clavacin, which are very effective against actinomycetes. Production of Antibiotics by Actinomycetes:— Prior to 1940, knowledge of the antibacterial properties of actinomycetes was limited to those of the living organisms. Only two antibiotic substances— one known as actinomycetin and the other as actinomyces h'sozyme— were recognized. Both had been isolated only in a crude state. Welsch reported recently (505) in detail upon the antibacterial properties of actinomycetin. The activity of this preparation was expressed in terms of mycolytic units per milliliter of culture filtrate of S. alhus. A unit was expressed in terms of lysis of a known suspension of heat-killed cells of E. colt. Mycolysis of the heated bacteria by actinomycetin took place at pH 3.5 to 11.0 (opt. 7.5-8.5); optimum temperature 38° to 40°. E. coli cells killed by chemicals are also dissolved by actinomycetin in a manner similar to the heated cells. The lytic principle is stable at pH 5.0 to Waksman — 118 — Actinomycetes 9.0; it is thermolabile and is destroyed by ultraviolet radiations. Ac- tinomycetin, as well as the living S. albus, has a lytic action upon living gram-positive and upon dead gram-negative bacteria. The bacteriolytic properties of the living S. alhiis and of the actinomycetin preparation upon dead or living cells of bacteria was said to be due to a lytic prin- ciple, designated as actinozyme. Isolation of antihiotics.— The first true antibiotic of actinomycetes was isolated in 1940 from a culture of Actinoviyces CStreptoviyces^ antihioticus. This substance was designated as actinomycin (490). It proved to be highly interesting from a chemical and biological point of view and it affected a large number of bacteria, mostly the gram-positive types. Unfortunately, actinomycin proved to be extremely toxic to the animal body (491) and did not offer, therefore, any chemotherapeutic potentialities. Later, two other substances, proactinomycin (129) and micromono- sporin (468), were isolated. These agents had limited antibacterial spectra and, for one reason or another, they too failed to offer promise as chemotherapeutic agents. Later, other antibiotics were isolated. Attention was concentrated upon the isolation of antibiotics active against gram-negative bacteria, an acid-fast group of bacteria which in- clude the tuberculosis organism. These substances varied greatly in their antibiotic spectra, in their chemical properties, and in their in invo activities. A number of antibiotics are now known to be produced by actino- mycetes, as shown in Table 22. Some of the substances listed are, no doubt, closely related to others or vary from them only in certain minor properties. Some of these substances are produced by different organ- isms; this is true, for example, of actinomycin, v/hich is formed by a great variety of cultures (465, 505). Some organisms, on the other hand, produce more than one substance; S. griseiis, for example, pro- duces 2 forms of streptomycin, actidione, and an antibiotic present in the mycelium of the organism, later designated as streptocin (466). Some of the antibiotics of actinomycetes are active largely on gram- positive bacteria. Others are also active against gram-negative forms. Some, like streptothricin, are active against fungi. Some, like actidione and antimycin, are largely active upon fungi. Some, like neomycin (471) and streptomycin are completely inactive upon fungi. Some are active against trichomonads, as is the case of streptocin. Some are active • against rickettsiae and even against certain viruses, including phages (392). These antibiotics also vary gready in their toxicity to animals. Some, like actinomycin and xanthomycin, are highly toxic. Others, like streptomycin, aureomycin, and Chloromycetin, are relatively non- toxic. The differences in antibacterial action are frequently quantitative rather than qualitative. Streptomycin and streptothricin, for example. Chapter VII — 119 — Antagonistic Properties show certain similarities in chemical nature and in their general anti- biotic spectra; they differ in their toxicity to animals, in their selective action upon certain bacteria, such as B. mycoides and S. marcescens, and in the greater action of streptomycin upon M. Uihercidosis haminis. Some of the antimicrobial spectra are very narrow, as shown by the so-called antismegmatis factor, which is active only against M. s^neg- luatis and certain other mycobacteria (215). On the other hand, strep- tomycin itself is produced, not only by S. griseiis, but also by S. hikini- Table 23 : Inhibition of different actinomycetes by their respective antibiotics (475) : — Organism producing it Activity of preparation per 1 gm Dilution units per mg, exj activity against pressed as Antibiotic S . antibioticus S . lavendulae S. griseus Actinomycin Streptothricin Streptomycin S. antibioticus S. lavendulae S. griseus 100,000* loot 125t 100 5,000 1,000 0.4 1,000 100 100 10 1.2 * S. lutea units; t E. colt units; ( :rude preparations. ensxs (194, 195). Streptothricin or similar substances are produced by a large number of organisms.- These substances show certain quanti- tative differences in their action upon different bacteria, in their activity upon fungi, in their toxicity to animals, and in certain chemical charac- teristics. Usually an organism producing a certain antibiotic is resistant to its antimicrobial action (Table 23). Methods of isolation and testing.— In a search for antibiotics pro- duced by actinomycetes, several steps are followed, namely, 1. The soil or other natural substrate is plated out on suitable media and the colonies of actinomycetes are picked and transferred to slants. 2. The cultures are tested by the agar-streak method (Fig. 24), using a series of test bacteria. Those that are found to possess the highest or more desirable properties are selected. 3. The selected cultures are grown on suitable media, under stationary and submerged or shaken conditions, and antibiotic spectra of the metabolite solution determined. 4. After suitable media and culture conditions have been established, for a particular organism, it is grown until a large quantity of the metabolic solution is obtained. 5. Methods are now developed for the isolation, concentration, and purification of the antibiotic. 6. The purified antibiotic is now studied for its chemical and physical, as well as its antimicrobial properties, since the antibiotic spectrum of the isolated anti- biotic may not correspond to that of the metabolite solution. 7. The antibiotic is now tested for its toxicity to animals and its in vivo activity. Waksman — 120 — Actinomycetes Such simple procedures as the agar streak method can be used for screening purposes. The nature of the medium is of great importance, however, as shown in Table 24. Although for most practical purposes, it is sufficient to use ordinary saprophytic bacteria as test organisms, it may become advisable to use in certain cases pathogens. This is true particularlv in the search for organisms active against the tuberculosis organism. Williston, Zia-Walrath and Youmans (510) have shown, for example, that for screening of actinomycetes for their anti- tuberculosis activities, the avirulent, rapidly growing strain 607 of M. tuberculosis is not suitable; some strains of actinomycetes which inhibit Table 24: Distribution of antagonistic properties among actinotnycetes (194): — Cross-streak method. Numbers reported in per cent of total cultures. Zone of inhibition Activity mm B. subtil is E. coli M. avium M. pblei Nutrient agar Strong 20-35 21 6 6 1-b Medium 10-19 46 3 35 35 Weak 1-9 3 13 29 16 None 30 78 30 26 Gl, jcose asparagine agar Strong 20-35 15 6 Medium 10-19 28 6 35 70 Weak 1-9 35 14 8 10 None 22 80 57 14 the virulent H37Rv do not inhibit, under the same conditions, strain 607 (Fig. 23fl and Fig. 2'h¥). The nature of the medium is also of great importance, as shown in Table 25. Isolation of streftothricin and streptomycin.— Streptothricin was the first substance that appeared to show distinct promise as a chemothera- peutic agent, since it was not very toxic to animals, and especially since it was active against gram-negative bacteria. It was obtained (493) from a culture of an organism found to be identical with Actinomyces CStreptomyces^ lavendulae that had been isolated in the same labora- tory, from the soil, in 1916 (443,460). The name was derived from Streptothrix, as the actinomycetes were designated bv Ferdinand Cohn in 1875. Other strains of the S. lavendidae group were later isolated and found capable of producing streptothricin or closely related antibiotics (179a, 204, 215). Streptothricin is water-soluble and fairly resistant to heat, and is Chapter VII — 121 — Antagonistic Properties active against bacteria over a wide pH range with an optimum at slight alkalinity. It is also active in vivo against various bacteria and fungi. It is not active against viruses. It is resistant to the action of different microorganisms and to enzymes. Unfortunately, it leaves in the animal body a residual toxic effect which precludes its parenteral administra- tion and limits its use to oral or topical applications. The experience gained in the study of streptothricin proved to be suggestive in planning a search for other antibiotics that would possess similar or even more desirable biological and chemical properties and that would be