ty tea apo Arete teas =? Sere! Perea cog ce hee, Ext pes b, TO tern) * N Sets ee “ ? 44%2eT200 TOEO O WACOM 0 0 1OHM/18lN The Chronica Botanica Co., International Plant Science Publishers Noteworthy Books:— Baldwin: Forest Tree Seed Bawden: Plant Viruses and Virus Dis- eases, third, entirely revised edition... . Camp et al.: International Rules of Botanical Nomenclature.......... Chardon: Los Naturalistas en la Ame- rica Latina, Vol. I Chester: The Cereal Rusts Clements, Martin and Long: Adapta- tion and Origin in the Plant World Condit: The Fig Copeland: Genera Filicum Correll: Orchids of North America (shortly) Crafts, Currier, and Stocking: Water in the Physiology of Plants Dachnowski-Stokes: Peat (in press)... . Darwin: A Naturalist’s Voyage with the Beagle (1839/1950) (in press) ca. Elliott: Bacterial Plant Pathogens (in press) ca. Erdtman: Introduction to Pollen Analysis Finan: Maize in the Great Herbals. . Foxworthy: Forests of Tropical Asia (in press) Frear: Chemical Insecticides Frear: Chemical Fungicides Plant Insecticides Fulford: Bazzania in C. Garrett: Root Disease Fungi Guilliermond: Cyptoplasm Plant Cell Gundersen: Families of Dicotyledens (shortly) Hoagland: Inorganic Nutrition of Honig, Verdoorn et al.: Recent Ad- vances in Tropical Biology and Agriculture (in press) Horsfall: Fungicides and their Action Howes: Vegetable Gums and Resins Jessen: Botanik der Gegenwart und Vorzeit in Culturhistorischer Ent- wickelung (1864/1948) Johansen: Plant Embryology (shortly) Kelley: Mycotrophy of Plants Knight: Dictionary of Genetics Lloyd: Carnivorous Plants Moldenke: Plants of the Bible (in ca. Murneek et al.: Vernalization and Photoperiodism Nickerson et al.: Biology of Patho- genic Fungi Sarton: Guide to the History of Sci- ence (in press) Schopfer: Plants and Vitamins Sirks: The Evolution of Biology (in ca. G. M. Smith et al.: Manual of Phy- cology (shortly) ca. Stevens: Agricultural Plant Pathol- ORY UINYPLESS) Or ieicrie ves aaa ate ca. Vaviloy: Selected Writings (in press) ca. Verdoorn et al.: Plants and Plant Science in Latin America Waksman: The Actinomycetes Wallace et al.: Rothamsted Sympo- sium on Trace Elements (shortly) Wilde: Forest Soils and _ Forest Wulff: Historical Plant Geography. . ZoBell: Marine Microbiology * * * CHRONICA BOTANICA, An Inter- national Collection of Studies in the Method and History of Biol- ogy and Agriculture (annual sub- scription) Special issues of Chronica Botanica which are available to non-subscribers:— Arber: Goethe’s Botany Asmous: Fontes Historiae Botanicae Rossicae BIOLOGIA, An International Year- Book (per annual volume) Browne: A Source Book of Agricul- tural Chemistry Browne: Thomas Jefferson and the Scientific Trends of His Time.... DeTurk (ed.): Freedom from Want, A Symposium Howard: Luther Burbank, A Victim of Hero Worship Jack: Biological Field Stations of the World Lanjouw—Sprague: Botanical Nomen- clature and Taxonomy, A Sympo- Merrill: Merrilleana—Selected Gen- eral Writings Rafinesque: A Life of Travels Reed: Jan Ingenhousz—Plant Physi- Rickett: Botanical Expedition to New Spain Saint-Hilaire: Voyages au Brésil Stevens: Factors in Botanical Publi- cation and other Essays Verdoorn et al.: Plant Science and Plant Scientists during the Second World War (in press) ca. Verdoorn (ed.): 21st World List of Plant Science Institutions and Societies (in press) Wyman: Arboretums of North Amer- Imports which we handle for the Americas only:— Lindquist: Genetics in Swedish For- estry Practice Meyer-Abich (ed.): Goethe-Zeit Nelson: Introductory Botany Verdoorn et al.: Manual of Bryology Verdoorn (ed.): Manual of Pteridology Weevers: Fifty Years of Plant Physi- ology Catalogue and Book Department List on Request The Chronica Botanica Co., Waltham, Massachusetts, U.S.A. 2.00 1.25 4.75 5.00 1.25 2.00 3.75 2.50 2.50 4.00 2.50 3.00 2.50 2.00 2.00 3.75 4.00 1.50 Serman A. WaxksMAN was born July 2, 1888, in Priluka, a small town in the Ukraine, Russia. His parents were JAcos and Frapt1a (Lon- poN) WaksMAN. After graduating in 1910 from the Fifth Latin School in Odessa, he left for the United States. He entered the College of Agriculture 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 appointed 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 Experiment 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 now is the head of the Microbiology Department of the College of Agriculture and Experiment Station at Rutgers University. In 1981, he was 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, honorary 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 which 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 Seciety 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. WaxsMAN lectured before scientific groups in Europe and was given an honorary degree of doctor of medicine by the University of Liége in Belgium. He holds also honorary degrees of doctor of science, awarded to him by Rutgers in 1942 and by Princeton Univer- sity in 1947, and an honorary degree of doctor of laws from Yeshiva Uni- versity, New York, in 1948. Dr, WAKSMAN’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 New Jersey Agricultural So- ciety medal; the Albert and Mary Lasker Award by the American Public Health Association, and the Amory Award by the American Academy of Sciences, all in 1948. He has published more than $00 scientific papers, and has written, alone or with others, eight books. Among these are Enzymes (1926), Principles of Soil Microbiology (1927, 1932), The Soil and the Microbe (1932), Hummus (1936, 1938), Microbial Antagonisms and Antibiotic Sub- stances (1945, 1947), and The Literature on Streptomycin, 1944-1948 (1948). Another recent work, edited by Dr. WaxsMan, is Streptomycin— Nature and Practical Applications. ANNALES CRYPTOGAMICI et PHYTOPATHOLOGICI Volume 9 ‘THE ~ACTINOMYCETES ANNALES CRYPTOGAMICI et PHY TOPATHOLOGICI (incorporating Annales Bryologia ) edited by FRANS VERDOORN, Pu.D. Managing Editor, Chronica Botanica Research Fellow, Arnold Arboretum, Harvard University Botanical Secretary, International Union of Biological Sciences Wij en konnen den Heer 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 volmaaktheit, met de wuiterste verwondering sien uit steken. Antoni van Leeuwenhoek 1950 WALTHAM, MASS., US.A. Published by the Chronica Botanica Company ‘THE ACTINOMYCETES Thar Nature, Occurrence, Actwities, and Importance by SELMAN A. WAKSMAN, PH.D. Profes of Microbiolo By, Rutgers ee Microbiolog ne ‘Net v Jersey Agricultural Exper oy Station 1950 WALTHAM, MASS., US.A. Published by the Chronica Botanica Company First published MCML By the Chronica Botanica Company of Waltham, Mass., U.S.A. Copyricnt, 1950, By Serman A. WAKSMAN All rights reserved, including the right to reproduce this book or parts thereof in any form Authorized Agents:— New York, N. Y.: Srecuert-HaFner, INnc., 31 East 10th Street. San Francisco, Cal.: J. W. Sracey, Inc., 551 Market Street. Ottawa, Ont.: THorBURN AND ABsortT, Lrp., 104 Sparks Street. Mexico, D. F.: AxEt Morten Sucrs., San Juan de Letran 24-116; Ap. 2762. Lima: LrsprertA INTERNACIONEL DEL PERU, Casa Matriz. Boza 879; Casilla 1417. Santiago de Chile: LiprertA ZAMORANO Y CAPERAN, Compania 1015 y 1019; Casilla 362. Rio de Janeiro: Livrarta Kosmos, Rua do Rosario, 135-137; Caixa Postal 3481. Sao Paulo: Livraria Crvitizacdo BRASILErRA, Rua 15 de Novembro, 144. Buenos Aires: AcmE AGENCY, Soc. DE Resp. Lrpa., Suipacha 58; Casilla de Correo 1136. London, W. C. 2: Wm. Dawson AND Sons, Ltp., Chief Agents for the British Empire, Cannon House, Macklin Street. London, W. C. 1: H. K. Lewis anp Co., Lrp., 136, Gower Street. Uppsala: A.-B. LuNDEQUISTSKA BOKHANDELN. Groningen: N. V. Erven P. Noorpuorr, Chief Agents for Continental Europe. Paris, VI: Lisrarrie P. RAYMANN & Cie., 17, Rue de Tournon. Torino: ROSENBERG & SELLIER, Via Andrea Doria 14. Madrid: Lisreria J. VILLEGAS, Preciados, 46. Lisbon: Livrarta SA pa Costa, 100-102, R. Garrett. Moscow: MrEzHDUNARODNAJA KNIGA, Kuznetski Most 18. Peiping: FrencH Booxksrore, 14 'T’ai-chi-ch’ang - Tung-chiao min-hsiang. Tokyo: MaruzeEn Company, Lrtp., 6, Tori-Nichome Nihonbashi; P. O. Box 605. Caleutta, Bombay, and Madras: Macmiiuan AnD Co., Lrp. Cape Town: Oxrorp Untversiry Press Markham’s Buildings. Sydney: ANGus AND RosBertson, Ltp., 89 Castlereagh Street, Box 1516D.D. G.P.O. Melbourne, C. 1: N. H. Sewarp, Pry., Lrop., 157, Bourke Street. Made and printed in the U.S.A. Designed by Frans Verdoorn PHEEACE Three and a half decades ago—in the spring of 1914—the writer, then a senior at Rutgers College, dug a spade into the earth of the New Jersey Agricultural Station experimental plots, to study the distribution of different groups of microorganisms occurring at different depths in the soil. This operation was repeated monthly, and sterile soil samples were removed 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 particular attention of the youthful investigator were the actinomycetes. Although 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 rough surface will disclose the fact that they are not bacteria. It requires careful observation to tell whether those white, pink or grey colonies are bacteria or not. Many counts 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 occurrence and abundance of actino- mycetes at different soil depths and in different soil types, the writer and his numerous associates and students have devoted much time to the study of the actinomycetes, their cultural 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 and animal residues in soils, peats, and composts, and finally their ability to produce antibiotic substances. The writer has thus been concerned, during 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, with somewhat greater emphasis upon the work done in the laboratories Waksman — x — Actinomycetes of the Department of Microbiology of Rutgers University and the New Jersey Agricultural Experiment Station. To his many associates, who have helped in making this work pos- sible, the author wishes to express his sincere appreciation for their unfailing enthusiasm and continuous interest and collaboration. The writer also wishes to express his gratitude to Lt. Col. M. L. Lirrman 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 XI, and to Dr. R. W. Goss of the Uni- versity of Nebraska for reading Chapter X. December 20, 1949 New Brunswick, N. J. THE COMPOST O how can it be that the ground does not sicken? How can you be alive, you growths of spring? How can you furnish health, you blood of herbs, roots, orchards, grain? Are they not continually putting distemper'd corpses within you? Is not every continent work’d over and over with sour dead? Where have you disposed of their carcasses? I 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 spring covers the prairies, The summer growth is innocent and disdainful 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 out 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. Wart WHITMAN. CO NAGE NGS TeETE NACE EO BRS 1S WAF VET Sete 2) Eee eRe ote da ee iv PREBAGE (7289 72") 1a Ma ee ei tinh a. eel Aes ane Smee 1x ‘Te: Concposr (by? Wis W ur MAN ewe ota ho nce OR me CONTENTS Tle co. ie de Bal ROE ia fe a te ae es et a TT (sis YOR ILLUSTRATIONS ht oy ie uk coe og ete tr ee ese mn aR [IST YOR WEARERS: = ose coy SRE eee ta on el =e XV TT INTRODUCTORY ie coor," hE OER Tg nO sie nar ane Ae eine Chapter I TERMINOLOGY, PHYLOGENY, AND TAXONOMY:-—Syn- ONYMS OF GENERIC NAMES OF ACTINOMYCETES—SYSTEMATIC PosITION AND CLASSIFICATION OF ACTINOMYCETES—IDENTIFICATION OF ACTINO- MY CETES( (2° ite C8 CARRS RAIS SAN he a ee Chapter II IDENTIFICATION AND DESCRIPTIONS OF IMPORTANT TYPES:—CLassiFICATION OF ACTINOMYCETALES—DESCRIPTION OF SEV- RRAL, INPORTANT JAC TINONYGETES 0 nila isen ee ee Chapter III MORPHOLOGY AND LIFE CYCLE:—Sraininc oF Actinomy- CETES—GENERAL MorpHoLocy—SPoRuULATION OF ACTINOMYCETES— ‘Types oF GrowTH ON SoLip AND Liourp Mepra’ . . . . +. 46 Chapter 1V VARIATIONS AND MUTATIONS:—Types oF VartaTtrion—Cuc- TURE CONSISTENCY . . ; net NY Mee 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 Opors—PrRopucTION OF PIGMENTS—IHERMOPHILIC Ac- THEN RVI CIETTES\ | IN rs bi TR einai SRM oes BAT uh Sek ray AM Cah any A Chapter VI PRODUCTION OF ENZYMES AND OF GROWTH-PRO- MOTING SUBSTANCES:—Propuction oF ENzymEs—PRopuUCTION OF VITAMINS—OXIDATIVE MECHANISMS .. . it Meee “te OO Chapter VII ANTAGONISTIC PROPERTIES OF ACTINOMYCETES AND PRODUCTION OF ANTIBIOTICS:—Anracontstic EFrects oF Ac- TINOMYCETES—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 WateER Basins—OccuRRENCE OF ACTINOMYCETES IN Dust AND ON ExposED SuRFACES OF PLANTS—OccURRENCE OF ACTINOMY- CETES IN FoopstuFFS—OcCURRENCE OF ACTINOMYCETES IN ANIMAL STEERS: Mc Ws SN EL ae OE a eae hr cae ie BE Pere ect (oi) Chapter 1X DECOMPOSITION OF PLANT AND ANIMAL RESIDUES:— Decomposition oF Pure Orcanic Compounps—DECOMPOSITION OF Compitex Pranr MarTertAts—DeEcomposiTion OF Humus—THER- MOPHILIC Composts Ri te hee dae. Ce I ee cee Chapter X ACTINOMYCETES AS CAUSATIVE AGENTS OF PLANT DISEASES:—Porato Scas—Sucar Beer anp MANcEL ScaB—SWEET Potato Pox or Sorr Rot—OruHer Piants INFECTED BY SCAB OrRGAN- IsMsS—OTHER PLant AcTINOMyYcosES—MeEtTHops oF ConTRoL . 155 Chapter XI ACTINOMYCETES AS CAUSATIVE AGENTS OF HUMAN AND ANIMAL DISEASES:—EttoLocy oF INFECTIONS—EARLIER IN- VESTIGATIONS—RECENT StrupreES— I RuE Actinomycosis—AEROBIC Ac- TINOMYCES INFECTIONS—CHEMOTHERAPY OF ActiNomMycosis . 170 Waksman — xiv — Actinomycetes Chapter XII SUMMARY:—Ro te oF ACTINOMYCETES IN NaturAL PRrocessEs— ACTINOMYCETES AS CauSATIVE AGENTS OF DisEASE—ACTINOMYCETES AS AGENTS OF SPOILAGE AND DETERIORATION—UTILIZATION OF ACTINO- MYCETES FOR THE PRODUCTION OF ENZYMES AND VITAMINS—PRopUC- TION. OF ANTIBIOTICS =: » Wye. cum ce poices ne ee a er Appendix MEDIA USED FOR THE STUDY OF ACTINOMY- CETES): oe ss oe BR Sa ee eS Bibliography io.) 2. "Se ee Cae ok cn ee eo General Index °°. gr. he ee eee eee Index-of Organisms’) “2120s faite Toma ae) eee ee 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 Conn, transl. by C. S. DoLtey). ErSt of- IE EUSTRALIONS Ficure 1.—Streptothrix of Ferprinanp Conn. Ficure 2.—First photograph of a species of Mi feromonospors (1899) . Ficure 3.—Structure of actinomyces mycelium . Ficure 4.—Typical growth of aerobic actinomycetes upon agar ante : Ficure 5 a-d.—Different types of branching of aerial mycelium of Eris of Streptomyces Ficure 6.—Nocardia paterordes: grown on n potato dexmase: pects ae ‘agar Ficure 7.—Nocardia asteroides, grown on potato dextrose-beef extract agar Ficure 8.—Branching and sporulation of different strains of Nocardia FicurE 9.—Sporulation of straight aerial hyphae of species of S treptomyces Ficure 10.—Streptomyces venezuelae, ee on potato dextrose-beef ex- tract agar . : : F Ficure 11. ” Streptomyces Spe; grown on potato denroee: meee agar : Ficure 12 a-d.—Different forms of sporulation of Micromonospora growing in composts, as shown by contact slide preparations : Ficure 13.—Details of sporulation and of spore germination Pgs S. griseus as shown by electron microscope Ficure 14. —Aerial mycelium of a Streptomyces, showing onan or “fairy ring” formation ae Ficure 15.—Electron micrograph of actinophage : Ficure 16.