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" 7 the As : Leary) F : J 7 Wy - Cis ve asi a ¥ a To An " na 7 7 1 | . 7 7 t ia _ CS I 4 Y i r , 1 7 one - + 7 : an i 6 a | wh: ' ut i i - j oe r : me ‘ y 7 - po ai, g € oe bday ’ : 7 : " ee J 7 a yj nA) ru hee iis een ai n P%eteoO THED 0 WNIT MY 1OHM/18W ef an ¢ nL y a | =e Lie a ; 7 . . @ ee a a av un A : ; : 7 : _ ; . 5 ? > - 7 s P 1 / ‘ ’ 7 =. { s ee , " - 7) io 1 if 1 ; ji we fe = » -~ @ i} ‘1a a \ fl i : 7 ] i oan ‘ ' ! } al - i ae 7 a : ss) oa 7 : ri / ‘ 7 é 7 : i, - © _ ; : > a. & es 7 - of it i i 7 >» 7 D e i { 7 . i a = >. a “yy 7 te ae a 5 , i a i = “1 |° i > i y : i 7 1 ~ ‘i ‘ : : iG 7 : 7) a i mu : _ a 5 i i 7 7 , ' io _ » d at ; - =) 7 we 7 ») a THE ACTINOMYCETES VOLUME TI Ferdinand Cohn (1828-1898), who was the first to observe and describe an actino- mycete (1875), under the name Streptothriz Foerstert. THE ACTINOMYCETES Volt CUA ssh CATLON 2 DENY EEDCA TON AN D DESGHRIPTIONS OF GCENERA AND SPECIES by Selman A. Waksman BALTIMORE iE Web ieh FAV Sivek WEE KILNS GCOMPAN Y 1961 THE ACTINOMYCETES Vou. II: CLASSIFICATION, IDENTIFICATION AND DESCRIPTIONS OF GENERA AND SPECIES Copyright ©, 1961 The Williams & Wilkins Company Made in the United States of America Library of Congress Catalog Card Number 59-9962 Composed and printed at the WAVERLY PRESS, INC. Baltimore 2, Md., U.S.A. PREFACE In 1922, Professor D. H. Bergey of the University of Pennsylvania wrote to me that he and the Committee on Characterization and Classification of the Society of American Bacteriologists were in the process of preparing a ‘‘Manual of Determinative Bacteriology’’; he asked whether I would be willing to under- take the preparation for that volume of a section dealing with the actinomycetes. This group of organisms had occupied my attention for the previous several years, and alone (1919) and with Roland Curtis (1916), I had deseribed a num- ber of new species; yet I hesitated to accept this assignment. There were several important reasons for this hesitation: (a) I was not at all sure that the descrip- tions of actinomycetes so far published provided sufficient information for the accurate identification of most of the species recorded in the literature; (b) only four years previously, | had been warned by the dean of American cryptogamic botanists, Roland Thaxter, not to make further descriptions of new species based solely or largely upon cultural and biochemical properties; and finally (¢) I was not even certain at that time whether the actinomycetes should be included with the bacteria. I told Professor Bergey all this and suggested that it would be better to wait a few years until more detailed information was obtained concerning this group of microorganisms, especially with regard to their morphological and biochemical properties, before an attempt was made to codify them. I received a curt and somewhat sarcastic reply that if I would not, for one reason or another, under- take this task, he would have to do it himself. My immediate answer was, ‘‘I will do it.”? The best that I could accomplish at that time was to use cultural and biochemical characteristics as a major basis for the classification of the actinomy- cetes and for the characterization of the known species. Since then, or for more than a third of a century and for seven consecutive edi- tions of ““Bergey’s Manual,” I have been largely responsible for the preparation of the descriptions of the actinomycetes. I have not, however, always had the final word in organization of the material for all the various editions. Alone (1940), and together with Professor A. T. Henrici of the University of Minnesota (1943), I proposed two systems for classification of the actinomycetes, the second of which consisted of a thorough revision of the group and its separation into four genera. The most significant change in this revision was the proposal, in 1943, of the new generic name, Streptomyces. This second system has been the basis for the organization of the material in the last two editions of Bergey’s Manual. In presenting this volume, I am now certain of one thing, namely, that the place of the actinomycetes is definitely among the bacteria and not among the fungi. Ample evidence of this belief has been presented in Volume I of this trea- tise. Unfortunately, the first reason for my hesitancy in 1922, I believe, remains valid; the accuracy of the information available for species identification is still Ve vl THE ACTINOMYCETES, Vol. II open to question. The chief reason for this uncertainty is that although much knowledge has since accumulated, especially during the last 20 years when many Streptomyces species became known as antibiotic-producing organisms, taxonomic work was largely neglected except by a few dedicated investigators. Recently, however, several important contributions (Hesseltine et al., 1954; Flaig and Kutzner, 1954; Kutzner, 1956; Waksman, 1957; Ettlinger et al., 1958; Pridham, 1959) to this subject have appeared. A survey of the recent literature shows that morphological characters are tending to replace physiological and cultural proper- ties as the leading criteria in species characterization. It may be said that we are now in a transitional stage in which our ideas are changing, not only concerning the usefulness of criteria for species differentiation, but also with regard to the species concept. Since a classification of a group of living organisms is always only ‘preliminary,’ based upon the current knowledge of these organisms, I believe that, in summarizing the subject at present, and in trying to combine the older and newer ideas, I have presented useful criteria for species differentiation and an outline of species concept for the genera Actinomyces, Nocardia, Streptomyces, Micromonospora, and certain others. The rapidly accumulating information about the separation of some of the genera into distinct groups or sections, the recent introduction of several new genera, and the description of numerous new species, all necessitated a complete recasting of the material presented in the last edition of Bergey’s Manual and in other treatises. This volume is largely the result. An attempt has been made to bring together in this volume all the information required for the identification of newly isolated cultures of actinomycetes. All descriptions and names for which insufficient data have been provided, especially when no reproducible media have been employed, have been placed in a separate chapter as ‘‘incompletely de- scribed.”? Descriptions in which excessive and often confusing information has been presented, have been abbreviated to fit a certain ‘‘standard.”’ Often, this standard has turned out to be a Procrustean bed. I beg forgiveness, both from the ‘‘reader” and from the preservers of the Code (International Code of Nomen- clature of Bacteria and Viruses). My sole apology is that it is my sincere hope that it would serve the purpose. The author wishes to acknowledge his sincere indebtedness to Dr. Norvel M. McClung of the University of Georgia, to Dr. R. E. Buchanan of Iowa State University, and to Dr. Ruth E. Gordon and Dr. Hubert A. Lechevalher of this Institute, for reading individual chapters and for making valuable suggestions; to Dr. Hans J. Kutzner of this Institute and Dr. Thomas G. Pridham of the Northern Regional Research Laboratory, for reading the major portions of this volume and for suggesting numerous corrections and modifications; to Miss Alma Dietz of the Upjohn Company, Dr. Edward J. Backus of the Lederle Labora- tories, and all others who kindly supplied photographs; to Mrs. Herminie B. Kitchen for editorial work, and to Mr. Robert A. Day for assistance in the prep- aration of the various illustrations and for reading the entire manuscript, Selman A. Waksman INTRODUCTORY This volume deals exclusively with the well recognized genera of the actino- mycetes. No consideration is given here to the various closely related genera that are often included in the order Actinomycetales, notably the genus Mycobacterium Lehmann and Neumann, 1896. The actinomycetes comprise three families, which are further subdivided into LO genera. A. Spores formed, but not in sporangia. I. Vegetative mycelium fragmenting into bacillary or coccoid elements. Family I. Actinomycetaceae Buchanan. 1. Anaerobic or microaerophilic, nonacid-fast. Ll. Actinomyces Harz 2. Aerobic, partially acid-fast or nonacid-fast. 2. Nocardia Trevisan II. Vegetative mycelium nonseptate, not fragmenting into bacillary or coc- coid elements. Family II. Streptomycetaceae Waksman and Henrici. 1. Aerial mycelium produced. a. Spores formed in chains. 3. Streptomyces Waksman and Henrici b. Spores formed singly. 4. Thermoactinomyces Tsiklinsky c. Spores occurring in pairs or in chains. a'. Mesophilic forms, in pairs. 5. Waksmania Lechevalier and Lechevalier b!. Thermophilic forms, in pairs or in chains. 6. Thermopolyspora Henssen 2. Aerial mycelium not produced. a. Spores occurring singly on short sporophores. al. Mesophilic forms. 7. Micromonospora Orskov b!. Thermophilic forms. 8. Thermomonospora Henssen B. Spores occurring in sporangia. Family III. Actinoplanaceae Couch I. Aerial mycelium usually not formed, coiled conidial chains lacking, spor- anglospores motile. 9. Actinoplanes Couch II. Aerial mycelium abundant, coiled conidial chains as well as sporangia formed in some species, sporangiospores nonmotile. 10. Streptosporangium Couch vil vill THE ACTINOMYCETES, Vol. IT Certain other genera, recently suggested, have been given tentative considera- tion. These genera comprise about 350 species. In addition to these, a large number of other species are listed as ‘incompletely described.” TABLE OF CONTENTS The Actinomycetes VoLuME II CLASSIFICATION, IDENTIFICATION, AND DESCRIPTION OF GENERA AND SPECIES PTE RC Cah Oreo Se Ba wig es. RPSL Aci: Ste Pant ess A Bn sk a a I Ae tne V MNT OCCU OTS chance co o.8 Soi a Pe te, a wna Neer Gik-oth a MeL eee vil 1. The Species Concept in Relation to the Actinomycetes.............. | Deal CUE E MUSA CLEMOMUCES 2:0 See or Pale a heaas ves seas Foe oes eee tad ee exe 12 “ah IU OEM (ey NILOTSENA'A(O170 1410 110 NEA ee Pe oe Be a oes ot mE cm ne 21 4. Characterization of Streptomyces Species.............. 02.0000 eee 61 5. Systems of Classification and Identification of Groups and Species of the GSMS HSU DLONUNCCG tice ole gts oie lois ijt Fe Boas e SeOPO ta Ae ne oe oR 82 6. Series and Species of the Genus Streptomyces... ........0.0...0.000-. LS fe Classiication ob wieplomiyces SPeCles.. - 2.6 ce. bebe ecw ove see nies 152 S. Deseriptionror Species of Streptomyces... o.oo wee ne 165 Omiihe: Genus Mecromonospordy, les ele. Slee els ca da See lasagne saan 293 LOMeithesGenusaw aksmanica (Microbtspora)): co ..08 2 fee le hs oso ed Ss 298 ea hiermopnilicy Actinomycetes. ..tsS sob. ae Oe bs bo awlegan eee ba bs 300 WAAC HINO DIAM ACCACUe +. oes teas es es Be aoe 2 eee 310 13. Incompletely Described Species of Actinomycetes................... 315 Appendix I. Color Designations for Describing Actinomycetes (Lin- GLEMDEII) Eee Ee UPA AI Mente sae. Slee CEs EO OS oat te eee 327 Appendix II. Certain Important Media for the Study of Actinomycetes 328 | 572) (EN 2) CET ARN Oa mane ae Maen Se rd OS eee ae ee) AE 335 InICexXAG MOTE AIMISMIS SSeS eo ENA Ee cl Meee 347 Crenerall Sinden... Mle tice, at teas: syns Neen e cae Uy cake mute clog CR Lt een eee 360 80982 - ¢ ‘ AN «SN we 12) NULL 5 Ji, Y), ie rrind of Sa & air s ree ag =F noes = ak ' hOv eee JO = 5 a 4 Pan al { ' - “aM — NN 1 0} Pay be “y, Ci25 ; i 41) 24 ; ATTY ies Chapter |! The Species Concept in Relation to the Actinomycetes Systematic Position of the Actinomy- cetes In the preface to this volume, the state- ment was made that ‘I am now certain of one thing, namely, that the place of the actinomycetes is definitely among the bac- teria and not among the fungi. Ample evi- dence of this belief has been presented in Volume I of this Nevertheless, some reiteration is warranted at this point. The taxonomic position of the mycetes, notably their relationship to the bacteria, on the one hand, and to the fungi, on the other, has been one of the most de- batable questions in microbiology. The size (width of thallus) and staining properties of the actinomycetes have usually placed them with the bacteria. Their branching and man- ner of sporulation have suggested their rela- tionship to the fungi. Still other properties of treatise.”’ actino- actinomycetes seemed to warrant their con- sideration as a transition group between the bacteria and the fungi. tecent evidence seems to point definitely to the fact that the actinomycetes are more closely related to the bacteria: 1. Some of the actinomycetes, such as species of Actinomyces and Nocardia, are closely related to true bacteria, notably spe- cies of Lactobacillus and Corynebacterium. 2. Neither have been shown to contain true nuclei; they both contain only chromatin granules dis- tributed through the hyphae or the cells. actinomycetes nor bacteria 3. The diameter of actinomycete my- celium and spores is similar to that of bac- teria. Actinomycetes also, as a rule, lack septa. 4. Actinomycetes are subject to attack by phages just as bacteria are; filamentous fungi are not. 5. Actinomycetes are usually sensitive (allowing for strain variability) to antibiotics that are active upon bacteria; they are usu- ally resistant to those antibiotics, like the polyenes, that are active upon fungi but not upon bacteria. 6. Chitin is absent from the cell substance of actinomycetes as well as from bacterial cells, but is present in fungus mycelium and In their lack of cellulose, actino- mycetes are also similar to most bacteria and unlike fungi. Avery and Blank (1954) con- cluded that “from the chemical point of view Actinomycetales have nothing in common with the true fungi, but rather with the bac- Cummins and Harris (1958) even further by suggesting that the order spores. teria.”’ went Actinomycetales be abolished altogether and that the families of the actinomycetes be included in the Hubacteriales. 7. Like bacteria, but unlike most fungi, actinomycetes as a rule are sensitive to an acid reaction of the medium. 8. The close relationship of the actino- mycetes to the bacteria is also evident from the work of Couch (1954), who found that certain J/icromonospora-like forms resemble 2 THE ACTINOMYCETES, Vol. II those of bacteria. Couch emphasized the re- semblance of the mycelium and sporangia of Actinoplanes to those of the chytrids; he con- cluded that this genus may represent a con- necting link between the bacteria and the lower fungi. The Generic Problem with Actinomy- cetes Prior to 1943, several systems of classifi- cation of actinomycetes had been proposed. In most instances, all the species were in- cluded in a single genus, which was fre- quently designated by different names. The most common of these names were the two oldest, Streptothrix and Actinomyces. / 7 Reena ay ‘ SF 2 a7 { ~ N ee Ne aN : oe ged — ey “NN te Pad ¥ é * * i 4 g. be + % 4 j ? én ee e * — . * ia & Cc * 4 : ? # Ficure 7. N. NOCARDIA 23 opaca: (a) grown for 4 days on n-dodecane and mineral salts; gram stain, X 960; (b) same grown 3 days; X 3700; (c) same as (a) but using fat stain, X 1920; (d) two-day growth, X 12,500 (Reproduced from: Webley, D. M. J. Gen. Microbiol. 11: 425, 1954). smooth, folded or wrinkled. Typical nocar- dias 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 oidiospores. * Many species of nocardias form pigments; their colonies are of a blue, violet, red, yellow or green color; more often the cultures are colorless. The color of the culture serves as a stable character. The type species is Nocardia farcinica Trevisan.” Classification of Nocardia Species De Toni and Trevisan (1889) described five species of Nocardia: N. farcinica, N. actinomyces, N. foerstert, N. arborescens, and N. ferruginea. * See the work of Gordon and Mihm (1958). Jensen (1932a) found that a number of organisms previously described as species of Mycobacterium actually belong, on account of definite mycelial growth in the initial stages of their life cycles, to the genus Nocar- dia.* Mycobacterium agreste Gray and Thorn- ton and B. mycoides corallinus Hefferan were found to be similar to one another and were The same was true of JJ. salmonicolor den Dooren regarded as one species, N. corallina. de Jong, which was designated as N.. sal- monicolor. Mycobacterium opacum den Dooren de Jong and JM. crystallophagum Gray and Thornton proved to be identical and were named N. opaca. Mycobacterium erythropolis, a closely related form, was des- * Generic name Proactinomyces used. Ficure 8. N. paraffinae, showing a section of a colony covered with mature aerial mycelium (Re- produced from: Hirsch, P. and Engel, H. Ber. Deut. Botan. Ges. 69: 454, 1956). Fiacure 9. Nocardia, strain 70, showing the de- velopment of the aerial mycelium on mineral agar (Reproduced from: Hirsch, P. and Engel, H. Ber. Deut. Botan. Ges. 69: 454, 1956). LI; THE ACTINOMYCETES, Vol. II A. Unstable mycelium (a-form), with short mycelium (if formed at all), bacterial (diffuse) growth in liquid media, bacteria-like colony. Stable mycelium (8-form), producing long hyphae, colony growth in liquid media, and Streptomyces-like type of colony. Umbreit (1939) modified the system of B. Jensen as follows: I. Partially acid-fast, nonproteolytic, non- diastatic; constantly utilize paraffin. 1. a-Mycelium type: N. opaca,* N. eryth- ropolts. . B-Mycelium type: a. Red-colored: N. polychromogena N. asteroides. b. Yellow-colored: NV. paraffinae. Nonacid-fast forms, constantly diastatic 1. a-Mycelium type: a. Nonproteolytic: N. mesenterica. b. Proteolytic: N. actinomorpha. 2. B-Mycelium type: a. Yellow-colored: N. flavescens. b. Red to orange: N. maculata. Krassiinikov (1938) divided the genus Nocardia* into two groups: re) I. Well developed aerial mycelium, with ignated as N. erythropolis. Microbacterium mesentericum Orla-Jensen was renamed N. mesentericus. Jensen divided the genus Nocardia into two distinct groups: I. Nonproteolytic organisms with strongly refractive cells showing a partial acid- fastness in milk and sometimes in other media; capable of decomposing paraffin. Some species of this group form a transi- tion to the genus A/ycobacterium. II. Mostly proteolytic forms with weakly refractive, nonacid-fast cells. This group forms a close transition to the forms now included in the genus Streptomyces. A further separation of the genus was based upon the structure of the aerial myce- lium. Ale substrate mycelium seldom producing cross walls. The hyphae break up into long, thread-like rods. Branches of the aerial mycelium produce segmentation spores and oidiospores; the latter are eyl- indrical with sharp ends. No spirals or fruiting branches. This is the same as group 8 of Jensen. Typical nocardial forms. Mycelium de- velops only at early stages of growth, then breaks up into rod-shaped and coccoid bodies. Smooth and rough col- onies, dough-like consistency, similar to bacterial colonies. Aerial mycelium not formed or only around colonies. * Generic name Proactinomyces used. THE GENUS NOCARDIA 25 TABLE | Summary of growth characteristics of 18 strains of Nocardia (McClung, 1949) Fragmentation Organism Scion |branchiae | branching Age Type 1 Type 2 Type 3 hr hy hr hr q % % Group I WieBie ae SRL We le Sach toh 2 8 16 0 13 70 30 0 INESOORLOSUUS Hera ee Stef 6 12 0 12 60 30 10 SU bo ial ene ea 9 15 0 13 50 50 0 BB eeeaer ete tees er 10 36 0) 14 50 50 0 N. erythropolis...... ; 7 1] 14 0 14 69 40) 0 N. polychromogenes... ‘rE 1] 13 0 14 40 30 30 Group II NSO Ree eae ec tes Sate 1] 12 5 19 6 28 66 ‘oleae SA ae meee 10 12 7 1S 5 40 50 INGETALD Chetan esc 10 14 1 20 5) 30 65 N. polychromogenes............ 14 30 20 120 Unknown IN@rastenovdes:s.<..)... 10 28 96 96 Unknown Group III ees Met ewer Paes ee 529s ah Sache ee oe: 10 16 4 0 0 0 Pedal errant erty t ora cat's loc). -yerantontrs 9 1] 6 0 0 0 tse O08 Sie) fue ae eae a 10 12 S 0) 0 0 NOE Zeta eaten! Staves ne Seetehehs aust 11 15 6 0 0 0 WINS) Shh alk o Aleantsi eo eeee ecere ee 10 15 5) 0 0 0 Hato goa ra Riek Oke i eres ee pate DR a eee 10 13 9 8) 0) 0 2) = OST O IE T TN Eco cous ys chen epes sis anes Bes Ss 13 7 0 0) 0 MeClung (1949) divided the genus Nocar- dia into three groups: I. Scant mycelial development, sparse branching. Colonial texture soft, pasty, and sometimes mucoid; pigment intra- cellular and insoluble. Extensive mycelial development, straight branches which do not over- lap. Colonial texture soft and pasty; pigment intracellular and insoluble. oxtensive mycelial development, fragmentation of hyphae, contorted and profusely produced branches which overlap. Colonial texture ‘artilaginous; generally both intracel- lular and soluble pigments are pro- duced (Figs. 8, 9). The pattern in Nocardia fragmentation can be separated into three types. In Type 1, an acute angle is formed in a hypha pre- ceding division, which occurs at the apex of II. III. no Waxy or the bend; following division the new hyphal tips grow out parallel to each other. In Type 2, division occurs in a straight or slightly curved portion of a hypha; following di- vision, the newly formed ends bend slightly and grow past each other. In Type 3, di- vision occurs in the parent hypha close to or at the juncture of a branch; a new hypha may grow from the place of division at the base of the branch; the newly formed hyphal tip bends and continues to grow. Type 1 fragmentation is characteristic of Group I, and Type 3 of Group II. Type 2 occurs in both groups, and Group III lacks fragmentation (Fig. 10). A summary of the growth characteristics of various strains of Nocardia belonging to these three groups is presented in Table 1. In an attempt to find a group of depend- able properties for the separation of the genera Nocardia, Streptomyces, and Mvyco- 26 THE ACTINOMYCETES, Vol. II Fieure 10. N. rubra: (above) 24 hr, glycerol nutrient agar, methylene blue; (below) same, stained with Sudan black B, X 1600 (Reproduced from: McClung, N. M. Lloydia 12: 165, 1949). bacterium, Gordon and Smith (1955) exam- ined 152 cultures labelled Streptomyces and 99 cultures labelled Nocardia; those strains of the latter that formed soft, fragmenting, vegetative mycelium were excluded. Of the cultures designated as Streptomyces, 83 per cent produced an aerial mycelium typical of this genus; 13 per cent failed to produce aerial mycelium, although a few formed rudi- mentary aerial hyphae; inability to form spores was considered as a lost property, the physiological reactions of the strains were the same as in the previous since group of sporulating cultures; only five strains, or + per cent of the cultures possessed nocardial properties. Of the cultures desig- nated as Nocardia, 68 produced aerial hy- phae, varying from rudimentary to luxuri- ant, some even forming chains of spores. According to their physiological properties, 24 of these cultures should have been desig- nated as Streptomyces. A few of the strains could be considered as intermediate between the two genera. Gordon and Mihm (1957) further reported the results of an examination of 219 cultures labelled Streptomyces, 214 Nocardia, and 243 TABLE 2 Certain physiological and biochemical characteris- tics of various strains of two species of Nocardia (Gordon and Mihm, 1959) N. asteroides N. brasiliensis (98 strains), (50 strains), Property positive positive strains strains Decomposition of : Casein 0 98 Gelatin 36 100 Tyrosine ) 100 Xanthine 0 0) Hydrolysis of starch 58 56 Acid from: Adonitol 0) 0 Arabinose 0 0 Erythritol 6 0 Galactose 24 92 Glucose 100 96 Glycerol 9S 98 Inositol 2 100 Lactose 0 0) Maltose 6 ) Mannitol ) 94 Mannose 19 68 a-Methyl-p-glucoside ) 0 Raffinose 0) 0 Rhamnose 33 0 Sorbitol 0 0 Xylose ) 0) Nitrite from nitrate Sd 92 Growth at: 50°C 24 8) 40°C 90, 56 35°C 100 100 28°C 100 100 10°C 12 30 Utilization of: Acetate 100 100 Citrate 33° 98 Malate 100 100 Propionate 100 100 Pyruvate 100 100 Succinate 100 98 Benzoate 0) 0) THE GENUS NOCARDIA TABLE 3 Comparative properties of certain acid-fast nocardias (Suter, 1951) Aerobic . fastidiosa . leishmanii . caprae 4 . pretoriana . pulmonalis . paraffinae . transvalensis 4 4 baths TE tis . polychromogenes 4 . minima 4 . coeliaca . rubropertincta . asteroides ‘ . salmonicolor 4 rubra . farcinica b+++1+++++4+4+4+44 1 4 aie 98> | Pigment production ;t++++t++4+4t4+ F441 Growth at room temperature Aerial mycelium Growth on potato +++ 4+ 44444 +++ _ Mycobacterium. In the case of the Strepto- myces-designated cultures, 83 per cent pro- duced sporulating aerial hyphae, 9 per cent nonsporulating aerial hyphae, and 8 per cent formed no aerial hyphae. The Nocardia- designated cultures gave, with regard to production of aerial hyphae, 24, 47, and 10 per cent, respectively. Among the 214 Nocar- dia-designated cultures, 79 were recognized as representing N. (Eppinger) Blanchard. They all produced acid from glu- cose and glycerol, and utilized acetate, mal- ate, propionate, pyruvate, and succinate. They all grew well at 28 and 35°C, and 88 per cent grew at 40°C. Eighty-six per cent reduced nitrate to nitrite, 54 per cent hy- drolyzed starch, and 34 per cent decomposed gelatin. A large number of cultures desig- nated as Nocardia (N. corallina, N. ery- thropolis, N. globerula, N. lutea, N. opaca, N. rhodni, N. rubra) were tentatively signed by Gordon and Mihm to the myco- bacteria under JW. rhodochrous (Overbeck) asteroides as- nov. comb. (Table 2). Of five species of aerobic actinomycetes associated with various mycetomas, Mariat (1957) recognized only N. asteroides and N. brasiliensis as nocardiae; Streptomyces ma- durae, S. pelletiert, and S. somaliensis were considered streptomycetes, although Mariat was not quite certain of their exact systematic position. Bojalil and Cerbon (1959) divided the genus Vocardia into two different metabolic groups: (1) Produces round colonies, adher- ing to wall and bottom of tube; utilizes gelatin as the only source of N and C, break- ing it down into amino acids and giving an alkaline reaction. NV. brasiliensis belongs to this group. (2) Produces flaky growth easily as dispersed through medium; poor growth on gelatin. N. asteroides belongs to this group. A detailed examination of the variability of different strains of two species of Nocar- dia with regard to their ability to utilize different well as in certain other physiological and ‘carbon and nitrogen sources, as biochemical properties, is reported in Table 2. Some comparative properties of several nocardiae are given in Table 3. Spalla (1958, 1959) eriticized the various descriptions of Nocardia species on the basis of an insufficient number of characters. He suggested that the following properties be used for characterization and classification of nocardiae: TABLE 4 Cultural and biochemical properties of certain species of Nocardia (Spalla, 1959) Growth* on dese auiseied -Sse-asoonyyy VSR [OIIIA[H umtpaut auIWIe-7-N Other properties uint,aoAUL [euey a0uR\sIsal ploy Shae. jo uoTVR[NSRO) ++t+4+ 01+ uljejas JO SISA[OIPAPHL ++4++4+11+ yorrys Jo SISA[OIPAP{ ayei}iu jo_"uoTjONpay Acid production from: [Oq1oS-P jouuopy oat te oer ere abe atl jouuuryy-p ++4+4+4 JOIII AT) +++++4+4+4+ uypnuy Pecilee ae oR lh ele st asouyjey asoyeyatL, aso}IV'T 9s0}[P asoiong asoulqeiy-/ +++4+111 asouuem-p | | | | ++ I + ssoumvyy | +++ | + | S800 P |e ar ae oN Pl se asoyElRet)-p Organism . asterotdes........ blackwellit ....... DUPURIYRIR Pl cen Strain 959) 09224. 424 Strain 2p. soe oe = = > S S = Zi Sk apricot; S = salmon. « c rose; L = lilac; A = colorless; R yellow; O = orange; C + Now recognized as a Streptomyces. Fy THE GENUS NOCARDIA 29 TABLE 5 Serological relations of major pathogenic actinomycetes (Gonzdlez Ochoa and Vasquez Hoyos, 1953) Antigens N. brasilien- 1 sis No. 447 VAD ODUSINIOR Od lteeace ne, shetssertan Sk Shy bo ee + N. brasiliensis No. 447.............. + N. brasiliensis No. 468 + N. mexicana No. 414... ...........5.55: + INES OSTETOUACSRINO:, NSir. oe.ctescye oes oi0mrs, 6 a oes + INPASEENOUGES INO ANG). a ipttcaysc sauce cess + Nie astern ovdesiNOe20) .f0n5ecsb es ss a a + IN asteTvovdes NiOn O02b 6. 1355006050 +-00- + N. asteroides No. 694................... + INENGUPSOUEES NO. DOLL. oo. ek ete nates os + INEACOnNCG NO G16). 2. 5. es ws oe eh + N. leishmanii No. 1030............. + DSEMITLA UT LCUN Oma Diet. 1. 2 5 he tna: sie ee cise ts Se pelletventiNO., WISS.2 we... went ee _ SME PRUGANGUNO: GID. «ices icesicsue owes s = S. somaliensis No. 1065............... — SS; somalvensis No: 1064............-... = ISIS OMMALLENSUSMINO. LOGOR 5. cad a+ cle See ee - — S. paraguayensis No. 285.............. _ SPE OLOUISS NOM OU Siedanvaiee ss ur:.c sere hun ee ae — GHUSC US NO OhOy tty: bee idem aes syle as - im. wavenautae No: 9963... ......6.+:-:- — ’. asteroides S. madurae Precipitin formation S. pelletiert S. somaliensis S. paraguay- No. 19 No. 412 No. 1185 No. 1065 ensts No. 285 a — — — — aL == = = = _ — — — ae a = — — — aa — — — a a — — — = 4. =— — — — + — — — = aa = — — — a = = = = + — — = = — — — = — — — + — == — — — — = - - + | | | | ap ct cea al 1. Color of growth on several synthetic and at least one organic medium. 2. Acid production from various carbon sources. 3. Heat resistance. 4. Size of terminal fragments. 5. Staining properties, notably acid-fast- ness. 6. Diastase formation. 7. Gelatin liquefaction. 8. Reduction of nitrate to nitrite. 9. Coagulation and peptonization of milk. 10. Formation of aerial mycelium. By utilizing these properties and consider- ing the high degree of similarity, Spalla was able to conclude that three mutants of NV. rugosa belonged to the same species as the parent, and that N. rugosa, N. rubra, and N. blackwellii represent distinct species, with a low degree of similarity. asteroides, N. A summary of some of the properties of N. rugosa and three of its mutants, as well as of certain other Nocardia species, is given in Table 4. The marked difference between the culture designated as NV. gardneri and the other nocardiae may be noted, particularly in utilization of carbon sources, starch hy- drolysis, and formation of aerial mycelium. These differences account for the fact that N. gardneri is now recognized as a Strepto- myces and not as a Nocardia. Various serological reactions of different species of Streptomyces and Nocardia are given in Table 5. Further information on the relation of Mycobacterium to Nocardia is found in the work of Haag (1927) and Gordon (1937). Of the Nocardia, the following three systems of classification of species are presented here: various treatments of the genus 30 THE ACTINOMYCETES, Vol. II 1. Classification of Nocardia* Species, According to Jensen (1932a) A. Partially acid-fast organisms with strongly re- fractive cells; nonproteolytic, generally non- diastatic; capable of utilizing paraffin. I. Initial mycelium limited, rapidly dividing into rods and cocci. 