less toxic to the animal bodv. After an extensive ex- Table 25: Inhibition of grotvth of i indent huviati tubercle bacilli by different actinomycetes (510) : — Inhibition, in millimeters, by agar streak method Medium 1 Medi um 2 Medium 5 Medium 6 Actinomyces ■ CULTURE H37Rv* H37Rv H37RvRt H37Rv H37Rv 1 25 25 13 32 2 15 18 21 3 17 4 9 11 27 5 11 6 15 6 16 18 10 17 7 3 4 14 8 23 9 20 15 15 10 16 18 15 18 11 12 20 3 4 8 12 13 15 15 20 18 14 S. griseus 20 12 12 20 * Streptomycin-sensitive strain of M. tuhenulosis . t Streptomycin-resistant strain of M. tuberculosis. amination of many cultures of actinomycetes, representing a number of species and strains, two freshly isolated cultures of an organism similar to one isolated from the soil in 1916 and described as Actinomyces CStre^toviyces} griseus (460) were obtained and were found to yield an antibiotic which did not possess the toxicity of streptothricin and had an even broader antibacterial spectrum. Since the generic name of this group of actinomycetes had recently been changed from Actinomyces to Streftoviyces (467), the new antibiotic was called streptomycin. Dif- ferent strains of S. griseus were later found to vary greatly in their ability to produce streptomycin and in their sensitivity to this anti- biotic. The course of growth of this organism, change in the con- Waksman — 122 — Actinomycetes stituents of the medium, and production of streptomycin are illustrated in Tables 26 and 27. A brief antibiotic spectrum of streptomycin as compared to that of another antibiotic produced by another strain of S. griseiis, namely grisein, is given in Table 28. The isolation, purification, and practical utilization of streptomycin in clinical medicine have had a most inter- esting history (390). Of particular interest vi'as the discovery that the J -^ \ Fig. 25.— Streptomyciii-piocluciiig strain ot S. ur/.scns, showing vegetative and aerial mycelium Cfrom Waksman and Schatz, 479). new antibiotic is also active against acid-fast bacteria (394), that it is not very toxic to animals, and that it is active both in vitro and in vivo against infections caused by various bacteria, including the organism that causes tuberculosis. Before many months had elapsed, strepto- mycin was tested clinically, and found to be effective against gram- negative bacteria causing a variety of human infections. It was also established that it is effective, not only in experimental tuberculosis, but Chapter VII — 1 23 — Antagonistic Properties in many forms of this disease affecting the human body. The cul- minating point of these studies was reached in 1946, with the publica- tion by the Committee on Chemotherapy (212) of the reports of one thousand cases in the clinical evaluation of streptomycin and of the first one hundred cases of tuberculosis treated with streptomycin (174). Streptoviycin-'prodzicing strains of S. grisetis.— An organism under Table 26: Growth and chemical chanties produced hy S. griseus under submerged conditions (10]): — Calculated as milligrams in 100 ml culture Incubation, DAYS 1 2 3 5 8 PH 7.4 7.3 7.6 7.5 8.3 8.9 Mycelium - 40 510 580 480 380 Streptomycin - 3.7 19.4 23.1 26.7 Glucose 900 880 800 240 60 - Soluble carbon 1,020 860 700 510 440 460 Lactic acid 29.2 32.8 11.4 1.3 1.6 1.5 Soluble nitrogen 148 130 110 76 73 114 Inorganic phosphorus 11,8 10.8 3.4 0.1 0.2 3.4 Ammonia-nitrogen 6.6 7.0 7.5 6.3 11.5 26.5 the name Actinomyces griseus was first described by Krainsky in Russia in 1914. In studies of the soil actinomycetes carried out by Waksman and Curtis in 1915, an organism was isolated from a Cali- fornia soil. This organism appeared to be similar to A. griseiis Krain- sky, so far as could be determined by comparison with the published description of the organism, but not by comparison of the actual cul- tures. In September 1943, two strains of S. griseus were isolated (390) Table 27: Nitrogen distribution in cultures of S. griseus (481): — Per 250-ml portions of broth* Nitroge ■n NH3-N NHo-N Total N L>TCUBATION in myceli ium in broth in broth accounted forj dajs per cent mg mg mg 7ng 10.0 35 4 35 4 10.0 35 23 57 115 5 9.7 40 38 70 148 7 10.0 56 56 73 185 10 8.9 62 63 67 193 15 9.6 55 93 79 227 21 7.2 38 95 71 204 J* Broth contained per liter 5 gm peptone, 5 gm meat extract, 5 grn glucose and 5 gm NaCl. t Total nitrogen in original broth 280 mg. Waksman — 124 — Actinomycetes and found to be very similar to the 1915 culture. One of these strains (No. D-1) was isolated from an agar plate streaked with the swabbing of a chicken's throat and the other (No. 18-16) from a heavily manured field soil. The two strains were identical in their morphological and cultural characteristics. They were isolated within two or three days of each other. Although it was believed at first that the second culture could not have arisen from the first, the possibility was not entirely eliminated. Both strains were very potent producers of streptomycin, but they differed in the relative amount of the antibiotic produced un- Table 28 : Antibiotic spectra of streptomycin and grisein (357) : — Units per gram of crude preparations* Streptomycin X 1,000 Grisein X 1,000 Bacillus subtilis 125 Bacillus megatherium 100 Bacillus mycoides 20 Bacillus cereus 30 Staphylococcus aureus 15 Sarcina lutea 100 Micrococcus lysodeikticus 150 Escherichia coli W 25 Serratia marcescens 25 Proteus vulgaris 10 Vseudonionas fluorescens 2 Ps. aeruginosa 1 Aerobacter aerogenes 10 Salmonella schottmulleri 15 Salmonella aertrycke 3 Eberthella typhi 25 Shigella sp. 25 Klebsiella pneumoniae 25 Mycobacterium phlei 100 * 1 unit of streptomycin is equivalent t o 1 M? of pure base a standard strain of £. call. 10 to 30 10 to 20 <.l <.l 30 to 100 0.5 200 to 300 25 10 <.l 3 <.l <.l 10 <.l <.l 30 5 <.l der different conditions of culture. No. DT was at first the more active strain, but later it declined in activity, whereas No. 18-16 continued to retain its high potency. The latter became the progenitor of all the cultures that are being used at the present time for the industrial pro- duction of streptomycin. When the 1915 isolates of A. griseus were later tested for their ability to produce streptomycin and for their sensitivity to the actino- phage of S. griseus (355), the former were found to produce no strep- tomycin and to be resistant to the phage. Upon the irradiation of this culture, Kellner (p. 73) succeeded in obtaining a mutant which had the capacity of forming typical strep- tomycin. This mutant was also sensitive to the phage which is active Chapter VII — 125 — Antagonistic Properties upon the streptomycin-sensitive strains. The conclusion was reached, therefore, that the streptomycin-producing cukures isolated in 1943 were identical with the 1915 isolate, that the latter has undergone con- siderable change in culture when grown for 30 years upon synthetic media, and that the 1915 isolate probably possessed the capacity for producing streptomycin, but has lost such capacity upon continuous growth upon artificial media (465a). CULTURE BRQTH I ADSORPTION ON CHARCOAL I ELUTION BY CH.OH-HCOoH I PRECIPITATION-PICRIC ACID I CONVERSION TO HYDROCHLORIDE CHROMATOGRAPHY ON ALO. OR CHARCOAL PRECIPITATION BY METHYL ORANGE CRYSTALLINE HELIANTHATE STREPTOMYCIN HYDROCHLORIDE- C01H3T.30N7O10.3HCI Fig. 26.— Method of isolation of streptomycin from metabolite solution. Many other strains of S. griseiis have now been isolated (65, 475) from soils, waters, river muds, animal excreta, dust, and other natural substrates. Only very few of these have been found capable of produc- ing streptomycin, the majority being inactive or producing another anti- biotic, such as grisein (357). Ability to form streptomycin may, there- fore, be considered as a strain rather than as a species characteristic, in Waksman — 126 — Actinomycetes contradistinction to ability to form penicillin, which is a characteristic property of the Penicillhim notatum—P. chrysogeniim group of fungi as a whole. The streptomycin produced by the active strains of S. griseiis was found to be made up of several chemical entities, namely, strepto- mycin and mannosidostreptomycin. Certain other species of Stre-pto- myces, such as S. hikiniensis (194), appear to produce an antibiotic which is identical with streptomycin. Certain actinomycetes produce a mixture of antibiotics, as streptomycin and streptothricin (428). One of the methods of isolation of streptomycin is presented in Fig. 26. In order to select the more active streptomycin-producing strains it is necessary to plate out the culture and pick colonies. These show con- siderable variation in streptomycin production. Several substrains ob- tained from No. 18-16, such as No. 4 and No. 9, are now largely used, one being more active in certain laboratories, and the other in others. Substrain No. 9 is also more susceptible to the actinophage. Strains which gave an activity of 100 to 200 [J-g/ml. of streptomycin have been developed to provide strains which give 400 to 500 [J-g/ml. and even 1,000 [;.g/ml. Irradiation with ultraviolet light, followed by picking cf colonies, has given cultures which yield 600 to 800 [J.g/ml. The high- est producing cultures in one medium are not always the highest in another. A suitable medium must also be selected for inoculation pur- poses in order to get high yields in the fermenter. The cultures are kept in a lyophilized state or are first grown on