—Method of measuring actinophage concentration . Ficure 17.—Variants of Streptomyces griseus growing on yeast extract- glucose agar . Ficure 18. cohol changes produced by S. Teeccebare, in ented and stationary cultures Ficure 19.—Metabolic changes produced by Ss aaibicnous in actated and stationary cultures Ficure 20.—Influence of temperature upon growth and carbon! diecde production by actinomycetes . , Ficure 21.—Decomposition of hemiceliuleses by actinomycetes, as meas- ured by CO: evolution . Ficure 22.—The use of the agar cross- eal method for testing the ability of actinomycetes to produce antibiotic substances . Ficurr 23 a.—The use of M. tuberculosis for testing production Ay, anti- biotic substances by actinomycetes . Ficure 23 b.—Inhibition of Sea ae ens but not of streptomycin: resistant strain Ficure 24.—Method of measuring prebacrenall or “antifungal potency of an antibiotic, by the agar streak method : Ficure 25.-Streptomycin-producing strain of S. griseus, showing vegeta- tive and aerial mycelium ; Ficure 26.—Method of isolation of streptomycin fom daetabolite ‘solution Ficure 27.—Crystals of the calcium chloride double salt of streptomycin Ficure 28.—Metabolic nee produced in the medium by Streptomyces S ee pom hec course OF development ‘of S. alas And bacteriolytic Aen: ity of the culture filtrate, actinomycetin 106 110 iu ts 122 1s) 127 128 130 Se LIBRARY] sa =) Waksman — xvi — Actinomycetes FicurEe 30.—Typical growth of soil species of Sere on synthetic media ; Ficure 31.—Relation between density of vegetative mycelium and plate counts of actinomycetes . 2 Figure 32.—Different forms of scales produced by S. scnbies : Ficure 33.—The relation of soil reaction to the occurrence of potato istak Ficure 34.—Influence of the hydrogen-ion concentration on the ae of the potato scab organism . Ficure 35.—Vegetative mycelium of a pathogenic annerabie actinomyces Ficure 36. —Actinomycosis of lymphatic gland showing Eau with my- celial network and peripheral growing edge Ficurr 37.—An early study of the structure of an actinomyces colony om a bronchopneumonic nodule : ee 38.—The plata of a Nocardia in ‘he ‘sputum of an anfected atient pice 39.—First use BE ihe generic - name Actinomyces (1877/ 78) Mycology is the Cinderella of Botany and has suffered the dis- advantages of step-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 fully 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 full of rations for the general good. May those who have served her faithfully benefit for their devotion. (J. Ramsgorrom). 132 LISh of PABLES Tasie 1.—Types of actinomycetes recognized in 1894 . 9 Taste 2.—Comparison of cultural, morphologic and staining Ten CHOnE of species of Nocardia . : 30 Tasie 3.—Comparison of the parasitic aid. saprophytic actinomycetes 35 TABLE 4.— —Streptomycin production and streptomycin sensitivity of differ- ent strains of S. griseus and their variants . 37 Tasie 5.—Cultural and physiological characteristics Bf ae streptomycin producing strain of S. griseus and its inactive variant. . 63 TasLeE 6.—Effect of phage upon the growth, phage multiplication, Sond streptomycin production 2 different oe was in east aes cul- tures . : eee: 66 TABLE 7. Stability of phage i in aqueous suspension upon storage at eeyerall temperatures 67 Tasre 8.—Production of streptothricin by two cine of St Wavendalne Al their variants . : WH. TasLe 9.—Decomposition ‘of different amino acids by microorganisms . 81 Tasie 10.—Decomposition of glycine by different Seen parin in pres- ence of glucose . 81 Taste 11.—The utilization of Cahn and nitrogen sources by S. Coeleolen 83 TasLeE 12.—Metabolic changes and efficiency of carbon utilization of S. lavendulae in aerated caleues 84 Taste 13.—Acid production by an actinomyces on meat extract- peptone- glucose medium... 86 Tasie 14.—Acid formation fom glucose in Aeaed ealmee of S. (ever dulae . . 87 Tasie 15.—Influence ne reaction on fie decomposition of a ‘protein- ich material by actinomycetes 90 TasLeE 16.—Rate of growth of S. prises and streptomycin production in in shaken cultures 90 TasLeE 17.—Metabolic changes produced by Ss arisen “with different sources of nitrogen 90 TasieE 18.—Proteolytic activity af actinomycetes in 2 per cent "gelatin solution 101 Tasre 19. Sp ierabuton Gf bacteriolytic properties among actinomycetes ; 102 Tasie 20.—Occurrence of antagonistic actinomycetes in different soils 109 TaBLe 21.—Distribution of antagonistic actinomycetes in nature . 2, Tasie 22.--Classification of antibiotics of actinomycetes . . . . 116/117 TasiE 23.—Inhibition of different cee 2 their ae anti- biotics 119 Taste 24. Dembution of antagonistic properties among actinomycetes 120 Tasie 25.—Inhibition of growth of virulent human qaberele bacilli by different actinomycetes ; 121] TasLe 26.—Growth and chemical changes produced by S. griseus under submerged conditions 123 TagLe 27.—Nitrogen Aeciution i in Galenres of Ss. griseus . , 123 Taste 28.—Antibiotic spectra of streptomycin and grisein . 124 Waksman =v Actinomycetes Tasie 29.—Antibiotic spectra of streptomycin, streptothricin, and anti- biotic 136 Taste 30.—Numbers of Racer ad actinomycetes in he eal developing on albumen agar . TaBLeE 31. _ Diss bifon of actinomycetes in various Gils : ia Tasie 32.—Distribution of microorganisms in different soils from Bie and Rongelap Islands Taste 33.—Influence of | per cent ged bleed upon fie microbiological population of the soil Taste 34.—Influence of addition of ‘CaCO; on ike AIS of sctinomy- cetes in acid soils : : Tasie 35.—A list of typical actinomycetes occurring in aheeile and: in com- osts : 3 Taste 36. maTndence of temperature upon ‘le development of microorgar isms in manure composts Taste 37._Numbers of microorganisms in an madre peat bog in Florida : ; Taste 38. —Decomposition of xylan by actinomycetes 3 Nae: Taste 39.—Decomposition of wheat straw by different microorganisms Taste 40.—Decomposition of sedge and reed peat by microorganisms Tasie 41.—Decomposition of stable manure by pure cultures of ther- mophilic microorganisms and by a mixed thermophilic population Tasie 42.—Certain characters of scab-producing actinomycetes 4 Taste 43.—Effect of competition of soil microorganisms poe occurrence of scab TasLe 44. — Comparative morphological and physiological properties AR two common types of pathogenic actinomycetes Sey ee Les S. chromogena et alba sont des microbes trés répandus dans la terre, surtout abondants dans les racines végétales et ad leur sur- face. Je les trouvai dans le terreau de jardin jusqu’a 1 m. de pro- fondeur; plus bas encore le nombre absolu de ces organismes n’est guére considérable, mais dépasse néanmoins celui des autres microbes du sol. Cela démontre leur résistance a l’égard des conditions défa- vorables pour leur nutrition. (CM. W. Brtyerinck). 129 Si 138 140 141 141 143 15 147 150 152: 152 153 158 168 171 figures ever to have first ‘The Z q fe) O a Z < g a Fe wy aa — S z lan N ~™~ wy if 2] vo oO ma £ (e) c es) 3) es c ee oe ° lan In ~~ co eo VU 31 © | 0) S| Be aa) =| jon f=} oO o ple) ptothri Stre BiG. = INTRODUCTORY Actinomycetes! comprise a group of branching unicellular organisms, which 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 Hyphomycetes, 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, ly. 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 mycelium 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 Eubacteriales, 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 physiology, 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 — 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 variety 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. ‘G hapter if 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 actinomycetes This has been further complicated 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, Ferprnanp Coun (72) designated a culture of a filamen- tous organism found by R. Forrsrer in the concretions of the lacrymal duct as Streptothrix Foersteri. Coun 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 Coun (Fic. 1) leaves no doubt that this was a true ac-. tinomyces. Soon afterward, in 1877, an infectious agent in cattle dis- covered by Botiincer (42) was named by Harz (166) Actinomyces bovis, 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 (Actino- mryces) has been meeting with much criticism, because the description of the organism was based on its etiology rather than its morphology 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 consideration 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 difh- culties, however, was the problem of digesting a most extensive litera- ture. Until very recently, too little was known of the morphology and physiology 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. “Ihe 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 previously 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 morphology 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 types, which could be raised to the status of genera. No attempt will be made to review in detail the early literature on the actinomycetes. Such reviews may be found in the monographs of Lieske (260), Orsxov (328), Ducué (98), Kriss (242), and Kras- Chapter I i Taxonomy SILNIKOV (234, 236), and in many of the earlier (310) and more recent papers (17-20, 106-108, 111-113, 185-192). It is sufficient here to sum- marize briefly some of the outstanding facts which led to our present knowledge of the terminology and classification of the actinomycetes. 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 concerning 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 albus 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 bovis 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 ot enaton 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: 1. Actinomyces Harz (1877).—The most widely used generic name for the actinomycetes is Actinomyces. 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 actinomyces or an actinomycete, 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 Actinomyces bovis. More detailed descriptions were pre- sented in 1890 by Bostroem (44) and by Wo ts and Isrart (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 received wide rec- ognition. ‘This is due largely to the fact that the name had been pre- empted: Corpa used it in 1839 for a true fungus, which he designated Streptothrix fusca. Some of the early students of the actinomycetes (32) recognized this and insisted upon the greater justification of the designation Actinomyces. Another reason why the generic name Strep- tothrix has not been generally accepted is that Coun himself failed to differentiate sufficiently between the organism to which he gave this name and the forms designated as Cledoune 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 dichotoma) and are often found in mouths of animals. The use of this term by many of the early students of actinomycosis 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 actinomycetes, 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 CL. ochracea) and various mouth-inhabiting bacteria CL. buccalis), 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, SauvacEau and Rapars (384) attempted, in 1892, to describe the actinomycetes under this genus. ‘THaAxrErR (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 Ae actinomycetes which was isolated by Nocarp from “farcine du boeuf.” Wricnut (519) proposed limitation of this name to a disease condition which is accompanied by Chapter I a a Taxonomy inflammation and which was, therefore, called nocardiosis, as distinct from actinomycosis. Prvoy (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 little used. 9. Micromyces Gruber, M. (1891).—The generic name Micro- myces 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 clearly recognized type (159). 10. Actinobacterium Haas, E. (1906).—To designate organisms that are intermediary between the true actinomycetes and the corynebacteria, the name Actinobacterium was suggested. It has not been generally accepted, although existence of these intermediate forms is not denied. 11. Actinobacillus Lingiéres and Spitz (1904) and Actinobacillus Brumpt (1910) were names applied to nocardia-like organisms, the true nature of which was not sufliciently recognized. ‘The generic name was also used by BrryErrnok, in 1914, for an organism which he had origi- nally described in 1903 as Bacillus oligocarbophilus and for another called Actinomyces (Streptothrix) paulotrophus. 12. Cohnistreptothrix Pinoy, E. (1911).—In order to differentiate anaerobic actinomycetes from the aerobic forms, Pinoy used this name to designate the former. CasTELLANrt 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 Mycobacterium 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 Ligniéres (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 — 8— 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 Corynebacterium and Mycobacterium. ‘The organisms belonging to the genus Proactinomyces were later included by LEHMANN and Haac in a separate family Proactinomycetaceae. ‘This group includes such important forms as A. hominis 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. Micromonospora Orskov (1923).—The generic name Micro- monospora was used to designate those actinomycetes that produce single spores on side*branches. Streptothrix chalcea of FouLerron and A. monosporus of LEHMANN and ScHtrze belong to this group. Fic. 2.—First photograph of a species of Micromonospora (1899). ‘This organ- ism was a thermophilic form growing in hot manure compost and called by Tstktinsky Thermoactinomyces vulgaris (429 ). 20. Thermoactinomyces ‘Tsiklinsky (1899).—This name was first used to designate thermophilic actinomycetes. Although one of the forms included in this group is definitely a Micromonospora, as shown in Fic. 2, the fact that forms producing the long-chain type were also in- cluded would preclude the use of Thermoactinomyces 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 KrassILntkov (234) to certain bacteria which appear to be related to the actinomycetes. 22. Asteroides Puntoni and Leonardi (1935).—This name was also Chapter I eat: pe 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 have been most commonly employed in micro- Namz OBSERVER SYNONYM OBSERVER A. bovis sulphureus Rivorra A. bovis @) - A. foersteri Coun Streptothrix foerstert - A. canis V ACHETTA A. pleuriticus canis RIVOLTA familiarts A. canis RaBe A. bovis farcinicus NocarpD Bacillus farcinicus - A. cati Rivo.ra - = A. bovis albus GaSPERINI Streptothrix 1,2,3 ALMQUIST S. alba Rosst-Dorta A. astercides EpPINGER Cladothrix aster:ides ~ S. astercides GaAsPERINI S. eppingerii Rosst-Dor1A A. chromogenus GASPERINI S. chromogenus = S. niger Rossi-Doria Oospora metschnikowi (2) SauvaGeau and Rapats O. guignardi (?) SauvaGEAu and Rapais A. bovis luteo-roseus GASPERINI - = A. cuniculi ScHMORL S. cunicult - A. hoffmanni GRUBER Micromyces hofmanni - A. albido-flavus Rosst-Dor1a_ S. albido-flava = A. violaceus Rosst-Dor1a_ S. violacea - A. carneus Rosst-Dorta_ S. carnea ~ A. citreus GASPERINI - - A. pluricolor (2) TERNI = - A. arborescens EDINGTON - = A. ferrugineus Naunyn — = biological literature, Streptothrix and Actinomyces. Tasie 1: Types of actinomycetes recognized in 1894 (132):— This new name obviates the need for using the name Streptothrix, 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- phyta, Bollingera, Indiella, Indiellopsis, Microsiphonales, Microsporum, Oidium, and others. Either these names proved to be mere synonyms o they could not be given serious consideration for various other rea- sons. 