1. Slowly growing organism; cells 0.5 to 0.7 » in diameter. Nocardia minima 2. Rapidly growing organisms; cells 1.0 to 1.2 win diameter. a. Cystites not produced; rapid forma- tion of cocci. Nocardia corallina b. Cystites produced; less rapid forma- tion of cocci. Nocardia salmonicolor II. Initial mycelium well developed, richly branching, dividing into rods and generally into cocct. 1. Substrate growth soft, without macro- scopically visible aerial mycelium. a. Substrate growth red; may produce variants with undivided substrate mycelium and visible white aerial mycelium, or yellow and white vari- ants. Nocardia polychromogenes b. Substrate growth white to pale pink. al. Growth in nutrient agar opaque, cream-colored; coeci in broth culture. Nocardia opaca b!. Growth on sugar-free nutrient agar watery; no cocci in broth culture. Nocardia erythropolis 2. Substrate growth hard, yellow, with white aerial sporophores divide into chains of acid-fast cocci. Nocardia paraffinae B. Nonacid-fast organisms with weakly refractive cells; no distinct formation of cocci. Diastatic. I. Nonproteolytic; no aerial mycelium; marked production of cystites. Nocardia mesenterica mycelium ; Il. Proteolytic organisms. 1. Growth on nutrient agar with rapid formation of unbranched diphtheroid- like rods; no typical cystites; broth tur- bid. Nocardia actinomorpha * Designated by Jensen as Proactinomyces. bo Growth with extensive mycelium on nu- trient agar; simple unbranched rods not formed; cystites present; broth clear. Nocardia flavescens 2. Classification of Nocardia,* Accord- ing to Krassilnikoy A. Cultures colorless, some excreting a brown sub- stance into the medium. I. Aerial mycelium and spore-bearing hy- phae produced in culture media. 1. Substrate and aerial mycelium ocea- sionally forming septae; the hyphae break up into long rods, 15 to 30 y; spherical bodies not formed. a. Saprophytes, found on dead sub- strates. Nocardia actinoides b. Parasites, living in bodies of man and animals. Nocardia gedanensis 2. Mycelium producing frequent septae; hyphae break up into short rods and cocci. a. Saprophytes. al. Grow on protein media. Nocardia actinomorpha b!. Grow on paraffin. Nocardia paraffinae b. Parasites. Nocardia bovis Il. Cultures produce aerial sporophores on the surface of the colonies, but no aerial mycelium; sporophores are short, straight, covering the surface of the colonies with a thin, pale layer. 1. Cultures grow in organic media. Nocardia albicans ~o) 2. Cultures grow in inorganic media. Nocardia oligocarbophilus III. Cultures not forming any sporophores or any aerial mycelium; colonies smooth or lichenoid. 1. Saprophytes found on dead substrate. Nocardia albus 2. Parasites or symbionts living within plants, animals, or man. a. Organisms living in symbiosis with plants, forming nodules on their roots. Nocardia alni Nocardia myricae Nocardia elaeagnii * Generic name Proactinomyces used. THE GENUS NOCARDIA 31 b. Organisms living in the bodies of man and animals. al. Anaerobes, living in absence of oxygen. Nocardia anaerobicus b!. Aerobes, or microaerophilic forms. a2. Strict aerobes. Nocardia lignierest b?. Facultative aerobes. a3’. Cells nonacid-fast. Nocardia israeli* b3. Cells acid-fast. Nocardia muris B. Cultures pigmented. I. Cultures pigmented violet or blue, the pigments diffusing into the substrate. 1. Well produced, hyphae forming occasional developed substrate mycelium septae and breaking up into long rods, 20 to 30 w; colonies form a faint aerial mycelium with straight sporophores; spores cylindrical. Nocardia gabritschewski 2. No aerial mycelium produced, hyphae fre- or substrate mycelium forming quent and breaking up into short rods and cocci. septae Nocardia cyaneus Il. Red or orange pigment produced. 1. Mycelium forming occasional septae and breaking up into long rods; some give rise to a faint aerial mycelium and short straight sporophores. a. Saprophytes. al. Cultures not forming any soluble pigment in medium. Nocardia fructifert b!. Cultures producing a brown sub- stance. Nocardia polychromogenes b. Parasites living in bodies of man and animals. Nocardia freerv 2. Hyphae forming frequent septae and breaking up into short rods and cocci; no aerial mycelium produced. a. Saprophytes living on dead sub- strates. Nocardia ruber b. Parasites living in bodies of man and animals. * See Chapter 2 for description of Actinomyces israelit. FiGuRE 11. N. soil extract agar (Reproduced from: Gordon, R. E. and Mihm, J. M. J. Bacteriol. 75: 240, 1958). asteroides, strain 730, grown on al. Cells acid-fast. Nocardia asteroides bl. Cells nonacid-fast. Nocardia variabilis III. Cultures citron-yellow or bright yellow. 1. Faint sporophores aerial mycelium with straight and cylindrical spores produced. a. Saprophytes. Nocardia flavescens b. Parasites. Nocardia somaliensis* 2. No aerial mycelium produced. a. Saprophytes. al. Cultures yellow or bright yellow. Nocardia flavus bt. Cultures citron-yellow. Nocardia citreus b. Parasites. al. Cells acid-fast. Nocardia farcinica bl. Cells nonacid-fast. Nocardia putoriae IV. Cultures pigmented green. 1. Saprophytes. Nocardia viridis 2. Parasites. Noca rdia pyogenes V. Cultures black. 1. Saprophytes. Nocardia nige | 2. Parasites. Now a rdia Sé ndate ns 1s * Now recognized as a Streptomyces. 32 THE ACTINOMYCETES, Vol. II 3. Classification of Nocardia, According to the system of Waksman and Henrici* A. Partially acid-fast organisms with strongly re- fractive cells; nonproteolytic and generally nondiastatic; capable of utilizing paraffin. I. Initial mycelium fully developed, well branching, dividing into rods and generally into cocci. 1. Substrate growth soft, without macro- scopically visible aerial mycelium. a. Substrate mycelium yellow, orange, or red. al. Pathogenic. a2. Substrate mycelium buff, or pale yellow. 18. Nocardia farcinica b2. Substrate mycelium yellow to red. 6. Nocardia asteroides b!. Not pathogenic. a2, Paraffin decomposed. 42. Nocardia polychromo- genes b?. Cellulose decomposed. 13. Nocardia cellulans b. Substrate mycelium white to pink. al. Gelatin not liquefied. a2. Growth on nutrient opaque, cream-colored. 38. Nocardia opaca b?. Growth on nutrient agar pink. 10. Nocardia calcarea a3, Aerial mycelium on milk white. 31. Nocardia leishmanit b’. Pellicle on milk pink. 11. Nocardia caprae e3, Pellicle on milk yellow. 9. Nocardia brasiliensis white, agar d’. Causing galls on blueberry plants. 53. Nocardia vaccinit b!. Gelatin liquefied. 43. Nocardia pulmonalis 2. Substrate mycelium hard, yellow. a. Aerial mycelium white; hyphae di- vides into chains of acid-fast cocci. 40. Nocardia paraffinae * This system was used, with certain minor omissions and additions, in the last edition of Bergey’s Manual. b. Aerial mycelium not produced on or- ganic media. 41. Nocardia petroleophila Substrate growth cream colored, later becoming yellow. 51. Nocardia serophila Substrate growth hard, orange-yellow. 58. Nocardia variabilis II. Initial mycelium very short, rapidly divid- ing into rods and cocci. a Growth pink. a. No cystites (swollen cells) formed. al. No indigotin from indole. 16. Nocardia corallina b!. Indigotin from indole. 26. Nocardia globerula b. Cystites formed. 49. Nocardia salmonicolor . Growth coral-red. 47. Nocardia rubropertincta Growth white, tan, or pink. a. No aerial mycelium. a!. Growth tan. 15. Nocardia coeliaca b!. Growth white. 28. Nocardia intracellularis b. Aerial mycelium produced. al. Growth frequently pinkish. 53. Nocardia transvalensis b!. Growth never pink. 50. Nocardia sebivorans Produces no pigment, no growth on potato, coagulates milk. 19. Nocardia fastidiosa . Nonacid-fast organisms with weakly refrac- tive cells; no distinct formation of cocci; di- astatic. I. Nonproteolytic, although some give gela- tin liquefaction. i Growth on agar pale cream. a. Gelatin not liquefied; starch hy- drolyzed. 35. Nocardia mesenterica b. Gelatin liquefied; starch not hy- drolyzed. 48. Nocardia rugosa Growth on agar whitish. 4. Nocardia albicans Growth on agar yellow. 20. Nocardia flava Growth on agar green. 59. Nocardia viridis Growth on agar yellow-green. 14. Nocardia citrea 6. 10. 14. Il. Proteolytic, . Growth THE GENUS Growth initially colorless, producing a yellow-green pigment in 2 to 4 days. 54. Nocardia turbata . Growth on agar dark brown and even black. a. No liquefaction of gelatin. 37. Nocardia nigra b. Gelatin liquefied. 29. Nocardia tvorensis . Growth consistency soft; aerial my- celium sparse. 33. Nocardia lutea consistency medium; aerial mycelium profuse. 8. Nocardia blackwellit Growth cream-colored to pink; aerial spikes produced. 52. Nocardia sumatrae . Growth grayish-yellow. 36. Nocardia muris . Growth yellowish-orange. 55. Nocardia uniformis Pigment on protein media deep brown. 44. Nocardia rangoonensis Pigment on protein media light brown. 12. Nocardia caviae although some are only weakly proteolytic. Ie Growth on nutrient agar with rapid formation of unbranched diphtheroid- like rods; no typical cystites; broth turbid. 1. Nocardia actinomorpha . Growth white, shiny or pale. a. Dough-like consistency; breaks up into short rods. 3. Nocardia alba b. Membranous, myceloid growth. 32. Nocardia listert Growth on nutrient agar with extensive mycelium; simple unbranched rods not formed; cystites present. Broth clear. 21. Nocardia flavescens . Growth cream-colored. a. Rapid liquefaction of gelatin. al. No aerial mycelium. 25. Nocardia gibsonit bl. Aerial mycelium scant, white. 56. Nocardia upcottit b. Slow liquefaction of gelatin. 17. Nocardia dicksonii . Growth rose-colored to bright red or red-orange. 24. Nocardia fructifera 6. Growth pink to red. NOCARDIA 33 FIGURE 12. N. produced from: McClung, N. M. First Reg. Conf. Asia and Oceania, Tokyo, 1956). rubra, electron micrograph (Re- a. Gelatin not liquefied. 2. Nocardia africana b. Gelatin slowly liquefied or not at all. 46. Nocardia rubra c. Rapid liquefaction of gelatin. 39. Nocardia panjae 7. Pigment on protein media light brown; color of growth pink. 45. Nocardia rhodnii 8. Growth yellowish to golden brown. 22. Nocardia fordir 9. Growth yellow to reddish-brown; solu- ble pigment brown to red. 30. Nocardia kuroishi 10. Growth tan to buff-colored. 23. Nocardia formica 11. Growth very limited on various media, except potato. 27. Nocardia hortonensis 12. Occur in the sea; liquefy agar. a. Growth yellow. 34. Nocardia marina b. Growth yellow-orange. 7. Nocardia atlantica 13. Produce nodules on roots of plants. 5. Nocardia alni In addition to the species included in the and described below, many more species of Nocardia have been above classification recorded in the literature, either under this or under other generic names. Some are listed in Chapter 13, under the incompletely described forms. Others are synonyms. There is no question that some of the Streptomyces species described in Chapter 8 could just as 34 THE ACTINOMYCETES, Vol. II well have been included among the Nocardia forms. It is also possible some of those listed as Nocardia could just as readily have been included in the genus Streptomyces. Fre- quently, the decision of the investigator as to whether a certain culture should be in- cluded in one genus or another was perfectly arbitrary. Descriptions of Nocardia Species* 1. Nocardia actinomorpha (Gray and Thornton, 1928) Waksman and Henrici, 1948 (Gray, P. and Thornton, H. Centrl. Bacteriol. Abt. II, 73: 88, 1928). Morphology: Growth colorless, smooth, consisting of long, branching filaments and rods, 0.5 to 0.8 by up to 10 yw. In older cul- tures, rods 2 to 3 uw long generally predomi- nate. On some media, extensively branching hyphae occur. Not acid-fast. Nutrient agar: Round colonies, 1 mm in diameter, convex, white, granular or resin- ous; long arborescent processes from the edge. No aerial mycelium. Potato-glycerol agar: wrinkled, pink to orange. Egg medium: Growth raised, dry, smooth, Growth dry, salmon-buff. Gelatin: Colonies round, white, raised rim, edges burred. Liquefaction saucer-like, positive. Nutrient broth: Turbid. Milk: Coagulation and peptonization. Starch: Hydrolyzed (diastase produced). Sucrose: Inverted. Nitrate reduction: Positive. Phenol and naphthalene: Utilized. Temperature: Optimum 25-30°C. Source: Soil. Remarks: Differs from N. liquefaction of gelatin. No acid from glucose, lactose, sucrose, or glycerol. coeliaca in 2. Nocardia africana Pijper and Pullinger, * For further details concerning some of the species, the last edition of the Bergey Manual should be consulted. 1927 “(Piper “As-andPullinger,-B. DD. 23: Trop. Med. Hyg. 30: 153-156, 1927). Synonym: Actinomyces africanus (Pijper and Pullinger) Nannizzi Pollaceci, 1934. Morphology: Substrate growth consists of unicellular branching mycelium. Aerial my- celium sparse, consisting of short, straight hyphae. Not acid-fast. Glucose agar: Colonies minute, red, dis- crete, round and piled up into a pale pink mass. Aerial mycelium thin, white. Nutrient agar: Colonies discoid, flat, pink. Glycerol agar: Growth made up of small, heaped-up, colorless masses with pink tinge; later, growth abundant, piled up, pale pink. Potato agar: Growth bright red, made up of small, round colonies with colorless sub- merged margins, and piled up patches. Aerial mycelium stiff, sparse, white. “ge medium: Colonies small, colorless, blister, partly confluent ; becoming wrinkled, depressed into medium. Liquefaction shght. Gelatin: Irregular pink flakes. No liquefac- tion. Milk: Surface growth bright red. Medium gradually becomes opaque, reddish-purple, with slow peptonization. Source: A case of mycetoma in South Africa. 3. Nocardia alba (Kxrassilnikov, 1941) Waksman (Krassilnikov, N. A. Actinomyce- tales. Izvest. Akad. Nauk. SSSR, Moskau, 1941, p. 1). Morphology: Growth smooth or folded, made up of white colonies of a dough-lke consistency; shiny or pale. Substrate myce- lium breaks up into short rods 2.7 by 0.7 to 0.8 uw, later changing into a mass of coccus- like cells, 0.7 to 1 uw. Many cells are swollen, others form side buds. Not acid-fast. No aerial mycelium. Synthetic agar: sources of nitrogen; sugar, starch, or organic Inorganic salts used as acids utilized as sources of carbon. Nutrient agar: Good growth. No aerial mycelium. THE GENUS Gelatin: Growth good. Positive liquefac- tion. Milk: Coagulation and peptonization. Starch: Rapid hydrolysis. Cellulose: No growth. Paraffin: No growth. Nitrate reduction: Negative. Sucrose: Inverted. Source: Soil. Remarks: Several subspecies were also listed: N. chromogena, N. paulotropha (Ac- tinobacillus paulotrophus Beijerinck, 1914), N. alba lactica, N. diastatica, N. hoffmanni. 4. Nocardia albicans (Krassilnikov, 1941) Waksman (Krassilnikov, N. A. Actinomyce- tales. Izvest. Akad. Nauk, SSSR, Moskau, 1941). Morphology: Growth red, hyphae break- ing up into rod-shaped cells, 12 to 25 by 0.6 to 0.7 uw, up to 50 uw in length. Cells straight or slightly curved, branching. Aerial myce- lium not observed, except surface layer of sporophores, which produce a velvety ap- pearance. Multiplication by fission, seldom by budding. Nutrient shiny. Gelatin: No liquefaction. Milk: No change. Starch: Hydrolyzed. Cellulose: No growth. Nutrient broth: Growth poor; produces agar: Growth good, smooth, faint turbidity, which settles on bottom and leaves a surface ring. No true mycelium. Cells rod-shaped 5 to 10 uw, seldom 15 to 20 u. Nitrate reduction: Negative. Sucrose: Inverted. Paraffin: Not utilized. Source: Soil. Remarks: Glycerol used as a ‘arbon, and nitrate as a source of nitrogen. 5. Nocardia alni (Peklo emend. v. Plotho, 1941) Waksman (von Plotho, O. Arch. Mikrobiol. 12: 1-18, 1941). Morphology: Mycelium contains fatty NOCARDIA source of ey) ~ globules; cells filiform, branching, disinte- Aerial mycelium usually absent, but may be formed grating into short rods and cocci. on cultivation. Sporulating cultures form white, spherical to oval spores. Agar media: Substrate growth compact, shiny, colorless or slightly brownish. Gelatin: pellicle. positive. Soluble pigment brownish. Liquid media: Slimy surface film. Tyrosine: Utilized as source of nitrogen; color turns red-brown. Cellulose: Not utilized. Carbon sources: Produces lactic acid from Surface Liquefaction glucose and lactose. Optimum reaction for growth: pH 6.0. Habitat: Roots of the alder. Remarks: Produces nodules on the roots of the host plant. Said to bring about nitro- gen fixation in symbiotic culture with the plant. 6. Nocardia asteroides (Kppinger, 1891) Blanchard, 1895 emend. Gordon and Mihm, 1959 (Eppinger, H. L. Beitr. Pathol. Anat. 9: 287, 1891; Blanchard, R. In Bouchard. Traite Pathol. Gen. 2: 811, 1895; Gordon, R. E. and Mihm, J. M. J. Gen. Microbiol. 20: 129, 1959). Synonyms: Cladothrix asteroides Mppinger, 1890; Streptothrix eppingeri 1891; Actinomyces asteroides Gasperini, 1892; Lossi- Doria, Oospora asteroides Sauvageau and Radais, 1892; N. asteroides R. Blanchard, 1895. Ac- cording to Ochoa and Sandoval (1956), N. leishmanii. Chalmers and Christopherson, and N. phenotolerans Werkam and Gammel are synonyms of NV. asteroides. According to Gordon and Mihm (1959), V. caprae (Silber- schmidt) Waksman Henrici, N. ep- pingert, N. minima, and N. sylvodorifera are also synonyms. Morphology : growth, usually yellow to orange to orange- red. Mycelium straight and fine; it breaks up into small, coceoid forms and rods. Some and Typical actinomycete strains are acid-fast ; others are only partially 36 THE ACTINOMYCETES, Vol. II so. Aerial hyphae produced; they vary from rudimentary to long branching. Some may produce chains of spores (Fig. 11). Sucrose nitrate agar: Growth thin, spread- ing, orange. No aerial mycelium. No soluble pigment. Peptone-beef extract agar: Growth much folded, light yellow, becoming deep yellow to yellowish-red. No soluble pigment. Yeast-glucose agar: Growth flat to wrinkled, beige to dark pink. Some produce white aerial hyphae. Potato: Growth much wrinkled, whitish, becoming yellow to almost brick-red. Gelatin: Growth yellowish on surface. No liquefaction. Milk: Orange-colored ring. No coagula- tion; no peptonization. Starch agar: Growth restricted, orange. No diastatic action. Blood serum: No liquefaction. Carbohydrate utilization: See Table 2. Nitrate reduction: Positive. Oxygen demand: Aerobic. According to Chalmers and Christopherson (1916), it may also grow anaerobically. Temperature: Optimum = 37°C. strains grow readily at 28°C. Pathogenicity: Transmissible to rabbits and guinea pigs, but not to mice. Source: Human infections and soil. Remarks: A number of strains of acid-fast scant, Some actinomycetes isolated from human lesions have deviated in certain particulars from the description of N. asteroides, but not sufficiently to warrant separation as different species. According to Gordon and Mihm, all strains of N. asteroides form whitish aerial hyphae, these varying from rudimentary to much branching. The following characteris- tics were considered the most valuable in the identification of the species: development of filamentous colonies with aerial hyphae; fail- ure to hydrolyze casein and to dissolve the crystals of tyrosine and xanthine; acid pro- duction from glucose and glycerol; failure to form acid from arabinose, lactose, mannitol, inositol, and xylose; utilization of acetate, malate, propionate, pyruvate, and succinate, but not benzoate. Numerous varieties of this species have been described. It is sufficient to mention N. crateriformis, N. gypsoides, and N. pseudo- carneus (Gordon and Mihm, 1957). Type culture: IMRU* 3308; also 504. 7. Nocardia atlantica (Humm and Shep- ard, 1946) Waksman (Humm, H. J. and Shepard, K.S. Duke Univ. Marine Sta. Bull. 3: 78, 1946). Synonym: Proactinomyces atlanticus. Morphology: Hyphae long, branching, breaking up into rods and cocci, 0.5 to 0.7 pu. Involution forms in old cultures. Nonacid- fast. Aerial mycelium not produced. Synthetic and organic media: Growth bright yellow or yellow-orange, smooth, com- pact, of a doughy consistency. Colonies flat with shghtly raised center. Soluble pigments none. Mineral sources of nitrogen utilized. Gelatin liquefaction: Positive. Agar: Liquefied. Milk: Coagulation rapid, acidified; pep- tonization slow. Nitrate reduction: Positive. Starch: Hydrolyzed. Cellulose: Decomposed. Chitin: Decomposed. Agar: Slowly digested. Alginie acid: Decomposed. Carbon sources: Acid produced from arab- inose, xylose, rhamnose, raffinose, fructose, galactose, gum arabic. No acid from lactose, dulcitol, mannitol, or sorbitol. Organic acids utilized: gluconie, lactic, malo- not utilized: acetic, sucrose, nic. Organie acids butyric, citric, ete. Optimum temperature: 28-30°C. Habitat: Marine algae and marine sedi- ments. * These designations represent the various cul- ture collections where the type cultures are de- posited. THE GENUS Remarks: Another closely related culture has been described as Proactinomyces flavus (see N. marina). Nocardia blackwellii (Erikson, 1935) Waksman and Henrici, 1948 (Erikson, D. aa ee irch Council Spec. Rept. Ser. 203: , 1935). es rod-like filaments, sparsely branching hyphae. Aerial mycelium short, straight; frequently large, round ovoid cells are interposed in the irregularly Growth consisting of short, growing out into longer segmented chains of cells. Glycerol nitrate agar: granular, irregular, thin, pinkish. Growth extensive, Nutrient agar: Growth confluent, wrin- kled, with small, round, pinkish, discrete colonies at margin. Glucose nutrient agar: Growth abundant, pale pink, in form of small conical colonies, piled up, convoluted. Potato agar: Colonies small, round, color- less. Aerial mycelium white. Later, colonies dull pink, submerged margins; few aerial spikes, moderate aerial mycelium at top of slant. Gelatin: Colonies few, along line of inoculation. colorless colonies to 10 mm_ below larger pink-yellow surface colonies white aerial mycelium. No liquefaction. Milk: Surface pellicle heavy, convoluted, bright yellow. No coagulation; no peptoniza- tion. Spalla states that milk is coagulated. Carbon utilization: See Table 4 Source: Hock joint of foal. Type culture: ATCC 6846; colorless, minute, Later, abundant, surface; with NCTC 630 9. Nocardia brasiliensis (Lindenberg, 1909) Castellani and Chalmers emend. Gordon and Mihm, 1959 (Lindenberg, A. Arch. Parasitol. 13: 265-282, 1909; Castellani, A. and Chal- mers, A. J. Manual of Tropical Medicine, 2d Kd. William Wood & Co., 1913, p. 816; Gordon, R. E. and Mihm, J.M. J. Gen. Microbiol. 20: 129, 1959). NOCARDIA 37 Synonyms: Gonzalez Ochoa (1945, 1953), Gonzalez Ochoa and Sandoval ee and Gordon and Mihm (1958) consider bra- siliensis as the proper name for ae organ- ism. Or ints In characterizing (}) Formation of antibiotics and vita- mins. (k) Formation of H»S in peptone-iron agar. Sensitivity to antibiotics: (a) Sensitivity to pure antibiotic prep- arations. (b) Phenomena’ of “‘cross-resistance”’ and ‘‘cross-sensitivity”’ on artificial media. Sensitivity to phages. Serological reactions. Chemical composition. Ecological properties. Genetic relationships. Age of culture. Information should be submitted concerning the age of the cul- ture when the particular properties were studied and the manner in which the culture has been kept in the laboratory. Type cultures. The culture should be deposited in a recognized collection and the assigned number reported. Every possible means for preservation of the culture should be used. Preservation of strains by lyophilization, soil culture, mineral oil seals on active slants, or storage in deep freeze is believed to re- duce physiological changes to a mini- mum. With the lyophilization technique within the reach of even the small lab- oratory, there is no excuse for an inves- tigator, particularly one publishing on designated strains, to ‘‘lose’’ his strains. Streptomyces species, only certain media should be used and well— 62 THE ACTINOMYCETES, Vol. II defined conditions of growth recognized. Un- necessary media and nonessential details had better be left out to avoid cumbersome de- scriptions and nonduplicable characteris- tics that may apply to varieties or strains rather than to species. A larger number of media and more detailed descriptions may not only fail to give additional information but may complicate the description of the species to such an extent as to render the identification of freshly isolated cultures difficult. The composition of the media is usually given first consideration for descriptive pur- poses. According to Waksman (1958), Shi- nobu (1958), and others, these media should include: (a) at least three synthetic media, preferably sucrose-sodium-nitrate-salt or su- crose-ammonium-salt agar, glucose-.or glyc- erol-asparagine agar, and calcium malate or ‘alecium citrate agar; (b) two or possibly three organic media, such as nutrient. (pep- tone-beef extract) agar, yeast extract-glucose agar, potato-glycerol-glutamate agar, or oat- meal agar; (c) three or four complex natural media, notably potato plugs, gelatin, and milk; (d) peptone-iron-yeast extract agar for HS production; (e) tyrosine medium for the tyrosinase reaction; and (f) a synthetic me- dium for carbohydrate utilization. Very few, if any, other media are required. Liquid media, with the exception of those previously listed, are better left out. Morphological Properties The method of study of the morphological properties of the streptomycetes would in- clude visual microscopic examination versus electron microscopic studies; direct exami- nation versus study of stained preparations; and hanging drop versus agar surface cul- tures. STRUCTURE OF SUBSTRATE MYCELIUM The substrate mycelium of a Streptomyces does not, as a rule, segment spontaneously into bacillary or coccoid forms. It produces leathery or tough-textured growth, remain- ing nonseptate and coherent even in old cultures. Although no true septa are ob- served in young cultures, it has recently been reported that older cultures show at least occasional septation. The compactness of this substrate growth is responsible for the fact that hquid media are always clear, un- less the culture has been subject to phage or lytic action. NATURE AND LIUM PROPERTIES OF AERIAL MYCE- The aerial mycelium is usually thicker than the substrate mycelium. While the morphology of the substrate mycelium is usually undifferentiated, the aerial mycelium of streptomycetes, under fixed conditions of culture, shows sufficient differentiation that a miscellaneous assortment of isolates can be segregated into a number of groups hav- ing like morphological characteristics. This is one of the most important criteria for classification in the genus Streptomyces. Sev- eral aspects relating to the aerial mycelium may be considered: a. Gross macroscopic appearance. The rela- tive abundance, structure (cottony, velvety, powdery), formation of rings or concentric zones, and pigmentation of the aerial my- celium are important diagnostic criteria. b. Microscopic properties. The microscopic structure of the aerial mycelium gives a clear picture of the morphology and reproductive structures of the organism. The hyphae may be long or short, with extensive or little branching. The branching may be simple or complex, monopodial or sympodial, broom- shaped or verticillate. The fruiting bodies or sporophores are short or long, occurring sin- gly, in clusters, or as verticils; they are straight, wavy, or spiral-forming. The spirals or coils are either long and open or short and compact. Spiral formation may take place on one medium and not on others. CHARACTERIZATION OF | i STREPTOMYCES SPECIES 63 FiaureE 16. Schematic representation of different types of spirals produced by various Streptomyces species; the spirals range from long to short, from compact to irregular (Reproduced from: Shinobu, R. Mem. Osaka Univ. Lib. Arts and Ed. B. Nat. Sei. 7, 1958). Before a culture is pronounced as forming no spirals, therefore, it must be grown on a variety of selective media that will allow optimum sporulation. Drechsler (1919) sug- gested use of the right-hand or left-hand curvature of the spirals as a diagnostic fea- ture, but composition of the medium (Ettlinger ef al., 1958: Shinobu, 1958). Verticil formation is also an important characteristic of certain this, too, is influenced by the species; it can be simple or branching (pri- mary or secondary verticils), the branches being straight or forming spirals; but this property as well is influenced to some extent the medium. Al- though nocardiae may produce sporulating by the composition of aerial filaments, these are never spiral- shaped (lig. 16). Waksman (1940, 1950) divided the or- ganisms belonging to the genus Actinomyces (largely the forms now included in the genus Streptomyces) into the following five sub- groups on the basis of the structure of the sporulation hyphae. I. Straight sporulating hyphae, monopo- dial branching, never producing regular spirals. Il. Spore-bearing hyphae arranged in clus- ters, or broom-shaped arising from compression of the sporophores. III. Spiral formation in aerial mycelium; long, open spirals. IV. Spiral formation in aerial mycelium; short, compact spirals. V. Spore-bearing hyphae arranged on my- celium in whorls (verticils) or tufts. (1941, 1949) the genus Actinomyces (largely forms included Krassilnikoy divided in Streptomyces) on the basis of the following properties: (1) spiral forming versus straight sporophores; (2) alternate distribution of sporophores on aerial mycelium versus verti- ) cil formation; (8) spherical versus oval spores; (4) colorless versus pigmented cul- tures; (5) white versus colored aerial my- celium; (6) saprophytes versus parasites. Aiso et al. (1948) divided the genus Strep- tomyces on the basis of the structure of the aerial mycelium into six types: I. Spirals not formed. 64 THE ACTINOMYCETES, Vol. II F Piate I. Diagrammatie representation of the morphology of the sporophores of Streptomyces (Re- produced by special permission from Ettlinger et al. Arch. Mikrobiol. 31: 336, 1958). a. Sporophores produce straight branching verticils on sterile aerial hyphae; S. retdculi type. b. Sporo- phores produce open spirals as side branches on sterile aerial hyphae; S. purpurascens type. ¢. Sporo- phores produce on sterile aerial hyphae verticils with open, more or less regular spirals; S. nowrsei type. d. Sporophores formed as side branches on sterile aerial hyphae, straight or slightly wavy; S. phaeo- chromogenes. e. Sporophores produced as verticils on sterile aerial hyphae, with open, irregular spirals; S. echinatus. {. Sporophores monopodially branched, forming irregular open spirals at the end of long hyphae; S. lavendulae. g. Sporophores monopodially branched, with open, regular spirals; S. parvullus. CHARACTERIZATION OF STREPTOMYCES SPECIES 65 mM n fe} Pp Prats II. Diagrammatic representation of the morphology of the sporophores of Streptomyces (Re- produced by special permission from Ettlinger ef al. Arch. Mikrobiol. 