soil then dried, or are continuously transferred on ordinary agar media (65). Various procedures can be used for the isolation of fresh cultures of S. griseiis from natural substrates. Ordinary agar media are usually employed, and colonies picked and tested. The S. griseiis strains can be readily recognized by the pale green to grayish green shade of their aerial mycelium. The agar used for plating purposes may also be en- riched with streptomycin, as 25 or 50 mg/ml., to eliminate from the plate the great majority of bacteria and other actinomycetes. To estab- lish the identity of such cultures with streptomycin-producing strains of S. grisens, the cultures are treated with S. griseiis actinophage (474). The following method also offers certain advantages: A suitable agar medium is seeded with living cells of a nonpathogenic strain of M. tu- berculosis. The diluted soil suspension is added, and the plates are incubated at 28°-30°C. to enable the actinomycetes to develop. This is followed by incubation of the plates at 37 °C. to favor development of the M. tuherculosis. The antagonistic colonies of the actinomycetes will be surrounded by clear zones, free from the growth of the acid-fast bacteria. By the use of this method. Woodruff and Foster (516) iso- lated a substance, designated as streptin, which was similar in many respects to streptothricin. Despite the fact that it is possible to isolate large numbers of S. griseus strains, only very few of them will be found capable of produc- Fig. 27.— Crystals of the calcium chloride double salt of streptomycin (478). Waksman — 128 — Actinomycetes ing streptomycin. Some produce grisein (358), some produce other antibiotics, and some produce no antibiotics at all. Production of mutants of S. griseiis.— When an active streptomycin- producing culture of S. griseiis is plated out and individual colonies are picked and transferred to agar slants, various inactive strains can be ob- tained. One such type was found to differ from the mother culture by being free from aerial mycelium (395). This strain undergoes more rapid lysis, especially when grown in submerged culture. It produces Strep to Ur, , Urzi bs y /O -*/A/2L Glucosc. MG/ML Lactic Ac^, MG/ML Fig. 28.— Metabolic changes produced in the medium by Stre^ptomyces sp. RivETT and Peterson, 362). Cfro an acid reaction in the medium, and yields a more viscous broth. It is sensitive to the antibiotic action of streptomycin, whereas the mother culture is highly resistant to the action of this antibiotic. The active culture and the inactive variant are similar in many of their cultural characteristics, such as lack of dark pigmentation on organic media, pro- teolytic action, and hemolytic capacity. By proper cultivation and se- lection, the inactive asporogenous strain can be made to revert to an active sporulating form which will also produce streptomvcin. Another type of inactive variant or mutant was found to differ from the mother culture in the production of a pink or vinaceous pigmenta- tion in the vegetative growth (475). Some of the cultures of S. griseus Chapter VII — 129 — Antagonistic Properties are more variable in this respect than others and give rise continuously to strains that appear to be physiologically different, or at least to vary in their quantitative production of streptomycin. The fact that inactive strains are sensitive to streptomycin, whereas the streptomycin-producing cultures are resistant, would tend to bring about the continuous self- purification of streptomycin-producing strains from the nonproducing strains, as long as they are growing under conditions favorable to strep- tomycin production. Production of other antibiotics by actinomycetes.—A number of other antibiotics are known to be produced by actinomycetes (482). Some have been crystallized; others ha\'e ne\'er been obtained in even a concentrated form. Some have wide antibiotic spectra; others act only Table 29: Antibiotic spectra of streptomycin, streptothricin, and antibiotic 136 (40): — Dilution units per mill: igram Test organisms Streptomycin Streptothricin* Antibiotic 136 B. subtilis 40,000 6,000 140,000 B. cere us 3,000 50 2,600 S. albus 10,000 1,500 160,000 S. aureus 30,000 7,500 160,000 E. colt 6,000 1,200 17,000 A. aero2fnes 5,000 1,200 5,000 Pr. vulgaris 2,000 900 6,000 A. vis cos us 8,500 3,750 60,000 Ps. aeruginosa 550 165 1,400 S . marcescens 9,000 1,125 8,500 * This preparation assayed 150 £. coli uni streptothricin should he divided by 8 to make rations. On the basis of this comparison, the units for streptomycin an( comparable with standard streptomycin and streptothricin prepa against very few organisms. There is also a marked difference in their chemotherapeutic potentialities. Some of the antibiotic-producing organisms are widely distributed in nature. This is true particularly of such groups as S. lavendulae and S. griseiis. One would expect that some of the strains of these organisms would produce antibiotics which differ in chemical structure and, therefore, in their biological activities. Attention has already been called to the fact that some antibiotics actually obtained from different organisms may either represent a mixture of compounds or a single type compound, which varies, however, in its antibiotic spectrum and in its toxicity to animals. This variation depends upon the strain of organism producing the antibiotic, composition of the medium in which it is produced, and conditions of growth. This can be illustrated by antibio- tic 136, which is produced by a strain of S. lavendnlae, but which differs frc^m streptothricin in its antibiotic spectrum, in toxicity to mice and in the ratio of activity in broth and in agar (Table 29). Waksman — 1 30 — Actinomycetes The fact that certain actinomycetes are capable of producing more than one antibiotic frequently tends to confuse the recognition of the identity of any single constituent. The literature on the antibiotics produced by actinomycetes continues to accumulate rapidly. New agents are being isolated, as in the case of the pigmented substances actinorhodin (46a) and actinomycelin (66a). New light is being thrown upon the composition and activity of agents previously an- nounced, as in the case of neomycin (179a, 469a). New fractions are being isolated from older agents, as in the case of antimycin A (lOll?) Dai^s c^ /ncuba-tion S lO Rate, o/: Gnov\/th Sacterioly tic Activ-itu Fig. 29.— The course of development of S. a\hui and bacteriolytic activity of the culture filtrate, actinomycetin Cf^om Welsch, 505). and neomycin A (332a). New methods are being developed for the isolation and purification of unknown agents (461?). Antibiotics of actinomycetes and chemotherapy.— Among the various antibiotics produced by actinomycetes, some have already occupied a prominent place as chemotherapeutic agents. It is sufficient to mention streptomycin, aureomycin, and Chloromycetin. The most important applications are their use in the treatment of infections caused bv gram- negative bacteria, infections by gram-positive bacteria made resistant to penicillin, rickettsial infections, and tuberculosis. Several volumes have alreadv been devoted to the clinical use of streptomycin. A most extensive literature has accumulated on the use of streptomycin covering nearly 2,000 titles. Comprehensive surveys Chapter VII _ ] 3 1 _ Antagonistic Properties of the literature (455) and of the use of this antibiotic in the treatment of numerous infections (456) have already been published. The ex- tensive utilization of streptomycin in tuberculosis has stimulated numer- ous surveys of the production of antitubercular agents by actinomycetes. Both members of the NocanUa (105, 258) and of the Strepomyces (193, 471) genera have been investigated in detail. Among the most important problems that have arisen in connection with the use of streptomycin is the development of resistance among bacteria on contact with this antibiotic (476), and especially the develop- ment of streptomycin-dependent strains (300, 331). Chapter VIU DISTRIBUTION OF ACTINOMYCETES IN NATURE Actinomycetes are among the most widely distributed groups of microorganisms in nature. Very few natural substrates are entirely free from them. In some of the substrates, as in soils, in lake water and in lake bottoms, in composts, they lead a normal existence. In other substrates, as in sea water and in dust, they are only in a transitory state. The ability of actinomycetes to survive for a long time is indicated by the fact that Omelianski isolated an actinomyces (A. elephantis frimigenW) from the slime of a mammoth's nose. This culture showed no particujar properties which would distinguish it from the common actinomycetes found in soils. It must be added, however, that the slime removed from the mammoth, upon its discovery, was kept, without spe- cial precautions of preservation, for several months previous to the iso- lation of the organism (323). Actinomycetes are found abundantly in all soils throughout the world; they make up, in many cases, especially under dry alkaline con- ditions, a large part of the microbial population of the soil. They also occur on plant residues and upon and in various foodstuffs, such as fruits, vegetables, milk and milk products, and cacao. The open sea is the only important natural habitat where they, like most of the true fungi, are almost entirely