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 I. 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- ties: 1. The manner of brafhching of the aerial mycelium of many representative groups of actinomycetes, especially the genera Streptomyces and Micromonospora, 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.—Many 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 —— ee Taxonomy tin liquefaction, or upon their morphology, especially the manner of sporulation. BucHanan (52) suggested placing the actinomycetes in the order Actinomycetales with a single family Actinomycetaceae. ‘The latter was divided into four genera, Actinobacillus, Leptotrichia, Actinomyces, and Nocardia. Later (53), in the early editions of Bergey's Manual, the Actinomycetales were divided into two families, the “Actinomycetaceae with the genera Actinobacillus, Leptotrichia, Actinomyces, and Ery- Fic. 3.—Structure of actinomyces mycelium: (a) S. albus Gasperini; (Cb) S. aurantiacus Gasperini (from KrasstLnikov, 234). sipelothrix, and the Mycobacteriaceae with eight genera, including My- cobacterium. In later editions of Bergey's Manual, Actinobacillus was dropped from the first family and Proactinomyces added; the second family was divided into the genera Corynebacterium 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 Corynebacterium and Myco- bacterium, and the Actinomycetaceae with a single genus Actinomyces. KLUuYVER pid van Niex (224) suggested that eine Mycobacteriaceae be removed altogether from the oulee “Actinomycetales. Waksman — 12— Actinomycetes Several of the systems for classifying the true actinomycetes may be listed as follows: A. Classification of Schabad (1904): I. Non-acid-fast types, liquefying gelatin, producing granules in lesions, with typical. “swelling of “hyphae. \-).)-).05) et easiest eterna A. typica. II. Acid-fast types, not producing granules in lesions, without typical swell- ings: Of lesions; '. Scare, Fohoenk Se ey ety ote cE ee ee ales A. atypica. I. "Gelatin ‘liquefied Som: <5 32) .-qene nyse eae A. atypica simplex. Paar ES AIGA SRN VAC PRE els pee be ad Ss te diek Cals Be A. alba. CPU PR aR se MMT Re ud stem ty Ratan, aa MLA cnc ely ea Ter) eM A. flava. 2. Gelatin not diquefiedia. 5 a1: A. atypica pseudo-tuberculosa. B. Classification of Krainsky (1914): 1. Large colonies (3-5 mm.) produced on solid media; aerial mycelium typically, pigmented; ovaléspotesiy (ss... a4 asa Macroactinomyces. 2. Small colonies (<3 mm.) produced on solid media; pigmented aerial mycelium; sphericall-sporesip wane -era sete eas Microactinomyces. This system has been applied only to the saprophytic aerobic forms. C. Classification of Chalmers and Christopherson (1916): lee Grantlessblackesnoncultivablentociicea ene ieee einen naan ae Actinomyces of Bapes and Mrronescu. II. Granules white, yellow, orange, or red: 1. Cultivated with difficulty, anaerobic types, no arthrospores; granules LIMITA SSCS hm mcs chock sin ee eae ONT AER eee Cohnistreptothrix. as (Granules-vellows' ny 0 o.com snes ee orice a ere C. israeli. b: Granules'very small whiter) 5.5 cnet ae C. thibiergi. 2. Cultivated easily, aerobic types, arthrospores produced. ... Nocardia. a. (Clubs present. manag ti srnr meee manatee ene Site tice em N. bovis. b. No clubs produced: a’. Granules surrounded by a hard shell. ........ N. somaliensis. b'. Granules without shell: a’. JNorerowth onigelatinis Se ccis -1-)--s1ete ee ctenee N. krausei. b*. Growth on gelatin: a®. Serum coagulated, liquefied: a‘. Pathogenic to laboratory animals. .... N. garteni. b‘. Nonpathogenic to laboratory animals: ay. Gelatineliquefied. yoemerateua a: N. liquefaciens. b®.*Gelatminot liquetied.s 9) - 2:4... N. convolutus. be a not liquefied: . Culture yellow orange to red. ...... N. asteroides. be Culture*white: thenzeds.4.—. +e 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 i Taxonomy Actinomyces and Nocardia, the cultures classified in this system (D) are now included under the genus Streptomyces. E. Classification of Wollenweber (1921): I. Weakly growing strains; aerial mycelium and conidia lackimpis wise as 3: : 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 Aerothrix. 1. With sclerotial or spirodochial stroma. ........ Section Sclerostroma. This group included A. bovis, A. foersteri, A. scabies, and A. aerugineus. 2. Sclerotial or spirodochial stroma not significant. Qe VVathe brown) comidiays- 44. ss sens. Section Poliophaerospora. b. 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. aly Win jolie wonrélins 64 4625onnunne0nceonee Section Glaucospora- F. Classification of Langeron (1923): PAN CTODICELOTINS arIA SE) sie Were carne haere ae aoe: Euactinomyces. 1. 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. ........ Minores. c. Forms difficult to cultivate, not growing on potato, not liquefying SE MATUENE Olas SCIEN iotacays tee te A ckchs seh Sis, ork ot eachedee: Breviores. leet Aero DIGHEOLIMS ya Sa cscisote s Seton of Sipe aio Sele ohne Cohnistreptothrix. Facultative anaerobic forms, difficult to cultivate; no arthrospores pro- duced. G. Classification of Orskov (1923): I. Typical conidia formation in aerial mycelium. ..... Cohnistreptothrix. I> Spore-formation by segmentation. ........-.......:--- Actinomyces. III. Spores produced singly on branches of mycelium. ... Micromonospora. H. Classification of Ligniéres (1924): I. Aerobic, long mycelium, not breaking up into rods. ..... Actinomyces. II. Anaerobic, short mycelium, breaking up into long rods............... Brevistreptothrix. Wiese mycelium; cells rod-shaped: ~...... 2... 72 -/n- Actinobacillus. I. Classification of Jensen (1931): PEM OMSDIORCS ECENICC 15 302 orcs, (olay enctiele Wins rate, «fev nim oy Proactinomycetaceae. } I. No mycelium formed: Ie NGG rasta OLroamismnSsners tote Sects Anche ete) Mycobacterium. Waksman a Actinomycetes 2: WNon-acid-fast organisms. sar ate pees eke aie el Corynebacterium. LU Gehibyeclliitone twoentes Bui Gobnagc godcoudyhuade sooo Proactinomyces. |3uers ess, HOMME Sibe Sob ay cuoacee joc Caco cocoa Actinomycetaceae. I “Spores, im/aeriallsmyceliana.) < agak stim? «o> oar Actinomyces. II. Spores terminally on branches of vegetative mycelium. ............ Micromonospora. J. Classification of Duché (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 albus group. I. Vigorously growing forms. 1. Mycelium yellowish, no exopigment. Species included in this group, based on pigmentation of the mycelium, were A. albus, A. alboviridis, A. roseus, 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 efHorescence. .. A. reticuli. 4. Mycelium yellow-red, no exopigment, white efHorescence ........ A. albosporeus. 5. Mycelium yellowish-clear, no exopigment, yellow efHlorescence..... 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. ........ Actinomyces. 2. Unicellular mycelium, later breaking into rods and cocci. .......... Proactinomyces. 3. No mycelium, elongated rod-shaped, branching and breaking into COCCOIGS OTITIS, eee tene ey ony esse A cece er oaeen ee Mycobacterium. 4. Cells coccus-like, seldom rod-shaped; resting cells develop in a manner similar to ACHMOMY.CESESPOLES ri-dnare ai-in tee eneleh eke eee 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. at, (Gellsichusitortmts <2 sageites siege eneearernteemic arate caeaene Fusiformis. bi. (Gells rod-shaped! or ‘coccus-like!\. 2 2c) ae Actinobacillus. c’. Cells rod-shaped, sometimes filamentous; branched. Pfeifferella. b. Hyphae frequently present, gram-positive. a’. Filaments branched, thickened, showing characteristic granules. Erysipelothrix. Chapter I ee Taxonomy b'. Filaments unbranched, fragmented into short rods, sometimes witheoramules\ and septa... chs. a4s ee deh els Leptotrichia. 2. Filamentous, readily dividing into bacterial SERED (Seen. na. hatc hess Proactinomycoideae. a. Filaments with angular growth, dividing into bacteria-like segments. PipNCIC PAS te Neath aes ae ce erie ge =, ce a Mycobacterium. Dam NOC ACIAEEAStS 1c tk OMS che Marne Hee eens Corynebacterium. b. Long branching hyphae, filaments as in 2a; aerial mycelium may be present but not different from vegetative mycelium: Agee ATIACLODICN Rae tae Lie nce h Ss hae ee Actinobacterium. b'. Microaerobic, sometimes with sclerotial masses. Cohnistreptothrix. c’. Aerobic, well-developed mycelium, aerial and vegetative myce- lit unedterentiated. 2.0.28. 240). . ene Proactinomyces. II. Conidia always produced, with distinct aerial TAY. Celiummeeeys citer te Actinomycetaceae. ie@onidia sproduced singly. 2.2 2 oe a care oe ooo Micromonospora. 2, Gonidiay-seriated and multiples }..5.05. 4.40250 ce Actinomyces. M. Classification of Waksman II. (1940): i) Mycelium: rudimentary or absent: %)....-..+2-0.: Mycobacteriaceae. 1. Nonmotile. AEE ANGI CEASE aut war GReeei n tena eee Ia: enn iter Se ee an Mycobacterium. DUMINONR-ACIG-tAStts 3 Secor ciel exciecee otiee os Corynebacterium. ESIC fil Coa xn oe are ee ave aie, Sie aang Se Sener ef Mycoplana. II. Mycelium produced. Il Spores formed by segmentation. see e crave eee Proactinomycetaceae. Al, /NDAEROO Sa, Sooanacanunoac wseeeeeess Cohnistreptothrix. bear Crobesseanne neds cisiaats iets AAU ted aes ies 2 i Proactinomyces. III. Vegetative mycelium normally remaining undivided. 1. Conidia formed in chains from aerial hyphae. .. . Actinomycetaceae. SPOKES PLOU MCE IME Chalns gets aley cir cket ayelas sees Actinomyces. 2. Conidia formed terminally and singly on short conidiophores. ...... Micromonosporaceae. Spores produced esingly. Garter wey feiss ates © tes Micromonospora. N. Classification of Waksman and Henrici (1943): A. Mycelium rudimentary or absent. .......... Mycobacteriaceae Chester. I. Acid-fast organisms. ..... Mycobacterium 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- ATIENNES ta aha eee Streptomycetaceae Waksman and Henrici. a. Multiplication by conidia in chains from aerial hyphae. ........ Streptomyces Waksman and Henrici. b. Multiplication by single terminal spores on short sporophores. ... . Micromonospora Orskov. we uodn sajooXkurounse stqoree Jo MOIS ead —+ “Oty “(Q97 ‘HuSaITT wmof) suis Chapter I =e 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 Krassi_nikov'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 Micromonospora 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 WoxrF-IsraEL 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 basis for classification.—Although 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 a Actinomycetes sion 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 Waxksman, and more recently by Jensen, Kriss, and Krassiinixov 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 freshly isolated, as well as cultures Fic. 5 a-d.—Different types of branching of aerial myce- lium of species of Streptomyces: above, long open spirals; Fig. 5b Gp. 19), tuft formation of sporulating hyphae; Fig. 5e (p. 20), short compact spirals; Fig. 5d Cp. 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. ‘“Uhose 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 DreECHSLER (97) suggested that the nature of the curvature of the spirals can be utilized as a distinguishing character. KrassiLnrkov, 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 Streptomyces are spherical, oval, and elongated, whereas the seg- mentation spores or the oidiospores, characteristic of the Nocardia, are Fic. 5 b (see p. 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 synthetic and on organic media are given con- Fic. 5 c (see p. 18). sideration. Because of this, a number of descriptions have béen 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, whether 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 may form a basis for more detailed differentiation of specific types. Aoxr (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 a Taxonomy reaction corresponded well to the agglutination reaction. The agglutinating re- ceptors were present more abundantly in the spores than in the mycelium. V. Maenus (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 the 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. griseus, and not others. : : Biochemical characteristics.—This group of properties comprises quantitative rather than qualitative differences. The S. coelicolor group, for example, was found (78) to include forms which differ greatly in type of pigment produced. Fie? 5d Gee p: 15). 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; (b) those that give moderate reduction; (c) those that give strong reduction. A similar basis of separation might be suggested for the properties of proteolysis, amylolytic 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. Chapter II IDENTIFICATION AND DESCRIPTIONS OF IMPORTANT TYPES Classification of Actinomycetales:— The following classification of the actinomycetes is based entirely upon material included in BERcEy’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 1.54 in diameter, mostly about ly 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 Mycobacteriaceae. I eAcid-fastaorgamisms, .yek.10s «note ie ieee tore Mycobacterium. B. True mycelium produced: I. Vegetative mycelium divided by segmentation into bacillary or coccoid Glémentst es 4 oiss he ee he Ee are Rca oe Family Actinomycetaceae. 1. Anaerobic or microaerophilic, usually parasitic, non-acid-fast Actinomyces. Type species—Actinomyces bovis. 2. Aerobic, partially acid-fast or non-acid-fast.............. Nocardia. Type species—Nocardia farcinica. II. Vegetative mycelium normally remaining undivided—Family Streptomycetaceae. Chapter II ——_ Important Types (a) Conidia produced in chains, in aerial hyphae....... Streptomyces. Type species—Streptomyces albus. Cb) Conidia produced terminally and singly on short conidiophores Micromonospora. Type species—Micromonospora chalcea. Genus I. Actinomyces Harz (Streptothrix Cohn; Nocardia Toni and Trevisan; Cladothrix Eppinger, Wolf- Israel fungus; Anaeromyces Castellani; Brevistreptothrix Ligniéres; Cohnistrepto- thrix Pinoy). Actinomyces bovis Harz. (Discomyces bovis Rivolta; Bacterium actinocladothrix Afanasiev; Nocardia actinomyces ‘Trevisan; Actino- myces hominis Wolf and Israel; Streptothrix actinomyces Rossi-Doria; Cladothrix bovis Macé; Oospora bovis Sauvageau and Radais; Actinomy- ces bovis sulphureus Gasperini; Nocardia bovis Blanchard; Streptothrix israeli Kruse; Cladothrix actinomyces Macé; Actinomyces israeli Lach- ner-Sandoval; Streptothrix actinomycotica Foulerton; Streptothrix bovis communis Foulerton; Streptothrix bovis Chester; Discomyces israeli Gedoelst; Actinomyces sulphureus Sanfelice; Streptothrix sulphurea Caminiti; Sphaerotilus bovis Engler; Actinobacterium israeli Sampietro; Cohnistreptothrix israeli Pinoy; Proactinomyces israeli Negroni, Actino- myces wolf-israel and Corynebacterium israeli Lentze,; Proactinomyces bovis Henrici; Actinomyces israeli Rosebury ). According to Batpacct (17), most of the cultures listed as A. bovis comprise forms which have also been designated as A. albus, A. sul- phureus, etc. These include the four species or varieties of A. bovis created in 1894 by Gaspertni, namely, A. bovis sulphureus, A. bovis farcinicus, A. bovis albus, and A. bovis luteo-roseus. Waxsman’s de- scription of A. bovis (443) is said to be equivalent to A. bovis sulphu- reus. Batpacct recommends that this species be considered as A. sul- phureus Gasperini. Batpaccr further included among A. bovis, Strep- tothrix hominis Foulerton, Streptothrix luteola Foulerton, Actinomyces bovis Harz fide Waksman, Actinomyces hominis Waksman (sub. A. hominis Bostroem), A. bovis 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 2y. 