31: 337, 1958). h. Sporophores produce numerous short, monopodial branches on sterile hyphae; S. ramulosus. i. Sporo- phores sympodially branched, forming tufts upon short main axes; S. griseus. k. Sporophores produced upon a long, straight main axis, monopodially branched with frequent, regular spirals; S. fradiae. 1. Sporophores monopodially branched, straight or slightly wavy; S. antibioticus. m. Sporophores mono- podially branched along the whole axis with open, irregular spirals; S. erythraeus. n. Sporophores mono- podially branched, with narrow, compact spirals; S. violaceoniger. 0. Sporophores sympodially branched, in the form of trees with a long main axis; S. viridogenes. p. Sporophores monopodially branched, stiff and straight; S. venezuelae. 69 THE ACTINOMYCETES, Vol. II 1. Straight, very little branching. 2. Wavy, abundant branching. II. Spirals formed. 1. Spirals formed on the axis, irregu- lar branching. 2. Spirals formed on branches in clus- ters. Verticils produced. 1. Verticillate branches entangled like a net. 2. Verticillate branches formed on both axis and branches, making primary and secondary verticils. Okami (1952) grouped the genus Strepto- myces on the basis of formation of aerial mycelium into the following types: I. Spirals not formed. 1. Branches produced. 2. Branches not produced. II. Spirals formed. 1. Spiral form mostly compact. 2. Spiral form mostly loose. Shinobu (1958) eriticized the systems of Aiso and Okami on the basis that insufficient attention was paid to the nature of the me- dium. In the system of Aiso et al. the distine- tion between | and 2 of each type appeared to him to be unclear, many forms belonging to an intermediate type. Okami’s system was considered as incomplete because the 1 formation of verticils was not taken into consideration. Waksman’s system was_ be- lieved to be comparatively better, but even this system was criticized because cluster or broom-shaped sporophore formation was not considered as a sufficient characteristic, and because a strain does not necessarily have only one kind of spiral, but usually forms various kinds of spirals which coexist (PI. IT). Hesseltine et al. (1954) and Pridham et al. (1958) considered the sporophore morphol- ogy as reasonably stable under definite nu- tritional requirements of the organisms. Several morphological groups were sug- gested. The components of each group were considered as suggesting a logical natural arrangement. The physiological data can be ’ used to produce ‘“‘species’”? or ‘‘species- groups,” with morphology as a_ starting point. Seven morphological sections were created as subdivisions of the genus Strep- tomyces. However, this system as well was considered by Shinobu as having certain de- fects because (a) culture media for morpho- logical study were not examined thoroughly enough; and (b) some of the sections may often coexist In one strain. Shinobu (1958) emphasized the following morphological properties of the aerial my- celium: a. Outward appearance — of (powdery, cottony, leathery). b. Branching, especially the formation and nature of verticils. ce. Formation and nature of spirals. d. Formation and shape of spores. e. Thickness and length of mycelium. Shinobu examined in detail the various properties of the aerial mycelium, emphasiz- ing again the need for suitable synthetic media. He concluded that the nature of the aerial mycelium is one of the most important characteristics for taxonomic study, but that it should be considered in connection with composition of the medium. The aerial my- celium was classified into the following three mycelium groups, from the standpoint of branching and formation of spirals. Group I. Straight or wavy aerial my- celium, monopodial branching, never pro- ducing spirals or clusters. Group II. Spiral formation in the aerial mycelium; long or short; loose or compact; open or closed. Group III. Verticil or cluster formation in the aerial mycelium. The loss of ability to form aerial mycelium and sporogenous hyphae by certain Strepto- myces cultures, on the one hand, and the formation of aerial mycelium and_ sporo- phores by certain species and strains of Nocardia, on the other, led Bradley (1959) to question the distinction between these two genera. We have here simply another CHARACTERIZATION OF STREPTOMYCES SPECIES 67 vase of natural overlapping between man- made concepts of genera and species or the improper labeling of cultures. Gordon and Mihm (1957) emphasized that it is easy to understand how a culture of NV. that formed acid-fast coecobaecilli, rods, and short filaments and growth heavy, finely to coarsely wrinkled, cream- colored to orange, and without noticeable aerial hyphae, could be mistaken for a J yco- bacterium. A culture of V. asteroides, how- ever, that produced nonacid-fast, long, tan- gled filaments and a cream-colored, pale yellow, or beige growth thickly covered with whitish aerial hyphae, could just as easily be accepted as a Streptomyces. Numerous other studies have been made asteroides whose was of the micromorphology of the various spe- cies and groups of Streptomyces, as in the work of Burkholder et al. (1954), Hesseltine et al. (1954), Ettlinger et al. (1958), Flaig and Kutzner (1960), and others. c. Spores. The spores, also called conidia, produced from, or in, certain hyphae of the aerial mycelium, or the ‘‘sporogenous hy- phae,”” may be oblong, oval, or spherical. Krassilnikov (1949) attached great impor- ance to this character, as determined by the light microscope, as a diagnostic feature. Kriss et al. (1945) were the first to use the electron microscope for study of spores of Streptomyces. This was followed by the work of Carvajal (1946); Kiister (1953); Flaig et al. (1952, 1955, 1958); Baldacci and Grein (1955); Grein (1955); Vernon (1955); and others. Flaig et al. (1952) found that the spores of some strains had smooth surfaces while others had spiny surfaces. They later detected spores with hairy and warty sur- faces; the nature of the nitrogen source in- fluenced the appearance of the spore surface, organic nitrogen favoring spine formation. Kiister (1955) classified Streptomyces spores into two groups: (a) those producing a smooth surface and (b) those having a rough surface. Each of these groups was divided into three subgroups, based on shape of the spores. Thus there are spores with smooth surfaces, with spines, with hairs, or with warty protuberances, and spores that are globose, long-ovoid, and cylindrical. On the basis of a system of classification that they had outlined, Baldacci and Grein (1955) examined 50 strains of streptomycetes with the electron microscope. Three types of spores were recognized: (1) Oval, more or less transparent spores; these were either smooth or rough, the latter having a spiny or hairy surface; the spines were either short and thick or long and thin. (2) Round, opaque spores, usually smooth. (3) Poly- hedral spores, smooth and transparent, or shghtly curved, wrinkled, and opaque. The form of the spores was constant for the series in Baldacci’s system. It can hardly be used, however, as a species characteristic. A cor- relation was observed (Pridham, 1959) be- tween spore characteristics and sporophore morphology (Table 6). According to Preobrajenskaya et al. (1959, 1960), strains within one species as a rule have a similar type of spore surface. Cultures with a white, yellow, greenish-yellow, yellow- gray, pink, or lilae mycelium have smooth spores; those with a bluish aerial mycelium have spiny and hairy spores, and species with a gray aerial mycelium have spores of all types. The diagnostic value of spore sur- face characteristics was found to be dissimi- lar for the various sections. The correlation between the gray and bluish species and the character of the surface of the spores was considered as insignificant. Tresner ef al. (1960) also emphasized the importance of spore surface in classifying species of Strep- tomyces; size and shape of spores of most species were considered of limited usefulness in taxonomic differentiation. Lechevalier and Tikhonienko (1959) re- ported that the spores of S. viridochromo- genes were mostly elongated and those of S. violaceus, spherical. The spines formed by 68 THE ACTINOMYCETES, Vol. II TABLE 6 Morphology of sporophores and spores of streptomycetes* (Pridham, 1959) eee AIREE Original strain no. Sporophore morphologyt BAN Ce griseus B-1598 Carvajal SL 842 Straight to flexuous (RF) Smooth-walled bikiniensis B-1049 Waksman 3515 Straight to flexuous (RE) Smooth-walled CaNesCus 2419 Com. Sol. 811.0 Straight to flexuous (RF) Smooth-walled venezuelac B-902 Gottheb 8-44 Straight to flexuous (RF) Smooth-walled cinnamonen- _B-1588 Okami 154-T3 Hooks and open loops (RA) Smooth-walled sis with many straight (RF) sporophores flaveolus B-1334 ATCC 3319 Hooks and open loops (aber- Spiny to hairy rant) (RA) albus B-1685 Waksman (ATCC Spirals (aberrant) (S) Smooth-walled 618) hygroscopicus B-1865 NRRL isolate Spirals (8) Spiny chartreusts 2287 Upjohn K-180 Spirals (8) Spiny * Data taken from Carvajal, 1946; Vernon, 1955. All other data on spore morphology supplied by K. L. Jones. + Sporophore morphology determined according to the methods of Pridham ef al., 1958. Fiaurp 17. Coremia formation by certain Streptomyces species, X 500; stained by Corti’s method (Courtesy of Dr. J. Giolitti, Milan, Italy). CHARACTERIZATION OF STREPTOMYCES SPECIES 69 strains of both species differed cytologically. The spines of the first seemed to be part of the cell wall, whereas the spines of the sec- ond seemed to be very superficial, appearing only on the envelope. It was concluded that spine formation is a stable characteristic of the spores. The shape of the spores varied with the composition of the medium. It was suggested that complex organic media be avoided for spore study. COLONY STRUCTURE The nature of the Streptomyces colony growing on a standard agar plate has been considered as among the important criteria for characterizing and recognizing a particu- lar organism. One may question, however, the significance of this property in describing a species. The morphology of the colony, notably its general appearance, size, shape, and texture, can all be readily determined by superficial examination. Various other properties may be recognized from a study of the colony. Krainsky used the structure of the colony, especially its size and shape, as one of the major diagnostic criteria. The superficial examination of gross col- ony structure, particularly its texture, can be of some help. Pridham* and others have noted the following very general correlations: 1. Straight to flexuous cultures generally are flat with a velvety, granular, or powdery texture. 2. Loop cultures generally are flat with a velvety to slightly granular texture. 3. Spiral cultures generally are elevated with a somewhat floccose texture. Occasion- ally, spiral cultures that are flat with a vel- vety or granular texture may be seen. In spiral cultures that are somewhat floccose the sporulating aerial mycelium often con- sists of long sterile hyphae with sporophores branched oppositely, singly, or sometimes in an apparent verticillate fashion. * Personal communication. FicureE 18. Sclerotium formation in Strepto- myces (Prepared by H. Lechevalier, Institute of Microbiology). Figure 19. Sclerotium in species of Strepto- myces, designated as new genus Chainia by Thir- umalachar (Prepared by H. Lechevalier, Institute of Microbiology). 4. Verticillate cultures generally are ele- vated and floccose. Aberrant verticillate cul- tures generally are flat with a velvety tex- ture. 70 THE ACTINOMYCETES, Vol. II Fiaure 20. Sclerotium in species of Strepto- myces, designated as new genus Chainia (Prepared by H. Lechevalier, Institute of Microbiology). According to Krassilnikov (1955, 1959, 1960), the nature of the sporophore is a per- manent property, being straight in the S. globisporus group, spiral-shaped in the S. coelicolor and S. violaceus groups. The same constancy is true of the shape of the spores, cylindrical versus oval or spherical, and of the manner of spore formation, namely, frag- mentation versus segmentation. Branching of the sporophores, namely, vertical forma- tion versus monopodial branching, 1s also a constant, although a variable, property. No single property, however, 1s sufficient to characterize species. Coremia formation (Fig. 17) is of no taxonomic significance; however, production of sclerotia is believed to be a constant property, in agreement with Thirumalachar (1955), but not with Gattani (1957), who denied its significance (Tigs. 18-20). Cultural and Biochemical Characteristics FORMATION OF PIGMENTS Among the cultural properties of strepto- mycetes, the color of the substrate growth of the aerial mycelium and the spores and the formation of soluble pigments in organic and synthetic media play a major role in characterizing species. This fact is amply illustrated by the numerous specific epithets referring to color that have been used to designate various organisms. Unfortunately, color characteristics vary greatly with age of the culture, composition of the medium, temperature of incubation, and nature of the inoculum. Before the introduction of synthetic me- dia, it was a common practice to divide the actinomycetes into two groups: (a) colorless or nonchromogenic, and (b) pigment-produce- ing or chromogenic forms. The latter com- prised those organisms that produced deep brown to black diffusible pigments when grown on proteinaceous media. With the in- troduction of synthetic media, 1t came to be recognized that different organisms are able to produce a great variety of pigments, rang- ing from red to blue and from orange and yellow to brown and black. Some are single pigments, and others comprise two or more constituent pigments. Some are water-sol- uble and others are water-insoluble, as shown in detail in Chapter 13 of Volume I. The presence of oxygen is essential for pigment formation. The pH of the medium greatly affects the nature of the pigments, both in- soluble and soluble. When cultures are grown on optimum sporulation media, the pigmentation of the spores is highly significant; 1t may be ob- served at an early growth stage, at maturity, or only in old cultures, since changes in color may occur with age of culture. The formation of deep brown to black pig- ments on organic media containing proteins CHARACTERIZATION OF and protein derivatives, notably the amino acid tyrosine, 1s an important species charac- teristic. Certain species may produce only faint brown soluble pigments on organic media, as well as on synthetic media. Differ- ent cultures, especially on continued cultiva- ion artificial media, will variation in pigment production. Since about one third of all species of on show great Streptomyces now recognized are melanin- positive (Waksman, 1919; Skinner, 1938), and since this property has been utilized ex- tensively in the classification of actinomy- cetes, a knowledge of this reaction is of great importance. Gasperini (1891) first utilized this property in dividing the aerobic Actino- myces into A. chromogenus and A. albus. It was later recognized, however, that melanin production is characteristic of a large number of species, including such important forms as the plant pathogen S. scabies. Beijerinck (1900, 1911, 1913) designated as “melanin” the dark pigment produced by A. chromogenus from peptone, although this organism did not always produce the pig- ment from tyrosine. He considered the pig- ment as a catabolic product of the organic nitrogen. Lehmann and Sano (1908) first suggested the expression “‘tyrosinase reaction.”’? They used for their studies a tyrosine-containing medium, melanin being known to be an oxi- dation product of tyrosine. Waksman (1916, 1919, that the production of a soluble dark pig- ment on beef-peptone agar is due solely to this reaction. Gelatin, containing no tyro- 1920) expressed considerable doubt sine, gives the characteristic pigmentation. Some species producing a typical dark pig- ment on the beef-peptone agar may fail to do so on synthetic media containing tyrosine. Skinner (1938) recognized a difference be- tween the dark pigment produced in peptone media and not in tyrosine-containing syn- thetic media and the black pigment pro- STREPTOMYCES SPECIES rial duced in tyrosine (or protein containing tyrosine) media. The tyrosinase reaction was, therefore, considered to be the proper one Shinobu (1958) attached great importance to the “tyrosinase reaction”’ in the species characterization of Streptomyces. Ettlinger et al. (1958) also recognized the difference between melanin formation and the tyrosi- In line with the of Beijerinck, Waksman, and Ettlinger et al., nase reaction. ideas recognition will be given here to melanin formation rather than to the tyrosinase re- action. The formation of yellow, red, blue, green, and other soluble pigments is also highly characteristic of the species growing on syn- thetic media. There is considerable variation in the intensity of these pigments, depending upon the strain of organism. In view of the fact that color standards are not always available, Lindenbein (1952) suggested a series of color designations which are simple and convenient. This system in a modified form is given in Appendix I. Pigment formation is considered by Kras- sinikovy (1960) as a constant specific prop- erty, although the nature of the pigments varies with the composition of the medium. The color of the aerial mycelium is not con- sidered as constant and is greatly influenced by the composition of the medium (see also Conn and Conn, 1941). The variability in pigmentation of differ- ent strains of S. aureofaciens was studied in detail by Duggar et al. (1954) and Backus et al. (1954), and is illustrated in Table 7. UTILIZATION OF CARBON SOURCES The ability of different species of actino- mycetes to utilize as sources of carbon and energy various organic substances, such as ot acids, fats, and amino compounds, can be of carbohydrates, alcohols, salts organic considerable diagnostic value. These studies “UOTYBOTUNUTUTOD [eUuOsSTOd y quousid euou JueU | ABIS puw yuRpUNqe | | 18°9 ad ‘uotjeztu0}4 YSIUMOIG UOTy auou Juss (d apqnyos -std afqnyjos Spato |) wmMtpaoA [eioe | | -ded pure uorepnseoy | -ovjonbiy poor | fystumorq uly YyMOdy | -[Od-a8uR10 YIMOL) |S paloloo-WiRadd YAMOL peta Mor | ETE | UMOL | YSIUMOLG oI M -ysydand = yuawisid yurey quewisid afqnyo JO SeoBiy WM Ta OJIYM JO SooBIy WNIT =| aqnyOs fasor yureyoy | oc, Ad uoly SOU M WMNITPOOAUL [BLA -AUL[BLIOB SYstUMOIgq =| -adXU [RBLIeR fpato OULYM WNTpOOAT [BIE | ‘uoteztuojded Suo0iyg | -ovyonbiy poor) | -ak fystumorq YyMOI) | JURPUNGe YIMOL) | -[O0-oFUR10 YjMOdr) | -oB SqQuRpUNqE YIMOLr) | FLLE } Ystumorqg JUdUL DED ABAIB-ISNOUL OF -sid apqnyos ‘oy1m Hd ‘ouou uoryeziu04 ouoU JUeUT auou yuaUsId ofqn O}LYM WNTPOOAUL PRL juRpunqe UWUNToo -ded fauou uo0ryRy uoly -31d_ ayqnjos = {paao -[OS !poto[oo-wiReado -O8 SO9SUBIO JULB] OF -AUL [BLO SYSTUMOLG “NSBOD $SuULl ooVpINng | -ovjonbiy poor) | -[Oo-ureedo UIQ} YYMOL) | JURPUNGeR YIMOIL) | polo[Oo-Weald YIMOL) | JURpPUNGeE yyMorry | eple so’. Hd | ‘ouou uoTyeztuoydoad yoryq | -9UOU = UOTYRENSBoo you ynq ‘yaep snyjd auou WNT] SOUT[BY[B SYSTuMorg euoly euou Jueustd a_qnyos jo Ojo Sprousyory -ddAUL [BIIOR SUMOIG | ylvp sult aoeying | -ovsonbiy ON | SYystumorg uly} yyMory | ‘ystdind yaep qymouy | -ystpdand IMO) SS9[10]OD YIMOIH | TILE A ABIB-ISNOW OF Lie ria YSIUMOLG auou JUOUI OFM UWUNTPIOAUL [BLL ‘ouou uolyeztuoydad uol} queustd aTqnyos “sid ajqnyjos !po1o0 -OB FASUBIO YUTL] 04 :ouou UOLYBINSvOY | -oBjonbIft poor) | Systumorq uTYy YIMOT) | -[Oo-asuUR10 YIMOLL) | potopoo-WiRI1) FIMO) Sso[1O]OO YYMOIT) | [TLE ABIS-ISNOUL OF | OVIYM WMITPOAUL [VIL aya | G99 auou JueWISTd a_qnyos auou yuawUsId apqnyos -98 SasuRIO JUIB] OF Aavoy wuntpoodu pe | Hd ‘yoyo o[qista ON | ZuOTpORFoNbITON | Systumorsq urgy yyMON) | SYystuMOrG yYS YIMOL | pocopoo-weedo YIMOAy | -oB SYSTUMOIG YMOLD — OTLE uoly auou yuetustd afqn auou JUaUUSId spqnyos OUOU LUN(PODAUL [BLO é -oBjenbIT poor) | -[Os SYystuMorg YIMOL) | SYSTUMOIG JUST YIMOIL) | ‘poaso[Oo-WRII) YYMOTL) Ssop{O]OO YAMOIY) — _GOLE yoryq | you ynq ‘yaep snyid auou WHI] | €0'°9 Hd ‘poqry SULL OOBJANS auou yususId afqnjos JO LO[ODO ‘prlousyory -d0AU [BLOB SUMOIG “13800 puB peoyIplwy poilojoo-lWibedg | SystuMOIG UIy) YAMoID | ‘ystjdind yaep yAMory | -Yystpdand YIMOL) | SSeP1O[OD YIMOLT) | SOLE YIUA uljRyas) Jese JUaTIINN 07810q dese auiseiedse-asoony‘y | {e3PR 9}RIVIU ASOIONG | ane (,1BSSNCT) SUdIOBjOIING “Gg fo suIDA]s JuaJaffip JO Su01]DIUDA JDLNZIN,-) 1 alavy CHARACTERIZATION OF the early work of Waksman (1919), who employed a synthetic solution to which he added various carbon- or ni- trogen-containing compounds. Liquid sub- date from strata were employed and cultures were incubated under static conditions. The use of static hquid substrata was later found to give misleading results. In some cases, uni- form inoculum distribution is not achieved unless considerable care is taken. Numerous studies (Pridham and Gottlieb, 1948; Bene- dict et al., 1955; ete.) indicate that solid substrates and different basal media later used (Table 8). Hata et al. (1953) found a correlation be- were tween the groups and types of organisms established on the basis of carbon utilization and their antistreptomytic and antibae- terial spectra. Zahner and Ettlinger (1957) did not at- tach major significance to the utilization of carbon sources for characterizing species of Streptomyces. They suggested that such in formation be combined with other criteria. None of the 125 cultures they studied could use dulcitol, for example. The best carbon sources for characterizing Streptomyces spe- cies were found to be raffinose, /-xylose, d- fructose, /-arabinose, and d-mannitol. Gor- don and Mihm (1959) considered as a species the malate, propionate, pyruvate, and succinate. characteristic utilization of acetate, None of the actinomycetes produce gas. Some are able to form acid, such as lactic, from certain carbon sources. Gordon and Smith (1954) used acid production from lae- tose, maltose, xylose, and mannose as one of the criteria for differentiating Nocardia and Streptomyces species. Gordon and Mihm (1959) later suggested for species separation the use of acid formation from glycerol, glu- cose, arabinose, erythritol, inositol, lactose, maltose, mannitol, and certain other carbon sources. Carbon source utilization by 12 natural STREPTOMYCES SPECIES TABLE 8 variant strains of S. aureofaciens (Backus ef al., 1954) No apparent utilization by Utilization by all variants Utilization variable with strain any variant Posi- | Nega- tive tive Sodium Glucose Galactose 11 i acetate* Sucrose Sodium 10 2 Sorbose Maltose citrate* Glycine Starches Levulose 9 3 Mannitol Dextrin Mannose 9 3 Arabinose Trehalose Lactose i) 3 Glycerol Magne- 7 5 | Sodium sium lac- succl- tate* nate* Xylose 6 6 Inulin * Used at 0.4 per cent level, all others at 1 per cent. PROTEOLYTIC ACTIVITIES Among the proteolytic activities of diag- nostic value in separating genera, liquefac- tion of gelatin, hydrolysis of casein, and pep- tonization of milk are very important. Species of Nocardia effect little, if any, liquefaction of gelatin, whereas most species of Streptomyces bring about liquefaction. The rapidity of liquefaction varies greatly. Some species show strong activity, and others give only limited liquefaction. This property, as well as milk peptonization, when combined with the ability of the species to produce brown to black pigments, provides significant criteria for species characteriza- tion. In a study of 477 cultures of Streptomyces, Stapp (1953) found only one that did not liquefy gelatin. Detailed studies on proteo- lytic activities of actinomycetes are found in the work of Waksman (1919), Jensen (1930), Gordon and Smith (1955), and Kutz- ner (1956), as well as in Vol. 1, pp. 183-186. Waksman (1919) reported that of 35 cultures tested, 33 liquefied gelatin more or less rap- 74 THE ACTINOMYCETES, Vol. II idly; when these tests were repeated three times, considerable variation in the degree of liquefaction was obtained. IXutzner (1956) kept gelatin cultures for 31 days; only four of 210 failed to liquefy the gelatin. Reports of inability to liquefy gelatin or coagulate or peptonize milk of certain species may often be questioned. Repeated tests with different inocula might have shown different results. Gordon and Smith (1955) suggested casein hydrolysis as one criterion for the separation of Streptomyces strains from those of Nocar- dia. Stapp (1953) reported further that in his collection 18 cultures brought about coagula- tion of milk without subsequent peptoniza- tion, 431 caused coagulation and peptoniza- tion, and 19 caused peptonization without previous coagulation. A few cultures ocea- sionally are found that cause no coagulation or peptonization. One wonders whether re- peated tests with different inocula might show different results. REDUCING PROPERTIES The reduction of nitrate to nitrite has been universally used among the criteria for species differentiation. In view, however, of the influence of nutritional factors upon this reaction, and its quantitative rather than qualitative nature, its significance in species characterization may be questioned. Proteolysis, starch hydrolysis, sucrose in- version, cellulose utilization, and nitrate re- duction were said (IXrassiinikov, 1960) to be characteristic of all actinomycetes and to have, therefore, no taxonomic signifi- almost cance. Sugar assimilation was considered, however, as a more or less constant property. UTILIZATION OF NITROGEN SOURCES As a rule, utilization of nitrogenous com- pounds has not been considered important for species characterization. Shinobu (1958) considered the utilization of urea, creatinine, and certain amino acids as of some impor- tance in species characterization. Gordon and Mihm (1959) suggested that the ability to attack casein, tyrosine, or xanthine can be considered of some significance in charac- terizing species. The use of hydrogen sulfide production as a taxonomic implementation in the differen- tiation of Streptomyces species has been sug- gested by Pridham (1948). Tresner and Danga (1958) later modified the peptone- iron agar medium. More than 900 strains belonging to one or another of 94 species were tested. There was a marked difference in response from strain to strain within a species; for example, 98 per cent of 227 strains of S. hygroscopicus were negative; 99 per cent of 112 isolates of S. lavendulae were positive. When employed in conjunction with other physiological, cultural, and mor- phological criteria, hydrogen sulfide produc- tion was said to give promise as an aid in the systematics of the genus Streptomyces. Sensitivity to Antibiotics Actinomycetes, especially species of Strep- tomyces, have been found in recent years to produce a series of highly valuable chemical substances, notably, antibiotics. This prop- erty has come to be considered as highly characteristic of a given species. The fact that a large proportion of all the cultures of Streptomyces isolated from natural strates show some degree of inhibition of growth of other microorganisms, when tested sub- on suitable media, suggested the ability to form antibiotics to be of potential diagnostic value. It is a question whether one is a “Jumper” or a ‘splitter’? when one regards the ability to produce an antibiotic as a species rather than a strain characteristic. Certain antagonistic strains belonging to the S. griseus group, for example, are able to produce various streptomycins and eyclo- heximide. Others may form various actino- mycins, grisein, streptocin, or candicidin. It has been suggested that because the CHARACTERIZATION OF STREPTOMYCES SPECIES growth of homologous strains of an organism is less inhibited than that of heterologous forms, added weight could be given to the potential diagnostic value of antibiotic pro- duction. The application of the concept of antibiosis as a major characteristic for the speciation of actinomycetes is not generally accepted, since the metabolism of these or- ganisms is too complicated to give sharp lines of autoinhibition. At most, it can be a varietal rather than a species characteristic. Krassilnikov (1950, 1958, 1960a, 1960b) tended to overemphasize the importance of antibiotics in species characterization of ac- tinomycetes. He made two unjustified as- sumptions: (a) every species synthesizes only one particular antibiotic, (b) antibiotics do not inhibit the growth of organisms belong- ing to the species producing such antibiotics. Many species and even individual cultures are able to form a variety of different anti- biotics; on the other hand, the same anti- biotic may be produced by different organ- isms. The growth of an organism may in some instances actually be inhibited by its own antibiotic, as with S. fradiae and neo- mycin. Finally, a single culture may produce mutants which either have lost the ability to form a particular antibiotic or have gained the ability to synthesize a totally different antibiotic. It is somewhat dangerous to use assumptions and generalizations as the basis for species characterization. The importance of considering antibiotic formation in the systematization of actinomycetes has also been emphasized by Gause (1955). Actinophage Sensitivity During the last 10 years several attempts have been made to determine whether “phage-typing” of actinomycetes might be of some help in identifying unknown isolates. The results obtained point to several facts which must be kept in mind if one tries to use this procedure for characterizing and Classifying Streptomyces species. ~J ) 1. Actinophages vary greatly in their host ranges. a. Most which tested against a large number of organisms proved to be polyvalent; that is, they lyse cultures that belong to different species or even genera (different according to our pres- ent species concept, which is based on the system presented here). The data presented by Bradley and Anderson (1958) might serve as an illustration (Table 45, Volume I). The activity of some phages upon members of the genera Streptomyces and Nocardia led these workers to question the validity of separating these two genera, which have ac- tually been placed in two different families within the order Actinomycetales. Activity of a polyvalent Streptomyces phage on two Nocardia species was also found by Gilmour et al. (1959). In a study carried out by St. Clair and McCoy (1959), however, nine phages which proved to be polyvalent against several Streptomyces species failed to attack any of the four Nocardia species tested. The polyvalent character of other phages tested against other species has been shown also by other investigators (Hoehn, 1949; Chang, 1953, Rautenstein and Kofa- nova, 1957; Gause et al., 1957; Mach, 1958; Shirling, 1959a, b; Kutzner and Waksman, actinophages were 1959a; Kutzner, 1960). Obviously, therefore, a phage characterized by a wide host range is usually of little value in species differ- entiation, unless one is inclined to be a “Jumper”? who demonstrates by the use of a polyvalent phage that he is right and the “splitters”’ are wrong. b. Some phages have been found to be specific, causing the lysis of strains of only a few species or of only certain strains of one species. In the latter case one might be justi- fied in doubting the uniformity of the spe- cies rather than in considering the phage as “‘superspecific.”’ This seems to be definitely true of the species ‘‘S. griseus,’’ various 76 THE ACTINOMYCETES, Vol. II strains of which show a very different sensi- tivity pattern against certain phages. It is true, likewise, of the separation of streptomycin-producing strains from grisein- producers and other members of the former S. griseus, which is now regarded as a species group rather than a single species (Waks- man, 1959). There have been various reports concerning phages which are active upon streptomycin-producing strains, but do not attack grisein-producers or nonantibiotic- forming cultures (Woodruff et al., 1947; Waksman et al., 1947; Reilly et al., 1947; Waksman and Harris, 1949; Hoehn, 1949; Carvajal, 1953; Burkholder eé al., 1954). Some streptomycin-producing cultures have been found, however, that are resistant to these specific phages (Okami, 1950; Carva- jal, 1953; Kutzner, 1960). Other phages have been discovered that are specific against S. coelicolor (Kutzner and Waksman, 1959a; Kutzner, 1960), S. lavendulae (Gause et al., 1957; Shirling, 1959), and SS. olzvaceus (KXha- vina and Rautenstein, 1958). 2. No general conclusion can be drawn from the spectrum of a polyvalent phage in regard to relationships between lysed strains. However, a polyvalent phage can be useful in taxonomic studies if it shows specificity within a particular group of organisms that are very similar in their other properties and therefore hardly distinguishable. Fur- ther, testing several polyvalent phages might result in typical sensitivity patterns of the organisms which might be of some value in recognizing whether one has to do with closely related or unrelated organisms. 3. Actinophages vary greatly when tested against numerous strains. In some cases dif- ferences in plaque counts might be due to host range mutants which are present at a concentration of 10° to 10° particles. These mutants would attack a ‘‘new host”’ resist - ant to the parent phage, as shown by Welsch (1954, 1957) and Welsch et al. (1957). In numerous other cases, however, the devel- opment of host range mutants cannot ex- plain the wide host range, and the phages retain their polyvalent nature even after several serial passages on heterologous hosts (Chang, 1953; Shirling, 1959a; Bradley, 1959; Gilmour et al., 1959). It is necessary to carry out phage tests with different dilu- tions of the original phage preparation, which should contain about 107 to 10° par- ticles per milliliter. 4. A survey of the literature shows that almost every investigator uses a different medium for phage typing. The methods comprise either spot tests or single plaque counts. In a comparative study of different media for phage typing, Kutzner (1960) found that some phages gave similar plaque counts on a variety of media. However, counts of phages that formed tiny plaques were found to be quite dependent on media composition. Inorganic salt content of media was found to influence plaque counts most strikingly. Some phages gave no plaques on media NaCl but gave high plaque counts when plated on the same me- dium without NaCl (with or without CaCl,), while other phages showed higher activity on NaCl than on CaCl, media. The expres- sion of phage activity is apparently influ- enced by a great many unknown factors. One of the phages lysed some strains with a medium containing NaCl and gave high plaque counts, but showed no activity against other strains on the same medium. These results suggest that a medium found containing optimal for one host-phage system might be quite unsuitable for another. Before a phage is typed against a large number of strains, an optimal medium must be developed. Bet- ter still, tests should be carried out with several different media, selected for their usefulness with particular strains. Serological Reactions Use of serological techniques, particularly those of agglutination and precipitation, has CHARACTERIZATION OF STREPTOMYCES SPECIES Ub been suggested for species identification of actinomycetes. Aoki (1935-1936) was thus able to differentiate between representatives of three genera, Actinomyces, Nocardia, and Streptomyces. By means of sonic vibrations, Ludwig and Hutchinson (1949) prepared an- tigen suspensions satisfactory for use in ag- glutinin and precipitin reactions and for the production of immune sera in rabbits. Use of such suspensions in the identification of actinomycetes was suggested by Yokoyama and Hata (1953) and Hata et al. (1953). A purified antigen of a streptomycin-producing strain was found active against immune sera of the same strain, but not against sera of other antibiotic-producing organisms. These investigators were thus able to establish the close relationship of luteomycin- and chlor- amphenicol-producing organisms. Ochoa and Hoyos (1953) found a correla- tion between microscopic morphology and serological reactions which made it possible to divide the actinomycetes into four groups: Group 1, including species of Ac- tinomyces and Nocardia; Group 2, made up largely of Nocardia; Groups 3 and 4, com- prising species of Streptomyces. Slack et al. (1951), however, found that antisera pre- pared with A. bovis brought about low titer agglutination of Nocardia and of two species of Streptomyces. They concluded that a close antigenic relationship exists between mem- bers of the genus Actinomyces and that there is a group relationship among Actinomyces, Nocardia, and Streptomyces. Okami (1956) found definite antigenic relationships be- tween strains of closely related forms of S. lavendulae. See also Tanaka et al., 1959. Chemical Composition A detailed study of the chemical composi- tion of cells of actinomycetes has been pre- sented in Volume I (pp. 158-163). The occurrence of specific chemical com- pounds in the cells of the organisms suggests possible differentiation between groups of actinomycetes. This is true, for example, of the occurrence of diaminopimelic acid, a constituent that may prove to be of generic rather than specific significance. Romano and Sohler (1956) and Sohler et al. (1958) have shown that cell walls of streptomycetes can be solubilized by lysozyme, suggesting the presence of a mucopolysaccharide; on the other hand, cell walls of nocardiae do not possess this property. Ecology The natural substrate of an organism, es- pecially diseased plants or animals, and com- posts of stable manures and plant residues at high temperatures, is of some systematic significance. Various attempts have been made to utilize the ecological characteristics of the actinomycetes as a basis of classifica- tion. Thus, the following rather broadly de- fined ecological categories have been pro- posed at various times to classify actino- mycetes: a. Animal parasites. b. Plant parasites. c. Soil inhabitants. d. Water inhabitants. e. Mesophilic forms. Thermophilic forms. Inhabitants of acidic (pH 3 to 6.5) sub- strates. h. Inhabitants of neutral to alkaline strates (pH 6.5 and above). The temperature at which an organism is oq sub- erown greatly affects the nature and amount of growth, the nature and extent of sporula- tion, and the degree of formation of soluble pigments. The optimum temperature for the growth of most species of Streptomyces is be- tween 25 and 30°C. Only a few of these or- ganisms are thermophilic. Abilities to grow under mesophilic and thermophilic condi- tions have been recognized as important criteria for establishing species and even genera of actinomycetes and other microor- ganisms. 78 THE ACTINOMYCETES, Vol. II The optimum reaction for the growth of actinomycetes is pH 6.8 to 7.5. When these organisms are grown on complex organic media, and on many synthetic media, the reaction usually becomes alkaline. Some actinomycetes, however, are able to grow at pH 4.5 to 6.5 and even at pH 3.0 to 4.5. Such forms are not common, but the reaction of the substrate has been recognized as a potential diagnostic property. On the basis of their effects on dead resi- dues and upon living forms of life, actino- mycetes have been grouped as saprophytes and parasites, the latter being further grouped into plant and animal parasites. Thus we speak of ‘‘actinomycosis,” caused by A. bovis and A. 7sraelzi, and “nocardio- sis,’ caused by different species of Nocardia. We associate S. scabies with the ‘“seab”’ of potato tubers, and S. zpomoeae with a dis- ease of sweet potato roots. Genetics Little is known about the genetic proper- ties of actinomycetes and their possible bearing upon problems of classification. Certain observations have been made re- cently, however, which offer rather promis- ing leads in establishing species relationships. The concept of vegetative hybridization of Streptomyces cultures has been suggested. By repeated growth of a culture in a sterile fil- trate of sand-macerated mycelium of another culture, the former undergoes morphological and physiological changes. The significance of this phenomenon and its potential utiliza- tion for species characterization are still to be elucidated. Sermonti and Spada-Ser- monti (1956) demonstrated several types of recombination among ‘wild’? and mutant strains of S. violaceoruber). It has been brought out in Chapter 6 of Volume I that true hybrids can be obtained by mating two different mutant coelicolor (most probably S. strains of an actinomycete. Welsch (1958) suggested that mating may offer a conven- ient criterion for the practical definition of a species. The assumption was thereby made that a species is distinct if it does not cross, or gives only unfertile crosses with other similar species. Type Cultures An important, and often-used technique in species characterization of actinomycetes is that of comparing fresh isolates with type cultures. For the higher forms of plant life, species characterization is facilitated by study of preserved herbarium specimens. For microorganisms, special collections of named cultures are available for study. These cul- tures allow comparisons of living material, since dead or dried cultures are of but little significance. In establishing type cultures of actino- mycetes it Is important to keep in mind the fact that such cultures undergo considerable variation when grown for a long time upon artificial media. Some of the cultures may lose their ability to produce aerial mycelium and are thus deprived of properties of major diagnostic value. Unknown strains of Strep- tomyces free from aerial mycelium may even be considered as species of Nocardia. According to Pridham, * if reasonably fresh isolates were maintained on the proper media and preserved by lyophilization, the indi- vidual laboratory would experience far fewer difficulties than have been experienced in the past. Pridham reported that since 1953, with the use of these media and techniques, very rarely have strains been found that produce no aerial mycelium (which is generally well sporulated) on the isolation media; a very low incidence of strain degeneration has been noted in active cultures as determined by the methods of assessment, and all isolates have been routinely lyophilized. These lyophil tubes, opened from time to time, have been found to give cultures that are * Personal communication. CHARACTERIZATION OF STREPTOMYCES SPECIES 79 Fiaure 21. Formation of straight sporophores by Streptomyces sp., X 1500 (Courtesy of Miss A. Dietz, Dept. of Microbiology, Upjohn Co., Kalamazoo, Mich.). equivalent to the original soil isolates, as determined by the methods of assessment used. The prior growth of the organism in soil media (sterile soil treated with a small amount of CaCO, , if acid, and with a half per cent of dried blood) or in carbon- or ni- trogen-poor media, its refrigeration or its lyophilization—each or all tend to prevent degeneration and thus preserve the original characteristics of the type culture. The cul- tures that have already degenerated will tend to regain their original properties as a result of such treatments. Shinobu (1958) suggested the following method for making a soil medium: Into a test tube of 1.5 em diameter place 7 g dried fertile soil; add 1.5 ml of 2 per cent solution 80 THE ACTINOMYCETES, Vol. II of glycerol; make up the water content to about 20 to 25 per cent. Sterilize the tubes at 20 pounds for 20 minutes. The culture strains are inoculated on this medium and incubated at 28-30°C. When the growth of the organism is successful, white aerial my- celium appears first on the surface of the soil; when the culture matures the charac- teristic color of the aerial mycelium is pro- duced. There is always the danger that an old culture, transmitted from one laboratory to another, may either have become modified or have lost some of its original properties. It may have become contaminated, and the contaminant may eventually replace the original culture. One must also remember that different investigators have often based their descriptions of a particular species not upon the original culture but upon subse- quent isolates, which may or may not repre- sent the same species. Finally, many holo-type cultures are not available at all. Therefore, in some cases type cultures are not reported. When re- ported, they usually refer to the Institute of Microbiology Collection (IMRU), the American Type Culture Collection (ATCC), or to the Agricultural Research Service Culture Collection of the Northern Re- gional Research Laboratory, U.S. Depart- ment of Agriculture (NRRL). Other collec- tions include Kidige Technischen Hochschule (ETH) Zurich; Centraalbureau voor Schim- meleultures (CBS) Baarn; and Institute of Applied Microbiology, University of Tokyo (IAM). Standard Media Some media are more favorable than oth- ers for sporulation of Streptomyces cultures. In view of the importance of sporulation in characterizing a species (also in placing an organism in the proper genus), it is essential to select favorable media. Furthermore, since some forms tend to lose the property of sporulation on continued growth, special precautions must be taken in preserving such cultures. The loss of aerial mycelium may be reversible or irreversible. Since non- sporulating streptomycetes may resemble nocardiae and since certain nocardiae have been reported to produce aerial mycelium and spores similar to those of typical Strep- tomyces cultures, the element of confusion between the two genera always exists. Description of Streptomyces Species It is commonly believed that to charac- terize a species it 1s essential to describe a large number of its morphological and phys- iological properties. This procedure is not always helpful, especially if based upon un- reliable criteria. The medium may not be readily duplicated, or conditions of growth may be different, or the inoculum may not be prepared in the same way. Because of these and other variations, many cultures recently isolated have been described as new species. Another reason is that it is much vasier to create a new species than to at- tempt to correlate the characteristics of a freshly isolated culture with those of known species already described in the literature. Numerous species also have been created to facilitate the obtaining of patents. Hesseltine ef al. (1954) suggested that the following steps be taken in the taxonomic hew study of a Streptomyces species: 1. Collection of strains on pigmentation of aerial mycelium. 2. Study of the morphology of strains the basis of erowlng on a number of media favorable to sporulation. 3. Examination of the color of spores of strains growing on optimum sporulating me- dia. Five color groups were recognized: (a) lavender, red, or pink; (b) blue, blue-green, or green; (c) yellow; (d) white; (e) gray, gray-brown, olive-gray, or dark gray. 4. Study of cultural characters of strains on various synthetic and organic media. CHARACTERIZATION OF 5. Analysis of certain physiological and biochemical properties, notably action on gelatin, starch, milk, and peptone-iron agar; nitrate reduction; utilization of carbon and nitrogen compounds; antibiotic action, com- prising formation of and sensitivity to anti- bioties. 6. Identification known species, and preservation of cultures. Pridham* emphasized that recently he has been placing principal emphasis on micro- of new strains with morphology, secondary emphasis on chro- mogenicity (deep brown to black diffusible pigments), and tertiary emphasis on color of aerial mycelium. In an effort to determine whether freshly isolated cultures can be identified on the basis of published descriptions and what con- ditions justify the creation of new species, several obvious comparisons were made (Waksman, 1957). Certain strains that might be included in various important species or group-species were critically ex- amined. The following conclusions were reached: At present, various morphological, cultural, and biochemical properties are known which make it possible to establish definitely certain distinct species among the actinomycetes. Some of these characters are constant within certain conditions of nutrition and environment, others are varia- ble. Certain additional properties may be re- quired in order to establish the degree of varia- tion of a culture before it can be recognized as a new species. Certain categories of relationships among the actinomycetes must be taken into consideration in order to establish definitely the systematic position of a given culture. These may be briefly summarized as follows: 1. On the basis of all the accumulated evi- dence, actinomycetes are shown to belong defi- nitely to the bacteria. 2. The position of the true actinomycetes in relation to related bacterial forms, notably the * Personal communication. STREPTOMYCES SPECIES SI mycobacteria and corynebacteria, must be recog- nized; this is true especially of certain nocardial types. 3. The generic interrelationships among the actinomycetes are highly significant. The separa- tion of members of the genus Streptomyces from those of Nocardia is difficult, especially when one is dealing with nonsporulating forms of the first and sporulating forms of the second. The recent addition of two new genera, Actinoplanes and Streptosporangium, and the recognition of certain thermophilic groups as separate genera add fur- ther problems to these generic interrelationships. 4. Within each genus, certain groups, species- groups, or series must be recognized. A combina- tion of morphological and cultural permits the establishment Some of these comprise a large number of forms with many variable characteristics. properties of species-groups. 5. Differentiation of individual species within each group is based upon a combination of cul- tural and biochemical properties. The production of specific antibiotics and the utilization of differ- ent sugars are ample illustrations of this. 6. Cognizance of the strains and varieties within each species must finally be taken. This may be based upon certain qualitative properties, such as sensitivity to phages, or quantitative properties, such as production of a given anti- biotic, vitamin, or enzyme, or sensitivity to a given antibiotic. The fact that a culture becomes important for the production of a particular metabolic product, such as an antibiotic, an enzyme, or a vitamin, may impart to the culture particular significance for characterization purposes. The existence of physiologic races or varie- ties among species of actinomycetes, espe- cially among those placed in the genus Streptomyces, has been fully recognized. Just as in improving higher forms of life one is always faced with the selection of varieties resistant to disease, or giving higher yields, or having other desirable qualities, so one must select strains of actinomycetes on the basis of resistance to phage or of pro- duction of higher yields of a given antibiotic or other metabolic product. Ghva ip tier 49 Systems of Classification and Identification of Groups and Species of the Genus Streptomyces Principles of Separation of Genera The historical background and various systems of classification of the order Actino- mycetales in general and of the actinomy- cetes in particular have been discussed in detail in Chapter 4 of Volume I. The princi- ples underlying the generic and specific separation of the organisms are presented in Chapter 4 of the present volume. The variability and overlapping among genera and species have been emphasized in Chap- ter 6 of Volume I. Certain important factors pertaining specifically to the genus Strepto- myces must be considered before any dis- cussion is presented of the separation of this genus into subgenera, series (sSpecies-groups), species, and varieties. Among the factors that must be empha- sized in any attempt to classify actinomy- cetes, the following three are most impor- tant: (a) the nature of the substrate (or vegetative) growth and the nature of the aerial mycelium, if any; (b) the degree of variability of the cultures; and (c) the effect of the composition of the medium. To facili- tate recognition of the organisms and to establish constant and variable differences fol purposes, well-defined media and standard conditions of cultiva- tion must be used. classification Actinomycetes are differentiated from the true bacteria by their filamentous growth and by their true branching. It is often dificult, if not impossible, to distinguish between the profuse branching of certain mycobacteria and the short-lived mycelium of the nocardias, except for the fact that the latter produce mycelium consistently in the early stages of their development. There is a gradual transition between the mycobacteria and the nocardias. It also is often difficult to differentiate between the nocardias and the streptomyces. The latter are characterized the constant and marked nature of their aerial mycelium, whereas by characterized largely by the transitory and undifferenti- ated nature of this mycelium. the nocardias are In establishing differences between no- cardias and streptomyces, one must con- sider the following factors: 1. Nocardias usually have been consid- ered incapable of forming aerial mycelum that could be differentiated from the sub- strate mycelium. It also has usually been assumed that no spirals are ever formed the mycelium. and Mihm that certain nocardias are able to form aerial from Recently, however, (Gordon (1958) have reported mycelium similar to that of streptomyces GROUPS AND SPECIES OF and that spirals also may be formed. A streptomycete forms a characteristic aerial mycelium. This property may be lost, how- ever, on continued cultivation or under special conditions of treatment. The aerial mycelium frequently develops characteristic spirals, tufts (Fig. 22), or verticils (whorls). 2. A streptomycete usually multiples by the concentration and fragmentation of the protoplasm within a filamentous cell, fol- lowed by the dissolution of the cell mem- brane. The fragmented portions of the my- celium usually develop, under favorable conditions, into fresh mycelium, either by germ tubes or by lateral budding. Spores or conidia are produced. The substrate my- celium does not segment spontaneously into bacillary or coccoid forms, but remains non- septate and coherent even in old cultures, thus forming the characteristic tough tex- tured, leathery growth. 3. In nocardias, the aerial hyphae are believed to represent an upward extension of the substrate mycelium, and usually do not exhibit any differentiated protoplasm. When a streptomycete loses its capacity to produce aerial hyphae, a form analogous to that of a nocardia may result, except for the structure of the mycelium that faculty of the degenerated culture to regain and the lost capacity. 4. Another difference between nocardias and streptomycetes is the acid-fastness or partial acid-fastness of some of the forme! when grown in certain media; the latter are never acid-fast. As pointed out in Chapter 1, Gordon and Smith (1955) proposed six distinctive char- acters for the separation of the two genera. These criteria are: (a) colony structure; (b) casein hydrolysis; (¢) dissolution of tyrosine and xanthine; (d) acid production from glu- cose and glycerol; (e) lack of acid formation from arabinose, xylose, lactose, mannitol, and inositol; and (f) utilization of acetate, propionate, pyruvate, malate, and succinate. Strains of organisms giving positive re- GENUS STREPTOMYCES 835 Ficure 22. Tuft formation in aerial mycelium of the Streptomyces griseus type. sults in five or six of the physiological tests belong to Streptomyces; strains with four to six negative reactions, to Nocardia. Although considered as somewhat arbi- trary, these criteria allowed clear-cut generic separation of 97 per cent of 251. strains studied, regardless of their morphological variation. Ninety-six per cent of the strains received as Streptomyces were positive in five or six of the following reactions: hy- drolysis of casein, dissolution of tyrosine, and acid production from xylose, mannose, maltose, and lactose. Strains that no longer formed aerial hyphae and spores, but known ot ones, also were positive in five or six of the beled Nocardia gave negative results in four to be descendants typical sporulating these tests. Two-thirds of strains la- to six of the same tests. Of the remaining third of the straims 24 had the strains of Streptomyces and were assumed to received Nocardia, as same reactions as accepted 84 THE ACTINOMYCETES, Vol. II be mislabeled; seven were listed temporarily as Intermediates between the two genera. The differentiation between Streptomyces and the other genera of actinomycetes is not very difficult. The formation of aerial mycelium and the manner of sporulation are markedly distinct for Streptomyces as compared to A/icromonospora. Species of Thermoactinomyces also produce an aerial mycelium, similar to that of species of Streptomyces, but they form single spores, similar to those of A/icromonospora. The other thermophilic genera, as well as the genera Waksmania (Microbispora*), Actino- planes, and Streptosporangium also can be differentiated from Streptomyces, as shown in Chapters 8 to 11. Among the numerous species belonging to the various genera of actinomycetes, those of the genus Streptomyces are by fai the most important, largely because of their wide distribution, their greater abundance, and their ability to produce antibiotics and vitamins and to carry out important chemi- cal conversions. Hence a detailed considera- tion of this genus is justified. Description of Genus Streptomyces Streptomyces species produce a_ well-de- veloped mycelium. The diameter of the hyphae seldom exceeds 1.0 u and is usually only 0.7 to 0.8 un. The hyphae vary greatly in length: some are long with limited branch- ing; others are short and much branched. The substrate mycelium does not form cross walls; it does not break up into rod-shaped and coccus-like bodies. Reproduction occurs by means of spores or by bits of mycelium. Spores or conidia are formed in_ special spore-bearing hyphae or sporophores which arise from the aerial mycelium either mono- podially or in the form of tufts or verticils. * Both designations were published, in different journals, the same month and the same year. Priority has not been definitely established. The sporulating hyphae are straight or curved. The curvatures range from mere waviness to perfect spirals, which may be compact, in the form of fists, or long and open (Fig. 23). The spores of streptomycetes comprise four types: smooth, warty, spiny, or hairy. About one-third of the gray- to brownish- spored species were found (Tresner et al., 1960) to form spiny, warty, or hairy spores; the remainder were smooth-spored. All the blue- to blue-green-spored forms had spiny spores. White, vellow, cream, or buff types had smooth-walled spores. All the pinkish- tan-spored group had smooth spores, with the exception of S. erythreus and S. pur- purascens Which had spiny spores. The con- clusion was also reached that, because of the variation of spore size and shape, those properties are of lmited usefulness for taxonomic differentiation. The growth of Streptomyces ‘‘colomies’ on artificial media is smooth or lichenoid, hard and densely textured, raised, and ad- hering to the medium. The colony is usually covered completely or partially (in the form of spots or concentric rings) by aerial my- ? celium, which may be variously pigmented, depending on the species and on the com- position of medium. In liquid media, es- pecially in shaken cultures, growth of strep- tomyces is usually in the form of flakes, which gradually fill the container, or in the form of spherical growths; the former type of growth is the more desirable from the point of view of antibiotic production. Many of the cultures, either in the form of colonies on the surface of solid media or as flaky growth in submerged culture, may undergo rapid lysis. The production of anti- biotics usually corresponds with the lysis of the cultures. Frequently, the lysis is brought about by a phage, known as actino- phage, which exerts an injurious or destruc- tive effect upon the mycelium. GROUPS AND SPECIES OF GENUS STREPTOMYCES 85 FIGURE 23. 3ull. Torrey Botan. Club 82: 111, 1955). Classification Systems of the Genus Streptomyces Early Systems of Classification Although the earlier systems of classifica- tion of actinomycetes were also supposed to be concerned with all the forms usually in- cluded in this group, they represented Various strains of Streplomyces californicus (Reproduced from: Jurkholder, P. Re el al. largely those forms that are now included in the genus Streptomyces. This is true, for example, of the first classification of San- felice (1896), and the subsequent ones of Krainsky (1914) Waksman Curtis (1916). Only the more comprehensive and of and and more recent systems are presented here. The earlier ones were given in Chapter t of Volume L. 86 THE ACTINOMYCETES, Vol. II 1. WAKSMAN AND CURTIS (1916) SYSTEM This system was based upon formation of soluble pigments in organic media, rate of gelatin liquefaction, and structure of aerial mycelium. A. Gelatin rapidly liquefied; no brown pigment. I. Spirals formed. 1. No soluble pigment on synthetic media. Actinomyces Rutgersensis 2. Pigment formed on synthetic media. a. Pigment dark blue. Actinomyces violaceus-Caesert b. Pigment brown. Actinomyces diastaticus II. No spirals. 