absent; whenever their presence has been re- ported, it was limited to waters close to shore or to waters subjected to land wash, or it was limited to growth upon submerged surfaces, notably piers and other landmarks. Actinomycetes are found in peat bogs, usu- ally in the surface layers where oxygen is present, although occasionally they are also found at greater depths. Comparatively few types of actinomycetes are known to be capable of causing plant and animal diseases, but both aerobic and anaerobic actinomycetes may be concerned with human and animal infections. Some of these are deep-seated and involve special methods of treat- ment. Our knowledge of the occurrence of actinomycetes in nature dates back to the early days of bacteriology. Following the early observations and descriptions of various actinomycetes by F. Cohn and by Bollinger and Harz, Miquel (301), in 1879, in connection with his work on the bacteria of dust over Paris, carefully described certain actinomvcetes. The full significance of the nature and importance of these organisms Waksman — 1 34 — Actinomycetes was not recognized for many years. Mace (278) reported, in 1888, that actinomycetes occur abundantly in water basins. Globig (138), in 1888, and Rossi-Doria (370), in 1891, made a detailed study of their occurrence and activities in different soil types. In 1900, Beijerinck drew attention to the fact that actinomycetes are widely distributed in nature. They were found not only in the surface layers of the soil but also in the subsoil to considerable depths; at depths of one meter in garden soil and two meters in sandy soils, they sometimes exceeded in numbers the other groups of microorganisms. They were also present in river mud below the river bed. Beijerinck emphasized the fact that actinomycetes represent a group of omni\'orous organisms capable of growing not only under conditions favorable to their development but even under certain unfavorable conditions. He even found them to grow in distilled water in ordinary laborator\' air. They were unable, however, to fix atmospheric nitrogen. Nadson (311) also studied in 1900 the occurrence of actinomycetes in nature and their role in natural processes and as geological agents. He isolated several cultures of these organisms from the curative mud of a salt lake, and established their ability to decompose proteins, to pro- duce ammonia and HoS, and precipitate CaCOa. Since the work of these pioneers, considerable information has ac- cumulated concerning the abundance of actinomycetes in various na- tural substrates, as determined by cultural and direct microscopic meth- ods. Although great advance has been made in the appreciation of the role of actinomycetes in many natural processes, no clear picture has been drawn of this function of so large and heterogeneous a group of microorganisms, and the information may still be considered as largely fragmentary. This is due largely to a lack of sufficient knowledge con- cerning the intermediary metabolism of actinomycetes, their relationship to other microorganisms growing in natural substrates, and their fre- quent confusion with the bacteria and with the fungi. The last is particularly important, since actinomycetes are capable of bringing about reactions, such as protein decomposition, ammonia formation, nitrate re- duction, and cellulose decomposition, which are commonly associated with activities of fungi and bacteria. Occurrence of Actinomycetes in the Soil:— The soil represents an ideal natural substrate for the development of actinomycetes. It is no wonder, then, that they are found so abundantly there, where they are represented by many genera and species. They are found in both virgin and cultivated soils, in fertile and in unfertile soils, in various regions throughout the world (126, 440, 461). They are particularly abundant in alkaline soils and in soils rich in organic matter. It has even been suggested that their major function in the soils consists in the decompo- sition of plant and animal residues (76, 484). Chapter VIII ^ ns — Distribution Methods of study.— Four methods can be used for determining the presence and abundance of actinomycetes in the soil: I. the direct micro- scopic method of stained soil; 2. the direct microscopic examination of undisturbed or unstained soils; 3. the contact slide method; and 4. the plate dilution culture method. Each of these methods has certain dis- tinct advantages and limitations. Conn (77) was able to demonstrate, by the direct staining of the soil, that the actinomyces mycelium is present abundantly in the soil, especially in soils rich in organic matter. This method does not permit the differentiation between actinomyces spores and certain bacteria. Since the mycelium is not uniformly distributed in the soil, the method does not permit an accurate quantitative evaluation of the abundance of the organism. Furthermore, recognition of individual forms is often limited, since in the process of staining, the mycelium is usually broken up, and both mycelium and spores of various forms will appear similar. The method may, therefore, be limited to the recognition of certain broad groups rather than of specifiic types. Direct examination of undisturbed natural soils presents certain marked advantages, since it gives a picture both of the relative abun- dance of this group of organisms in the soil and its distribution through the soil mass. Kubiena and Renn (245) used a vertically illuminated microscope. Actinomycetes were found growing in the soil spaces open- ing to the surface. Aerial tufts of hyphae in the form of more or less compact colonies with long twisted strands were found to bridge the gulfs between the soil crumbs. When the soil is enriched with organic materials, such as proteins and lignins, the growth of actinomycetes is greatly stimulated. The contact slide method offers certain advantages over the direct staining method. It permits development of specific organisms upon the slide, and even formation of fruiting bodies, thus making possible the differentiation and recognition of certain broad groups. One is also able to determine by means of this method, not only the gross effects of additions of organic matter and lime to the soil, but also the response in the development of actinomycetes to different types of fertilization and cropping. The relation between pathogenic and saprophytic forms to the root systems of plants can also be studied by the use of the con- tact slide method. Cholodny (68) demonstrated, for example, that the direct microscopic method gives a rather inaccurate picture of the abun- dance of actinomycetes, as compared to the contact slide method. The latter has, however, a marked disadvantage, since it gives no idea of the relative abundance of actinomvcetes in undisturbed soil. By the use of the contact slide method, Waksman, Umbreit, and Cordon (487) were able to demonstrate that, in composts kept at dif- ferent temperatures, the fungi and the bacteria were the first groups of microorganisms to develop at 50° to 65 °C.; however, these organisms Waksman — 136 — Actinomycetes were rapidly replaced by an abundant population of actinomycetes. Specific forms could be recognized, by means of this method, and studied in detail. The plate method has been used most commonly to study the abun- dance of actinomycetes in the soil and to isolate specific organisms. Synthetic media were found to be highly favorable for the development of these organisms. Some forms grow readily on virtually all common media, whereas others require either certain specific media or special con- ditions of growth. The actinomyces colonies can easily be distinguished from those of bacteria, a somewhat longer period of incubation usually 20 o - A ^^ ./. t / \ --' '' / /'\^ i -3 ^ /SO' / \ I / \ i % / \ /cv- ?j / ^ / / \ s 3Z?- / / ■•J >J ^ ^ / 1 J S /(? }s zo 25- /ncubation, Dai/S C^ensity of Mi/celium ' Plate counirs Fig. 31. Relation between density of vegetativi actinomycetes Qroni Jensen, 192). lyceli id plate counts of being required. This method also has certain limitations, the most im- portant of which is the lack of differentiation between spores and my- celium. A colony of an actinomyces may originate from a single spore or from several spores or from a piece of mycelium. Not all spores are capable of germinating on a given plate and developing into colonies. The numbers thus obtained represent only a minimum content of actino- mycetes in a given quantity of soil, or only a fraction of the mass of actinomycetes. This method has been used most extensively, however, for the evaluation of the actinomyces population of the soil. Ah^indance of actinomycetes.