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 — a 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 Stack (403) from glucose, maltose, mannitol, sucrose, and lactose; according to Nrecroni and Bonricriotr (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 hyphae 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 )s Further information on the morphology and physiology 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; Streptothrix Kruse, Caminiti, Rossi-Doria, Silberschmidt; Cladothrix Eppinger; Brevistreptothrix Ligniéres; Actinobacterium Haas; Actinocladothrix Afanassiev; Actinobacille Ligniéres 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 Mycobacterium. Paraffin, phenol, and m-cresol are frequently utilized as sources of energy. In their early stages of growth on culture media Cliquid or solid), the structure of a nocardia is similar to that of a streptomyces. Both form a typical mycelium: hyphae branch abundantly, the branching being true. ‘The hyphae vary in diameter between 2.5y. and ly, most of Fic. 6.—Nocardia asteroides, grown on potato glucose-beef extract agar, gram stain, X 100. (Prepared by liver enecacned jeanece InGaN of Pathology). Waksman Ao Actinomycetes them measuring 0.7-0.8», according to the species. ‘The mycelium is not septate. The further development of nocardias, however, differs from that of streptomyces cultures: the filaments soon form a transverse wall 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 3y.). 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 Streptomyces; 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. ane color of the culture serves as a stable character. KrassiLtntkov (234) divided the genus Nocardia into two groups: 1. 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 lucose-beef extract agar, bottom 8 Lirrman of Armed Forces Institute otato Pp (Prepared by Fie. 7.—Nocardia asteroides, grown on of colony, gram stain, X 975. of Pathology). Waksman Se 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: Partly acid-fast organisms, which are nonproteolytic, nondiastatic, and utilize res 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. (Streptothrix farcinica Rossi-Doria; Oospora farcinica Sauvageau and Radais; Actinomyces far- cinicus Gasperini; Actinomyces bovis farcinicus Gasperini; Bacillus far- cinicus Gasperini; Cladothrix farcinica Macé; Streptothrix farcini bovis Kitt; Streptothrix nocardii Foulerton; Discomyces farcinicus Geodoelst; Actinomyces nocardii Buchanan, and many others ). Filaments 0.254 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, etten 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 Rosesury (75) recently presented CTaBie 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 number of additional species only incompletely described. Genus III. Streptomyces Waksman and Henrici CStreptothrix Cohn; not Streptothrix Corda; Actinomyces Harz; Discomyces Rivolta; Actinocladothrix Afanassiev; Nocardia ‘Trevisan; Micromyces Gruber; not Micromyces Dangeard; Actinobacterium Haas; Carteria and Carterii Musgrave, Clegg and Polk; Euactinomyces Langeron). es Fic. 8.—Branching and sporulation of different strains of Micromonospora (from JENSEN, 186). Waksman — 30 — Actinomycetes Tase 2: Comparison of cultural, morphologic and staining reactions of species of Nocardia Ga) CZAPEK’S FRAGMEN- Cotor or Acip- SABOURAUD’S AGAR TATION OF SPECIES GRANULE FAST GLUCOSE AGAR (PIGMENT) MYCELIUM 1. Nocardia asteroides Yellowish- + Glabrous, rarely Yellow to + (Eppinger ) white, chalky. Moist, orange Blanchard, 1896 with or soft, folded or without wrinkled and clubs granular. Yel- low, orange- ochraceous, red 2. 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 3. Nocardia madurae Yellowish- — Glabrous. Moist, Creamcolored — (Vincent ) white, soft, wrinkled. at first; Blanchard, 1896 with or Cream colored later be- without coming clubs pinkish to red 4. Nocardia pelietieri Red, with ©— Small. Glabrous, Coral red - (Laveran) Pinoy, or with- heaped, wrin- 1912 out kled. Mucilagi- clubs nous. Coral pink to red 5. Nocardia Black, — Glabrous. Soft, Dark cream _ paraguayensis with white center. (Almeida) clubs Projecting bor- Conant, 1947 der adherent, darker Organisms growing in the form of a much-branched mycelium 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 hyphae arranged on mycelium in whorls or tufts. Chapter II =o 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, usually synthetic agar, but not on organic media. Type Species: Streptomyces albus (Rossi-Doria em. Krainsky) Waksman and Henrici. (Streptothrix alba Rossi-Doria; Cladothrix alba Macé; Nocardia alba Chalmers and Christopherson; Cladothrix dichotoma Mace, Streptothrix foersteri Gasperini; Streptothrix 2 and 3 Almquist; Actinomyces saprophyticus Gasperini; Oospora doriae Sauva- geau et Radais; Cladothrix liquefaciens Hesse; Cladothrix invulnerabilis Acosta e Grande Rossi; Actinomyces chromogenus Gasperini; Strepto- thrix nigra Rossi-Doria; Streptothrix gedanensis I Scheele et Petruschky; Streptothrix graminearum Berestneft; Actinomyces thermophilus (Berest- neff) Miehe; Cladothrix odorifera Rullmann; Actinomyces chromogenes Gasp. ( alba 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 albus Krainsky, Actinomyces sanninii Ciferri; Actinomyces almquisti Duché; Actinomyces gougeroti Duché, and numerous others). This is one of the most widely distributed and most widely described types in nature. It produces no soluble pigment and abundant white aerial mycelium. Various strains isolated by different investigators have been variously described. “The most complete recent study was made by Ducué (98) and by Barpaccr (20). Vegetative hyphae: Branched, lu in diameter. Aerial mycelium: Abundant white, 1.3 x 1.74, 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 BErcey’s Manual contains descriptions of 73 spe- cies of Streptomyces, and an additional large number of incompletely described species. Genus IV. Micromonospora Orskov (Thermoactinomyces Tsiklinsky ). Well developed, fine, nonseptated mycelium, 0.3-0.6y. 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 — 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 Micromonospora:— ey igorously growing organisms, typically copious spore formation on dextrose- asparagine agar. A. Vegetative mycelium pale pink to deep orange, no typical soluble pigment. ERR ne EN Nt ae cliche Cor cee 1. Micromonospora chalcea. B. Vegetative mycelium orange changing to brownish-black, brown soluble LTS Mis oodrts Apeig ad o dlocea cmaeclow nid.5 2. Micromonospora fusca. 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. Micromonospora parva. B: Vegetative mycelium yellow to orange-red 4 -). -y.jtia- tee erie eee 4. Micromonospora globosa. C. Vegetative mycelium blue....... aresaneiets 5. Micromonospora vulgaris. Type Species: Micromonospora chalcea (Foulerton) Orskov. (Streptothrix 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 difficulty, 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 = 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- fireanlokes! Each of these can be divided into three ; 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 bovis Harz. A. bovis 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. bovis is a gram-positive, branching, filamentous organism, non- acid-fast, and not producing spores. “The hyphae are usually less than lu 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 but 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 —— 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 morphology of the organism has been described as varying from a compact mass of branching mycelium of gram-positive filaments to a mass of short rods which may 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 fae ents 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 days. ‘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. Interment: strains tend to grow as smaller particles or gran- ules either at the bottom or along the ide 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 > per cent COs, for 4 to 6 days, rough or intermediate strains of A. bovis produce white-grayish to yellowish colonies having a diameter of not more than | to 3 mm. These colonies usually aaliere to the medium, so that they are hard to remove with an inoculating needle, often com- Chapter II = = 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- CO, plates, the colonies are very different from aheee 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 of the parasitic and saprophytic actinomycetes (367) :— Natural habitat: Cellular morphology. Character of growth: Temperature requirements. Relation to oxygen: Metabolism: Species recognized: Pathogenicity: PARASITIC ACTINOMYCETES 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. Bacteria-like colonies without aerial hyphae; no spores; no pigments. Optimum, 37°C.; DIRE: Oxygen tolerance limited; gener- ally fail to grow or grow poorly under aerobic conditions. no growth at Probably never proteolytic. Fer- ment carbohydrates with pro- duction of acid. One only: Actinomyces bovis. (Pro- visional; heterogeneous but not yet satisfactorily subdivided.) Causative agent of true actinomy- cosis in man and animals. SAPROPHYTIC ACTINOMYCETES 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. 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- ease. 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 2u (0.5-1.3y.). 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 brownish, 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 hyphae are often clavate and are continuous; transverse septae are laid down simultaneously, di- viding the hypha 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 0.7-1.9y, the variations being observed in the same chain, as shown in Fic. 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 a 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 mycelium, not producing any streptomycin and sensitive to this antibiotic as brought out in Taste 4. (b). A pigmented mutant, pro- ducing pink to vinazeous colored vegetative growth, but forming the Tasie 4: Streptomycin production and streptomycin 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 23,125 Strain No. 19 Sporulating active form 128 2 a3125 Variant 3 Non-sporulating form 0 20 Variant 4 Non-sporulating form 0 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 medium. Waksman — 5 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. griseus 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 mycelium tends to be higher during the active stages of growth. ‘The production and accumulation of strep- tomycin 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 streptomycin 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 synthesis of the streptomycin. Such a factor can gradually be synthesized by the organism, when it is provided in the medium ina 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 8m in diameter. Conidia oval, 1.0 to 1.2 by 1.6 to 2.0u. Gelatin stab: Creamy to brownish surface growth. Liquefied. Synthetic agar: Thin, spreading, colorless. Aerial mycelium cottony, white, becoming vinous- eee Cech agar: Restricted, glistening, transparent. Plain agar: Gray, wrinkled. Dextrose broth: Abundant, flaky sediment. u é F ? 3 # 3s i 2. Fic. 9.—Sporulation of straight aerial hyphae of species of Streptomyces (from KRassILNikey, 234). Waksman ao 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. lavendulae was designated as streptothricin (452). Since then, several other antibiotics have been isolated from members of this group. Some of these antibiotics, notably, 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 Paibianics produced by organisms belonging or closely related to the S. lavendulae group are distinctly different from streptothricin both chemically and in their antibiotic spectra, as is the case for chloromycetin. The streptothricin-producing S. lavendulae 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 acer a brown diffusible pigment appears, the growth becom- ing covered with abundant lavender-colored mycelium. On_ subse- quent transfer on fungus-agar slants, the two types of variants prove to be rather stable. Some of the strains isolated from an active 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 ce tionary and in shaken cultures growth and activity reach a maximum and then decline, the maximum foe 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 ee 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 | 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. Streptomyces venezuelae Ehrlich, Gottlieb, Burkholder, Anderson and Pridham S. venezuelae, 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 and the branches grow to about 150y 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 1.8y. in diameter, and vary in length up to about 350y. 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 0.9% in diameter and from 0.7 to 1.6%. in length. ‘The spores formed by fragmentation of hyphae in the substrate are generally smaller than those formed from the aerial hyphae. Individual spores are colorless at maturity but in mass appear tan to gray when viewed without magnification. They may be stained readily with crystal violet Waksman — 4) 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 melee 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 strains also differed from S. lavendulae in their sensitivity to actinophage and in serological reactions. Streptomyces antibioticus (Waksman and Woodruff) Waksman and Henrici A detailed description of S. antibioticus has been given by Waks- MAN and WoopruFF (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 PeacHon change, no coagulation of milk, no clearing; whitish Sediments at bottom of tube. Old cultures—heavy growth ring on ithaca 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. Streptomyces aureofaciens Duggar S. aureofaciens, the organism that produces aureomycin, was isolated from the soil (99a). Chapter II — AS 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 crystalline 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. Streptomyces scabies (Thaxter) Waksman and Henrici Morphology: wavy or slightly curved mycelium, with long branched aerial hyphae, showing a few spirals. Conidia more or less cylindrical; 0.8 to 1.0 by 1.2 to 1.5p. 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 Fic. 10.