1. No soluble pigment. a. Growth orange-red, aerial mycelium white. Actinomyces albosporeus Fraure 24. Sporophores of Streptomyces sp. producing spirals, X 1500 (Courtesy of Miss A. Dietz, Dept. of Microbiology, Upjohn Co., Kalamazoo, Mich.). ———— GROUPS AND SPECIES OF GENUS STREPTOMYCES 87 b. Growth rose-colored, aerial mycelium rosy. Actinomyces Fradit ce. Growth a mixture of white and yel- low. al. No conidia. Actinomyces albo-flavus bt. Abundant conidia. a2. Conidia rod-shaped, powdery, gray-yellow. Actinomyces griseus b?. Conidia spherical and oval, growth compact, citron-yel- low. Actinomyces citreus d. Growth at first colorless, then brown to black. al. Aerial mycelium white. Actinomyces alboatrus bt. Aerial mycelium dark gray. Actinomyces Lipmanit . Soluble pigment produced. a. Soluble pigment green. bo Actinomyces Verne b. Soluble pigment dark blue. Actinomyces violaceus-niger B. Gelatin liquefied; brown pigment formed. I. Spirals produced. a. Growth rose-colored; aerial mycelium rosy. rapidly Actinomyces roseus b. Growth colorless; aerial mycelium golden brown. Actinomyces aureus ce. Growth slightly brown; aerial mycel- ium white. Actinomyces Halstedii Il. No spirals. 1. No soluble pigment on synthetic media. a. Growth red to red-orange; no aerial mycelium. Actinomyces Bobili b. Growth white; aerial mycelium white. al. Aerial mycelium thin, rare, net- like. Actinomyces reticult b!. Aerial mycelium thick, white to gray. Actinomyces albus 2. Soluble brown pigment produced on synthetic media. a. Aerial mycelium white, abundant. Actinomyces diastato-chromo- genus b. Aerial mycelium white, late or not at all. produced Actinomyces group ce. Growth green; aerial mycelium white. chromogenus Actinomyces — virido-chromo- genus C. Gelatin slowly liquefied; no soluble pigment. I. Spirals produced in aerial mycelium. 1. Soluble red and blue pigments. Actinomyces violaceus-ruber 2. No soluble pigment; substrate growth red. Actinomyces Californicus Il. No spirals produced in aerial mycelium. 1. Growth yellow; no soluble pigment. Actinomyces parvus 2. Growth tends to crack; soluble brown pigment. Actinomyces exfoliatus D. Gelatin slowly hquefied; brown pigment pro- duced. I. Spirals produced; aerial mycelium laven- der. Actinomyces lavendulae II. No spirals. 1. Growth yellow; aerial mycelium gray. Actinomyces flavus 2. Growth colorless; aerial mycelium purplish-white. Actinomyces purpurogenus 3. Growth black; aerial mycelium scant. Actinomyces — erithrochromo- genus 4. Growth purple; no aerial mycelium. Actinomyces purpeo-chromo- Genus 2. WAKSMAN SYSTEM (1919) This was a modification of the previous system and was based upon a study of 41 species. An examination was made of the morphology of the aerial mycelium on two media; growth, aerial mycelium, and solu- ble pigment on 12 different media; various biochemical properties, such as carbon and nitrogen utilization, proteolytic activities, diastase and invertase formation, reduction of nitrate to nitrite, and change in reaction of medium. A brief outline is presented here: A. Soluble pigment produced on organic media. Py ia 4 {/ (/ P qi a - 5 turns; melanin-positive. 11. Streptomyces pilo- SUS * The verticillate nature of these organisms is open to question. FIGuRE 27. Sporogenous coiled hyphae of Strep- tomyces T 3110; taken from a gray area of a colony (Reproduced from: Duggar, B. M. ef al. Ann. N. Y. Acad. Sci. 60: 71-85, 1954). b®. Spirals with <5 turns, melanin-negative. 12. Streptomyces flaveo- lus B. Spores smooth. I. Aerial mycelium yellowish- to greenish- gray. 1. Melanin-negative. 13. Streptomyces — gri- seus 2. Melanin-positive. 14. Streptomyces michi- ganensis Il. Aerial mycelium white. 1. Sporophores in verticils. 15. Streptomyces rubri- reticult 2. No verticils produced. a. Sporophores form spirals. 16. Streptomyces nive- oruber b. Sporophores straight or wavy. al. Melanin-negative. 17. Streptomyces fulvis- STMUS b!. Melanin-positive. 18. Streptomyces phaeochromogenes LOO THE ACTINOMYCETES, Vol. II IIT. Aerial mycelium hght carmine to brown- ish. 1. Sporophores in verticils. a. Sporophores straight or wavy. 19. Streptomyces ne- tropsis b. Sporophores in spirals. 20. Streptomyces tendae 2. Sporophores not in verticils. a. Sporophores straight. 21. Streptomyces vene- zuelae b. Sporophores in spirals. al, Spirals at end of long, straight sporophores; melanin-positive. 22. Streptomyces laven- dulae b!. Spirals different; melanin-nega- tive. a®. Spirals closed. 23. Streptomyces — vio- laceoniger b?. Spirals open. a’. Spirals regular, usually >5 turns. 24. Streptomyces — fra- diae b*. Spirals irregular, usually <5 turns. 25. Streptomyces ery- thraeus IV. Aerial mycelium ash-gray. 1. Sporophores in verticils. 26. Streptomyces — reti- cult 2. Sporophores not in verticils. a. Sporophores straight or wavy. & FIGURE 28. Sporogenous coiled hyphae of Streptomyces T 3110; taken from a blue sector of a colony (Reproduced from: Duggar, B. M. et al. Ann. N. Y. Acad. Sci. 60: 71-85, 1954). GROUPS AND SPECIES OF al, Sporophores sympodially branched. 27. Streptomyces — viri- dogenes . Sporophores monopodially branched. 2. Sporophores as side branches of sterile hyphae. 28. Streptomyces ramu- es) « c losus 2. Sporophores different. a’, Melanin-negative. 29. Streptomyces oliva- ceus b’. Melanin-positive. 30. Streptomyces bioticus b. Sporophores in spirals. al. Spirals closed. 31. Streptomyces hygro- SCOptcus antt- b!. Spirals open. a®. Spirals irregular, usually >5 turns. 32. Streptomyces aureo- faciens b?2. Spirals regular, <5 turns. a’. Melanin-negative. 33. Streptomyces — par- vullus bs. Melanin-positive. 34. Streptomyces gali- laeus 8. SHINOBU SYSTEM (1958b) Shinobu proposed the following system pro} £ Sy} for grouping of the species of the genus Streptomyces: Group I. Monopodial branching, straight or wavy aerial mycelium; never producing spirals. Subgroup 1. Tyrosinase reaction: positive; ni- trite production: positive. Streptomyces olivaceus ~ positive ; nitrite production: negative. Streptomyces phaeopurpureus Subgroup 2. Tyrosinase reaction: Ow reaction: negative; nitrite production: positive. Streptomyces sp. No. 2 Tyrosinase reaction: negative; nitrite production: negative. Streptomyces sp. No. 232 Group IL. Spiral formation; long or short, loose or compact, and open or closed spirals. Subgroup 3. Tyrosinase Subgroup 4. ‘ GENUS STREPTOMYCES 10] Subgroup 5. Tyrosinase reaction: positive; nitrite production: positive. Streptomyces viridochromo- genes Subgroup 6. Tyrosinase’ reaction: positive; nitrite production: negative. Streptomyces sp. No. 2076. reaction: . Tyrosinase negative; “I Subgroup nitrite production: positive. Streptomyces sp. No. 236 negative; nitrite production: negative. Streptomyces scabies Subgroup 8. Tyrosinase reaction: Verticil formation; primary and verticils; rarely one tertiary verticil Group III. secondary (Fig. 29). Subgroup 9. Tyrosinase reaction: positive; nitrite production: positive. Streptomyces hiroshimensis Tyrosinase reaction: positive; nitrite production: negative. Streptomyces luteoverticillatus Tyrosinase reaction: negative; nitrite production: positive. Subgroup 10. Subgroup 11. Streptomyces roseoverticilla- tus reaction: negative; nitrite production: negative. Streptomyces olivoverticillatus Group IV. Intermediate group of Nitella-type and Anitella-type verticil. Subgroup 13. Subgroup 12. Tyrosinase Streptomyces spiroverticillatus Shinobu further emphasized that in the identification of species of Streptomyces other characteristics should be considered. These are the following: 1. Morphological properties: type of colony, shape of spiral, etc. 2. Physiological nase, and amylase reactions; utilization of carbon properties: cellulase, man- and nitrogen sources, etc. 3. Cultural properties: growth of colony, pro- duction of pigment, ete. 9. FROMMER SYSTEM (1959) This system does not apply to the genus Streptomyces as a whole but only to the actinomycin-producing species. A. Chromogenic group. I. Spirals not formed on aerial mycelium. Occasionally a few spirals are found. 102 THE ACTINOMYCETES, Vol. II Ficure 29. Verticil formation (Nitella type) including both primary and secondary verticils (Re produced from: Shinobu, R. Mem. Osaka Univ. Lib. Arts and Ed. B. Nat. Sei. 7, 1958). 1. Aerial mycelium on_ synthetic media gray. Tyrosinase-negative. Streptomyces antibioticus mycelium on agar bo Aerial synthetic media yellow. Tvrosinase-positive. agar Streptomyces michiganensis Il. Numerous spirals produced on aerial my- celium. 1. Yellow or yellow-green pigment duced on synthetic agar. pro- Streptomyces galbus 2. Soluble pigment on synthetic agar, dark brown. Streptomyces lanatus B. Nonchromogenic group. I. No spirals on aerial mycelium. 1. Strongly proteolytic. Aerial mycelium on synthetic agar white, yellow, or greenish. Streptomyces chrysomallus 2. Weakly proteolytic. Aerial mycelium on synthetic agar mouse-gray. Streptomyces chrysomallus v. fumigatus II. Numerous spirals on aerial mycelium. 1. Aerial elycerol-glycine agar grayish-rose. Practically no growth mycelium on on synthetic agar. Streptomyces murinus faciens, S. 2. Abundant growth on synthetic agar. Aerial mycelium on agar cream-colored. glycerol-glycine Streptomyces galbus v. achro- mogenes 10. MAYAMA SYSTEM (1959) Mayama (1959) concluded that morphol- ogy, and types. of growth on liquid media are the most impor- serological reactions, tant properties for the classification of Streptomyces. On the basis of these prop- erties, he divided the genus into seven groups: S. olivaceus, S. lavendulae, S. aureo- griseolus, S. albus, S. rimosus, and S. reticult. In addition to these, he also listed a number of species for which no group char- acteristics were known, notably S. anti- bioticus, S. fulvissimus, S. ruber, S. coelicolor, etc. Mayama (1959) and Mayama and Ta- wara (1959) classified the genus Strepto- myces into five sections and 14 series: Section I. Aerial mycelium — irregularly branched. Sporophores produced at the GROUPS AND SPECIES OF GENUS STREPTOMYCES FrGureE 30. Spore formation in Streptomyces species (Reproduced from: Shinobu, R. Mem. Osaka Univ. Lib. Arts and Ed. B. Nat. Sei. 7, 1958). terminal portion of the branching hyphae. Series 1. Sporophores straight to flexu- ous. Series 2. Sporophores form open loops. Series 3. Sporophores form spirals. Section II. Aerial mycelium branches in tuft formations. Sporophores produced at the terminal portion of the branching hyphae. Series 4. Sporophores straight to flexu- ous. Series 5. Sporophores form open loops. Series 6. Section ITI. main stem. Sporophores produced at the Sporophores form spirals. Aerial mycelium forms long terminal portion of side branches. Non- verticillate. Series 7. Sporophores straight to flexu- ous. Series 8. Sporophores form open loops. Series 9. Sporophores form spirals. Section IV. Aerial mycelium forms long main stem. Sporophores produced at terminal portion of side branches. Ver- ticillate. Series 10. Monoverticillate, straight to flexuous. Series 11. Series 12. Monoverticillate, spirals. Biverticillate, straight to flexu- OuS. Series 15. Section V. Series 14. Biverticillate, spirals. No aerial mycelium. 11. NOMI SYSTEM Finally a purely morphological system may Nomi (1959) proposed a division of the genus Streptomyces into eight be listed. morphological groups. He returned to an earher concept of Drechsler (1919) that the nature of the turn of the spirals, namely sinistrorse and dextrorse, is an important characteristic of Streptomyces species. He recognized, however, that some cultures may be rather indefinite in this respect. A. Aerial hyphae somewhat flexuous or straight; few long hyphae. The terminal filaments de- velop into spiral-shaped sporophores. 1. Spirals sinistrorse. 104 THE ACTINOMYCETES, Vol. II re te ME SR Y ob 10 11 12 13 oO Puiate III. Morphological groups in the genus Streptomyces (Mayama, 1959) (For details, see text, pp. 102-103). Section I: series 1—3 Section IT: series 4—6 Section IIT: series 7—9 Section IV: series 10—18 B: D. ee GROUPS AND SPECIES OF a. Spirals long, extended to compact. S. coelicolor, S. albogriseolus, S. fla- veolus, S. parvullus b. Spirals compact to compressed. None found. 2. Spirals dextrorse. a. Spirals long, extended to compact. S. viridochromogenes b. Spirals compact to compressed. Streptomyces sp. No. 189 Most aerial hyphae long, straight or slightly flexuous. They do not sporulate, but give rise to short side branches whose terminal filaments develop into spiral-shaped sporophores. 1. Spirals sinistrorse. S. purpurascens 2. Spirals dextrorse. Various unidentified forms. . Aerial hyphae irregularly flexuous or wavy; long hyphae absent. Terminal filaments form spirals. 1. Spirals sinistrorse. S. sulphureus 2. Curvature of spiral indefinite. S. griseoluteus Aerial hyphae long, straight, or wavy. They give rise to short side branches, which develop into spore-bearing hyphae containing spirals. 1. Spirals sinistrorse. S. hygroscopicus, S. violaceoniger, S. albus . Aerial hyphae in clusters. The terminal fila- ments develop into sporophores, both spiral and nonspiral forming. S. vinaceous, S. microflavus, S. fra- diae, S. lavendulae, S. roseochromogenes, virginiae, S. cinnamonensis, S. S. phaeochromogenes Aerial hyphae branch in clusters. No spirals or loops. S. venezuelae, S. tanashiensis, S. bikiniensis, S. antibioticus, S. aureo- faciens, S. olivaceus, S. nitrosporeus, S. griseus, S. lipmanii, S. rutgersensis, S. parvus, S. flavovirens, S. californi- cus, S. vinaceus, S. ruber, S. caeruleus Aerial hyphae long, straight or slightly flexu- ous. Verticillate. No spirals. S. reticuli, S. griseocarneus, S. echi- mensis, S. hiroshimensis, S. salmoni- cida, S. thioluteus, S. albireticuli, S. netropsis . Aerial hyphae somewhat flexuous or wavy. Long hyphae and spirals are not produced. S. albus (atypical), S. halstedii, S. GENUS STREPTOMYCES 105 scabies, S. verne, S. griseolus, S. erythreus (Plates IV and V) 12. OTHER SYSTEMS Other systems have been proposed for the the Some of these systems are modifications or supplementations of that presented in Ber- gey’s Manual (7th ed., 1957), or modifica- tions of one or the other of those outlined in classification of genus Streptomyces. this chapter. One of these is the system outlined by Routien (1959). The various species in- cluded in the Streptomyces divided into three major groups: (1) sapro- phytes; (2) plant parasites or cultures isolated from diseased plants or from soil in which diseased plants were grown; (3) cul- tures isolated from animal tissues. These genus were groups were subdivided on the basis of for- mation and color of aerial mycelium (green, brownish-purple to black, blue-gray or blue- green, yellowish to orange, pink to rose, ete.). The color of the substrate mycelium and the various biochemical properties were then used for further subdivisions. Morphology (spiral formation, shape of spores) played only a minor role in this system. See also Sakai, 1959. Summary of the Properties Used in Subdividing the Genus Streptomyces Evaluation of the above systems of clas- sification leads to the conclusion that sporo- phore morphology has been given first or second consideration by the great majority of investigators. Lesser attention was paid to the color of the aerial mycelium and the nature of soluble pigments. Chromogenesis, or pigment formation in protein media, Was often given first position. Antibiotic pro- duction and ecology received the least con- sideration. In Baldacci’s system of dividing the genus into groups or series, the color of the sub- THE ACTINOMYCETES, Vol. II 106 e ‘ a . y A ie a) ‘J | er ae 2x) Q 2 Puate IV. Morphological types, according to Nomi (1959). a. represents a schematic presentation of each type; b. gives the actual photograph. A. Aerial hyphae flexuous or straight; spirals extended to compact (S. coelicolor). B. Aerial hyphae straight; spirals on side branches (S. purpurascens). (See continuation, next plate). GROUPS AND SPECIES OF GENUS STREPTOMYCES LO7 PLate IV. (Continued) C. Aerial hyphae wavy; spirals on terminal filaments (S. griseoluteus). D. Aerial hyphae long, straight or wavy; sporophores as side branches; spirals produced (S. hygroscopi- cus). 108 THE ACTINOMYCETES, Vol. II ae ine a oi { é om Ly. - fi ; 4 y PLtate V. Morphological types, according to Nomi (1959). a. represents a schematic presentation of each type; b. gives the actual photograph. i. Aerial hyphae in clusters, terminal filaments developing into sporophores, both spiral- and nonspiral- forming GS. lave ndulae, Se roseochromogenes ). F. Aerial hyphae in clusters; no spirals or loops (S. antibioticus). (See continuation, next plate). GROUPS AND SPECIES OF GENUS STREPTOMYCES ef, lvl 30 Vv 50 20 | Nvvreluvistta quali Prats V. (Continued) G. Aerial hyphae verticillate; no spirals (S. hiroshimensis). H. Aerial hyphae flexuous or wavy; no long hyphae and no spirals (S. albus). 109 L10 strate mycelium was taken as the basis for the primary subdivision into sections, and the color of the aerial mycelium for the secondary subdivision into series. In the systems used by Flaig and Kutzner (1954) and by Ikutzner (1956), the color of the aerial mycelium was used in connection with that of the substrate mycelium. The system proposed by Yamaguchi and Saburi (1955) was based principally upon the structure of the sporophores, the color of the aerial my- celium being utilized in a secondary sub- division; in the final characterization, ad- vantage was taken of the production of soluble pigments. A similar system was used by Shinobu (1958). Krassilnikov (1941, 1949), Hesseltine ef al. (1954), Pridham e¢ al. (1958), Etthnger et al. (1958), Mayama (1959), and Nomi (1959) used morphological criteria for the primary subdivision of the genus. Each one of the above systems has in it- self certain serious limitations. It 1s neces- sary, therefore, to combine several prop- erties in order to bring out the characteristics of the group or series, and especially those of the species. Proposed System of Classification of the Genus Streptomyces into Groups or Series In presenting the following system, full cognizance is taken of the criticisms to be directed against it, especially that the for- mation of the melanin pigment is given leading consideration, and that the pro- duction of other soluble pigments as well as of antibiotics is also given important con- sideration. I have felt that because of my own previous proposals, especially those in- corporated in the various editions of Bergey’s Manual, and my own interest in antibiotics, the best I could do would be to modify this system slightly. I hope that it will serve its purpose in the future as it has done in the past. The suggested series further broaden my earlier concept of species-groups. In THE ACTINOMYCETES, Vol. II view of the fact, however, that it is desirable for each group to be designated by a repre- sentative species, it has been found neces- sary, In some cases, to use a more recent, well defined species rather than one used long ago, for which no well established species is now recognized. This is true, for example, of the “‘chromogenes” series, which has been designated as Phaeochromogenes, for which a well recognized type culture is available. I beheve that the system of classification of the genus Streptomyces into series proposed here is simple and convenient. The use of ecological properties as a basis for the major subdivision of the genus, as in the last edi- tion of Bergey’s Manual, has been discarded. The thermophilic forms have been, for the part, transferred to other genera (Chapter 11). The animal and plant isolates, including both pathogens and saprophytes, have been distributed throughout the genus, most among the various series, where they logi- cally belong on the basis of their morphologi- eal, cultural, and biochemical properties. Both morphological (structure of sporo- phores) and cultural (color of aerial myce- lium, melanin formation) characters are combined in the major subdivision of the genus into subgenera and into series. Each series is subdivided, on the basis of specific cultural and biochemical properties, into species. Formation of soluble pigments, pigmentation, and antibiotic production are also frequently taken advantage of in char- acterizing species. To identify a new culture properly, it is important to consider not only the series subdivision and species classifi- cation, but also the detailed description of each organism. Before it can be decided whether a newly isolated culture is different from one already described, a study should also be made of the varieties within the species previously created, as well as possible mutations and variations within the culture. The names given for the various series are GROUPS AND SPECIES OF GENUS STREPTOMYCES the names of the type species within the particular series. (See also Table 10.) Genus Streptomyces Waksman and Hen- rici, containing 16 series. Type species S. albus (Rossi-Doria) Waksman and Henrici. A. Subgenus Streptomyces Waksman, with 14 series. Type species Streptomyces (Strepto- myces) albus (Rossi-Doria) Waksman and Henrici. B. Subgenus Streptoverticillium Baldacci, with 2 series. Type species Streptomyces (Streptoverticillium) reticuli (Waksman and Curtis) Waksman. A. Sporophores straight, wavy, or spiral- forming. Subgenus Streptomyces Subgroup A. MESOPHILIC I. Melanin-negative Series 1. Albus. This series is character- ized by a white to light gray aerial myce- lium, covering the whole of the substrate growth; concentric rings may be formed. It is melanin-negative. A faint brownish pig- ment may be produced on organic media. Sporophores are spiral-shaped, occasionally broom-shaped. The species within this series are usually strongly proteolytic, without formation of bad-smelling products. It may be argued that the type species S. albus is no longer available and that many species possess similar properties. This series and this species must be recognized historically, whatever the final the type species to be adopted (Pridham and Lyons, 1960). Series 2. terized by a gray aerial mycelium, ranging in color from light gray to mouse-gray or smoke-gray to ash-gray to bluish-gray; it may be white at first, later turning various shades of gray. Substrate growth may be decision of Cinereus. This series is charac- colorless or yellowish, turning gray to dark. Frequently a soluble yellow pigment is pro- duced. The sporophores are either straight or spiral-shaped. Nel Series 3. Flavus. This series, as well, has a long historical background; it was one of the three groups so designated by Sanfelice in 1904. It is characterized by a yellow or vellow-orange to yellowish-brown substrate growth, and by an aerial mycelium which is white to yellowish to gray. A yellowish- green to golden yellow soluble pigment. is usually produced. straight, or spiral-shaped. Series 4. Ruber. This series is character- Sporophores are long, ized by a pink to orange to red substrate growth, and by a white to yellowish to red aerial mycelium. No soluble pigment is pro- duced; occasionally a yellowish to brownish pigment may be formed. Sporophores are straight or spiral-shaped. Series 5. Viridis. This series is character- ized by a green to dark green substrate growth, and by a white to gray to light green aerial mycelium. Usually there is no soluble pigment; occasionally a light green pigment is formed. Sporophores are straight or spiral-shaped. Series 6. Violaceoruber. Substrate growth is at first colorless, gradually becoming red or blue; aerial mycelium is white to gray with bluish tinge. The characteristic soluble pigment is blue, frequently changing in color with the reaction of the medium; it is blue at an alkaline and red at an acid reaction. Sporophores form spirals. Series 7. Fradiae. This series is character- ized by a yellow to orange substrate growth, and by a powdery pink to seashell pink to light orange aerial mycelium. Usually no soluble pigment forms on synthetic or or- ganic media; a pink pigment may occasion- ally be produced. Sporophores are straight or spiral-shaped. Species are strongly pro- teolytic and antagonistic. Series 8. Griseus. This series is character- ized by colorless substrate growth, becom- ing, in certain media, brown to almost olive- black. Aerial mycelium is yellowish with a greenish tint, or greenish-gray or sea-green. 112 No soluble pigment is produced. Sporophores are straight or flexuous, producing tufts. Series 9. Hygroscopicus. This series is char- acterized by a colorless substrate growth, which gradually becomes yellow, dark to almost black. Aerial mycelium is white to gray; it is often moist and even soft. Sporo- phores are straight and spiral-shaped. No soluble pigment is produced. Il. Alelanin-positive Series 10. white to gray to buff. Substrate growth is brown to black. Sporophores are straight or spiral-shaped. Series 11. Lavendulae. Aerial mycelium is Scabies. Aerial mycelium is lavender to rose or pink to vinaceous laven- der. Substrate growth is colorless to cream- colored. Sporophores are not flexuous, often forming loops and loose or open spirals. Series 12. Hrythrochromogenes. Aerial my- celium is white with brownish shade. Sub- strate growth is brown to black. Sporo- phores produce spirals. Series 13. Viridochromogenes. Aerial my- celium is light green to olive-green. Sub- strate growth is grayish-green to brown to black. Sporophores produce spirals. Subgroup B. THERMOPHILIC Series 14. Thermophilus. This series com- prises six species. These are listed in Chapter 1 B. Sporophores produce verticils. Subgenus Streptoverticillium AMelanin-negative This and the next series are largely characterized by the Series 15. Cinnamomeus. morphological structure of their sporulating bodies. The sporophores produce verticils on the primary or on the secondary branches of the aerial mycelium, or on both. The spore chains are straight or spiral-shaped. This THE ACTINOMYCETES, Vol. II group is further characterized by being melanin-negative. The aerial mycelium is white to pinkish to cinnamon-colored. Melanin-positive Series 16. Reticuli. This series is charac- terized by the same morphological proper- ties as Series 15, but it is melanin-positive. The aerial mycelium is white to gray. There is a considerable overlapping of the different series. Frequently a given culture may be placed in one series or another, de- pending on the media and the conditions used for growing the organism, not to mention the idiosyncrasies of the observer. Classification becomes particularly difficult when one bears in mind the marked varia- tions frequently observed between different isolates of the same species, and the tendency of individual cultures to mutate upon con- tinued cultivation in artificial media. The fact that identification is frequently based upon comparison with published descrip- tions rather than with type cultures has resulted in the tendency to create new species on the basis of minor differences, some of which may be simple variations. Most of the series are made up of non- chromogenic forms (or those that produce no melanoid pigments), although some of the constituent species may produce faint brown soluble pigments on certain media. Some of these pigments result from lysis of the my- celium of the organism; others may be quite distinet and chemically different from the typical melanoid or chromogenic pigments, eg. the olive-green to olive-buff pigment frequently produced by S. griseus. Fewer series are composed of truly chromogenic forms, those capable of producing brown to dark brown or almost black soluble pig- ments with protein-containing media. The various ‘ quite distinct from those proposed by Bal- dacci et al. (1954). They proposed, for ex- ‘series’? suggested here are GROUPS AND SPECIES OF GENUS STREPTOMYCES 113 ample, a ‘‘Bostroem’’ series, for which no true representative can be recognized at present. Their series ‘‘Antibioticus’? and “Caeruleus”’ reasons that need not be discussed further here. Certainly, the idea expressed by Bal- dacei et al. in 1955 that “it is not possible to speak of a natural systemization of these microorganisms at the present knowledge... for the time being, one must limit oneself to a classification aiming solely at diagnosis and nomenclature,’ represents a defeatist attitude. It is well illustrated by his creation of a series named ‘‘Diastaticus.”’ state of Here were included pigmented and nonpig- mented organisms, chromogenic and non- chromogenic, with such fantastic names as A. rubrocyanodiastaticus, and such varieties as atrodiastaticus. This is certainly a good cause for confusion. Similar criticism grouping of the species proposed by Gause et al. (1957). Whereas Baldacci used the color of the substrate mycelium for primary subdivisions of the genus into sections, and the pigmentation of the aerial mycelium for the further division of the into series, Gause et al. (1957) omitted the sec- tions altogether, and divided the genus directly into series largely on the basis of the pigmentation of the aerial mycelium. Descriptions of 37 old and 71 new species ‘an be apphed to the sections were reported by a group of six collabora- tors. The authorship was of a collective nature, with all the possibilities for confusing the credit to be assigned to each individual, since it is stated that “the study of the structure, classification, ecology and dis- the attention of large scientific collectives in a tribution of actinomycetes occupies number of institutes and universities.” Proceeding from the fact that so many new species have been recently created, in de- scribing producers of antibiotics, these in- vestigators assumed that this was further ‘cannot be accepted for other proof that the old systems of classification were insufficient. Although it was recog- nized that the pigmentation of the aerial mycelium, the major criterion for classifica- tion purposes, could change on continued incubation, as in the case of their Group I, “cultures with lavender and brownish-rose pigment may change in color to salmon, red, and pale terra cotta,’ nevertheless, 15 series were adopted. This fact alone would tend to cast doubt upon the significance of recog- nizing major groups solely on the basis of pigmentation of the aerial mycelium. In establishing the species, structure of the sporophores was used in some cases; in others, the pigmentation of a single medium, frequently unknown in composition, was used.