— Hiltner and Stormer (172), in 1902, made the first comprehensive survey of the abundance of actino- mycetes, as compared to that of bacteria, in the soil. In the spring of the Chapter VIII — By- Distribution year, 20 per cent of all the colonics developing upon an ordinary agar plate, when various soils were plated out in accordance with accepted bacteriological procedures, consisted of actinomycetes. In the fall, the number of actinomycetes increased to 30 per cent of the total microbial population developing on the plate. This increase was believed to be due to the greater amounts of fresh plant residues becoming available at that time of year. In the winter, there was a drop in the relative num- Table 30 : Numbers of bacteria and actinomycetes in the soil developing on albumen agar (460) : — Per 1 gm of soil Soil type Bacteria Actinomycetes Numbers Numbers Per cent 1 New Jersey Sassafras garden 5,300,000 900,000 14.5 2 New Jersey orchard 4,800,000 700,000 13.4 3 New Jersey clay meadow 8,100,000 550,000 6.3 4 New Jersey Sassafras forest 610,000 110,000 15.3 5 Iowa Carrington loam 1,764,000 236,000 11.8 6 Jamesburg Cranberry soil 204,500 7,500 3.5 7 Louisiana sandy loam 8,300,000 1,700,000 17.0 8 California fertilized soil 3,570,000 630,000 15.0 9 California unfertilized soil 580,000 330,000 36.3 10 California upland 2,220,000 1,238,000 35.8 11 California adobe 3,620,000 800,000 22.0 12 California sandy loam 6,010,000 1,430,000 19.2 13 Oregon adobe 13,100,000 2,400,000 15.4 14 Oregon white land 3,400,000 300,000 8.1 15 Porto Rico clay loam 2,140,000 960,000 31.0 16 North Dakota wheat soil 2,067,000 933,000 31.1 17 North Dakota flax soil 1,737,000 263,000 13.2 18 Hawaiian pineapple soil 4,334,000 666,000 133 19 Alaska soil 6,034,000 1,566,000 20.6 20 Texas Lufkin fine sandy soil 2,126,000 574,000 21.3 21 Colorado alfalfa soil 2,440,000 1,560,000 39.0 22 Maine Aroostook potato soil 4,650,000 250,000 5.1 23 Maine dark Aroostook infected 15,900,000 2,200,000 12.2 24 Alberta grass soil 1,110,000 760,000 40.6 25 Alberta garden soil 2,000,000 1,700,000 46.0 Average 4,245,000 870,500 17.0 ber of actinomycetes to 13 per cent, believed to be due to the effect of frost. When the soil was treated with stable manure, there was a marked increase in the total and relative numbers of these organisms. Fisher (122) reported a much smaller number of actinomycetes in various soils, seldom exceeding 15 per cent of the total number of organisms developing on the plate. This may have been due to different media used in the evaluation of the abundance 'of these organisms in the soil. Tusuhioavif -j- smitqoq ^ snuB'EoucoAcponsoA ■ ^ SVtpVAf -^ uuvmii] -^ 3tU9a '^ snssu'i -^ S713AitO J timtfSA -J ytufvoqjp -^ snqjp s stiotiPisvtp -J smd'ioiuoMpoputa -^ rnuB^oiuoAcposijtni -^ 9P9jlffBP0PdOU •j' AOJOOtJBOJ 'J + + + + + + + 4- ++++++ + + + + + ++ +++++ + + + + + + + + + + ++++++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -13 .J:J " 'o ^ E ^ ^ !;1|,^ llllllllllH ^N ?*^ P*^ ?*% rH ^ pJl^ n --^ -ic V- H V? ^ S 3 'S '" '- '- '^ .„ ^ ^ i; O --s— s— sr-s-^ "5; O ,0 ^O ^O S O O q jC Q Q .5 Z2ZMH-l(JUUUUOOa.ZZaC.: •■: •■■:■■• ",- '"^ ..:■ -...- 'u^v^V:'-k: W^^^J^^^ 1 -" K ^ j\ s***" '^" ■ „ ■ » Fig. 38.— The appearance of a Nocardia in the sputum of an infected patient Cfrom Davis, 88). infection at first based upon the work of Bostroem, who suggested that infection results from handling of straw or other plant materials, has b^en gradually replaced by the endogenous theory, as postulated by Lord, Naeslund, Emmons, Slack, and others. According to the Waksman — 182 — Actinomycetes newer theory, the mouth, including the oral cavities and pyorrhea pus, the pharynx, and other organs such as the tonsils, is the normal carrier of the infectious agent. In addition to the common abdominal, pulmonary, and cervico- facial, forms of actinomycosis, other types exist, such as cardiac in- volvements and subcutaneous infections. Myocardial and pericardial forms of actinomycosis may be considered clinically as cases of rheumatic heart disease (81). Two cases of actinomycotic endocarditis, due to aerobic forms, have been studied in detail by Wedding (496). Gins and Paasch (137) found that most of the clinical cases suspected of being actinomycotic turned out to be due to other causes; only 1 out of 14 cases was caused by a true actinomyces. Aerobic Actinomyces Infections:— In addition to the anaerobic forms of actinomycosis, many infections in man and in animals are caused by aerobic species of actinomycetes. "Streptothricosis" or "no- cardiosis" should always be differentiated from "actinomycosis," espe- cially in lung infections. BosTROEM (44) reported the discovery of an aerobic type of Actino- myces, designated as A. hominis, but, as pointed out above, his conclu- sions were erroneous. The significance of these results has frequently been questioned (170) for several reasons: (a) saprophytic aerobic ac- tinomycetes occur abundantly as air contaminants; (I?) Bostroem suc- ceeded in making only relatively few isolations; (c) he, as well as others, failed to obtain infection in experimental animals and in cattle; and (d) he made his isolations from the common "lumpy jaw" type of bovine actinomycosis known to be caused by an anaerobe, A. hovis. It may be mentioned here, in passing, that Bostroem's A. hovis and several of the forms accepted by Baldacci are probably species of Strep- tomyces. The aerobic nature of the actinomycetes causing certain infections, such as that of Madura foot spoken of as Mycetoma fedis and occurring largely in the tropics, is well established. The pus contains white or yellowish granules, similar to the sulfur granules of the lumpv jaw, from which as many as 13 strains of Nocardia have been isolated. The causative agent studied by Vincent (438) is considered to be the most common. The organism is readily cultivated and is now recognized as Nocardia madurae. These aerobic organisms cause specific types of mycetomas; their multiplicity has no bearing whatsoever on the early erroneous work of Bostroem on the etiology of "lumpy jaw," as brought out above. Among the aerobic forms, the acid-fast actinomycetes are particularly significant. Infections of the lungs and of the skin are frequentlv produced but no clubs are formed at the extremity of the hyphae in infected tissues. The aerobic tvpes are cultivated much more readily than the anaerobes and are pathogenic to laboratory animals. The or- Chapter XI — 183 — Human and Animal Diseases ganism can be demonstrated in the sputum of infected animals. N. farcifiica, isolated from cattle, forms a yellowish, wrinkled growth on solid media. N. cafrae, isolated from the lung of a goat (402), gives a more whitish growth and greater fragmentation of the mycelium. N. canis produces infection in dogs (310), and is similar to N. cafrae. Eppinger, in 1891, reported the isolation of an aerobic, gram-posi- ti^'e, acid-fast actinomvces from the cerebral abscesses and meningeal exudate of a man who became delirious and died in 2 weeks. This organism readily grew on ordinary media in the form of small colonies that were star-like because of the radiating filaments. It has been known since its isolation as Cladothrix asteroides, Streptothrix e-pfingeri, Streptothrix asteroides, Oosfora asteroides, Actinomyces asteroides, and Nocardia asteroides. This group of the aerobic pathogenic actinomy- cetes is the most common. It is the least proteolytic and produces a yellowish to orange, wrinkled growth on solid media; aerial mycelium is white and scant, if formed at all. In 1921, Henrici and Gardner collected 26 cases of infections with aerobic acid-fast actinomvcetes. They reported that the causative or- ganisms fell into three different types, which differed chiefly in the color of the growth on solid medium and in other minor biologic char- acters. Twenty-three of these cases were of pulmonary origin, and all but one were fatal. Another form was isolated from the sputum of a patient with a cough of 3 years' duration. It differed somewhat from the other three types, and because of the chalky white appearance of the growth, was named Nocardia gypsoides. After repeated subcul- tures, however, the strain became almost identical with N. asteroides. Gordon and Hagan (144) found that some acid-fast actinomycetes isolated from soils and plant material are similar to those found in lesions of men and animals. The pigments produced by these organ- isms range from yellow through orange to coral. One of the soil forms was pathogenic to rabbits soon after isolation, but not to guinea pigs (145). Various strains of Nocardia have been described as causative agents of madura foot, a disease usually referred to as "nocardiosis" or "madura- mycosis." These organisms include not only N. madurae, but also N. indica, N. pelletieri, N. mexicaniis, N. hrasiliensis, N. paraguayensis, etc. These strains were identified by their cultural characters on dif- ferent media, production of pigment, by their morphology and staining properties, some being acid-fast. They were all apparendy of the same general type as the other forms of Nocardia listed above. The same is probably true of the several strains isolated by Pipper and Pullinger (338), namely N. transvalensis, N. africana and N. pretoriana. These authors assumed that the natural habitat of No- cardia is grass. They ascribed, therefore, the relative frequency of Nocardia infections in South Africa to local conditions and habits, namelv abundance of grass, open-air hfe, scanty clothing and bare foot Waksman — 184 — Actinomycetes walking. The organisms were said to produce typical clubs, but their cultural characters and animal pathogenicity were different from those caused by species of Actinomyces. The nocardias were found to have a marked affinity for iron. Baldacci (18-20) suggested that the various strains of aerobic acid- fast actinomycetes represent only minor differences in their biologic characters, and must be considered as variants of N. asteroides. According to Drake and Henrici (96), Nocardia asteroides has little