—Streptomyces venezuelae, grown on potato glucose-beef extract agar, gram stain, X 975. (Prepared by Lrrrman of Armed Forces Institute of Path- ology ). Chapter II NS er 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. Maturity, 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 III MORPHOLOGY AND ALIFE 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 when grown on artificial media, have also contributed to the confusion. One of the early students of the actinomycetes, F. Conn, recorded in 1875 that the “ray fungi,” a common designation given to this group of organisms, are funguslike in nature. ‘This point of view was held by a number of subsequent investigators, notably THaxrer in 1891, LACHNER-SANDOVAL in 1898, BERESTNEW in 1899, Neuxircu in 1902, and more recently 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 methods of examining the structure of the actinomycete colonies and their growth characteristics, that is, the general methods developed by students of glucose-beef agar, gram stain, Fic. 11.—Streptomyces sp., grown on potato x 975. (Prepared by Lrrrman of Armed Forces Institute of Pathology). Waksman — Actinomycetes fungi and bacteria, certain special methods have also found application. Among these, it is sufhcient 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 be 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 formper sine above 50°C; Most actinomycetes are non-acid-fast. Some of the ees 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 Brussoy (49) and Drecusier (97); this was believed by some investigators to indi- cate that the cultures possess the property of pleomorphism. 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 frequently in the mycelium (242). The forma- tion of vacuoles has also been reported (97). Neuxrecn (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. Krassrint- 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 PrornHo (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 Rreret (361), who believed that the bodies stained by the Feulgen re- action are fats in nature. JENSEN described (186) the staining reactions of Micromonospora 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 NasCOs 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 xylose 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 — 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 Fic. 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 600». or more; others are only 50 to 100y. in length, and are much branched and curved. ‘The vegetative mycelium varies in diameter from 0.2 to 0.8. Occasion- ally, involution forms are produced which have even a greater diam- eter. ‘The structure of the hyphae 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, Lreske (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 usually 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 1 or less in length. Together with the cellular contents they form a granular mass aie deposits on the bot- tom of liquid cultures. Ginna, cultures undergo rapid lysis, especially Chapter TIT — 5] — 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. KrassirntKov (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 mycelium.—Many 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 hyphae vary considerably in length and may have a diameter of ly. or even 1.4y. 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, chalklike 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 differentiation. 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 mycelium which arises through the elongation of the growing filament tip shows very little subsequent increase in thickness, the sporogenous hyphae are, in the beginning, thinner than the axial hyphae 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 ae 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- Ne One A Fic. 12 a-d.—Different forms of sporulation of Micro- monospora growing in composts, as shown by contact slide preparations (from Waxsman, Corpvon and Hut- por, 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 powdery 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 by 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 sporophores produced on the aerial hyphae comprise several types, as pointed out previously Cp. 30). Chapter II — 93 —— 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 H.SO, it is first pigmented dark and is then dissolved. As the great majority of the cultures do not show such septa, it has Fic. 12 b (see p. 52). generally become recognized that an actinomyces colony represents a single-cell type. DrecusLer (97) considered the actinomyces 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. Krassm_n1Kov (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 formed early in the vegetative mycelium. This mycelium, however, although septated, never breaks up into single cells. Waksman i 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 Waxsman for S. reticuli; 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 Micromonospora. 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. [ ? | : be Fic. 12 ¢ (see p. 52). Among species of Nocardia, Lieske observed the production of swollen cells, which he considered as involution forms. KrassiLNrKOv 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 ainibes 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 formation.—The actinomyces spore has been described as con- taining a spherical, relatively large chromatin body which is surrounded Chapter III — 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 ee -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 chercres 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 fhe cell wall into particles: or fragments, more or less uniform in size. ‘These fragments are later liboried 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- al 3 Fie. 12°d (seexp. 52). sionally taken for cross walls. When the spores mature, the surface cover becomes less defined and may gradually disappear, as a result of autolysis. [he spore-bearing fede 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 Streptomyces. 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 and the hyphae break up into smell segments. ‘These are cylindrical in form, with sharp edges and are nioeni in size, usually 1-25 x 0.7-0.8u.. These often have been considered as true oidiospores Gay: a Fic. 13.—Details of sporulation and of spore germination by S. griseus as shown by electron microscope: Top, left, aerial sporogenous hypha showing septation prior to spore formation; top, right, more advanced 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 (from CarvaJjAt, 64). Note (top, left) mitotic division taking place in developing spores. Chapter III et Morphology The cylindrical oidiospores may swell, giving rise to spherical bodies. This manner of sporulation is characteristic of the genus Nocardia and of certain species of Streptomyces. DrecusLer recognized three types of sporulation: (a) by means of true fragmentation, (b) by means of doubling of the cell wall, Cc) by means of contractions similar to segmentation. According to Ducué, the last process alone results in the production of three types of spores: (a) regular and irregular arthrospores, (b) 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 (1.5-1.7y.), with 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 Krassitntxov (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 | 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 Cdextrorse)) or counterclockwise (sinistrorse). Drecus- 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- 0.3. in diameter ), oval, or cylindrical (0.8-1 0.74). ‘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 similar 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 Jo Actinomycetes Reproduction can also occur by the vegetative process, namely, through the growth of pieces of mycelium, 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). Fic. 14.—Aerial mycelium of a Streptomyces, showing zonation or “fairy ring” formation (from LresKE, 260). Sporulation of the Micromonospora 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—5-10y. 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 — oe Morphology duced by the splitting off of the tip of the hypha. The conidia are spherical (1.0-1.34 in diameter), oval, or oblong (1.3-1.5 & 1.2,). 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 Micromonospora. Anaerobic organisms.— The morphology of the anaerobic forms repre- sents a special problem. Erikson (112) made a detailed study of the morphology of 15 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 oxygen 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 =o Actinomycetes The colonies from the bovine strains were smoother and softer in consistency and did not adhere to the medium. Growth was scantier. The mycelium 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 actinomycetes.—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 Krassiintkov (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 different 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- Fic. 15.—Electron micrograph of actinophage (from Woopruer, et al., 518). Waksman — oo 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. DmirrieFF and SouTEEFF (91) observed that a culture of an or- ganism designated as Actinontyces bovis, 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 type 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 Scuatz and Waxsman (395) in the production of inactive strains by S. griseus. These strains were free of aerial mycelium, produced no streptomycin, underwent much more rapid lysis, and formed much more acid in the medium (Tase 5). Effect of actinophage upon actinomycetes.—WiEBOLS and WIE- RINGA (509) observed that cultures of actinomycetes isolated from in- fected potatoes underwent lysis. “This phenomenon was ascribed to the Chapter III 5 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- Tasie 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- 1. No streptomycin formed either in cin in both shaken and stationary cul- shaken or stationary cultures. tures. 2. Growth. Surface growth always heavily 2. No sporulating aerial mycelium; scant sporulated; grayish-green aerial myce- development of aerial hyphae with lium. slight tendency to form spores in some old cultures. 3. Reaction. Medium always changes to 3. Reaction of medium at first acid, pH alkaline; pH 7.5-8.5. 5.0-6.5; later becoming alkaline. 4. Glucose. Glucose completely consumed 4. Glucose utilized more slowly. in 6-8 days in stationary cultures and in 3-4 days in shaken cultures. 5. Lysis in shaken cultures. Shaken cultures 5. Cultures produce at first balls of growth produce very fine flocculant growth, which change into the turbid, floccu- tending to lyse slowly after about 15 lant type; rapid and complete lysis in days. 7-10 days. 6. Lysis in stationary cultures. Surface pel- 6. Stationary cultures produce no surface licles stable; any submerged, flocculant growth but flocculant, submerged my- growth tends to lyse as the surface pel- celial growth which lyses slowly, only licle develops. after a month or longer. 7. Viscosity. Culture filtrate not showing 7. Culture filtrate becomes viscous during any viscosity. or after lysis. 8. Reinoculation. Inoculation of cultures 8. Inoculation of cultures with spores of with lysed inactive culture induces no active strain produces growth and anti- lysis or reduction in activity. biotic activity if some glucose remains. 9. Variation. Sporulating strain gives rise 9. Asporogenous variants may reconvert to non-sporulating variants. to active, sporogenous forms. 10. Sensitivity to streptomycin. Very resist- 10. Very sensitive to this antibiotic. ant to this antibiotic. genic organisms A. bovis and N. farcinica. A polyvalent phage was obtained from one of the actinomycetes which was also active upon S. scabies, thus 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. Krassitnikov and Korentako (237) emphasized the resemblance of the process of autolysis among actinomycetes to the action of phage Waksman = G4 Actinomycetes upon bacteria. ‘They reported, however, that the lytic factor of actino- mycetes was highly specific, since it had no effect 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- fans 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 ath the various organisms. At temperatures of 60° to 70°C., lysis occurred in a few minutes. The lytic agent was resistant to a Fic. 16.—Method of measuring actinophage concentration (from Remuy, Harris and WaxsmaNn, 355) temperature of 80°C. for 1 hour but was destroyed 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 ne agent is different in its action foe that of true phage. With the discovery 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 ph ase. ‘The lysis of the actinomyces produced by the phage appeared to be quite distinct from the lytic phenomena. SaupEK and Corincswortu (383) were the first to report that S. griseus is subject to the action of a transmissible lytic agent which had Chapter HI 165 = Morphology all the properties of phage. In the presence of young cultures of S. griseus, the phage developed 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. WoopruFF and Foster (516) exposed to laboratory air for 24 hours a submerged culture of S. griseus 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. griseus, the phage multiplied. It was calculated that after six transfers, each phage particle increased to 75 > 107° 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. griseus 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 (Fic. 15). The following method can be employed (355) for assaying the activity concentration of phage in a given 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 days. The plaque counts are made, as shown in Fic. 16, and calculated for 1 ml. of the preparation. Some preparations gave 4 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 different concentrations of filtered 7-day-old shaken culture of streptomycin-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 39] } 1.0 698 0.1 756 Waksman =o Actinomycetes These results show that the plates do not have to be heavily inocu- lated with S. griseus 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. griseus or on other streptomycin-producing organisms such as S. Taste 6: Effect of phage upon the growth, phage multiplication, and streptomycin production by different actinomycetes in stationary cultures (355):— 9 days 13 days Phage Phage Phage per ml Strepto- per ml Strepto- OrGANISM added* 50 <5 370 <5 No. 3480 0 0) 31 0 189 sts 10 <5 30 28 No. 3481 0 0 13 0 174 “iF 50 <5 260 13 No. 4 0 0 43 0 201 ail 30 << 160 <5 3475-2PR 0 >0.01 40 40 129 ar > 50 16 370 75 Grisein-producing strain of 0 0 <5 0 <5) S. griseus 3478 ae @) <5) 0 <5 Inactive strain of 0 = 0 <5 S. griseus 3326a os = = <0.2 <5 Streptomycin-producing 0 0) <5) 0 30 S. bzkiniensis + 3 30 7 33 * Each 60-ml flask of culture received at start 0.1 ml of M-1 phage, amounting to7 X 107 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 —o7— Morphology 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 types.