* To complicate the situation further, authors of old species and emendations of species were incorrectly credited, providing a potential source of confused nomenclature. Lieske (1921) was the classical ‘‘tumper,”’ largely because of the limitations imposed by the use of complex organic media, and because he was not aware of some of the characteristic morphological and cultural properties of the organisms, brought out particularly on synthetic media. The “‘split- ting” attitudes of Baldacci, Gause, certain others have brought the system of classifying this important group of organ- 4 and isms to undesirable extremes. In the decision to classify the genus Strep- tomyces into 16 series, it is well understood that in time other series will be added; some of those presented here may eventually be spht into two or more series; some of the varieties may be raised to the status of species; or some of the species may be raised to the status of series. The problem of whether antibiotic produc- tion is a species characteristic is still un- settled. Undoubtedly, the different antibiotics can be combined with production of * Hottinger’s, to which no reference is given. 114 THE ACTINOMYCETES, Vol. II certain other distinct properties, such as pigmentation, morphology, and carbon utili- zation, to justify the creation of new species. This has actually been done for the separa- tion of S. griseinus from S. griseus and the raising of the latter to a series status. In other cases, however, the mere formation of a different antibiotic without other accom- panying differences hardly justifies, for the present at least, the creation of new species. Chapter 6 Series and Species of the Genus Streptomyces The genus Streptomyces was created in 1943, to separate certain aerial mycelium- producing actinomycetes from the rest of the order Actinomycetales. Although there is considerable overlapping between species placed in this genus and those of Nocardia and some of the thermophilic groups, there are certain important properties that may said to characterize this genus, thus separating it, if not for any other reason than that of convenience, from the others. The major important characteristic prop- erties that distinguish the genus Streptomy- ces from the others can be briefly summar- ized as follows: be 1. A more or less branched, nonseptate, substrate or vegetative mycelium (stroma) is produced. 2. Growth takes place either on the sur- face of agar or gelatin media or penetrates deep into the medium, forming a compact, often leathery mass, designated as a colony. During growth in stationary liquid media, no turbidity is produced except on 9 oO. lysis; the masses of growth appear as clumps or compact Masses. 4. The surface colony gradually becomes covered with an aerial mycelium, though this occasionally may not occur. 5. The aerial mycelium produces sporo- genous hyphae or fruiting bodies, which are straight, or in the form of tufts, or curved, spiral-shaped, or verticillate. ibs) 6. The sporophores carry chains of single- celled spores (or conidia), which vary in shape from spherical to oblong or cylindrical, and also in surface appearance when viewed with the electron microscope. 7. The vegetative growth, the aerial my- celium, and the spores en masse frequently are colored in a characteristic manner; the color may also dissolve into the medium, producing a ‘ ‘soluble pigment.”’ 8. The species are aerobic and meso- philic, nonacid-fast and gram-positive. The genus Streptomyces comprises, by far, the largest number of species of actinomy- cetes now known to occur in nature. The various species belonging to this genus differ in cultural, physiological, and biochemical properties. greatly their morphological, They include the majority of antibiotic- producing actinomycetes. The growing eco- nomic importance of these organisms has tended to increase the need for the separa- tion of the genus into groups, each of which would contain one or more species. This need has recently been further emphasized by the creation of numerous additional spe- cles. The color of the aerial and substrate my- celium, the morphology of sporophores, and the formation of melanin pigments have been largely used for the separation of the genus Streptomyces into series and species. lor the supplementary characterization of 116 THE ACTINOMYCETES, Vol. II the species, the formation of nonmelanin pigments, the ecology of the organisms, and some of the biochemical properties (notably antibiotic formation) have been utilized. This is also true of their practical utiliza- tion for the production of enzymes, vita- mins, or antibiotics. It would appear that morphological prop- erties might offer a natural and stable basis for a system of classification of these organ- isms. Unfortunately, certain characteristic morphological features of the genus Strepto- myces undergo variation, depending upon the nutrition of the organisms and upon the environment. This tended to suggest, at first, the inadvisability of considering mor- phology as the major basis for the classifica- tion of the genus Streptomyces. This was true, for example, of Drechsler’s idea of consider- ing the type of curvature of the spiral-form- ing aerial hyphae as a basis for classification. It was also true of Waksman’s suggestion that the mode of branching of the sporo- phores might be used for this purpose. The ideas of Krassilnikov (1941, 1949) in empha- sizing the size and shape of the spore would also meet with similar criticism. Flaig et al. (1955), as well as Ettlinger et al. (1958), pro- posed use of the nature of the spore surface as a species characteristic; unfortunately, this property, depending as it does upon the use of the electron microscope, has not been readily enough established to enable the sep- aration of the genus into groups and species. Certain morphological properties are now, however, well recognized and can be utilized for the separation of certain groups of organisms belonging to the genus Strepto- myces. Such groups possess sufficiently well defined morphological features to differen- tiate them from the rest of the genus. This is true particularly of those forms that produce radiating sporulating hyphae (verti- cils), with straight or spiral-shaped branches on the main sporophores or on the side branches. This property makes it possible to distinguish these particular forms from the majority of other species of Streptomy- ces, Which produce either straight, flexuous, curved, or spiral-forming sporophores. Sev- eral systems of classification of the genus Streptomyces into series (Hesseltine ef al., 1954; Shinobu, 1958b) took full advantage of the verticil-producing property; Baldacci (1959) went so far as to suggest placing the latter into a separate genus. The separation of the spiral-forming from the straight sporophore-producing types into separate groups has also been frequently suggested. In view of the above limitations, the only conclusion that can be reached is that, for the present at least, a logical system of separation of the genus Streptomyces into a number of distinct series should be based upon a combination of several of the mor- phological and physiological properties. It is proposed here to divide the genus into 16 series. This system, likewise, is open to criticism: (a) there is left, for example, con- siderable room for a certain amount of over- lapping in some of the major properties which characterize the various series; (b) the posi- tion of a species within a series is not always well defined, and some of the species could frequently be placed with as much Justifica- tion in one series as in another; (c) there is, further, a lack of uniformity in characteriz- ing the various series: In some instances color of the aerial mycelium or of the sub- strate growth is used, and in others the formation of soluble pigments is emphasized. Fully recognizing the above limitations, however, I feel that a sound basis has been laid, taking full advantage of the knowledge now available, for dividing the genus Strepto- myces into series. As further information accumulates, the system can easily be modi- fied, since it lends itself readily to various changes and modifications. A detailed characterization of the various series 1s presented here. Some of the series are described in greater detail than others. SERIES This is due either to their longer historical background or to their capacity to form important economic products, especially an- tibiotics. In characterizing each series, the follow- ing properties have been given special con- sideration. a. Morphological properties, — notably structure of sporophores and spores. b. Color of aerial mycelium on synthetic media. ce. Formation of soluble brown pigment (melanin) on protein media. d. Spore surface. e. Other characteristic properties, such as color of substrate growth, formation of soluble, nonmelanoid pigments, rate of proteolysis, or production of specific anti- bioties. A summary of the properties of the 15 series within the genus Streptomyces is given n Table 10. AND SPECIES OF GENUS STREPTOMYCES 117 I. Series Albus Characteristic Properties a. Sporophores produce spirals; spores spherical to oval. b. Color of aerial mycelium white. c. Melanin-negative. d. No soluble pigment produced (except a faint brown pigment on certain media). e. Weakly proteolytic and weakly antago- nistic. The Albus series comprises a large num- ber of organisms, characterized by the pro- duction of substrate a typical leathery and compact growth, various media. Aerial mycelium is snow-white to colorless on white in color, assuming various shades as the culture grows older. The sporophores are long and form spirals; the spores are spherical to ovoid. The various strains grow well on both organic and synthetic media. They vary greatly in their proteolytic and diastatic properties. As a rule, this group of TABLE 10 Characteristic properties of various series of Streptomyces SA Name of series Mee Aerial mycelium Color of growth forma Type species 1 Albus — White Colorless + .§. albus 2 Cinereus = White to gray Colorless to yel- | +— | S. craterifer low 3 Flavus — Mouse-gray Yellow + | 8S. flavus 4 Ruber — Rose Red +— | §. ruber 5 Viridis —_ Gray to green Green +— | S. viridis 6 Violaceoruber - Gray Red to blue + | S. violaceoruber 7 Fradiae - Pink to rose Yellow to orange +— | S. fradiae 8 Griseus — Grass-green Colorless to olive- — |S. griseus buff 9 Hygroscopicus — White to gray Dark gray to black + |S. hygroscopicus 10 | Scabies + Gray Brown to black + |S. scabies 11 Lavendulae a Lavender Colorless +— §. lavendulae 12 Erythrochromo- + Yellowish Orange +— |S. erythrochromo- genes genes 13 Viridochromo- + Green to olive- | Brownish to green + | S. viridochromo- genes green genes 14 Cinnamomeus — Pinkish Yellowish — SS. cinnamomeus 15. | Reticuli + | White to gray Colorless +— | S. reticulr 118 organisms, so far, has not been reported as containing any significant antibiotic-pro- ducing forms. Although one of the first preparations possessing certain antibacterial properties ever recorded for a culture of an actinomycete was said to have been obtained from a member of the Albus group (Gratia and Dath, 1925), it is open to question whether the particular culture was a true S. albus. According to Pridham and Lyons (1960), this organism should be considered as more closely related to the Griseus group. Species belonging to the Albus series are found in soil and in dust. Various early in- vestigators, notably Almquist, Gasperini, Rossi-Doria, Beijerinck, and Sanfelice, re- ported the isolation of organisms belonging to this series. Various systems of classification of the Albus series have been proposed. Attention may be directed here to the fact that at one time or another all the sporulating actino- mycetes, especially the saprophytic forms, mostly now recognized as belonging to the venus Streptomyces, were classified (see Bei- jerinck, 1900, for example) ito two groups: (1) A. albus (Streptothrix alba), comprising those forms that produce a white aerial mycelium and no soluble pigment; (2) A. chromogenus (Streptothrix chromogena), i- cluding those forms that produce a black pigment on protein media. Duché appeared to follow this system as late as 1934, since he included in his monograph (Duché, 1934) on the actinomycetes only those spe- cies that were said to belong to the A. albus group. In view of the significance of the specific name ‘‘albus,” representing the type eulture of the genus Streptomyces, it may be of interest to trace the usage of this name in the literature on the actinomycetes. In presenting this historical summary, the writer has taken full advantage of the com- ments this group made by Baldacci (1939), who is frequently quoted Baldacei concerning here almost verbatim. recorded THE ACTINOMYCETES, Vol. II about 30 some of which are listed in Table 11. (Others were not included, since they are not considered as typical of the group). The name ‘‘alba”’ was first applied to an actinomycete culture by Rossi-Doria (1891). He established the characteristics of this organism, indicating its synonymy with the cultures previously characterized by Alm- quist (1890); he also identified it with a culture designated as Streptothrix Foerster, isolated from the air by Gasperini (1890). Xossi-Doria refused to accept the identi- fication of this organism with Streptothrix Foersteri Cohn. He said: ‘Nothing in the description given by Cohn can justify such an idea. In that description, in fact, only generic characters are given; of spe- synonyms, cific characters there does not exist even a shadow.’ The three cultures of Almquist appeared to differ little among themselves. Since one (culture I) was said to form a white crust changing in time to gray, Bal- dacci preferred to exclude it from the syno- nymity with the A. albus. Gasperini (1894) recognized the difference between A. albus and A. chromogenus. The latter possessed chromogenic properties, the pigment diffus- ing into the substrate. Santelice (1904) was the first to divide into three groups the actinomycetes now recognized as belonging to the genus Strepto- myces, using S. albus as the representative of the first of these groups. He noted that some of the cultures belonging to this group may produce a black pigment when grown on potato. He added quite significantly: “On the basis of this observation, a super- ficial observer may create a new species out of a pigmented culture without considering the fact that it originated from Str. alba.” Krainsky (1914) isolated from garden soil a culture which he described as A. albus. This culture produced a well developed growth, white at first, then becoming gray on certain media such as glucose agar and SERIES AND SPECIES OF GENUS STREPTOMYCES TABLE 11 Nature and dimen- pcos ene Author sions of spore “ Cladothrix dichotoma | Macé a E Streptothrix Foersteri | Gasperini Oval (1.0-1.5) Streptothrix n. 2 Almquist + Streptothrix n. 3 Almquist + Streptothrix alba Rossi- Doria | + Actinomyces albus Krainsky Oval (1.0) A, albus Waksman and Curtis by 1.1-1.4) A. albus | Jensen Rectangular (0.4-0.5 by 2.4) A. albus Duché + A. chromogenus Gasperini Oval (1.5) Cladothriz odortfera Rullmann + (1.0) A. thermophilus Berestnew + A. thermophilus Gilbert + (0.5-0.6) A. thermodiastaticus | Bergey Oval A. sanninii Ciferri Round A. almquisti | Duché | + A. gougeroti Duché a Streptothriz gedanen- | Scheele and oo a | | sis I Petruschky Streptothrix candida | Petruschky + Streptothriz lathridii | Petruschky a Cladothrix invul- | Acosta and G. ao nerabilis Rossi Spherical, oval (1.2-1.6 Comparative characters of Streptomyces albus and related species (Baldacci, 1939) Mycelium 119 Proteolytic action Chrom- bes ogenesis Substrate Aerial Gelatin | Milk White a Colorless to yellow-| White _ + ish White ~ a White - ~ Colorless to black | White ~ _ Colorless White (less often = + gray) White or gray Cream or gray — + a Cream or yellow- | White > ocher White-yellow White = ~~ | Ocher to black White + | White or chalk | Weak | + white | White Gray-yellow Gray ar SF Colorless White ar = Colorless White or ivory oF Weak white Yellowish - ~ Greenish | White } — Colorless | White Weak Colorless | White 3 te Colorless White + + | White - + gelatin. The aerial mycelium was produced readily, the medium remaining colorless. The spores were oval, 1 yw in size. Gela- tin was liquefied. Nitrate was reduced. The culture had no diastatic action on starch. Waksman and Curtis (1916) isolated from soil cultures of an organism considered to be A. albus. It was similar to that of Kvain- sky, although the exact identity of the two was doubted. The diagnosis of Krainsky was, therefore, amended. The aerial my- celium appeared either white or gray, ac- cording to the composition of the medium; the substrate growth varied from white to gray. The sporophores produced short and rare spirals. The spores were 1.2 to 1.6 by 1.1 to 1.4 uw. The culture was nonchromo- genic, hydrolyzed starch, reduced nitrate, and liquefied gelatin. Jensen (1931) also isolated a culture of A. albus from soil. The aerial mycelium was constantly white, and the substrate growth cream-colored yellow-ochre. The culture was said to pro- duce cylindrical spores, 0.4 to 0.5 by 2.4 wu. Duché (1934) created excessively broad “A. albus group.’ He was severely criticized by Baldacci (1939), who said that ‘df it is meant by ‘albus group’ those species or an that produce white aerial mycelium in cul- ture, they are many more in numbers than those described by Duché. These are also the ‘viridis’ species and those that show analogy with the ‘flavus’ forms.’? Baldacci further emphasized that A. albus is a well characterized species that does not permit with the ‘‘albus group”? of Duché comprised 18 species, of confusion others, while which 15 were new ones; among these were 120 THE ACTINOMYCETES, Vol. II such forms as A. viridis, A. albidoflavus, and A. alboflavus, which definitely belong to other groups. In his morphological study of the actino- mycetes, Duché recognized five types of sporulation, none of which was used for systematic purposes. The relationship of aerial hyphae, spirals, and spores was not sufficiently emphasized. Duché stated that colonies may also originate from arthro- spores, implying thereby that any mycelial fragments will reproduce and multiply in the culture. He spoke, however, of ‘“‘mono- sporic”’ although he used the method of successive dilutions and not that of single-spore isolation. He documented the various interpretations of the species A. albus, stating at first that the description of Waksman “‘ne correspond pas tout a fait aux type albus de Gasperini, Rossi-Doria et Krainsky.”’ On comparing his own cul- ture with the preceding ones, he stated, “T’espece de Waksman and Curtis semble posséder toutes les propriétés de celle de Krainsky.... Notre espece resemble aux deux précédentes.”’ Baldacci concluded that the work of Duché, after trying to prove the diversity of the various interpretations in the literature, had not attained its purpose of establishing what the species A. colonies, albus should be. In proposing the genus Streptomyces in 1943, Waksman and Henrici stated: ‘‘We have selected as the type species of this newly named genus, Streptomyces albus (Rossi-Doria emend. Krainsky) comb. nov. This species was formerly known as Actino- myces albus WKrainsky and first described as Streptothrix alba Rossi-Doria. This is one of the commonest and best known species of the group, and, although it may later be subdivided into further species, it 1s at present as definite as any other. It has been recently studied intensively by Duché (1954) and by Baldacci (1939). It is colorless, with ovoidal white aerial mycelium, forming spores in coiled chains on lateral branches of the aerial hyphae. It is proteolytic, lhque- fying gelatin and peptonizing milk with the production of an alkaline reaction in the latter. It does not produce any soluble pig- ment either on an organic or synthetic me- dium, but does produce a characteristic earthy or musty odor.” Pridham and Lyons (1960) have recently made a comprehensive analysis of the pres- ent status of Streptomyces albus. Their study was based upon a detailed examination of 55 cultures collected from various sources. They came to the conclusion that ‘‘there has existed since about 1916, two entirely different concepts with regard to the nature of Actinomyces (Streptomyces) albus. One concept centers around strains with the following characteristics: flexuous fruiting bodies, colors of aerial mycelium in tints and shades of olive-buff (yellowish-gray or tan); nonchromogenicity (nability to form brown, deep brown, or black diffusible pig- ments in organic substrates); and marked abundance in nature” (these strains are now considered as comprising members of the Griseus group). “The other concept con- cerns strains that are characterized by coiled or spiralled fruiting bodies with catenulate ovoldal spores; by aerial mycelium colors generally interpreted as cretaceus (chalk- white, often with faint tinges of pink); by nonchromogenicity (inability to form brown, deep brown, or black diffusible pigments in organic substrata); and by their relative rareness in nature” (these strains are now considered as Albus group proper). Morphologic Characters Various methods were used in the study of the morphology of S. albus. Baldacci observed two types of mycelium. One was hyaline, not less than | uw in diameter, rami- fying more or less abundantly, and having an undulated appearance. The ramification starts perpendicularly from the point of SERIES AND SPECIES OF GENUS STREPTOMYCES 12] intersection, but it can follow in other di- rections or assume a Wavy appearance. This mycelium originates directly from the germi- nating spores and can be rather abundant. It corresponds to the first vegetative growth and is designated as ‘“‘substrate mycelium.” The second type of mycelium is more dis- tinctly visible than the first. It is larger in diameter (1.1 to 1.4 yw); it is subhyaline with a tendency to assume yellowish colora- tion. This mycelium carries abundant sporog- enous hyphae, scarcely ramified. The ex- tremities curve to hook shapes and succes- sively turn to spirals. This mycelium is white and is superimposed on the substrate growth; in time, it turns to dirty white or milky white, powdery or crusty. It is desig- nated as ‘‘aerial mycelium.” The branches of the aerial mycelium be- come sporophores and give rise to spores that are formed by contraction. The spores are short, oval in shape, white, and not without a certain polymorphism, appearing sometimes as short rods. One may also ob- serve round forms, but they must be inter- preted as spores seen in a vertical projection. The spore dimensions are 0.6 to 0.7 by 1.2 u. According to Baldacci, the spores are smaller than those observed by Jensen and longer than those described by Drechsler. Baldacci was not sure, however, that Drech- sler’s culture corresponded to S. albus. The difficulty in reaching an agreement con- cerning the shape and measurement of the spores is not due, as claimed by Duché, to their small size, but to the time at which the measurements are taken and observations made. When the spores are united in chains in the sporophores, they appear longer and rectangular; if measured when they are spread in the preparation, they appear shorter. It is natural, therefore, that only the shape and dimension in the latter case be accepted. It should be noted that the free spores are not distributed on the slide in a uniform manner, because many remain attached to the glass at their smaller sur- face, and thus appear round. Spirals are abundant. The degeneration of the hyphae can be observed in the old substrate mycelium: protoplasm zones of with empty simulating arthrospores can be seen. This spaces phenomenon led earlier investigators, includ- ing Gasperini, to make unjustified generali- zations. This is also evident in the claims of Lachner-Sandoval, Vuillemin, and Grigora- kis, who observed this false sporulation. Al- though these authors did not always specify the particular species used in their studies, Baldacci was inclined to think that they had to do rather with a Nocardia; the vege- tative mycelium of the latter, when its growth is arrested, subdivides into frag- ments that look like bacillary elements. This type of fragmentation was studied by Mrskov and by Jensen. The ‘ irregulars”’ of Duché and others bring out very clearly this type of degeneration, which was erroneously interpreted as a type of sporulation. ‘arthrospores Cultural Characters S. albus produces a colony in the form of a growth adherent to the substrate; it is wrinkled and colorless. The aerial mycelium appears first in the drier portion of the colony; it is chalk-white in color, as if lime had been sprayed over it. On aging, it turns to ivory-white; on some media, such as nutrient assumes a grayish tint. With age, growth of the culture becomes agar, it opaque or even yellowish, comparable to the color of the substrate, as can be ob- served in the cultures where the aerial my- celium is not produced. The formation of the aerial mycelium appears to correspond to the presence of water of condensation, the aeration of the cultures, and various other factors, independent of the strain or variety. The aerial mycelium is more or less abundant and may cover the entire colony. 122 THE ACTINOMYCETES, Vol. II Concentric ring formation may be observed in smaller colonies, as described by Rossi- Doria. The cultures produce the character- istic moldy or soil odor. On synthetic agar, the initial develop- ment of the culture is characterized by a dusty or powdery white, dry growth, form- ing furrows or concentric rings. The sub- strate mycelium is formed in a thin, scarcely visible layer over the agar. The colony does not assume a vigorous aspect. The white aerial mycelium appears late. Pigmentation of the agar is seldom observed, except for a few strains that show feeble chromogenesis in this medium. On potato, the development is rapid, with small, partly confluent colonies or in the form of extended membranous growth that becomes covered with white aerial myce- lium. Biochemical Properties Temperature: Optimum 24-28° (24— 44°)C, Gelatin: Liquefied. Starch: Diastatic action variable. Sucrose: Inverted. Nitrate: Reduced to nitrite. Antagonistic properties: The organisms belonging to the S. albus group are usually weak antagonists. Some cultures possess activity against gram-positive bacteria. Species KrassilInikov (1949) included 19 species in the Albus series. He used a combination of different criteria for their separation and identification. Tor separation of the cul- tures, he considered the odor produced as the major criterion, which is rather unrelia- ble. Use of this criterion is largely responsi- ble for the inclusion, in this group of species, of forms designated as aromaticus, odoratus, odorifer, putrificus, ete. IKrassilnikov also considered, for identification purposes, the shades of white in the aerial mycelium, tem- flocculus, A. perature relation, proteolytic and antago- nistic properties, the secretion of a brown substance, growth in acid media, production of ammonia and H.S, decomposition of rubber, and formation of coremia. He in- cluded in this group various thermophilic and thermotolerant organisms. Gause et al. (1957) divided the Albus series, on the basis of the color produced on a complex organic medium, into three sub- groups, comprising five species and one variety: a. Medium not pigmented: A. candidus, A. candidus var. alboroseus, and A. albidoflavus. b. Medium colored brown: A. longisporus and A. mirabilis. c. Medium rubidus. The above characterization fails to recog- nize some of the fundamental cultural prop- erties of actinomycetes, namely, the produc- tion of melanin pigments in protein media and the structure of the sporophores. The resulting subdivision of such a group into albo- colored brownish: A. subgroups thus loses all significance. Only one of the above species (A. albidoflavus) is found in’ Krassilnikov’s ‘‘albus’’ — series. Kutzner (1956) reported that he had ob- tained four strains of S. albus from different institutions and found them to be identical. Baldacci placed in the Albus series the following organisms, either because they were considered as synonyms or because they were believed to be closely related: Actinomyces albus, A. acidophilus, A. alm- beddardi, A. chromogenus, A. A. exfoliatus, A. farcinicus, A. gedanensis, A. gelaticus, A. gougeroti, A. heimii, A. kimbert, A. liesket, A. listeriit, A. malenconi, A. reticult, A. soma- liensis, A. sanninii, A. saprophyticus, A. thermophilus, A. wpcotti1, A. willmorec; Clad- othrix dichotoma, C. liquefaciens, C. imvul- nerabilis, C. odorifera; Oospora doriae, O. alpha; Streptothrix alba, Str. candida, Str. quisti, A. erythreus, SERIES AND SPECIES OF GENUS STREPTOMYCES 12 dassonvillei, Str. foerstert, Str. graminearum, Str. leucea, Str. lathridiz, and Str. pyogenes. Such a large conglomeration defeats com- pletely the purpose of grouping, since the above forms vary greatly morphologically, culturally, and physiologically. A number of species can, however, be in- cluded in this series. It is sufficient to men- tion S. albus, S. calvus, and S. niveus. Il. Series Cinereus Characteristic Properties a. Sporophores straight or spiral-shaped. b. Color of aerial mycelium white to gray; occasionally dark humid stains or guttation drops. ce. Growth usually colorless, occasionally yellow to tan. d. Melanin-negative. When aerial mycelium, the color is characteristi- ‘ally gray. Although it may be white at first, it changes to various shades of gray, members of this series form an ranging from light gray to mouse-gray to bluish-gray or even vinaceous-gray or black- ish-gray. Frequently white spots are pro- duced in the aerial mycelium. The substrate growth is often colorless or gray, occasion- ally becoming yellowish to buff-colored; it is either opaque or somewhat slimy; the reverse 1s usually colorless, occasionally turning yellow to tan. It is melanin-nega- tive. Occasionally a yellowish or brownish soluble pigment may be produced. The sporophores are straight, often formed in clusters or tufts; they may also produce spirals that are either open or compact. This series is widely distributed in nature. It comprises, in addition to well described species given here, a great number of incom- pletely described forms, as shown in Chapter i: A variety of antibiotics have been found to be produced by members of this series. Oo Some Characteristic Species The following species may be tentatively included in this series: S. craterifer, S. inter- medius, S. parvullus, and S. cellulosae. One may also include in this series various organisms described by Krassilnikov (1949), including his emendation of T a = De Sane Ay’ 288 oR gat ee = A nt cee S. purpurascens a a + tr ar Seed a daiz, eee en a S. bobiliae SI “F ls = a + she (=) C=). [Ge S. cinereoruber + —- (-— = 2 = = Ve) ae as S. cinereoruber var. fructofermen- + (+) + (4+) + (ey ee aS ae as tans + = good growth; (+) = — = no growth. V. Series Viridis Characteristic Properties a. Sporophores straight or spiral-shaped. b. Growth at first colorless, becoming green to dark green. Aerial mycelium white to gray to light green to light blue. ce. Melanin-negative. d. Soluble pigment absent or greenish. The species included in this series show considerable overlapping with the species in- cluded in the chromogenic series, such as S. viridochromogenes (syn. A. viridis (Lom- bardo-Pellegrino) Baldacci). Various other organisms that might be included in this series have been described. It is sufficient to list S. alboviridis, S. griseo- viridis, and S. dassonvillet. The following organisms may be included in the Viridis series: S. viridis, S. prasinus, S. hirsutus and S. prasinopilous. Several forms described by Gause ef al. (1957) could be ineluded in this series, notably A. malachiticus and A. olivaceoviri- dis. VI. Series Violaceoruber Characteristic Properties a. Sporophores produce spirals. Spores spherical to oval. Surface of spores smooth. b. Substrate growth colorless, becoming weak growth, questionable carbon utilization; (—) = very weak growth; red, later blue. Aerial mycelium white to gray with bluish tinge. c. Melanin-negative. Soluble red pigment in acid media, changing to blue in alkaline. A number of organisms belonging to the genus Streptomyces are able to produce a blue pigment when grown on certain media (Tables 16, 17). This pigment is either re- tained in the substrate mycelium or is readily dissolved in the medium; it is frequently ac- companied by a dark chromogenic pigment. The color of the pigment ranges, therefore, from light blue to dark blue or violet, and to almost black. The soluble pigment fre- quently changes in color with a change in reaction of medium, from red at an acid reaction to blue at an alkaline reaction. Be- ‘ause of this change in the color of the pig- ment, various names, indicating the red and blue color combinations, have been used to describe the species, such as ‘‘vzolaceus,” “violaceoruber,” ‘‘violaceoniger,” ‘‘tricolor,”’ and ‘“‘pluricolor.”’ The species capable of producing blue pigments are divided here into two distinct subgroups: S. violaceorwber and S. vzolaceoniger. The first comprises the forms that produce a litmus-like pigment, changing from red in acid media to blue in alkaline; the second includes those forms that produce violet to dark blue to almost violet-black pigments on synthetic and or- b) ganic media. SERIES AND SPECIES OF GENUS STREPTOMYCES TABLE 16 Organism Color of aerial mycelium Light grayish-blue Grayish-yellow Greenish-grayish-yellow Bluish-gray to blue Greenish-brownish-gray Gray, sometimes with _ brownish tinge White with purple tinge due to substrate myce- lium Whitish-gray S. caeruleus S. coelicolor S. cyaneofuscatus S. cyaneus S. cyanoflavus S. litmocidini S. novaecaesareae (= A. violaceus caesart) . pluricolor —R S. tricolor S. violaceoruber Light brown to light gray Ash-gray | Ash-gray Mouse-gray S. olivaceus Streptomyces sp. No. 169 —; seldom Streptomyces species, producing a blue pigment (Kutzner and Waksman, 1959) Melanin pigment Author Spirals Spore surface = — Baldacci, 1944 _ Smooth Miller, 1908 Gause et al., 1957 Krassilnikoy, 1949 Funaki et al., 1958 Gause et al., 1957 + Spiny +++ 1 atts + = Waksman and Cur- + | | tis, 1916; Waks- | man, 1919 ok Berestnew, 1897; | Krassilnikovy, | 1949 Wollenweber, 1920 Waksman and Cur- tis, 1916; Waks- man, 1919 Corbaz et al., 1957 oS Kurosawa, 1951 ++ Smooth _ — Smooth = The first organism belonging to this series was isolated, in 1891, by Rossi-Doria and described as Streptothrix violacea. It was later studied by Gasperini (1894), and by San- felice (1904) as one of the three important constituent groups of actinomycetes. Baldacci (1942) designated the series as “wolaceus,” which he did not differentiate, however, from the subgroup designated here as violaceoniger. Gause et al. (1957) created a new series, ‘‘roseoviolaceus,”’ which logically belongs in this series; they also included in their series “‘violaceus’? a variety of other forms that logically belong to this series. Ettlinger et al. (1958) designated as azureus the light blue pigmented forms. This series is not known for the production of any important antibiotics, although coeli- colorin has been reported for cultures of S. violaceoruber and chartreusin for S. char- treusts. The following species may be included in this series: S. violaceoruber, S. novaecaesareae, S. cyanofuscatus, and S. litmocidini. Some of the melanin-producing forms, such as S. violaceochromogenes, may also be included in this series. Many of the forms described by Gause et al. (1957) also belong here. These include A. coerulescens, A. glau- cescens, A. bicolor, A. coeruleofuscus, A. violaceorectus, A. color, A. litmocidini, A. griseoruber, A. coeruleorubidus, Hooper. i... Antibiotics: .& Chemotherapy 3: 1239-1242, 1953). Morphology: Aerial mycelium forms nu- merous loosely wound spirals. Spores spher- oidale 0 toa..2 by 1:6 towl.8: a: Sucrose nitrate agar: Growth moderate, wrinkled, yellow-brown. Aerial mycelium secant. No soluble pigment. Glycerol-asparagine agar: Growth abun- dant, cream-colored, turning russet-brown with aging. Aerial mycelium abundant, slate-gray. Soluble pigment amber. Calcium malate agar: Growth moderate, golden colored. Aerial mycelium scant. No soluble pigment. Nutrient agar: Growth abundant, yellow. Aerial mycelium white to light yellow. Solu- ble pigment faint yellow. Potato: Growth abundant, cream-colored. No aerial mycelium. Slight reddish-brown darkening of the potato. Melanin-negative. Gelatin: Moderate liquefaction at 26°C in 14 days. No soluble pigment. Milk: Alkaline with no coagulation; slight peptonization in 14 days. Starch: Hydrolysis in 96 hours at 30°C. Nitrate reduction: Positive in 96 hours at 30°C. Carbon utilization: Good growth with arabinose, rhamnose, xylose, dextrose, galac- tose, fructose, cellobiose, lactose, maltose, sucrose, dextrin, inulin, raffinose, soluble starch, glycerol, inositol, mannitol, and sodium salicylate. No growth observed with dulcitol, sorbitol, sodium acetate, sodium THE ACTINOMYCETES, Vol. II citrate, sodium formate, sodium malate, sodium oxalate, sodium tartrate, or sodium succinate. Antagonistic properties: Produces the antibiotic amphomycin, active against gram- positive bacteria. Habitat: Soil. Type culture: ATCC 12,237. 44. Streptomyces carnosus (Millard and Burr, 1926) Waksman and Henrici (Millard, W. A. and Burr, S. Ann. Appl. Biol. 13: 580, 1926). Morphology: Spores cylindrical, 1.0 by OFT amis. Sucrose nitrate agar: Growth pale smoky- gray to olive-gray. Aerial mycelium abun- dant, gray. Colorless guttation drops appear over the whole surface. Soluble pigment ivory-yellow to cartridge-buff. Potato: Growth lichenoid. Aerial celium gray to brownish with white spots. Plug becomes colored gray to black. Gelatin: Surface growth. Aerial mycelium white in center, gray at margin. Rapid lique- faction. Soluble brown pigment. Milk: Surface growth good. No aerial my- celium. Positive coagulation and peptoniza- tion. Starch: Hydrolysis. Tyrosinase reaction: Negative. Nitrate reduction: Positive. Habitat: Potato scab. my- 45. Streptomyces catenulae Davisson and Finlay, 1959 (Davisson, J. W. and Finlay, A. C. U.S. Pat. 2,895,876, July 21, 1959). Morphology: Growth with smooth edge. Sporophores in form of short wrinkled clusters; a few tight spirals. Spores oval to cylindrical, 1.0 by 1.3 wu. Agar media: Growth dark brown to dark greenish-brown. Aerial mycelium dark olive- gray to brown. Sucrose nitrate agar: Growth transparent, white. Aerial mycelium light gray. No solu- ble pigment. DESCRIPTION OF SPECIES OF STREPTOMYCES 19] Calcium malate agar: Growth poor. Aerial mycelium mouse-gray, with some white. Cal- cium malate digested. Nutrient agar: Growth pale yellow. Aerial mycelium white, turning pale gray. Soluble pigment pale yellow. Nonchromogenic. Glucose-yeast extract-beef-peptone agar: Growth dark brown. Aerial mycelium olive- gray with white. Soluble pigment medium brown. Starch agar: Growth Aerial mycelium poor, mouse-gray. Good hydrolysis. Gelatin: Growth moderate. Aerial myce- lium buff, some white. No soluble pigment. Poor liquefaction. Potato: Growth good, greenish. Aerial my- celium pale olive to smoke-gray to brown. Soluble pigment dark greenish or absent. Nitrate reduction: None. Antagonistic properties: Produces biotic catenulin. Habitat: Soil. Type culture: ATCC 12,476. 46. Streptomyces cavourensis Giolitti, 1958 (Giolitti, G. Belgian Pat. 560,930, March 18, 1958*). Morphology: Aerial mycelium produces spirals on certain media. Spores spherical to elliptical. Sucrose nitrate agar: Growth yellowish. Aerial mycelium chalky white to yellowish. brownish-orange. anti- No soluble pigment. Glycerol-asparagine agar: Growth hazel- colored. Aerial mycelium whitish. Soluble pigment faint brown. Calctum malate agar: Growth scanty, yellow-brownish. Aerial mycelium scanty, white. Soluble pigment scarce, yellow-brown- ish. Nutrient Aerial mycelium scanty, chalky white to yel- agar: Growth orange-brown. lowish. Soluble pigment light brown. Glucose agar: Growth brown, wrinkled. * Supplemented by personal communication. Aerial mycelium white-yellowish. Soluble pigment dark brown. Potato agar: Growth dark brown. Aerial mycelium gray with dark yellow dots. Solu- ble pigment brown. light brown, wrinkled. Aerial mycelium gray with brown patches. Soluble pigment light brown. Starch agar: brownish. Aerial mycelium scanty, white- grayish. Soluble pigment brownish. Strong hydrolysis. Gelatin: Growth scanty, brown, wrinkled. Aerial mycelium scanty, gray. Soluble pig- Oatmeal Growth agar: Growth scanty, yellow- ment brownish. Liquefaction fairly good. Potato: Growth brown with yellow edges, wrinkled. Aerial mycelium grayish. Soluble pigment brown. Milk: Growth consists of white to yellow ring around surface. Positive coagulation and peptonization. Nitrate reduction: Negative. Antagonistic properties: Produces flaven- somycin, an antibiotic active against fila- mentous and yeast-like fungi, and to a certain extent some gram-positive bacteria. Very active against some insects. Type culture: IMRU 3758. 47. Streptomyces celluloflavus Nishimura et al., 1953 (Nishimura, H., Kimura, T., and Kuroya, M. J. Antibiotics (Japan) 6A: 57— 65, 1953). Morphology: Sporophores straight with a few flexible, hooked spirals. Spores nearly spherical, 1.0 by 0.9 x. Sucrose nitrate agar: Growth glossy, de- veloping deep into medium, later becoming yellow. Soluble pigment faint sulfur-yellow. malate agar: Growth yellow, Glycerol later turning white to pale olive-buff with blackish center. Aerial mycelium cottony, white, with grayish patches, later turning olive-buff. Soluble pigment yellow. Glucose-asparagine agar: Growth cream to yellow. Aerial mycelium scant, cottony, 192 THE ACTINOMYCETES, Vol. II white to gray. Soluble pigment sulfur-yel- low. Nutrient agar: Growth olive-buff, turn- ing colorless. Aerial mycehum scant, cot- tony, white to grayish. Soluble pigment yellow with tinge of green to gold. Potato: Growth wrinkled, deep olive-buff. Aerial mycelium white to olive-buff. Soluble pigment deep olive-buff. Gelatin: Growth ivory-yellow to olive-buff on surface of liquefied layer. No aerial my- celium. Faint brownish pigment. Rapid to medium liquefaction. Milk: Growth yellow to dark olive-buff. Aerial mycelium white. Soluble pigment red- dish-brown. Coagulation and rapid peptoni- zation. Tyrosine medium: Growth ivory-yellow to cream-buff. Aerial mycelium absent or scant white. Soluble pigment greenish-yellow. Cellulose agar: Growth poor. Soluble pig- ment yellow. Production of HS: Negative. Antagonistic properties: Produces thio- lutin, aureothricin. Habitat: Soil. 48. Streptomyces cellulosae — (IXrainsky, 1914) Waksman and Henrici, 1948 (x ay AO} at o¢ Antagonistic properties: Antagonistic ef- fect strongest in freshly isolated cultures. Property lost on cultivation; activity lost first against gram-negative, rod-shaped bac- teria, coccl remaining most sensitive. Pro- duces antibiotic miramycin. temarks: Highly proteolytic and lipoly- tic. Grows best on complex organic media, at slightly acid reaction, pH 6.0 to 6.6. 157. Streptomyces mitakaensis Arai et al., 1958 (Arai, M., Karasawa, K., Nakamura, S., Yonehara, H., and Umezawa, H. J. Antibiotics (Japan) 11A: 14-20, 1958). Morphology : Sporophores short, branched; spirals produced. Spores spheri- eal, 1.2: to 1.5: a: Sucrose nitrate agar: Growth good, color- less or white to dark yellowish-brown. Aerial mycelium powdery, abundant, light gull- eray. No soluble pigment. Glucose-asparagine agar: Growth good, white to brown; becomes dark brown. Aerial mycelium powdery, abundant, gray. No soluble pigment. Glycerol citrate Growth colorless or white to brownish-white; later brownish-yellow. Aerial mycelium powdery, abundant, whitish-gray in center, gray in the edges. No soluble pigment. Nutrient agar: Growth good, colorless to reverse agar: good, pale yellowish-brown. Aerial mycelium pow- dery, abundant, white, with or without eray parts. No soluble pigment. Starch agar: Growth good, colorless to pale yellowish-brown. Aerial mycelium pow- dery, abundant, light gray in center, gray at the edges. No soluble pigment. Hydroly- sis strong. Potato plug: Growth eream-buff. Aerial mycelium poorly devel- good, wrinkled, DESCRIPTION OF SPECIES OF STREPTOMYCES oped, white or grayish-white. No soluble pigment. Gelatin: Growth colorless. Aerial myce- lium pale gull-gray. Soluble pigment absent, yellow. Liquefaction moderate. Milk: Growth good, colorless to white. Peptonization and coagulation rapid. Nitrate reduction: Negative. Antagonistic properties: Produces — an antibiotic, mikamycin, active against gram- positive and acid-fast bacteria. 158. Streptomyces = murinus Frommer (Frommer, W. Arch. Mikrobiol. 32: 198, 1959). Morphology: Sporophores small, mono- podially branched, tree-like; spirals com- pact, with 1 to 3 turns. Glycerol nitrate agar: Growth greenish- yellow. Aerial mycelium thin, white. Soluble pigment greenish-yellow. Glycerol-glycine yellow to brown-yellow. Aerial mycelium white- gray to gray-brown. Soluble pigment golden agar: Growth yellow. 0 Growth yellow, occasionally brown-violet. Aerial mycelium powdery white to gray-white. Soluble pig- Glucose-asparagine agar: ment yellow to greenish-yellow. Calcium malate agar: Growth colorless. Aerial mycelium white. No soluble pigment. Nutrient agar: Growth yellow to green- ish-yellow. Aerial mycelium lacking or white. Soluble pigment lacking or golden yellow. Starch media: Growth colorless to yellow- ish. Aerial mycelium gray-brown. No hy- drolysis after 10 days. Potato: Growth abundant, golden brown. Aerial mycelium cream-colored to yellow. Soluble pigment questionable. Gelatin: Growth abundant, golden yellow. Aerial mycelium eray. Soluble pigment yellow to golden yellow. Liquefaction limited. Melanin-negative. Milk: Growth abundant, golden yellow to cream-colored to 245 Aerial mycelium powdery, cream-colored. Questionable coagulation and vellow-brown. liquefaction. Cellulose: Growth weak. Aerial mycelium gray-brown. Soluble pigment yellowish. Antagonistic properties: Produces actino- mycin. 159. Streptomyces naganisht Yamaguchi and Saburi, 1955 (Yamaguchi, T. and Saburi, Y. J. Gen. Appl. Microbiol. 1: 201- 230 L955): Morphology: Sporophores straight with many compact spirals and a few open spirals; spores oval to short rods, 0.8 to 1.4 by 0.5 to 0.7 up. Sucrose nitrate agar: Growth colorless, thin. Aerial mycelium powdery, at first white, later colored buff. No soluble pig- ment. Calcium malate agar: Growth is at first pinkish-white to pinkish-gray, later becom- ing whitish-brown. Aerial mycelium whitish. Soluble pigment light pink, but soon disap- pears. Nutrient agar: Growth at first colorless to dark cream, later becoming yellowish- brown to brown. No aerial mycelium. Solu- ble pigment light brown. Melanin-negative. Starch agar: Growth colorless to creamy with reddish-purple portion. Aerial myce- lium abundant, white or smoke-gray to light drab. Soluble pigment absent or faint pink. Good hydrolysis. Potato: Growth vigorous, at first yellow- ish-gray. Aerial mycelium white to grayish- white. Soluble pigment deep purple to black. Gelatin: Growth dark brown with some tint of olive. Soluble pigment deep brown and a more diffusible yellowish-green. Lique- faction moderate. Milk: Growth vigorous, yellowish-brown, with white aerial mycelium along the glass. Soluble pigment hght reddish-brown. Coagulation and peptoniza- brown, sometimes tion. 246 Carbon utilization: Utilizes p-xylose, L-arabinose, L-rhamnose, D-galactose, lactose, raffinose, mannitol, inositol, salicin, acetate, citrate, and succinate; does not utilize su- crose, inulin, sorbitol, or cellulose. Antagonistic properties: Active against gram-positive and acid-fast bacteria, fung), and trichomonads. temarks: Related to S. antimycoticus. 160. Streptomyces narbonensis Corbaz_ et al., 1955 (Corbaz, R., Ettlinger, L., Gau- mann, E., Keller, W., Kradolfer, F., Ky- burz, E., Neipp, L., Prelog, V., Reusser, R., and Zahner, H. Helv. Chim. Acta 38: 935- 942, 1955). Morphology: Sporophores spirals. Spores smooth, cylindrical, 0.8 to Lil byaO2760.0.9 ju: Glycerol nitrate agar: Growth thin, color- less to yellowish-brown. Aerial mycelium velvety, whitish-gray. No soluble pigment. Glucose-asparagine agar: Growth thin, at first colorless, then yellowish-brown. Aerial mycelium sparse, chalk-white. No soluble straight; no pigment. Calcium malate agar: Growth colorless. as FiGguRE 43. isolate AA 877, re- sembling S.nelropsis, showing character of verticils Hyphae of of sporogenous branches (Reproduced from: Dug- gar, B. M. etal. Ann. N. Y. Acad. Sei. 60: 85, 1954). THE ACTINOMYCETES, Vol. II Aerial mycelium white-gray. No soluble pigment. Glucose-peptone agar: Growth light yel- lowish, punctiform. Aerial mycelium pro- duced late, powdery, gray-white. No soluble pigment. Nutrient agar: Growth punctiform, yel- lowish. No aerial mycelium. No soluble pigment. Melanin-negative. Starch agar: Growth thin, colorless to vellowish. Aerial mycelium powdery, white. No soluble pigment. Hydrolysis good. Gelatin: Growth yellowish-white. Aerial mycelium snow-white. Soluble pigment light reddish-brown. Liquefaction slow. Potato: Growth lichenoid, bluish-gray to reddish-gray. No aerial mycelium. Soluble pigment dark brown. Milk: Surface ring whitish-yellow. Pep- tonization without coagulation. Production of H.S: Positive. Antagonistic properties: Produces basic antibiotic, narbomycin, related to picromy- cin. Carbon utilization: Utilizes xylose, arabi- nose, rhamnose, fructose, galactose, sac- charose, maltose, raffinose, inulin, salicin, sodium acetate. Does not utilize mannitol, sorbitol, dulcitol, mesoinositol. Habitat: Soil. femarks: Ettlinger et al. (1958) consider this organism as belonging to S. olzvaceus. 161. Streptomyces netropsis Finlay and Sobin, 1952 (Finlay, A. C. and Sobin, B. A. U.S. 2.586.762. 1952). Morphology: Sporophores in form of ver- ticils or terminal clusters on tips of short hyphae (Fig. 43). Spores short, cylindrical, 0.7 by 1.8 uw, smooth (Pl. I a). Sucrose nitrate agar: Growth thin, pale olive-buff. Aerial mycelium pale vinaceous- fawn. No soluble pigment. Glucose-asparagine agar: ate, wrinkled. Aerial mycelium white. Solu- Growth moder- ble pigment brown. Calcium malate agar: Growth moderate, DESCRIPTION OF SPECIES OF STREPTOMYCES 247 cream to buff. Aerial mycelium white. No soluble pigment. Nutrient agar: Growth moderate to good, light brown. Aerial mycelium white. Solu- ble pigment light brown. Starch agar: Growth moderate, thin; pale olive-buff reverse. Aerial mycelium white. No soluble pigment. Strong hydrolysis. Potato: Growth poor, waxy, wrinkled, brown. No aerial mycelium. Soluble pig- ment dark brown. Gelatin: Moderate surface growth. Aerial mycelium white. Soluble pigment dark brown. No liquefaction. Milk: Growth poor. No peptonization. Nitrate reduction: Negative. Production of HS: Variable. Antagonistic properties: Produces a basic antibiotic, netropsin. Remarks: Ettlinger et al. (1958) report this organism to be melanin-negative. They also consider S. cznnamomeus as closely re- lated. 162. Streptomyces niger (Rossi-Doria, 1891; emend. Krassilnikov, 1949) Waksman (Rossi-Doria, E. Ann. igiene, 1: 399-43 1891; Krassilnikov, N. A. Actinomycetales. Izvest. Akad. Nauk. SSSR, Moskau, p. 53, 1941). Morphology: Substrate growth of soft consistency. Aerial mycelium produced only on potato and synthetic agar. Sporophores formed only seldom; open spirals, with 3 to 5 turns. Spores oval. Synthetic agar: Growth black. Aerial mycelium dark gray. No soluble pigment. Nutrient agar: Growth black. Soluble pigment brown. Gelatin: Slow liquefaction, Melanin-negative (?). Milk: No change. Starch: No growth. Cellulose: No growth. Nitrate reduction: Negative. Sucrose: No inversion. Temperature: Optimum 25-30°C. in 30 days. Antagonistic properties: None. Remarks: This is a very unstable species which dies out rapidly. It easily mutates, giving rise to colorless cultures, producing no aerial mycelium. It appears transition form, if not a true Nocardia. A. niger aromaticus Berestnew and A. nigrificans (Kriiger) Wollenweber are listed by Krassil- nikov as varieties of A. niger. In view of the formation of a soluble brown substance on to be a certain protein media, this organism may belong to one of the chromogenic groups. 163. Streptomyces nigrifaciens Waksman, 1919 (Waksman, 8. A. Soil Sci. 8: 167-168, 1919). Morphology: Sporophores branching with tendency to curl; no true spirals. Spores oval-shaped to elliptical. Sucrose nitrate agar: Growth colorless. Aerial mycelium thin, gray. No soluble pig- ment. Glucose-asparagine agar: Growth cream- colored. Aerial mycelium mouse-gray with white patches. Nutrient agar: Growth thin, cream-col- ored. Aerial mycelium gray. Soluble pigment brown. Starch agar: yellow. Aerial mycelium light Hydrolysis imperfect. Ege media: Growth abundant, dark brown. No aerial mycelium. Purplish zone around growth. cream-colored to buff-gray. Growth Potato: Growth gray becoming dark. Aerial mycelium white, appearing late. Soluble pigment black. Gelatin: Growth cream-colored to brown- ish. Aerial mycelium white. Soluble pigment brown. Liquefaction slow. Milk: Surface growth dark brown. Aerial mycelium white. Coagulation and slow pep- tonization. Nitrate reduction: Positive. Sucrose: No inversion. Cellulose: No growth. Habitat: Pineapple soil in Hawaii. 248 Remarks: This organism had been de- scribed by Waksman (1919) as Actinomy- ces 145, but never named before. Type culture: IMRU 3067. 164. Streptomyces nitrificans Schatz et al., 1954 (Schatz, A., Isenberg, H. D., Angrist, A. A., and Schatz, V. J. Bacteriol. 68: 1-4, 1954). Morphology: Sporulating hyphae straight, branched. Most solid and liquid media: Growth gray, with a pink to buff reverse. No solu- ble pigment. Blood agar: Growth brick-red. No hemoly- sis. Potato: Growth wrinkled. Nitrate reduction: Positive. Milk: No coagulation; slow peptonization. Starch: Hydrolysis. Gelatin: No liquefaction. Cellulose: Not attacked. Remarks: S. nitrificans grows well on a variety of substrates, such as ethyl carbam- ate. With ammonia providing nitrogen in the basal medium, glucose, sucrose, mannitol, sorbitol, glycerol, ethanol, n-propanol, ace- tate, lactate, succinate, fumarate, and citrate permitted good growth. In a glucose con- taining medium, ammonia, nitrite, nitrate, urea, and guanidine were satisfactory sources of nitrogen. Several amino acids, purines, and miscellaneous other nitrogenous com- pounds, supped alone or with glucose in the basal medium, supported growth. The organism grew as well on carbamate when first isolated from a carbamate-enrich- ment culture as it did after serial transfer over a 2-year period on various simple and complex media containing no carbamate. In addition to its apparently unique abil- itv to grow on carbamate as sole substrate, this culture also produced nitrite from car- bamate. It did not oxidize the carbamate nitrogen beyond the nitrite stage. Hirsch (1960) considers this organism as a Nocardia THE ACTINOMYCETES, Vol. II (N. nitrificans) capable of utilizing petro- leum. 165. Streptomyces nitrosporeus Okami, 1952 (Okami, Y. J. Antibiotics (Japan) 5: 477-480, 1952). Morphology: Aerial mycelium. straight, formed in clusters or tufts. Spores elliptical to oval. Sucrose nitrate agar: Substrate growth colorless, grayish. Aerial mycelium blackish- oray. Gelatin: Limited growth in liquefied zone. Liquefaction rapid. Soluble pigment yellow- ish-brown. Milk: Growth cream-colored to brownish. Strong coagulation and peptonization. Starch: Strong hydrolysis. Cellulose: Attacked. Nitrate reduction: Vigorous. Tyrosinase reaction: Negative. Production of HoS: None. LoefHler’s media: Growth thin. Aerial mycelium gray. Soluble pigment limited. Rapid liquefaction of serum. Carbon utilization: Utilizes arabinose, galactose, glucose, maltose, rhamnose, xy- serum lose, and glycerol; does not utilize sucrose, fructose, inulin, lactose, mannitol, raffinose, or sorbitol. Antagonistic properties: Produces an anti- biotic, nitrosporin (proactinomycin?). Habitat: Soil in Japan. Remarks: Resembles S. griseolus and S. cellulosae. Type culture: IMRU 3728; ATCC 12,769. 166. Streptomyces niveoruber Ettlinger et al., 1958 ‘(Bttlinger, L., Corbaz,.-Resand Hitter, R. Arch. Mikrobiol. 31: 350, 1958). Morphology: Long, straight sporophores, monopodially branched, forming open, regu- lar spirals. Spores smooth (PI. II k). Glycerol nitrate agar: Growth hght yellow or carmine-red. Aerial mycelium sparse, chalk-white. Glucose-peptone agar: Growth white-yel- DESCRIPTION OF SPECIES OF STREPTOMYCES low to carmine-red. Aerial mycelium abun- dant, white. Soluble pigment carmine-red. Calcium malate agar: Growth light yellow to ight carmine. No aerial mycelium. Starch agar: Growth light yellow-red to carmine-red. Aerial white. Limited hydrolysis. Potato: Growth light brown. Aerial myce- lium powdery, chalk-white. Melanin-nega- tive. Gelatin: Growth carmine-red. Aerial my- celium sparse. Trace of liquefaction. No soluble pigment. Milk: Pellicle hight yellow. Aerial myce- lium sparse. Coagulation limited; no pep- tonization. Antagonistic properties: biotic cimerubin. Habitat: Soils in England and Germany. somewhat mycelium abundant, Produces anti- 167. Streptomyces niveus Smith et al., 1956 (Smith, C. G., Dietz, A., Sokolski, W. T., and Savage, G. M. Antibiotics & Chemo- therapy 6: 135-142, 1956). Morphology: Sporophores straight at the base, corkscrew-coiled at tip, occur in clus- ters and bear oblong spores. Sucrose nitrate agar: Growth cream-col- ored. Aerial mycelium white. Soluble pig- ment yellow. Calcium malate agar: Growth cream- colored. Aerial mycelium white. Soluble pigment yellow. Nutrient agar: Growth cream-colored. Aerial mycelium trace, gray-white. Soluble pigment yellow. Gelatin: Growth good. Liquefaction par- tial. No soluble pigment. Nutrient starch agar: Growth vellow. Aerial mycelium cream-pink. Hydrolysis good. Tyrosine agar: Soluble yellow pigment. Milk: Ring on surface; floeculent growth at bottom. Positive peptonization. Production of H.S: Negative. Carbon utilization: b-xylose, b-arabinose, 249 rhamnose, D-fructose, D-galactose, D-glucose, D-mannose, maltose, sucrose, lactose, cello- biose, raffinose, dextrin, inulin, soluble starch, glycerol, duleitol, bD-mannitol, b-sorbitol, im- ositol, salicin, sodium formate, sodium ox- alate, sodium tartrate, sodium acetate, so- dium citrate, and sodium succinate utilized. Phenol, cresol, and sodium salicylate not utilized. Nitrate: No reduction. Antagonistic properties: Produces strepto- nivicin, a form of novobiocin. Habitat: Soil. Remarks: According to Kuroya et al. (1958), this organism is related if not identi- cal to S. griseoflavus. 168. Streptomyces noboritoensis Isono. et al., 1957 (Isono, K., Yamashita, S., Tomi- yama, Y., Suzuki, S., and Sakai, H. J. Anti- biotics (Japan) 10A: 21-30, 1957). Morphology: Aerial mycelium long and wavy; no regular spirals. nitrate Growth Aerial mycelium slight. Soluble pigment Sucrose agar: colorless. absent or pale yellow. Glucose-asparagine dark pale violet-gray. slightly brownish. Nutrient agar: Growth flat, pale gray, smooth and restricted. No aerial mycelium. Soluble pigment dark red-brown. Starch agar: Growth dry, wrinkled, pale agar: Growth pale brown. Aerial mycelium No soluble pigment or brown. to grayish-brown. Aerial mycelium pale-gray, cottony. Weak diastatic action. Gelatin: Growth dark brown. Soluble pig- ment dark brown. Liquefaction absent or slight. Potato: Growth flat, wrinkled, black. Aerial mycelium grayish-white in some strains. Color of plug black. Carbon utilization: Glucose, lactose, man- nitol, trehalose, and raffinose well utilized. Utilization of arabinose, inositol, salicin, and xylose limited. Rhamnose and sucrose not utilized. 250 Antagonistic properties: Produces anti- biotic homomycin-hygromycin. Remarks: S. noboritoensis belongs to the group of chromogenic actinomycetes closely related to S. cinnamonensis, S. flavochromo- genes, S. phaeochromogenes, S. aureus, and S. tanashiensis. They differ in spiral forma- tion, pigmentation on synthetic media, ni- trate reduction, and production of anti- biotics. 169. Streptomyces nodosus Trejo, W. nov. Sp: Morphology: Aerial mycelium forms open and closed spirals, the latter predominating as tightly knotted coils. Spores spherical to oval, 0.5 to 1.0 by 1.0 p. Sucrose nitrate agar: Growth white-green- ish. Aerial mycelium pearl-gray to dawn- eray. Nutrient agar: Substrate growth scant. No aerial mycelium. No soluble pigment. Melanin-negative. Oatmeal agar: Growth black with a buff margin. Aerial mycelium deep olive-gray. Reverse: olivaceus to black with a peripheral ring of cream-buff to chamois. No soluble pigment. Potato: Growth buff. Aerial mycelium light olive-gray. No darkening of plug. Milk: Rapidly peptonized. Gelatin: Rapidly hydrolyzed. Nitrate reduction: Positive. Starch: Strong hydrolysis. Tyrosine: Utilized with no melanin forma- tion. Carbon utilization: Utilizes mannitol, in- ositol, rhamnose, xylose, D-fructose, treha- lose, and melibiose. Does not utilize adoni- tol, sorbitol, arabinose, cellulose, sucrose, lactose, sodium acetate, esculin, or dextrin. Antagonistic properties: Produces an anti- fungal antibiotic, amphotericin. * Personal communication from Squibb Insti- tute for Medical Research (1958). THE ACTINOMYCETES, Vol. II Source: Isolated from soil in South America. Remarks: This culture was specially de- scribed for this treatise. It appears to be closely related to S. rutgersensis. 