invasive power for rabbits and guinea pigs. Large doses intra- peritoneally in guinea pigs, smaller doses intravenously in rabbits, pro- duced death with acute peritonitis and multiple miliar)^ abscesses, re- spectively. But smaller doses produced neither lesions nor death. Subcutaneous and intramuscular injections did not spread, but healed. All attempts to produce a progressive disease similar to tuberculosis failed. An allergic state in rabbits and guinea pigs against N. asteroides could be induced with regularity only by the intratesticular injection of oil suspensions of live organisms. Various other strains of aerobic actinomycetes have been isolated from human infections. An actinomyces isolated from the ear in- volved a disease resembling the ordinary type of chronic suppurative otitis media (327). The organism was said to have maintained itself in the middle ear for 10 or more years. Two cases of N. asteroides in- fection with pulmonary and multiple subcutaneous abscesses and si- nuses, with a cerebral abscess suspected in one case, were observed (30). The organisms were isolated from the sputum and from the subcutaneous lesions of both. N. asteroides was also isolated (37) from a case of chronic suppurative pneumonitis and massive cerebral abscess in a man under observ^ation for a brain tumor. KiRBY and McNaught (220) studied two cases which showed gross and histologic signs of specific lesions as of an acute pyogenic inflam- mation with a central zone of liquefactive necrosis and numerous polymorphonuclear leucocytes. About the area of liquefaction there was a zone of granulation tissue with neutrophils, Ivmphocytes, and plasma cells and, at times, varying amounts of slightly more dense fibrous tissue. The dispersed mycelia of the organism were not seen in hematoxylin and eosin stains but were obser\'ed in sections stained by Gram's method. In one case, the etiologic agent was isolated from the sputum, sub- cutaneous abscesses, and blood stream; at autopsy, the lungs, peri- bronchial lymph nodes, heart, thyroid, kidneys, spleen, intestines, muscles, and subcutaneous tissues contained abscesses produced by N. asteroides. The second case was hospitalized for 5 days prior to death with a clinical diagnosis of an intracranial lesion without localizing signs. Autopsy revealed an abscess in the cerebellum caused by N. asteroides. The lungs were suspected as the primary focus, and no other metastases were found. Chapter XI — 185 — Human and Animal Diseases N. asteroides, or closely related strains have been isolated from cases of diffuse peritonitis, of pseudotuberculosis with cerebrospinal menin- gitis, and of brain abscesses. Sartory and Bailly (382) isolated a cul- ture from the urine of a patient suspected of renal tuberculosis. The organism was acid-alcohol-resistant; was cultivated on ordinary solid or liquid media only with difficulty; and grew well on serum and blood media at 35°-37°C. The organism, described as A. sero-^jJiilus, was be- lieved to be the causative agent of renal actinomycosis. Various attempts have been made to study the immunological re- actions of actinomycetes. Goyal (151) examined 11 cultures obtained from collections and as fresh isolations. Most of them appeared to be members of the genus Nocardia. When inoculated into rabbits, they proved to be either entirely non-pathogenic or only slightly virulent, except N. efpingeri. These cultures were grown in glycerol broth at 38 °C. for 30 days. Extracts were prepared in a manner comparable to tuberculin. These extracts were designated as streptothricine. Their antigenic reactions were very similar to tuberculin. Animals sensitized to the nocardia extracts were also sensitive to tuberculin, and xnce versa. Serologic studies confirmed the conclusions reached on the basis of allergy tests; a common antigen was demonstrated for the tubercle bacil- lus, the diphtheria organism and the nocardias. These results led to the conclusion that there is a definite antigenic relationship between the actinomycetes and the mycobacteria. Chemotherapy of Actinomycosis:— In addition to the application of vaccinotherapy, radiotherapy, and chirurgy of actinomycosis, subjects which need not be discussed here, extensive use is made of chemo- therapy. A detailed survey of the various clinical aspects of actinomycosis in man and of methods of treatment was made by Colebrook (73), Cope (80), and others. Lyons, Owen and Ayers (273) and others (128) reported favorable results from the treatment of actinomycotic cases with sulfonamides, especially sulfadiazine, or with thymol (248). Long- continued drug therapy is required, and the danger of recurrence is always present. The favorable effect of massive doses of penicillin has also been observed in a number of cases. Cutting and Gebhardt (82) found sulfadiazine and sulfathiozole more effective than sulfonamide in inhibiting the growth of both an- aerobic strains of both laboratory and freshly isolated strains of an or- ganism designated as A. hoviinis. DoBSON, Holman and Cutting (93) obtained an apparent cure from the use of sulfanilamide, iodides, and roentgen rays in the treat- ment of three cases of actinomycosis. Dobson and Cutting (92) treated 16 cases with sulfonamide or penicillin. In 7 cases, the disease was considered as cured and as ar- rested in another 7. In three cases, penicillin alone was effective; in Waksman — 186 — Actinomycetes three, penicillin and sulfadiazine were required; and six cases were cured by sulfonamide drugs. In two cases in which sulfonamide medication was given, the disease ended fatally. The conclusion was reached that both penicillin and sulfonamides are highly effective drugs in the treat- ment of the anaerobic forms of actinomycosis. A detailed study of the effectiveness of penicillin on various actinomycetes, including the repre- sentatives of the different genera, has recently been made by Drake (95). Patients suffering from infections due to the aerobic N. asteroides have benefited from treatment with the sulfonamide compounds and penicillin; this benefit was similar to the effect upon patients infected with the anaerobic A. hovis. Surgical drainage, iodides, and roentgen ray therapy are recommended as the indications arise (92). Two cases of pulmonary and chest wall infections with acid-fast Nocardta gave good response to rest treatment, surgery, vitamins, sulfonamides and iodides (30). Numerous contributions have recently been made concerning the treatment of actinomycosis with penicillin, alone (248) or in combina- tion with sulfadiazine (93, 119, 209, 345). However, in a case of nocardiosis which resembled pulmonary tuberculosis, only intensive therapy with sulfadiazine was recommended; penicillin and strepto- mycin failed (139). Actinomycosis of the central nervous system responded clinically to treatment with sulfadiazine, penicillin and streptomycin (183). Holm (177) surveyed the penicillin-sensitivity of anareobic actinomycetes. Their sensitivity was found to be similar to that of staphylococci. If whole colonies were used, however, in making the tests, some were found to be more resistant. The resistance of the typical "sulfur gran- ules" in the pus to penicillin may be due to this phenomenon. The dosage and mode of administration of penicillin should therefore be controlled by the presence of such granules. Various strains of A. hovis, including both human and bovine iso- lates, were found to be highly sensitive to penicillin, all being inhibited by a concentration of 0.5 unit per milliliter. They developed only slight resistance to penicillin upon continuous transfer in media con- taining this antibiotic. The strains were inhibited by 30 units per milhliter of streptomycin. All strains rapidly developed a high degree of resistance upon consecutive transfer in media containing strepto- mycin. Both resistance and reversion to original sensitivity occurred in a step-wise manner suggesting the possibility of genetic changes in the organism (39a). Chapter XII SUMMARY Actinomycetes are among the most widely distributed groups of microorganisms. Thev are of universal occurrence and they play an active part in the cycle of life in nature. One of the early students of the group, Beijerinck, recognized that they are omnivorous organisms and that they are capable of living both in a nutrient-rich and in a very poor en\dronment. Water and air were said to supply nutrients for the modest needs of these organisms. Actinomycetes are- able to utilize both inorganic and organic forms of nitrogen. The extent of their growth upon artificial media is gov- erned bv the available energy, the supply of oxygen, available nitrogen, and certain other nutrient elements. One of the greatest contributions to a better knowledge of these organisms was their cultivation on syn- thetic media, upon which they form characteristic morphological struc- tures and upon which thev develop a variety of specific biochemical characteristics. Actinomycetes are capable of breaking down proteins to amino acids and to ammonia; frequently, active proteolytic enzymes are produced. They are able to utilize a large variety of organic compounds for nutri- tive purposes and grow under various favorable and adverse conditions. Manv actinomvcetes are strongly diastatic, and many are capable of at- tacking various hemicelluloses. Some are able to utilize cellulose, some