—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 Streptomyces, the combined morphological and cultural properties well characterize each genus. Very often a species of Streptomyces may lose, by selection or by mutation or natural variation, the property of forming aerial mycelium; it may then appear to become a typical 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. Tasie 7: Stability of phage in aqueous suspension upon storage at several temperatures (355):— Phage particles X 107 per ml, after storage* TEMPERATURE OF STORAGE, 3 days 12 days 29 days BG 6 44 - 60 28 31 20 0.00005 37 37 15 0.0000009 56.5 18 0.001 0) * At start all preparations contained 36 X 107 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. Apparently 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 mycelium. 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, Ertrkson (115b) 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 mycelium and very sparse aerial mycelium 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 Streptomyces 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- dosfores; the broken bits of mycelium also have the capacity of growing into a fresh mycelium. The genus Micromonospora 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. Micromonospora may be looked upon as the most highly developed group among the actinomycetes, placing the whole order Actinomy- 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 1V 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 readily subject to variations and mutations, the actinomycetes 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, (hb) 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 iafienee of various environmental condi- tions but even on continued cultivation under the same conditions. The soluble pigment may be lost or changed in color; the color of the aerial mycelium may change; even the property of forming aerial Fic. 17.—Variants of Streptomyces griseus growing on yeast extract glucose agar (from Duraney, Rucer, Hiavac, 101a). Chapter IV i 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; (b) continuous or fluctuating, as shown by differences 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. Tempet (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 were confirmed, but these changes were not permanent in nature. RrppeL and Wirrer (361) could not obtain any variability 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; Cb) trans- formation of an actinomyces into diphtheroid organisms (211); (c) transformation of anaerobic, short hyphal-producing forms into aerobic, long hyphal forms (328); Cd) change of aerial mycelium and strepto- Waksman i Actinomycetes mycin-producing strains of S. griseus into inactive strains free- from aerial mycelium (395); Ce) change of strain of S. griseus from a color- less vegetative 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 effects, 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 modiecedone athe 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 my- celium from strains producing such mycelium or vice versa; formation Chapter IV ee 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 Krassitnikov (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 typical species of Streptomyces and others of which were closer to the mycobacteria. JENSEN (191) also observed that under the influence of LiCl mycobacteria gave rise to forms that might be con- sidered as species of Nocardia. Recently, extensive investigations have been made of the effect of ultraviolet radiations and x-rays in inducing mutations of various species of Streptomyces. Savace (385) reported that ultraviolet rays were less mutagenic than the x-rays, the harder rays of 0.710A and 0.210A wave lenoths 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 streptomycin production. By means of x-ray and ultraviolet light irradiations, KELLNER (213) found that most antibiotically inactive peulenres gave rise to antibiotic- producing mutants. Of the greatest interest was the fact that a strain of S. griseus kept for a long time in the culture collection and which was inactive antibiotically 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 may 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 streptomyces; the reverse phenomenon occurs but seldom. This reasoning led Krasstinrkov 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, id applied. ‘These may be briefly summarized as follows: Morphological variations.—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 poly- chromogenes of two different Caan 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. “Iwo subtypes were recognized for the R-form: the soft or s-type 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 stype. 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 hyphae, 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 Henricr (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 Krassitnikov 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 Sanrexice, Ducué, and others. Evidence of this is found in Chapter IV —75— Variations and Mutations the designation of such groups as albus, flavus, and violaceus. Waxs- 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 Cor 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. griseus, can hardly be recognized either by comparison with the original cultures of Waxksman and Curtis or from the original description of Krarnsky, 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 Cp. 60). ‘The phenomenon of lysis, 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 development of resistant strains. Marked variations in agar-decomposition and pigmentation of S. coelicolor have also been observed (408). Erimson (115a) found that the major variations of S. coelicolor 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 pigment 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 =o Actinomycetes ScHaat (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. Tuomas (421) isolated six physiologic races of S. scabies which distinctly differed in pathogenicity on ten different potato varieties or selections. [he 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. [he 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. griseus. ‘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. griseus 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 a Variations and Mutations streptomycin. On the other hand, the nonsporulating variant produced no aerial mycelium, formed no streptomycin, was sensitive to the antibi- otic action of this substance, was characterized by a type of growth that in shaken culture 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 Streptomyces? 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 Tase 8: Production of streptothricin by two strains of S. lavendulae and their variants (478 ):— STRAIN OR VARIANT STREPTOTHRICIN®* Strain No. 8 25 Variant 8a 40 w — DextroseConsumption 50 % oO c Nae Streptethricin /20 Gos SS=e cv % 7 >~e 8 : 80 Q 20. So Ze B g S is Dry Weight Sh Days 5 rf) Days Shaker; as —Statwnary Fic. 18.—Metabolic. changes produced by S. lavendulae in aerated and stationary cultures (from WooprurrF 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 (Tasre 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 Tare 12 and in Fic. 18 (517).. The efficiency of carbon utiliza- tion is greater in stationary than in submerged cultures. The maximum growth is attained, however, in submerged and aerated cultures. ‘The carbon efficiency A 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 efh- ciency drops. After growth of the organism reaches a maximum, less synthesis takes place. If lysis sets in, the mycelium is destroyed and CO, and NHs 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 tryptone for cell synthesis. ‘This is true especially for submerged cultures, where the abundance of available oxygen leads to greater oxidation of carbohydrate as compared to the tryptone consumed. Acid production by actinomycetes.—As a result of the growth of ac- tinomycetes in different media, there is always a tendency for the reac- Tasie 13: Acid production by an actinomyces on meat extract-peptone-glucose medium (340) :— Strain No. Final pH* Lactic acid 3 8.4 = 4 8.6 = 5 8.5 = 6 4.9 + 7 4.7 asst 8 4.8 =F 10 152 ++ * Original pH 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 — 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. acidophilus. The fact that various actinomycetes are able to produce organic acids form carbohydrates has been long recognized (470). Macnus (281) observed that many of the actinomycetes found in the larynx are able to produce acid of the lactic type Cether-soluble) even in sugar-free media. PrornHo (341) confirmed these observations and definitely established the fact that the acid produced by various actinomycetes is of the lactic type, as shown in Tasre 13. WooprurtF 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 Taste 13. In the presence of Tasre 14: Acid formation from glucose in aerated cultures of S. lavendulae (517):— GLUCOSE pH of medium* after CONCENTRATION per cent 3 days 4 days 5 days 6 days 0 8.2 8.6 8.7 8.8 1 6.8 6.9 7.0 7.4 2 6.5, 6.5 (S53 6.5 5 6.2 6.1 Suey 5.7 * Initial pH 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 pH levels may go down as low as 3.2 in 2 days. ‘The changes in reaction are thus parallel to the concentrations of sugar CTasxe 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 tryptone 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 —o— 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 consumption.—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 synthesis and to the formation of incom- 8 betes 8 SS 8 Milligrams per 85 ML. Culture ~ 8 &o X —_---.— 2 oh 200: Acti TLOMmYCIIL /00 / § 50 ye A ip S LEZ wey Weight ™ o idee SB te Days 4 3 Days 4 8 —Shakerr —-— ——Stationery Fic. 19.—Metabolic changes produced by S. antibioticus in aerated and stationary cultures (from WoopruFF 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; @-alanine was deam- Chapter V =o Metabolism inated only about one-third as readily as d-alanine. Leucine, isoleucine, and certain other amino acids were not deaminated at all or only in mere traces. Utilization of complex organic compounds.—It 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 11. 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.—Many 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 (2NO.- + 4NH,. — 3N,. + 4H.O), 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 actinomycetes.—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 Tasre 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 destroyed at the higher temperatures, the resistance of the spores being only slightly greater than that of the mycelium. When a culture is kept for 10 minutes at 70°C., not only the mycelium but even the spores lose their viability. TaBe 15: Influence of reaction on the decomposition of a protein-rich material by actinomycetes (445) :— 100 gm soil + 1 gm dried blood NH;-N produced in 28 days pH of soir eo S. scabies S. viridochromogenus S. griseus mg mg mg 322 (0) 0 0) 3.6 0 Shall 1.0 4.0 DED 0.9 ilgil 5.0 28.9 9.4 i153) 5.8 68.3 47.5 40.9 6.4 64.3 65.2 60.0 2 66.9 63.3 62.0 WG 62.6 63.6 53.0 8.8 0.4 = 2.8 9.6 ) 0 0.4 Tasie 16: Rate of growth of S* griseus and streptcmycin producticn in shaken cultures (481):— Incubation Growth pH of filtrate Residual glucose Streptomycin days gm mg/ml pg/ml 0 6.8 10.2 0 1 0.048 6.9 S25) <5 2 0.237 8.5 7.6 5 3 0.394 8.6 5.6 63 5 0.370 8.4 0.5 84 7 0.248 8.7 0.5 62 10 0.140 8.9 0.5 51 Tasie 17: Metabolic changes produced by S. griseus with 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 mt =100 mt* 100 Mi puG /ML pH Sodium nitratef 5 208 580 - 21 WA 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 2: 60 8.7 * Control = 980 mg glucose per liter. T Mineral sources of nitrogen, 3 gm per liter; organic sources, 5 gm per liter, Chapter V — i Metabolism The optimum temperature for the production of streptothricin by S. lavendulae (452) lies between 20° and 28°C.; at 37°C. very little of the antibiotic is produced. The course of growth of an actinomyces, consumption of energy and metabolic changes, as influenced by different sources of nitrogen are brought out in Tasres 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 ere 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. bovis 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. bovis grows in the absence of a carbohydrate, it is greatly favored by the presence of glucose. It produces acid from carbohy- drates. A. bovis 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. LrEskE, 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. Rutimann (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- duce chalky white aerial mycelium with round spores are capable of forming this odor; the nonsporulating forms of the Nocardia type and Waksman = Actinomycetes those that produce cylindrical spores do not give rise to any odor. ‘The presence of carbohydrates in the medium favors odor production. ‘The thermophilic 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 actinomycetes. ‘The odoriferous substance is soluble in ether (373, 376). NES 9 Q Sy Milligrams COz Per 100 Grams § Density of Myceliurn, Per Cert- aw a es 10) oO 2 /0 IF 20 aa IO Incubatior , Days Density of Myceiium ——--CO;, Evolution Fic. 20.—Influence of temperature upon growth and carbon dioxide production by actinomycetes (from 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 ——95,— 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 60: t 8 15- Carbon Dioxide Evolved, Mil ligrarns 46 /8 28 ee Days Of Incuéation Fic. 21.—Decomposition of hemicelluloses by actinomycetes, as measured by CO: evolution (from WaxsMan and Dreum, 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 cacao beans (55), milk (120), and other foodstuffs, rendering them inferior in quality or totally unsuitable for human consumption. Waksman — 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 intensity 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. violaceus-ruber 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 Coun, in 1875, and later studied ex- tensively by BetyERINCK 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, mostly 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 Beryertnck (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 mee 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 Mriiarp and Burr (298) reported that nonchromogenic actinomycetes may also cause Chapter V 952 = Metabolism scab formation, AFANASIEV questioned these reports, since the cultures 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. M@ULier observed that this pigment was produced on synthetic media only with starch as a source of carbon. Waxsman, 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, Beryertnck (26) described, under the name A. cyaneus, 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 litmocidin and was found to possess antibiotic properties (133). LreskeE distinguished two groups of pigments produced by actino- mycetes: (a) the “chromophores or a which is not excreted from the mycelium into the medium, and (b) the chromopars or pigment which is readily excreted. ‘The first group comprises various pigments produced in the vegetative mycelium grown on synthetic media, namely, yellow, orange, red, blue, violet, brown, black, and green; the aerial mycelium 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 LirsxKe’s separation of actinomyces pigments into the above types or the classification of Ducné 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 pigmenis are capable of passing through the living cell plasma. ‘They have been subdivided into, (a) anthocyanins, soluble only in water, and (b) hydroactinochromes, soluble in water and in alcohol. By Lipoactinochromes, insoluble in water but soluble in alcohol and in other organic solvents. , C. Pigments insoluble in water and in organic solvents. D. A combination of water-soluble and water-insoluble pigments. Waksman — Actinomycetes Only a few of the pigments produced by actinomycetes have been studied from a chemical viewpoint. Krarnsky (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- lulosae. It became violet-red in an alkali solution and blue-green in concentrated H,SO,. ‘This, as well as the red pigment, was considered to be acarotin. ‘The green pigment of S. viridochromogenus was found to change to red on treatment with concentrated H2SO,. Waxsman demonstrated that the pigment produced by S. violaceus- ruber 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 Streptomyces, S. coelicolor and S. violaceus-ruber, 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 Oxrorp (330), since the pigment contained too little nitro- gen; neither could its phenazine (116) or anthocyanidin nature be ac- cepted. LirskE 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 H2SO, it is changed to a blue-green pigment. N. polychromogenes 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 H2SOx4. 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 H2SOu,, first to green, then to dark brown. According to LirsKe, 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 H.SO,. The yellow-red pigment of N. corrallina 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 Taste 11. 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. longisporus ruber. Although several pigments were thus recog- nized, they were apparently related. The blue pigments of S. coeli- Chapter V a 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, fhe pigment was believed to belong to the p mecy ans, a fact not confirmed (116) by further study. pene (234) confirmed the observations of Kriss, that an- thocyanins 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- chromogenus, and A. verne. ‘The pigment is also soluble in glycerol and in alkali solutions, but not in organic solyents. 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). Reaper (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. Scarpata (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 known since the work of Grogsic (138), in 1888. Tstxiinsky (429) was the first to establish, in 1899, that composts contain an abundance of actinomycetes. The normal temperature for their growth ranges from 50° to 70°G. Waksman oo Actinomycetes Thermophilic actinomycetes in culture can be isolated by one of several simple procedures. ‘’stkLinsky 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. ‘Iwo cultures of actinomycetes were thus obtained, one of which produced chains of spores and may, therefore, be dopuidered as a spe- cies of Streptomyces, 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 Temamed 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 Thermoactinomyces I, was less proteolytic, and the spores were ies resistant to heat. Gipert (135) isolated several thermophilic actinomycetes from various soils. He included them under one species as A. thermophi- 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. Mrene (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. thermophilus 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. Mreve reported, however, that some of the thermophilic actinomycetes produced spores in a manner similar to that described by Grrpert. Scuttrze (399) reported the presence in decomposing clover hay of representatives of two types of thermo- philic actinomycetes, one of which was designated as A. thermophilus Berestneff and the other as A. monosporus Lehmann and Schiitze. The latter may be definitely considered a member of the Micromono- spora group. Chapter V Metabolism In a more recent review of the literature (36) on the occurrence of thermophilic actinomycetes, some 20 species were listed, namely: ORGANISM: Thermophilic forms Cladothrix thermophile Thermomyces lanuginosus Thermoactinomyces vulgaris Streptomyces sp. Streptothrix thermophile, No. 12 Streptothrix thermophile, No. 20 Actinomyces sp. Actinomyces thermophilus Actinomyces thermophilus AUTHOR: GLoBic KeEpzror ‘TsIKLINSKY ‘TstKLINSKY ‘TsIKLINSKY ‘TstKLINSKY ‘TsrKLINSKY SAMES GILBERT Mirene ORGANISM: Thermomyces lanuginosus Actinomyces monosporus Streptothrix No. 8 Streptothrix No. 9 Streptothrix No. 12 Actinomyces spinosporus Actinomyces thermodiasta- ticus Actinomyces cus Streptothrix thermophilus Actinomyces thermophilus Actinomyces casei nondiastati- AUTHOR: Mrener ScHuTZzE Brunt Brunt Brunt VELICH BERGEY BERGEY Eckrorp KRoun BERNSTEIN and Morton Chapter VI PRODUCTION OF ENZYMES AND OF GROWTIL 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 variety of bacteriostatic and bactericidal substances, or antibiotics, which are discussed in Chapter VI. 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 pH 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 by the presence of glucose or other available carbohydrates. ‘The breakdown of the protein proceeds, through the amino acid stage, to ammonia. ‘This is brought out in Taste 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 Lreske, the resistance of the enzymes to the effect 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 ‘hee a long time at 40°-45° C., the reproductive capacity of the cells is destroyed, Bae 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. BrrjERINcK 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. Taste 18: Proteolytic activity of actinomycetes in 2 per cent gelatin solution (187) :— Results in milligrams of nitrogen per 25 ml of medium 10 days 30 days Liquefaction SPECIES Formol Formol of gelatin NHa-N titration NHa-N titration S. griseus 6.4 16.5 16.2 30.7 Very rapid S. griseoflavus Hell 17.1 22bs 36.6 Very rapid S. cellulosae 5.0 16.0 14.5 27.4 Rapid S. olivaceus Dad) 13.2 9.2 24.0 Rapid S. fulvissimus 255, 11.8 7.0 29.5 Fairly rapid S. violaceus-ruber 2.6 14.5 9.2 39.2 Rapid S. roseus Dep 11.4 10.2 20.2 Slow S. bobiliae DES, iia) 6.2 26.9 Slow S. viridochromogenes pe) [3a 10.6 19.8 Slow S. erythrochromogenes 3.5 13.0 Weil 20.5 Slow S. phaeochromogenus 2.0 9.3 4.4 DDE Slow S. diastatochromogenes Sl il Ia 21.5 Slow S. aureus Dell 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). Bacteriolysis may often parallel growth inhibition CTaBie 19). The ability of certain actinomycetes to cause the lysis of various bacteria is characteristic of certain species of Streptomyces. ‘This effect directed upon plant pathogenic bacteria may be of considerable eco- nomic importance. A culture designated as Streptomyces 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 lytic agent. Cultures of Streptomyces 105 Waksman I Actinomycetes added to soil infected Phytomonas tabaci 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. Taste 19: Distribution of bacteriolytic properties among actinomycetes (503) :— Total Activity against Growth-inhibition of PREPARATION strains S. aureus B. subtilis He Be 0 Number of strains 164 24 54 86 22 54 88 Per cent of strains 100 14.6 32.9 52.4 13.42 32.9 S%is7/ Number of filtrates 67 9 ila 47 5 4 58 Activity on heat-killed E. coli and S. aureus Number of filtrates 67 23 25 19 us a a 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. ruber, N. viridis, and others. Some of the yellow species CN. flava) are weak liquefiers. ‘The lemon-yellow forms (CN. citrea) and the white types CN. alba), 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 — 103 — 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 (377), and later confirmed and extended by Krarnsky, Waxsman, Lieske, and others. The ethod 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 IE 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 igeeane 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 heen pointed out by Krainsxy, Caminitr (61), and Waxsman. ‘The ability of some species to wiilize 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 Oa 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 lipolytic enzymes has been established (260). ‘The activity of these enzymes 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 autolytic 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 Lreske, 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. albus was designated by Wetscu (505) as “actinomycetin.” ‘This property is widely distributed among the actinomycetes, as shown in Tasie 19; as many as 50 per cent of all cultures have been found active against heat-killed cells of E. coli and against living S. aureus. . Boroputina (43) and Naxuimovskata (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 enzyme systems or with true antibiotics. _ These are actinomyces lysozyme 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 actinomycetes 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 synthesize 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 blakesleeanus. 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 By, has also been established Cp. 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 Streptomyces to transform aestradiol to oestrone (507 ). According to ‘Turrirr (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). ii eeOO PML iy ee "te ra hte, % sane eas ett, ee a a “8 4 Fic. 22.—The use of the agar cross streak method for testing the ability of actinomycetes to produce antibiotic substances.—Upper two plates, S. lavendulae 3516; middle two plates, S. lavendulae 3440; lower two plates, S. griseus 3496.—Test bacteria, from top to bottom: (a) right column, M. ranae, M. avium, M. tuberculosis 607, M. tuberculosis 607R; Cb) left column, B. mycoides, B. subtilis, E. coli W, E. coli R, S. aureus. (CR = forms resistant to streptomycin. ) (Original. ) Chapter VII 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 observed that some of the colonies of actino- mycetes on the plate are surrounded by clear zones free from the growth of bacteria and fungi (483). In 1917, Greic-Smitn (157) observed, for example, that when a soil is plated out on a nutritive agar the growth of certain spreading colonies of B. mycoides and B. vulgatus may be in- hibited by other colonies on the plate. These will ie 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 types 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 of soil with specific patho- genic bacteria, such as M. tuberculosis 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 =| (03) —— Actinomycetes parasite, as a result of the faculty that the actinomycetes possess of di- gesting the membrane of these lower fungi. Gretc-Smiru 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. aureus and that different actino- mycetes vary greatly in this respect. Lirsxke believed, however, that the antagonistic effect of actinomycetes may be due to a specific bacteriolytic enzyme, namely: “Ein bestimmtes bakterienlésendes 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. albus) at first considered to be of the nature of an endo- and exo-bacteriolysin (499). It was later designated by Wetscu 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 Naxurmovsxkara (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 (Taxsre 20). ‘These actinomycetes pos- sessed the property of inhibiting the growth of gram-positive bacteria Chapter VII = [Ke 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 light, 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 Boroputina (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 Tare 20: Occurrence of antagonistic actinomycetes in different soils (316):— Total number of Number of Strains producing NaTuRE OF SOIL strains tested antagonistic strains antibiotics Chernozem 24 10 9 Podzol 11 7 3 Solonets 4 4 4 High altitude soil 9 6 5 Sandy soil 6 5 1 Dry desert soil 5 4 1 River bank meadow 14 7 2 Cultivated soil 7 4 2 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. KrasstLnikov and Korentako (237) also reported that many species of actinomycetes, notably members of the genus Streptomyces, but not of Nocardia, produce a substance that is strongly bactericidal 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- Fic. 23 a.—The use of M. tuberculosis for testing production of anti- biotic substances by actinomycetes. Upper horizontal streak H37Rv strain; lower streak H37RvR Cesistant to > 1000 ug/ml streptomycin ): inhibition of streptomycin-sensitive but not of streptomycin-resistant strain (from WixusTon et al., 510). sistant to the effects of light, air, and high temperatures. ‘The optimum reaction for its production by Streptomyces violaceus 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 —1ll— Antagonistic Properties Waksman et 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- Fic. 23 b.—No inhibition of streptomycin-sensitive or cf streptomycin- resistant strains (from WI .istTon et al., 510). lection (503). ‘The antagonistic forms were most abundantly repre- sented by the genus Streptomyces CT Asie 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. aureus in agar streak plate tests, 261 inhibited E. coli, and 514 showed an antagonistic effect against Candida albicans. -, Various other surveys have been conducted dealing with the capacity of large numbers of actinomycetes to inhibit the growth of bacteria as Waksman ee 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 Garpner (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). Tasie 21: Distribution of antagonistic actinomycetes in nature (468) :— Group I* Group II Group II Group IV Number ——-2777]77—. + -- —- —— of No.of Per No.of Per No.of Per No.of Per SouRcE OF cultures cul- centof cul- centof cul- centof 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 13 48 64.9 Infertile, unma- nured, limed soil 75 11 14.7 8 10.7 Potted soil 13 1 Vea il Asif Potted soil, en- riched with E. coli 21 1 4.8 4 19.0 4 19.0 122 Si /e74 Potted soil, en- riched with mixtures of 5.2 Sy 69.3 11 84.6 ont [o) bacteria 15 12 80.0 2 13.3 (@) 0) 1 6.7 Lake mud 9 3 33.3 4 44.4 @) (@) 2 WD Stable-manure compost 37 1 7] 20 54.0 4 10.8 12 32.4 Total 244 49 20M! 44 18.0 13 3155) 138 56.6 *I—Most active antagonists; II and II—more limited antagonistic properties; [V—no antibacterial effects with methods used. ALExopouLos (8, 9) made a survey of the antagonistic properties of 80 cultures of actinomycetes, using Colletotrichum gloeosporioides as the test organism. ‘The following seaulls 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. MerepitH (291) surveyed the distribution of organisms antagonistic to Fusarium oxysporum cubense 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 —— Antagonistic Properties that were antagonistic to the Fusarium 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 Fusarium. When spores of both or- ganisms were mixed in an agar medium, the fungus at first developed Fic. 24.