attack lignins, paraffins, fats, and rubber-like materials. Manv actinomvcetes are able to reduce nitrate to nitrite, but not to ammonia or atmospheric nitrogen. Beijerinck believed, however, that under certain conditions this reduction may lead to losses of nitro- gen through the interaction of nitrites with ammonium compounds. Actinomycetes are unable to fix atmospheric nitrogen, certain re- ports to the contrary notwithstanding (32). These reports were based upon the observation that many actinomyces colonies develop on media to which no fixed form of nitrogen has been added. The limited growth produced by such colonies can easily have obtained their nitro- gen from various impurities in the medium or in the atmosphere. Ac- tinomvcetes do not nitrify ammonium salts, although detection of small amounts of nitrites has been reported (308) in media containing am- monia; this may have been due to the sensitivity of nitrite reagents. ^ Certain actinomycetes can develop at temperatures as high as 60° Waksman — 188 — Actinomycetes to 65 °C., especially in composts, whereas others, such as those found in abundance in muds and in lake and river bottoms, thrive at rather low temperatures. Under unfavorable conditions, actinomycetes grow only slowly and poorly; this has often raised the question concerning their active participation in a given process. Most of them are sensitive to an acid reaction (pH 6.0) and are favored by an alkaline reaction (pH 7.0 to 7.5) of the medium. Actinomycetes produce a variety of pigments. The black pigment formed on protein media may function as an oxidizing agent, and on this basis, the suggestion has been made that actinomycetes play an im- portant role in the formation of humus in the soil. They occur in soil at considerable depths, where they may exceed in numbers the other groups of microorganisms. These facts have led to the suggestion that actinomycetes play an important role in soil processes. Of the four genera now recognized among the actinomycetes— Actinomyces, Nocardia, Streptomyces, and Micromonos'pora— the animal pathogens are found largely in the first, the anaerobic genus, and to some extent in the second. The plant pathogens are found in the third. The water forms and the high-temperature compost forms are found largely in the fourth. The last three genera occur in great abundance in soils, where they make up nearly 25 per cent of the total population of microorganisms developing on the ordinary agar plate. They occur in the dust and on the surface of grasses and foodstuffs. Their relative abundance in close proximity to the roots of plants is due not so much to their particular preference for living roots as to the fact that they find nourishment in the dead residues and excreta of the roots. With the rapid progress, within recent years, of our knowledge of antibiotic substances, and with the recognition that actinomycetes may play an important role in the production of such agents, new interest was aroused in the nutrition of these organisms. The introduction of the submerged culture method for their cultivation has made possible not only their rapid and abundant growth but also the study of many physiological reactions not previously recognized. In order to obtain abundant growth, sufficient energy material must be applied by proteins, carbohydrates, or organic acids; proper sources of nitrogen, either organic or inorganic; and certain minerals, notably potas- sium, magnesium, phosphorus, sulfur, and iron, are also necessary. Certain forms are capable of producing vitamin-like substances favoring the growth of other microorganisms. Many are able to produce anti- biotic substances injurious to the growth of other organisms. Under comparable conditions of nutrition, actinomycetes may pro- duce as much growth and decompose as much of the substrate as do some of the common fungi and bacteria. Carbohydrates, such as glu- cose, favor the growth of the organism and the utilization of proteins and amino acids. The efi^ect, however, is different from that upon fungi, since the latter prefer the carbohydrates to the nitrogen com- Chapter XII —189— Summary pounds as sources of energy, whereas the actinomycetes prefer to utiHze for this purpose the organic nitrogenous compounds. The glucose may thus serve as a buffer, since the acid produced from it tends to neutralize the excessive amounts of ammonia which are produced by actinomycetes and which would soon bring growth to a standstill because of a rapid change in reaction of the medium to highly alkaline, pH 8.6-9.0. The metabolic changes produced in the medium by actinomycetes are greatly influenced by the nitrogen source. In general, however, actinomycetes are similar to bacteria and to fungi in their nutrition, in their energy utilization, in the transformation of nitrogenous com- pounds, in the liberation of ammonia, and in cell synthesis. Actinomycetes thus form a major group of microorganisms, and com- parable to the other two major groups, the bacteria and the fungi, their activities can be summarized under the following five headings: Role in natural processes. Causative agents of disease. Agents of spoilage and deterioration. Utilization for production of enzymes and vitamins. Production of antibiotics. Role of Actinomycetes in Natural Processes:— The general occur- rence of actinomycetes in all soils and their omnivorous nature suggest their probable importance in soil processes. The facts that they make up as many as 15 to 40 per cent of all colonies developing on the plate, that they occur in the soil at great depth, and that they are favored by arid soil conditions and by an alkaline reaction suggest that, under cer- tain conditions, actinomycetes are concerned in a number of important processes. The following soil reactions may be due, to a considerable extent, to the activities of actinomycetes: I. Decomposition of complex plant and animal residues in soils and in composts. 2. Liberation of ammonia from complex proteins. 3. Humification processes accompanied by the formation of black coloring substances, the decomposition of humus com- pounds, and the synthesis of cell material, which further contributes to the formation of soil organic matter or soil humus. 4. Reduction of nitrate to nitrite, but not to atmospheric nitrogen. 5. Favorable effects upon plant growth. For example, when a soil was enriched with actino- mycetes, plant roots were longer. This effect was explained by the greater decomposition of organic soil constituents. The effect was greatest on legume bacteria, which suggested possible assistance to legume bacteria in infecting the plants and causing greater nodule devel- opment (125). Actinomycetes appear to be important geological agents, although thei^ role in this respect has not been fully established. Nadson (311), who isolated several actinomycetes from lake muds, found that they Waksman — 190 — Actinomycetes could reduce gray mud to black mud, a process accompanied by move- ment of calcium and iron to the upper mud layers. The precipitation of CaCOs was said to be fa\'ored by the production of ammonia, which changes the reaction of the medium to alkaline. Molisch (302) included an Actinomyces among the organisms contributing to the pre- cipitation of CaCOs. Sawjalow (386) also isolated from lake mud an actinomyces (y4. pelogenes') which was believed to be capable of reduc- ing sulfate to hydrogen sulfide. Actinomycetes as Causative Agents of Disease:— Actinomycetes, unlike the bacteria and the viruses, are not responsible for any of the great plagues that affect mankind and his domesticated animals. Neither are they as universal agents of plant destruction as are many fungi. Still, they are capable of causing certain important deep-seated diseases that affect both the animal and plant kingdoms. The actino- mycotic diseases of man and animals and the scab diseases of certain plants, notably potatoes and mangels, point to their great potential im- portance as disease-producing agents. Among the animal diseases, those brought about by anaerobic organ- isms (actinomycosis) and those brought about by aerobic forms (no- cardiosis) are frequently confused. The introduction of penicillin as a chemotherapeutic agent has served to reduce the danger from these infections, at least so far as man is concerned. The problem of plant diseases may sometimes reach alarming pro- portions in connection with the highly important economic crop the Irish potato. Scabbiness is favored by dry soil conditions, by an alkaline re- action, and by a high humus content of the soil. On the other hand, the use of organic fertilizers and green manures, especially under humid conditions, serves to control this infection. In addition to potato scab and mangel scab, a few other plant diseases, such as those of the sweet potato, are caused by actinomycetes, but these are only minor in nature. Actinomycetes as Agents of Spoilage and Deterioration:— Ac- tinomvcetes may play a far more important role as agents of spoilage than is commonly appreciated. This includes two phenomena: 1. Deterioration of certain foodstuffs, which is largely caused by the im- parting of characteristic earthy and pungent. flavors and odors to milk, cacao, potable waters, and fish. In the case of the latter it is not the direct infection of the fish but the tainting of their flesh due to the absorption of the odoriferous substance from the water. 2. Staining and actual destruction of certain fabrics, notably, woolens, cotton goods, and paper. Actinom^'cetes cannot compare with the fungi as agents of destruction of textiles under humid and high temperature conditions. But even as slower-growing organisms, they can produce on cloth, cither woolen or cotton, and on paper, especially in books, stains which reduce considerably the value of the material. Chapter XII _ 191 — Summary Utilization of Actinomycetes for the Prockiction of Enzymes and Vitamins:— Comparatively little use has been made so far of aetinomy- cetes for production of chemical compounds that hnd application in in- dustry or in nutrition. Only one attempt has been made to utilize the diastatic enzyme of an actinomyces; this has been produced under the name "superbiolase," because of its ability to withstand higher tempera- tures than the corresponding enzymes of barley and of certain micro- organisms. Of much greater importance is the recent finding (359a) that certain strains of S. grisens (grisein-producing) are capable of pro- ducing vitamin Bi2- The red crystalline material isolated from these cultures had all the properties of the compound isolated from liver. These crystals possessed optimal "animal protein factor" activity for the chick at a level of 30 [^-g/kg of diet, similar to that found for vitamin Bio. Production of Antibiotics:— Among the various groups of micro- organisms that have the capacity to produce antibiotic substances, or agents vi'hich have the capacity to inhibit the growth of and even to destroy bacteria and other microorganisms, the actinomycetes occupy a prominent place. Within the last 7 or 8 years, nearly 30 antibiotics have been isolated. They vary greatly in their antibacterial properties or in their antibiotic spectrum, in their chemical nature, in their toxicity to animals, and in their chemotherapeutic potentialities. Some, like actinomycin, are highly toxic; others, like streptomycin, possess only a very limited toxicity. Some are produced by more than one organism; and some organisms produce more than one antibiotic. Of the various antibiotics produced by actinomycetes, streptomycin occupies a leading place. First announced in January 1944, it was used clinically within less than 2 years. Among its most striking properties are its action against gram-negative bacteria and the bacteria causing tu- berculosis. Thus, a chemotherapeutic agent that has marked effects against the "white plague" of man has been discovered. What ap- peared only a few years ago to be one of the greatest scourges affecting millions of human beings has been subjected to control by the product of an actinomyces. Within 5 years after its announcement, the produc- tion of this antibiotic has risen to nearly 8 million grams per month. Some of the newer antibiotics, notably aureomycin and Chloromy- cetin, have also attained remarkable production records. The possibility of discovering other antibiotics that would supple- ment streptomycin or take a place by its side as an important therapeutic agent appear very promising. Although some agents, like streptothricin, appear to be too toxic to offer great immediate promise, others, like grisein, are highly active and possess only very limited toxicity. These, therefore, appear promising. J Thus, the actinomycetes have contributed important tools for com- bating human and animal infections. The end of these possibiUties is Waksman —192— Actinomycetes not yet in sight. Of what significance these reactions are to soil proc- esses still remains to be determined. The actinomycetes can take their place among the major groups of microorganisms affecting the economy of man in numerous ways. Their importance in the cycle of life in nature and in the control by man of natural processes can hardly be exaggerated. APPENDIX MEDIA USED FOR THE STUDY OF ACTINOMYCETES 1. Czapek's agar: NaNOa 2gm K.HPO4 1 gm i\IgSO,.7H.O 0.5 gm KCl 0.5 gm FeSO. 0.01 gm Sucrose 30 gm' Agar 15 gm Distilled water 1000 ml pH 6.6 2. Glucose-asparagine agar: Glucose 10 gm Asparagine 0.5 gm K.HPO. 0.5 gm Agar 15 gm Distilled water 1000 ml pH 6.8 3. Glycerol agar: Glycerol 10 gm Sodium asparaginate 1.0 gm K.HPO. 1.0 gm Agar 15 gm Tap water 1000 ml pH adjusted to 7.0 4. Tyrosin agar: Glucose 10 gm Tyrosin 1 gm (NH0.SO, 0.5 gm K.HPO. 0.5 gm Agar 15 gm Distilled water 1000 ml Reaction made neutral with NaOH 5. Meat-peptone agar: Peptone 5gm Meat extract 5 gm NaCl 5 gm Agar 15-20 gm ) Tap water pW 7.2-7 A 1000 ml Waksman — 194 — Actinomycetes 6. Glucose-peptone agar A: Peptone Glucose 5 gm 20 gm NaCl Agar 5gm 15 gm Distilled water 1000 ml pH 7.2 7. Glucose-peptone agar B: Peptone Glucose 5gm 10 gm KH2PO. MgS04.7H20 Agar 1 gm 5 gm 15 gm Distilled water 1000 ml 8. Meat-peptone gelatin: Peptone Meat extract 5gm 5 gm Gelatin 100 to 200 gm Tap water Adjust to pH 7.4 Sterilize 30 minutes at 110°C. 1000 ml 9. Peptone gelatin: Peptone Glucose 5gm 20 gm Gelatin 100 to 200 gm Tap water 1000 ml Adjust to pH 7.2 Sterilize 30 minutes at 1I0°C. 10. Starch agar A: Potato starch 10 gm (corn starch or soluble starch) K.HPO. 0.3 gm MgCO. NaCl 1.0 gm 0.5 gm NaN03 1.0 gm Agar 15gm Distilled water 1000 ml Neutralize Sterilize 30 minutes at 110°C. 1 1. Starch agar B: Soluble starch 2gm K.HPO, 0.5 gm MgSO,.7H.O 0.2 gm CaCl2 0.05 gm NaNOs 0.05 gm Asparagine Fe^CSOO 0.05 gm Trace Washed agar 20 gm Distilled water 1000 ml pH7A Appendix _195_- Media for Study 12. Egg albiiiiioi agar: Glucose 10 gm K.HPO, 0.5 gm MoSO,.7H.O 0.2 om Fe^CSO.).-. Trace Egg albumen 0.15 om Agar 1 5 gm Distilled water 1000 ml Egg albumen is first dissolved in water and made neutral "to phenolphthalein with N/10 NaOH. 13. Potato-nutrient agar: Peeled potatoes 500 gm Peptone 10 gm Meat extract 10 gm NaCl 5 gm Agar 1 5 gm Tap water 1000 ml pH 7.0 The potatoes are cut into small cubes to which 350 ml of water is added and the whole steamed for three- quarters of an hour. The extract is strained through fine muslin without squeezing the pulp. The other nutrients are dissolved in 350 ml of water which is then added to the potato extract, and the whole steamed for three-quarters of an hour. The mixture is then made up to bulk, standardized and filtered, after which the agar is added. 14. Potato-glucose agar: Peeled potatoes 300 gm Glucose 5 gm Agar 20 gm Tap water 1000 ml 2^H 6.8 15. Starch nitrate agar: Formula same as in medium 19, with addition of 1.5 per cent agar. 16. Glucose broth: Glucose 10 gm Peptone 5 gm Meat extract 5 gm NaCl 5 gm Distilled water 1000 ml pH 7.1 Frequently tap water is used, as in media for the produc- tion of streptomycin. 17. Nutrient hroth: As above, but free from glucose. 18. Sticrose solution: Same as for No. 1, free from agar. Waksman 196 Actinomycetes 19. Starch solution: Soluble starch K,HP04 MgSO,.7H.O KCl NaNOs CaCOs Distilled water Starch is made into a paste and boili 20 gm Igm 0.5 gm 0.5 gm 2gm 2gm 1000 ml 2 water added. Mixture steamed for an hour to give a clear solution. 20. Yeast extract medium A: Autolyzed yeast extract Glucose Distilled water pH 6.0 to 7.0 (383) 21. Yeast extract medium B: Yeast extract Glucose NaCl MgS04.7H.O FeS04.7H.O Distilled water 22. Yeast extract agar: Yeast extract Glucose, technical Agar Tap water pH 6.8 23. Emerson's medium: Beef extract Peptone NaCl Yeast extract Glucose Distilled water 2.5 gm 5gm 1000 ml 10 gm 10 gm 5 gm 0.25 gm 0.01 gm 1000 ml 10 gm 10 gm 15 gm 1000 ml 4.0 gm 4.0 gm 2.5 gm 1.0 gm 10.0 gm 1000 ml 24. Com steep medium: Peptone Corn steep NaCl Glucose Distilled water 5.0 gm 15.0 gm 5.0 gm 10.0 gm 1000 ml 25. Soybean medium: Soybean meal Commercial glucose NaCl Curbay B.G. CaCO,, Distilled water 10.0 gm 10.0 gm 5.0 gm 0.5 gm 1.0 gm 1000 ml Appendix — 197 — Media for Study 26. Synthetic lactate medium: Glucose 7.4 gm KH.PO4 2.38 gm K.HPO4.3R.O 5.65 gm NH4 lactate 5.4 gm iMgS0..7H.O 0.98 gm ZnS04.7H.O 11.5mg FeSO,.7H.O 11.1 mg CuSO. 6.4 mg MnCl,.4H.O 7.9 mg Distilled water 1000 ml pH 6.95 27. Potato flugs. 28. Carrot ^lugs. 29. Brom-cresol milk: Prepared according to formula of Clark and Lubs. Me- dium sterilized by steaming for 20 minutes on three successive days. 30. Dorset's egg or glycerol egg medium. 31. Brain-heart infusion with 2 fer cent agar (368). A number of other special media are used, either for the growth of organisms or for the production of special substances, notably antibiotics. In several media, the peptone is replaced by casein hydrolysate and the meat extract by various digests of plant residues, such as soy bean meal or by waste products of manufac- turing industries, such as corn steep liquor or by fermentation residues, such as distillery slops. Aetin<»!ny«M'> Uovis, ein ueucr Sehiiuiiiel hi iU:n vt.u dt-r E>i- .-.-r 1' ; I- !!'■!•*' der Oew, ' lU!;.;.-:. M'ili, _ t!H», ■ _ ■ ■ ' ' Form : ^ l.-irht Krank- thi.Tlx !.. dunh ■ \«-i Fig. 39.-First use of the generic name Actinomyces (C. O. Harz: Aciinotuyces hovis, ein neuer Schimmel in den Geweben des Rindes in Deut. Zeitsch. Thicrmed. 5:125-140, 1877/78). BIBLIOGRAPHY 1. Abbott, E. and Gildersleeve, A., 1902: On the actinomyces-like de- velopment of some of the acid resisting bacilli (streptothrices?). Centrbl. Bakt. I 31: 547-550. 2. Abramov, S., 1921: Zur Frage iiber die Streptothrichosen des Zentral- nervensystems. Centrbl. Bakt. I, 61: 481-494. 3. 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