—Method of measuring antibacterial or antifungal potency of an antibiotic, by the agar streak method (from Rritty, Scuarz and Waxksman, 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 Fusarium was later designated as musarin. It was found to be an optically active acid, of the probable composition of CCasHeoO14Ne) 72, and had an activity of 1:80,000 to 1: 100,000 Gy» Waksman |) Actinomycetes Lepen and Kerrr (254) isolated a culture of Streptomyces 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 active substance extracted from the precipitate with ethanol. A preparation was obtained which completely inhibited Venturia inaequalis 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 antimycin. 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 feotareds from antimycin preparations and designated as A, B and C. The A was a nitrogenous phenol (Cys- Hyo9Q9N2). The substance inhibits the respiration of Saccharomyces cerevisiae, of cytochrome oxidase and succinic dehydrogenase. Actinomycetes also exert marked antagonistic effects against species of Pythium, as in the case of root rot of sugar cane. Of 3,788 cultures isolated from soil and tested against a parasitic strain of Pythium, 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 actinomycetes were found (51la) to be responsible for the destruction of the mycelium of Ophiobolus 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 Ophiobolus in natural soils. Actinomycetes possess antagonistic properties not only against bac- teria and fungi but also against other actinomycetes Cis: = The more aerobic species are antagonistic to the less aerobic types. Mr_iarp (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. scabies was reduced by the simultaneous inoculation of the soil with Streptomyces praecox, an obligate saprophyte (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 contol potato scab by in- oculation, with S. scabies and S. praecox, of either steam-sterilized or natural soil containing different amounts of green plant materials. Chapter VII aie 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. prae- 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 Behece Goss (146, 147) observed ee 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. Kiesztinc (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. Konisnr and Fuxucur (229) have shown that cer- tain actinomycetes are able to inhibit the growth, on the plate, of root- nodule bacteria; some of the or ganisms, like S. flavus, were particularly inhibiting. In association waite 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 heencd by Nrxorateva (323) in 1914. Nuickert 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 aritibiasis may be responsible for development of these organisms in the soil. die. antagonistic effects of actinomycetes upon plant pathogenic bacteria has alle been well established. Hrno (173) isolated several actinomycetes active against Ps. solanacearum. Corynebacterium. se- pedonicum, 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. mega- Waksman we ep Actinomycetes Tasre 22: Classification of antibiotics of actinomycetes:— A. Soluble in ether and in other organic solvents: I. Pigmented substances: il We Orange colored; somewhat soluble in neutral aqueous solution; nitrogen- bearing ring compound, highly toxic, Cs,HssOuNs; largely active against gtafi-positive bactehia.« 2 At tes eo eee en ee eects eae Actinomycin . Yellow pigment, active against both gram-positive and gram-negative bac- qarila lavtedmMbycorole wo) choilallS: 45 Goaacaudesancocpodae Xanthomycins A and B =. Compounds related to actinomycin sos .o tes oe aia oe en Actinoflavin Red-blue pigment; soluble in aqueous alkaline solution; active against gram- POSHEVEDactetla senha vegy roe ae eae eey Ie eee wie eae Litmocidin Compounds related to litmocidin...... Me or no are Actinorhodin Orange colored; extracted from charcoal nets and from mycelium by ether-alcohol mixture; active largely against M. tuberculosis......... Nocardin Green pigment, active against gram-positive bacteria........ .Actinomycelin II. Non-pigmented substances: 1. Organic base; soluble in acidified aqueous solution, inhibits mostly gram- POSIELVEDACCH AM iojat as i ine eee ita Raa teh eee Eee Proactinomycin 2. Largely fungistatic, not bacteriostatic, Co7H4,.N2O7...............-. Actidione 3. Soluble in water; active against various bacteria and fungi...... Mycomycin 4. Insoluble in water, present in mycelium; active largely against gram-positive ACTER Aes, 52, tacrten cite cal age MACS cae RI rain EAE oe Sean ere Streptocin 5. Neutral compound; slightly soluble in water, readily soluble in organic sol- vents, contains nitrogen (8.6%) and non-ionic chlorine (21.7%); active against Vatioussbacteriakandanickettsiaenn. si enen hae nie eerie Chloromycetin 6. Amphoteric; active against bacteria, rickettsiae and certain viruses Terramycin Fea bleatelabilestlarcelysactivelacainse unciee se eee ere aee Antimycin Si iHeat stable; active against fungi... nese: ae i ee ere Fradicin g. An acid; active i2 viva 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: Tx Violet-bluespiemented substances. 2 hoa joe ine seremtetseieiemiciest- eine Mycetin I Golorlessy'sulphur-contaimine;substancer en secre iy tise Sulphactin III. Colorless, nitrogenous body, active against parasitic fungi............ Muasarin 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. dk. Ds Little activity against Bacéllus mycotdes, Serratia marcescens; active against Bodenheimer oreanismiana ite. eee en ee iee ea eee 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 Cd) Actinorubin (e) Antibiotic 136 C£) Streptothricin VI and VII Active against B. mycoides and S. marcescens, little activity against fungi, no activity against Bodenheimer organism; glycoside (streptidine-streptobiosa- 120k) (SER tend Aa Ar eee is ab aria, schanae Gale Streptomycin complex Chapter VII ne Antagonistic Properties Tase 22 (Cont.) a. Constituents of streptomycin complex: (a) Streptidine-streptobiosamine, Cx)H3gN7Ow................ Streptomycin (b) Mannose derivative of streptomycin. ........... Mannosidostreptomycin CopReducedistreptomycia... 7.0 20. cndcc seek oe one Dihydrostreptomycin b. Streptomycin-like materials: (a) Antibiotic F, (b) Streptomycin 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; Marrow antibiotic spectrum against certain gram-positive and gram-negative DAGte rT are ertnre: Vetch hn einer PRR OS Nee ae cea Orin Gh gets pone Dern de Grisein 1. Grisein-like material, still narrower spectrum than grisein, mostly enteric Pidetetaremin eet. seer le ie antec ae huwn ae arcane a tra Antibiotic 5310 D. Proteins and polypeptides: I. Colourless preparation, possessing lytic properties against living gram-positive bacteria and dead gram-negative bacteria................000.00005 Actinomycetin leeAccive traction Ob actinomycetini. soe oF) fakin owiw. coat ase eh Actinoz yme II. Active largely against micrococci; lyses cell membrane. ..... Actinomyces lysozyme III. Combined with orange pigment; largely bacteriostatic against gram-positive BAGte ria ct tte rtek ce mts coc Rt eee kh en ines aS lL Micromonosporin E. Incompletely described agents: IPACtivedcainststhesmermabacillussarcre. dee seem secre ee Smegmatis factor Le Active against pactertophages.. nies acne oes a Re sees Antiphage factors MPR NERV. Cea AINISELVALUSES 5c, Wes ahs and ca te cals acta aMsyek HEY Antivirotics F. Agents not produced readily in liquid media; activity obtained only on agar streak. Peletetlegkn ov ySUPStanGest eo qi a ahsn Oke ooteeh Asta ani aera Insoluble factors therium, may be antagonistic to certain species of actinomycetes but can be antagonized by others. Certain bacteria, like Ps. 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 lysozyme—were recognized. Both had been isolated only in a crude state. WetscH 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. albus. A unit was expressed in terms of lysis of a known suspension of heat-killed cells of E. coli. 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 So Actinomycetes 9.0; it is thermolabile and is destroyed by ultraviolet radiations. Ac- tinomycetin, as well as the living S. albus, hhas a lytic action upon living gram-positive and upon dead gram-negative bacteria. The bacteriolytic properties of the living S. Fildine and “oh the actinomycetin preparation upon dead or living celle of bacteria was said to be due to a lytic prin- ciple, designated as actinozyme. lsallaeee of antibiotics.—The first true antibiotic of actinomycetes was isolated in 1940 from a culture of Actinomyces (Streptomyces ) antibioticus. ‘This substance was designated as actinomycin (490). It proved to be highly interesting from a nehertical and biological point of view and it affected a large spanan be of bacteria, mostly phew gram-positive types. Unfortunately, actinomycin proved to Ae extremely toxic to the ial 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. [hese substances varied greatly in their antibiotic spectra, in their chemical properties, and in their in vivo activities. A number of antibiotics are now known to be produced by actino- mycetes, as shown in ‘Taste 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, which is formed by a great variety of Gultaes (465, 505). Some organisms, on the other hand, produce more than one substance; S. griseus, 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 greatly 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. tuberculosis hominis. Some ae the antimicrobial spectra are very narrow, as shown by the so-called antismegmatis factor, which is active only against M. smeg- matis and certain Worter mycobacteria (215). On the other hand, strep- tomycin itself is produced, not only by S. griseus, but also by S. bikini- Taste 23: Inhibition of different actinomycetes by their respective antibiotics (475) :— Dilution units per mg, expressed as Organism Activity of activity against producing preparation ANTIBIOTIC it perl gm S. anttbioticus S. lavendulae S. griseus Actinomycin /S. antibioticus 100,000* 100 5,000 100 Streptothricin S. lavendulae 100F 1,000 0.4 10 Streptomycin S. griseus 125T 1,000 100 i172 * §. lutea units; crystalline material. T E. coli units; crude preparations. ensis (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 CTas.e 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 (Fic. 24), using a series of test bacteria. ‘Those that are found to possess the highest or more Slestaitlic 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 ie 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 Tasie 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 particularly in the search for organisms active against the tuberculosis organism. Wiriiston, Zia-WarratH 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 Tasre 24: Distribution of antagonistic properties among actinomycetes (194):— Cross-streak method. Numbers reported in per cent of total cultures. Zone of inhibition ACTIVITY mm B. subtilis E. coli M. avium M. phlez Nutrient agar Strong 20-35 21 6 6 23 Medium 10-19 46 3 35 35 Weak 1-9 3) 13 29 16 None 0 30 78 30 26 Glucose asparagine agar Strong 20-35 15 0 0 6 Medium 10-19 28 6 35 70 Weak 1-9 35 14 8 10 None 0) 22 80 >y/ 14 the virulent H37Rv do not inhibit, under the same conditions, strain 607 (Fic. 23a and Fic. 23b). The nature of the medium is also of great importance, as shown in ‘TABLE 25. Isolation of streptothricin 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 (Streptomyces) lavendulae that had been isolated in the same labora- tory, from the soil, in 1916 (443,460). The name was derived from Sireptothrix, as the actinomycetes were designated by Ferprinanp Conn in 1875. Other strains of the S. lavendulae : 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 — 12] — 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 body. After an extensive ex- Tasie 25: Inhibition of growth of virulent human tubercle bacilli by different actinomycetes (510):— Inhibition, in millimeters, by agar streak method Medium 1 Medium 2 Medium 5 Medium 6 ACTINOMYCES CULTURE H37Rv* H37Rv H37RvRt H37Rv H37Rv 1 37 Ds 25 13 32 . 17 15 18 iT 21 3) 20 0 0 22 17 4 Dif 9 0 11 27 5 14 lil 6 7 15 6 20 16 18 10 17/ 7 13 3 4 2 14 8 0 0 0 23 (0) 9 17 20 (0) 5 15 10 16 18 15 3 18 ili 0 10) 0) 0 0 12 20 3 4 8 12 13 15 15 20 18 14 S. griseus 20 12 0 12 20 * Streptomycin-sensitive strain of M. tuberculosis. fT 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 (Streptomyces) 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 Streptomyces (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 eee Actinomycetes stituents of the medium, and production of streptomycin are illustrated in TasBies 26 and 27. A brief antibiotic spectrum of streptomycin as compared to that of another antibiotic produced by another strain of S. griseus, namely grisein, is given in Taste 28. ‘The isolation, purification, and practical utilization of streptomycin in clinical medicine have had a most inter- esting history (390). Of particular interest was the discovery that the Fic. 25.—Streptomycin-producing strain of S. griseus, showing vegetative and aerial mycelium (from Waxsman and Scnatz, 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 ne Antagonistic Properties in many forms of this disease affecting the human body. The cul- minating point of these studies was Paced 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). Streptomycin-producing strains of S. griseus.—An organism under Taste 26: Growth and chemical changes produced by S. griseus under submerged conditions (101 ):— Calculated as milligrams in 100 ml culture INCUBATION, DAYS 0 1 2 3 5 8 pH 7.4 753) 7.6 US: 8.3 8.9 Mycelium = 40 510 580 480 380 Streptomycin = 0) Sp7/ 19.4 23}. 26.7 Glucose 900 880 800 240 60 = Soluble carbon 1,020 860 700 510 440 460 Lactic acid 29°2. 32.8 11.4 113} 1.6 1.5 Soluble nitrogen 148 130 110 76 7S 114 Inorganic phosphorus 11.8 10.8 3.4 0.1 0.2 3.4 Ammonia-nitrogen 6.6 7.0 US 6.3 TUES 26.5 the name Actinomyces griseus was first described by Krarnsxy in Russia in 1914. In studies of the soil actinomycetes carried out by Waxsman and Curtis in 1915, an organism was isolated from a Cali- fornia soil. ‘This organism appeared to be similar to A. griseus Krain- sky, so far as could be determined by comparison with ake 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) Tasre 27: Nitrogen distribution in cultures of S. griseus (481):— Per 250-ml portions of broth* Nitrogen NH3-N NH2-N Total N LycuBATION in mycelium in broth in broth accounted fort days per cent me 28, mg mg 0 10.0 35 4 35 4 10.0 35 23 57 TTS 5 9.7 40 38 70 148 7 10.0 56 56 73 185 10 8.9 62 63 67 193 15 9.6 35) 93 79 227 21 UP 38 95 71 204 )* Broth contained per liter 5 gm peptone, 5 gm meat extract, 5 gm 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 CNo. 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- Tasie 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 10 to 30 Bacillus megatherium 100 10 to 20 Bacillus mycoides 20