170. Streptomyces noursei Hazen and Brown, 1950 (Hazen, E. L. and Brown, R. Science 112: 423, 1950; Proc. Soc. Exptl. Biol. Med 76: 93, 1951; Science 117: 609, 1953). Morphology: Sporophores produced as side branches of sterile aerial hyphae; occa- sionally produce open spirals and, according to Ettlinger et al. (1958), also some verticils. Spores round to oval, with thin long spines CPE ey Sucrose nitrate agar: Growth scanty, col- orless, flat. No aerial mycelium. Glucose-asparagine agar: Growth wrin- kled, tan-colored, with gray and white knob- like projections. Reverse of growth dark eray. Aerial mycelium white, then reddish- eray, finally ash-gray; limited shell-pink diffusible pigment. Glucose-peptone agar: Growth good, folded, brown. Aerial mycelium white, turn- ing gray. Soluble pigment brown or pome- eranate-purple. Starch agar: Growth in form of discrete colonies. Aerial mycelium white in center, periphery colorless and embedded. Hydroly- Sis. Potato: Growth folded. Aerial mycelium chalky white. At 35-36°C a reddish-purple pigment is formed. Gelatin: Rapid negative. Milk: Coagulation, followed by peptoni- zation. Cellulose: Growth poor. Nitrate: Traces of nitrite produced. Production of HS: Negative. Blood agar: Growth consists of convex, lobate colonies, with central perforation. Aerial mycelium heavy, chalky white. No hemolysis, but darkening of blood. liquefaction. Melanin- DESCRIPTION OF SPECIES OF STREPTOMYCES Antagonistic properties: Produces an antifungal agent, nystatin. Type culture: IMRU 3771. 171. Streptomyces novaecaesareae (Waks- man and Curtis, 1916) Waksman and Hen- rici, 1948 (Waksman, 8. A. and Curtis, R. E. Soil Sci. 1: 111, 1916, 8: 158, 1919). Morphology: Aerial mycelium forms both straight and spiral (dextrorse) sporophores. Spores oval to elongate. Sucrose nitrate agar: Growth gray, be- coming bluish, glossy, much wrinkled. Aerial mycelium white, appears late. Soluble pur- ple pigment formed. Glucose-asparagine agar: stricted, gray, becoming red. Nutrient agar: Growth thin, cream-col- ored. Potato: Growth wrinkled, cream-colored, turning yellowish. Melanin-negative. Gelatin: Surface colonies small, cream- colored. Liquefaction slow. Milk: Gray ring. Coagulation slow; pep- tonization slow. Starch agar: Colonies restricted, circular, bluish-violet. Positive hydrolysis. Nitrate reduction: Positive. Production of H.S: Negative. Temperature: Optimum, 37°C. Antagonistic properties: Negative. Remarks: At first this organism was des- ignated as A. violaceus-caesari. This species is considered by Krassilnikov as synonymous with A. violaceus (Rossi-Doria) Gasperini. It appears to be related to S. violaceoruber. Growth — re- 172. Streptomyces odorifer — (Rullman emend. Lachner-Sandoval, 1898) Waksman (Lachner-Sandoval, V. Ueber Strahlenpilze. Strassburg, 1898). Morphology: Sporophores long, straight, branching, forming spirals. Spores spherical. Sucrose nitrate agar: Growth cream-col- ored, with trace of brown. Aerial mycelium abundant, cream-colored. Glucose-asparagine agar: Growth cream- 251 colored to brownish. Aerial mycelium abun- dant, cream-colored. Soluble pigment faint brownish. Nutrient agar: Growth folded, brown. Aerial mycelium white around edge. Soluble pigment faint brown. Starch agar: brown. Aerial mycelium abundant, cream- colored to straw-colored. No soluble pig- ment. Hydrolysis good. Growth cream-colored to Maltose-peptone agar: Foulerton and Price-Jones (1902) described growth as “raised, drab-colored, semi-translucent, the surface becoming reticulated; soluble pig- ment deep brown; gelatin liquefied, with light brown pigmentation.” Potato: Growth folded, brownish. Aerial mycelium cream-colored. Soluble pigment faint brown. Gelatin: Surface ring cream-colored. Ae- rial mycelium thin, white. No soluble pig- ment. Liquefaction slow. Milk: Surface ring colorless to brownish. No aerial mycelium. No coagulation; some peptonization. Cellulose: Good growth. Sucrose: Inversion. Paraffin and fats: Good growth. Nitrate reduction: Positive. Production of HS: Negative. Odor: Strong, characteristic of soil. Antagonistic properties: Some strains give positive effects, others are negative. Habitat: Soil. Type culture: IMRU 3334. 173. Streptomyces oidiosporus (Krassilni- kov, 1941) Waksman (Krassilnikov, N. A. Actinomycetales. Izvest. Akad. Nauk. SSSR, Moskau, p. 23, 1941). Morphology: Sporophores — straight or wavy, never forming spirals; short or long, frequently forming broom-shaped structures. Spores 1.0 to 1.8 by 0.5 to 1.0 u, frequently appearing as double cocci or segmented spores (oidiospores). Agar media: Growth red or rose to pale; 252 THE ACTINOMYCETES, Vol. II pigment insoluble in medium. Aerial myce- lium poorly developed, velvety, rose-white. Gelatin: Aerial mycelium weakly devel- oped, frequently lacking; aerial hyphae short, rose-white. Liquefaction weak. Milk: No coagulation; peptonization posi- tive. Starch: Rapid hydrolysis of starch. Cellulose: No growth. Nitrate reduction: Positive. properties: positive None. Jolly effects for his Antagonistic (1956) obtained strain. Habitat: Rarely found in soil. Remarks: The resembles SS. ruber and S. longispororuber. Some strains were obtained as variants of Nocardia rubra. Jolly (1956) reported the isolation of a strain of S. otdiosporus from an Italian organism soil. 174. Streptomyces olivaceus (Waksman, 1919) Waksman and Henrici, 1948 (Waks- man, S. A. Soil Sci. 8: 168, 1919). This organism was first described as strain No. 206 by Waksman (1919). It was used by Jensen (1930) for comparison with his own isolates. It was studied more re- cently by Shinobu (1958) and Ettlinger et al. (1958). Morphology: § Sporophores — branched monopodially, straight or somewhat wavy; no true spirals on most media; a few long, open spirals on calcium malate agar. Spores spherical and oval, 0.8 to 1.2 uw; surface smooth (Pl. III). Sucrose nitrate agar: Growth abundant, yellow to olive-ocher, reverse yellow to al- most black. Aerial mycelium ash-gray to light drab. Glucose-asparagine agar: Growth yellow to light olive to olive-gray. Aerial mycelium light olive-gray to light brownish-gray with greenish tinge. No soluble pigment. Calcium malate agar: Growth greenish- yellow to yellow. Aerial mycelium yellowish- white to yellowish-gray. Soluble pigment yellow. Nutrient agar: Growth white, glistening. No soluble pigment. Starch agar: Growth brownish-yellow to yellowish-green. Aerial mycelium brownish- white. Hydrolysis strong. Potato: Growth abundant, much wrin- kled, elevated, gray, turning sulfur-yellow on edge. Melanin-negative. Gelatin: Liquefaction rapid. No soluble pigment. Milk: Growth faint, pinkish; coagulation and peptonization rapid. Cellulose: Growth good. Mannase: Reaction strong, according to Shinobu (1958). Nitrate reduction: Positive. Production of HS: Negative. Tyrosinase reaction: Although this organ- ism has been considered as melanin-nega- tive, Shinobu (1958) reported a positive reaction. Temperature: Optimum 25°C. Carbon According to Shinobu (1958), S. olivaceus rapidly utilizes xylose, rhamnose, fructose, sources: galactose, sucrose, lac- tose, and mannitol; slow utilization: treha- lose, raffinose, and inositol. Antagonistic properties: Various strains produce a variety of antibiotics, including streptomycin, olivacein, and granaticin. Habitat: Very common in soil. Remarks: Krassilnikov (1949) placed the organism in the A. flavus group. Ettlinger et al. (1958) considered the following organ- isms as belonging to S. olzvaceus: S. felleus, S. flavus, S. griseolus, S. halstedii, S. nar- bonensis, S. scabies (sic), and S. verne. Type culture: IMRU 3335. 175. Streptomyces olivochromogenes (Waks- man, 1919) Waksman and Henrici, 1948 (Waksman, S. A. Actinomyces No. 205, Soil Sci. 8: 106, 1919). Morphology: Sporophores form numerous closed spirals. Spores oval or elliptical. DESCRIPTION OF SPECIES OF STREPTOMYCES nitrate Growth white, spreading. Aerial mycelium ash-gray with brownish tinge. No soluble pigment. Glycerol malate agar: Growth colorless. Aerial mycelium light grayish-olive to dark oray. Glucose-asparagine agar: Growth abun- dant, natal-brown to almost black. Aerial mycelium white with gray tinge. Soluble Sucrose agar: pigment brownish. Nutrient agar: Growth wrinkled, brown, becoming gray-green. Aerial mycelium white. Soluble pigment brown. Starch agar: Growth transparent, spread- ing. Aerial mycelium buff-gray. Rapid hy- drolysis. Potato: Colonies small, wrinkled, black. No aerial mycelium. Soluble pigment black. Gelatin: Surface growth cream-colored, spreading. Aerial mycelium white. Soluble pigment dark brown to deep olive-green. Slow liquefaction. Milk: Dark brown ring. Coagulation and peptonization. Cellulose: Growth faint. Nitrate reduction: Faint reduction to ni- trite. Sucrose: Invertase positive with good growth. Temperature: Optimum, 37°C. Antagonistic properties: Positive. Habitat: Soil, water, river mud. Remarks: Ettlinger et al. (1958) consid- ered this organism as a strain of S. griseus. KrassilInikov (1949) considered it as a vari- ety of A. chromogenes. 176. Streptomyces olivoreticuli Arai et al., 1957 (Arai, T., Nakada, T., and Suzuki, M. Antibiotics & Chemotherapy 7: 435-442, 1957). Morphology: Sporophores form primary and secondary verticils; secondary may also be formed as tip clusters. Spores spherical to oval. Sucrose nitrate agar: Growth thin, yellow to brown. Aerial mycelium scant, later be- 253 coming cottony, white with yellowish tinge. Soluble pigment faint brown or absent. Glucose-asparagine agar: Growth thin, hight brown to olive-drab. Aerial mycelium cottony, white with faint yellow to grayish- pink tinge. Nutrient agar: Growth limited, brownish. Aerial mycelium grayish-white. Soluble pig- ment light brown. Gelatin: Surface growth poor. Liquefac- tion slow, later becoming rapid. Soluble pig- ment brown. Blood agar: Strong hemolysis. Potato: Growth wrinkled, dark brown. Aerial mycelium abundant, powdery, cream- colored to tea-green. Soluble pigment brown. Milk: Ring on surface brown. Coagulation with limited peptonization. Soluble pigment brown. Nitrate reduction: Negative. Starch: Hydrolysis. Cellulose: No decomposition. Antagonistic properties: Produces anti- biotic viomycin. 177. Streptomyces olivoverticillatus —Shi- nobu, 1956 (Shinobu, R. Mem. Osaka Univ. B (N. 8.) 5: 84-93, 1956). Morphology: Sucrose-ammonium agar most suitable for microscopic study. Pri- mary and secondary verticils produced, branches issuing sometimes closely, near the the sporulating hyphae, forming tuft-like spherical to elliptical, 0.6 to 0.8 u. top of cluster-like or branches. Spores Sucrose nitrate agar: Trace of growth. Glucose-asparagine agar: Growth thin, moderate, pale olive to pale dark yellow. Aerial mycelium thin, partially yellowish- oray. Nutrient brown. Aerial mycelium olive-gray to yellow agar: Growth heavy, deep to green. Soluble pigment brown. Potato: Aerial mycelium yellow-white to yellow-gray. Solu- Growth heavy, brown. ble pigment brown. Milk: Growth brown. Aerial mycelium scant, yellow-white. Soluble pigment brown. Gelatin: Liquefaction weak. Starch: Rapid hydrolysis. Tyrosinase reaction: None. Nitrate reduction: Negative. Cellulose: No growth. Carbon utilization: Fructose and inositol utilized. Xylose, rhamnose, sucrose, lactose, raffinose, and mannitol not utilized. Habitat: Soil in Japan. 178. Streptomyces omiyaensis Umezawe et al., 1949 (Umezawa, H., Tazaki, T., Okami, Y., and Fukuyama, 8. J. Antibiotics (Japan) 3: 294-296, 1949). Morphology: Aerial mycelium scant branching. Sporophores straight, no spirals. Spores 1.0 to 1.2 by 2 to 3 u. Sucrose nitrate agar: Growth thin, trans- parent, cream-colored to dark. Aerial my- shows celium absent, or scant, white. No soluble pigment. Nutrient agar: Growth wrinkled, white to cream-colored. No aerial mycelium. No soluble pigment. Melanin-negative. Starch agar: Colorless thin colonies, al- most all submerged. No aerial mycelium. No soluble pigment. Hydrolysis. Gelatin: Growth on surface white. No soluble pigment. Liquefaction slight in crateriform. Potato: Growth white to cream-colored. No aerial mycelium. No soluble pigment. Milk: Growth white. Peptonization rapid. Acid formed. Antagonistic properties: Produces the antibiotic chloramphenicol. Habitat: Soil. Remarks: Related to S. cacaoz. 179. Streptomyces orientalis Pittenger and Brigham, 1956 (Pittenger, R. C. and Brig- ham, R. B. Antibiotics & Chemotherapy 6: 642-647, 1956). Morphology: Substrate growth made up of typical prostrate, much-branched myce- THE ACTINOMYCETES, Vol. II lium. Aerial mycelium abundant if starch is used as carbon source. Straight or irregularly branched sporophores made up of cylindrical to ovoid spores, 0.7 to 1.0 by 1.4 to 1.8 up. Sucrose nitrate agar: Growth secant to moderate, pale cream color. Aerial mycelium trace of off-white. No pigment or pale yel- lowish-brown to light brown soluble pigment may be formed. Glucose-asparagine agar: Growth moder- ate to good, cream-colored. Aerial mycelium pale to cream-colored, powdery. Soluble pig- ment pale greenish-yellow. Glycerol malate agar: Growth pale cream to intense cream-yellow. Aerial mycelium whitish in color. No soluble pigment. Insolu- ble malate cleared in agar around growth. Nutrient agar: Growth cream-colored. Ae- rial mycelium whitish. No soluble pigment. Starch agar: Growth moderate, cream-col- ored to buff to brown. Aerial mycelium white, becoming pale cream and _ finally grayish. Soluble pigment cream-yellow, be- coming pale brown. Hydrolysis limited. Potato plug: Growth shows slightly rough surface. Aerial mycelium white. Slight to moderate amount of brown discoloration of plug. Gelatin: Growth flocculent, not forming intact pellicle. Aerial mycelium scant, white. No soluble pigment. Liquefaction moderate. Milk: Heavy wrinkled pellicle, with dull gray aerial mycelium. No coagulation. Pep- tonization begins in 11 to 14 days and is complete in 14 to 21 days. Very dark soluble pigment obscures litmus color. Cellulose: Growth good. Antagonistic properties: Antibiotic vanco- mycin produced. temarks: S. orientalis is most closely re- lated to species intermediate between S. albus and S. flavus, such as S. alboflavus, S. fllobisporus, and S. longisporoflavus. S. albo- gavus cannot utilize cellulose, hydrolyzes gelatin far less effectively than S. orzentalis, but attacks starch readily. Milk is feebly DESCRIPTION OF SPECIES OF STREPTOMYCES 2595 digested by S. alboflavus but rapidly hy- drolyzed by S. orzentalis. Production of ae- rial mycelium by the two cultures differs on several media. 180. Streptomyces paraguayensis — (Al- meida, 1940) nov. comb. (Almeida, F. Mycopathologia 2: 201-203, 1940). Morphology: Thin, ramified mycelial fila- ments; aerial mycelium consists of thicker and darker filaments, | u in diameter. Gram- positive and nonacid-nonalcohol resistant. Glucose-peptone agar: Growth hard, ad- hering to the medium; white with dark center, gradually changing to dark yellow to almost chocolate. Nutrient agar: Growth rough, adhering to the medium; dark gray in color. Potato: Colonies cerebriform, growth dry and friable. Gelatin: Growth on surface. No liquefac- white; tion. Milk: Surface membrane, the milk colored pink; no peptonization. Habitat: Thoracic mycetoma; dark heavy erains. 181. Streptomyces parvullus Waksman and Gregory, 1954 (Waksman, S. A. and Greg- ory, F. J. Antibiotics & Chemotherapy 4: 1050-1056, 1954). Morphology: Sporophores long, podially branched, twisting into long closed spirals. Spores spherical, smooth (Pl. I g). Sucrose nitrate agar: Growth abundant mono- with yellow reverse. Aerial mycelium ash- eray. Soluble pigment yellow. Glucose-asparagine agar: Growth yellow. Aerial mycelium abundant, gray. Soluble pigment yellow. Nutrient agar: Growth yellowish, covered with thin white aerial mycelium. Soluble pig- ment yellow. Melanin-negative. Potato: Growth orange-colored, covered with white to gray aerial mycelium. No solu- ble pigment. Gelatin: with Surface pellicle covered heavy gray aerial mycelium. Liquefaction slow. Soluble pigment yellow. Milk: Surface growth heavy, greenish-yel- low. Aerial mycelium abundant, gray. Solu- ble pigment brown. No coagulation, very slow peptonization. Production of HoS: Negative. Antagonistic properties: Produces actino- mycin D. Habitat: Soil. Type culture: IMRU 3677. 182. Streptomyces parvus (Xrainsky, 1914) Waksman and Henrici, 1948 (Krainsky, A. Centr. Bakteriol. Parasitenk. Abt. II., 41: 685-686, 1914). Morphology: branched, or wavy; no true spirals; some strains, however, produce spirals. Spores oval, 0:9. to L.d:by 1.2 to-1-8 pu: Sucrose nitrate agar: Growth yellow, rose or red. Aerial mycelium light yellow to white-rose. Soluble pigment rose-colored to Sporophores straight, bright yellow. Calcium malate agar: Small yellow colo- nies. Light yellow aerial mycelium. Nutrient agar: Growth yellow. Aerial my- celum light yellow. Soluble pigment bright vellow. Potato: Growth yellow to brown-yellow. Aerial mycelium white to yellow. Melanin- negative. Gelatin: Growth yellow. Soluble pigment bright yellow. Liquefaction slow. Milk: No coagulation; rapid peptoniza- tion. Starch agar: Growth rose-colored. Aerial mycelium light gray. Hydrolysis positive. Cellulose: Growth good, rose-colored. Ae- rial mycelium yellowish-gray. Nitrate reduction: Weak. Production of H.S: Negative. Antagonistic properties: Produces actino- mycin. Habitat: Soil. Remarks: Ettlinger et al. (1958) con- sidered S. parvus as belonging to the S. griseus series. According to Gause ef al. (1957), S. parvus is a member of the series Fradiae. Type culture: IMRU 3686. 183. Streptomyces pelletiert (Laveran, 1906) nov. comb. (Laveran, 8. Compt. rend. soc. biol. 61: 340, 1906). Morphology: Growth red, smooth, con- sisting of small, dense, pink colonies. Myce- lium nonsegmented, branched; hyphae slender, straight, and not very long. Aerial hyphae few, straight. Glucose-asparagine agar: Growth in form of small, hard, red or purple adherent col- onies. No soluble pigment. Glucose agar: Growth poor, in form of minute, pink colonies. Glycerol agar: Growth poor, as few moist, pink colonies. Nutrient agar: Colonies minute, colorless, piled up into pale pink masses. Potato: Growth sparse, yellowish-pink, irregularly piled up; later, abundant, small, rounded, pink Aerial mycelium scant, white. Blood agar: Colonies at first a few pin- head, cream-colored; no hemolysis. Later, dense, button-shaped, with narrow, fringed margin. Dorset’s egg medium: Growth abundant, wrinkled, pink skin with small discrete colonies at margin; later, surface rough, masses. colonies are mealy, with considerable liquefaction. Gelatin: Few pink flakes. At first slow, later almost complete liquefaction. Milk: Soft curd; gradual peptonization. Starch: No hydrolysis. Production of H.S: Negative. Source: Mycetoma in Nigeria. Remarks: In the original description of this culture by Laveran, the organism was called Micrococcus pelletiert, because no my- celium was seen, only coccoid bodies. N. indica was regarded as identical by Pinoy. N. genesti Froes was described as closely THE ACTINOMYCETES, Vol. II allied (Erikson, 1935); the distinction was founded upon the fact that the red grains were smaller and much more numerous. A. africanus is considered as a synonym of this organism. According to Mariat (1958), S. pelletiert hydrolyzes gelatin, serum albumin, casein, and egg albumin; it utilizes urea but not (NH4).SO, and KNO; as_ nitrogen sources; it does not utilize xylose, galactose, maltose, starch, mannitol, or paraffin as car- bon sources. The species S. africanus is indistinguishable from S. pelletier?. 184. Streptomyces pentaticus Umezawa and Tanaka, 1958 (Umezawa, 8. and Tanaka, Y. J. Antibiotics (Japan) 11A: 26- 29, 1958). Morphology: Straight sporophores pro- duce primary and secondary verticils. Spores can scarcely be observed. Sucrose nitrate agar: Growth poor, trans- parent, penetrates deeply into medium. No aerial mycelium. No soluble pigment. Glucose-asparagine agar: Growth less, becoming purplish-pink to dull red- purple, deep into medium. Aerial mycelium white, sometimes pink. Soluble pigment faint brown. Calcium malate agar: Growth red, irreg- color- ular margin. No aerial mycelium. Soluble pigment faint brown. Nutrient agar: Growth wet, colorless or brownish-white. No aerial mycelium. Solu- ble pigment brown. Starch Growth yellow, penetrates deeply into medium. Ae- rial mycelium white, partially pinkish cot- agar: colorless or pale tony colonies. No soluble pigment. Starch hydrolyzed. Gelatin: Growth consists of reddish colo- nies produced on surface. No aerial myce- lium. Soluble pigment deep brown. Rapid liquefaction. Potato: Growth wrinkled, wet, grayish- brown. No aerial mycelium. Soluble pig- ment brownish-black. DESCRIPTION OF SPECIES OF STREPTOMYCES Milk: Surface ring dull yellow. Coagula- tion and peptonization. Antagonistic properties: Produces an anti- fungal polyenic antibiotic, pentamycin. Habitat: Soil in Japan. Remarks: Resembles S. which differs from the strain producing rubrireticuli, pentamycin in the following ways: spirals are formed; growth on nutrient agar is red; growth on milk is abundant and red; cellu- lose and sucrose are utilized. 185. Streptomyces phaeochromogenes (Conn, 1917) Waksman and Henrici, 1948 (Gonn: HH. J. (N. Y: Agr.. Expt: Sta. Tech. Bulle 60.1917). This culture has been studied by Conn (1917), Waksman (1919), Jensen (1931), Krassilnikov (1949), Kutzner (1956), and Ettlinger et al. (1958). Morphology: Sporophores form narrow, open, elongated, sinistrorse spirals (Conn, Waksman, Jensen, Krassilnikov). Kutzner (1956) examined 25 strains belonging to this species; only five of them produced spirals. Ettlnger et al. (1958) could not find any spirals on any of the strains obtained from various culture collections. Spores spherical to short rods; surface smooth (Pl. I d). Sucrose nitrate agar: Growth brown to almost black. Aerial mycelium abundant, white with brownish shade. Soluble pigment brown to dark brown. buff to brown. Aerial mycelium white. Soluble pig- Calcium malate agar: Growth ment brown. Nutrient later turning nearly black. Aerial mycelium agar: Growth gray to brown, white to gray, often absent. Soluble pigment deep red-brown. Starch agar: Growth brown. Hydrolysis medium. Potato: Growth brown to almost black. No aerial mycelium. Soluble pigment dark brown to black. Gelatin: Surface growth abundant, spreading, cream-colored, becoming brown. Liquefaction slow. Soluble pigment brown. Milk: Dark, almost black ring: tion with slow peptonization. Nitrate: Reduction limited. Production of H.S: Positive. Temperature: Optimum 25°C. Antagonistic properties: Strong. Habitat: Soil. Type culture: IMRU 3338. coagula- 186. Streptomyces phaeopurpureus Shi- nobu, 1957 (Shinobu, R. Mem. Osaka Univ., B. Nat. Sci. 6: 638-67, 1957). Morphology: Substrate mycelium mono- podial, 0.4 to 0.6 uw in diameter; no frag- mentation. Aerial mycelium straight, usu- ally short. Spores spherical to elliptical, 0.6 to 0.8 uw; rarely 1 yu. Sucrose nitrate agar: Growth good, brown to dark red. No aerial mycelium. Soluble pigment brown. Glycerol malate orange to purple. Aerial mycelium powdery, vellowish-gray to pinkish-gray. Soluble pig- ment red-purple to brown-purple. Glucose-asparagine agar: Growth moder- agar: Growth good, ate, orange to red-brown. Aerial mycelium moderate, in patches, pinkish-gray. Soluble pigment reddish-orange to reddish-brown. Nutrient agar: Growth good, deep brown. No aerial mycelium. Soluble pigment brown to deep reddish-brown. Potato plug: Growth wrinkled, reddish to yellowish-brown. Aerial mycelium absent or scant, light brownish-gray. Plug colored brown. Milk: Growth in form of deep brown ring. Soluble pigment brown. No coagulation; peptonization uncertain. Tyrosinase reaction: Positive. Gelatin: Liquefaction fairly strong. Diastase: Weak. Cellulose: Negative. Nitrate reduction: Negative. Carbon sources: Utilizes xylose, rhamnose, 258 THE ACTINOMYCETES, Vol. II fructose, sucrose, lactose, raffnose, mannitol, and inositol. Habitat: Soil in Japan. 187. Streptomyces phaeoviridis Shinobu, 1957 (Shinobu, R. Mem. Osaka Univ., B. Nat. Sci. 6: 67-70, 1957). Morphology: Growth monopodial, hyphae 0.4 to 0.6 uw in diameter, no fragmentation. Aerial mycelium short; monopodial branch- ing; some spirals, sinistrorse, 1 to 8 turns. Spores elliptical, 0.6 to 0.8 uw. Sucrose nitrate agar: Growth pale yellow to dark brown. Aerial mycelium scant, brownish-white. Soluble pigment yellow to brown to dark blue. Malate-glycerol agar: Growth yellowish- brown to dark brown. Aerial mycelium scant, white to brownish-white. Soluble pigment brown to dark brown with blue tinge. Glucose-asparagine agar: Growth thin brown to yellow-orange; aerial mycelium scant, white to brownish-white. Soluble pigment pale brown to yellow-orange. Nutrient agar: Growth thin, yellow-orange to brown. No aerial mycelium. Soluble pig- ment brown to dark red with purple tinge. Potato: Growth poor, pale brown. No aerial mycelium. Soluble pigment uncertain, probably pale brown. Milk: Growth good, pale yellow, partially blue, sometimes grayish-green. Soluble pig- ment absent or pale orange. Coagulation and peptonization. Tyrosinase reaction: Negative. Gelatin: Liquefaction variable. Diastase: Strong. Nitrate reduction: Negative. Cellulose: No growth. Carbon sources: Utilizes xylose, rhamnose, sucrose, fructose, raffinose, and mannitol; lactose and inositol uncertain. Habitat: Soil in Japan. I88. Streptomyces pilosus Ettlinger et al., 1958 (Ettlinger, L., Corbaz, R., and Hitter, R. Arch. Mikrobiol. 31: 347, 1958). Morphology: Sporophores monopodially branched, with long, regular, open spirals. Spores covered with fine long hair. Glycerol nitrate agar: Growth yellow to yellow-brown. Aerial mycelium powdery, chalk-white to gray-blue. Glucose-asparagine agar: Growth light yellow. Aerial mycelium powdery, white to ash-gray. Calcium malate agar: Growth white-yel- low to yellow-brown. Aerial mycelium white- yellow to white-gray. Starch agar: Growth yellow-brown to red- yellow. Aerial mycelium scant, white to ash-gray. Limited hydrolysis. Gelatin: Growth yellow-brown. Aerial mycelium powdery, chalk-white. Liquefac- tion slow. Soluble pigment dark brown. Potato: Growth golden yellow. Aerial mycelium ash-gray. Soluble pigment dark brown. Milk: Surface growth white-gray to gray- ish-blue. Aerial mycelium ash-gray. No co- agulation; peptonization weak. Antagonistic properties: Positive. Habitat: Soil from Rome, Italy. 189. Streptomyces platensis Pittenger and Gottlieb, 1954 (Pittenger, R. C. and Gott- heb, D. Brit. Pat. 713,795, August’ 18, 1954*). Morphology: Aerial mycelium forms loose to tight spirals on its sporophores. Spores ovoid, 0.7 to 0.9 by 0.8 to 1.2 yu. Sucrose nitrate agar: Substrate growth deep olive, reverse becoming dark olive. Aerial mycelium pale smoke-gray with tufts of white; areas of black pigmented aerial growth may also be found, giving effect of a mosaic. Glucose - asparagine agar: Substrate erowth ochraceous-buff becoming tawny. Aerial mycelium white becoming grayish- olive to almost black. Soluble pigment ab- sent or shght, brown. * Supplemented by personal communication. DESCRIPTION OF SPECIES OF STREPTOMYCES 259 Calcium malate agar: Growth ochraceous- salmon, becoming cinnamon-buff. Aerial mycelium quaker-drab with areas of black and white. Soluble pigment slight, greenish- yellow. Nutrient agar: Poor substrate growth, cream-yellow becoming buff to drab. No aerial mycelium. Slight soluble brown pig- ment. Starch agar: Growth cream- to buff-col- ored. Aerial mycelium white, becoming mouse-gray with patches of black. Slow hydrolysis. Potato: Excellent growth. Aerial myce- lium white to pale mouse-gray. Soluble pig- ment brown. Gelatin: Very slow liquefaction. Melanin- negative. Milk: Growth secant, forming partial ring at surface. No coagulation or peptonization. Blood: Hemolysis. Cellulose: Growth slight. Aerial mycelium gray to black. Nitrate: Reduction to nitrite, especially with starch as source of carbon. Carbon utilization: Starch, malic acid, inositol, sodium succinate, sodium citrate, sorbitol, mannitol, maltose, arabinose, lac- tose, galactose, fructose well utilized. Dul- citol, raffinose, cellulose, sodium formate, sodium tartrate, xylose poorly utilized. Asparagine, rhamnose, o0-cresol, m-cresol, sodium acetate, inulin, sodium. salicylate not utilized. Antagonistic properties: Produces oxytet- racycline. Remarks: Tresner and Backus (1956) con- sidered this organism as a variant of S. hygroscopicus rather than a separate species. Ettlinger et al. (1958) came to similar con- clusions. 190. Streptomyces pluricolor (Berestnew, 1897 emend. Krassilnikov, 1941) Waksman (Krassilnikov, N. L c S ~~ y vag * jp , ba’ 5. re be } ys : Za ne 2 a >. 4 = S a | 5 gm COO eye e arent hee 1.0 gm ENO Trae Dost) ge eNiy sn = BA 15.0 gm 329 Distilled waiter.) sa. 7 0002 0emil pH 7.0 The ammonium salt may be replaced by KKNO;. This medium was proposed by Masumoto (1952). 10. Glycerol-urea agar: Glycerol 724. 4: arte ee 15.0 gm Wrens 4. lato ia see ee 2.0 gm Ke O 2. yeti ea 0.5 gm INTE SOA Fs ede ans ue tomer 0.5 gm NS Cte is lo Ohh oo 0.5 gm HES Oe Misriens has mee 0.1 gm ENE) IA Oe eee ERE a Ona y 15.0 gm Distilled water: «52.0. se. 11. Glucose-tyrosine agar: GrliGoses 7 4.9 oan Her ee 10.0 gm ‘Eyrosiie: Oi Saee oe eee 1.0 gm CIES OM fakin serine alee 0.5 gm GELB Ose xk xe oitangt Abe 0.5 gm INGA T Rss be SPs cet ae Be goed Be 15.0 gm Distilled water teaction made neutral with NaOH. 12. Glucose-peptone agar: Glucose, crude 40 gm 10 gm PEplOne cso passe eee Agar. powdereds =. 4 24208 Distilled waters =..5: sce pH 5.6 Dissolve glucose and peptone in 500 ml dis- tilled water. Dissolve agar, by heating at 100°C, in another 500 ml water. Mix the two solutions, cool, add 10 gm egg-albumin dissolved in 50 ml water. Shake, steam for 30 min, allow clot to settle, filter, distribute in tubes, and autoclave at 115°C for 10 min. This medium, also known as Sabouraud’s agar, is fairly acid. 20 gm 1000.0 ml 13. Tyrosine-casein-nitrate agar: AVPOSING aid te Sete S ae Oo Sodium caseinate.......... Sodium nitrates. see. LCE Ae ae a, Tee ae 1.0 gm 25.0 gm 10.0 gm 15.0 gm Tapmweiler..2:2.. 55... 252 8 LOUORO ml This medium has been recommended (Men- zies and Dade, 1959) for the isolation of Streptomyces scabies from potato scab lesions 500 THE ACTINOMYCETHS, Vol. II or from soil. It inhibits spreading bacteria and favors the production of a dark brown pigment closely encircling colonies of the pathogen. Since almost all pathogenic iso- lates of S. scabies produce a dark pigment, the selection of the probable pathogen from dilution plates containing other actinomy- cetes is favored. 14. Peptone-beef extract or nutrient agar: PEPDOMNG 2 tbs. jee ome als ether 5.0 gm BGEISEXUEA CU oi incon = 5.0 gm NBIC] ees eh oo cath noel’ oa 5.0 gm J3\(037) ie AA ee a 15 to 20 gm Distilled water....... 1000.0 ml pH 7.2 to 7.4 Irrequently 10 gm of peptone is used. Glu- cose (10 gm) or glycerol (15 gm) may be added to give glucose- or glycerol-peptone agar. Tap water is often used in place of distilled water. In a liquid state without agar, these media are designated as nu- trient broth, glucose-peptone broth, or glyc- erol-peptone broth, respectively. 15. Peptone-beef-salt agar: Gincose, oa Maan O22 oe eae 10.0 gm BEDtONG: Sou ae. Sa Anes 5.0 gm Beehextract. a. i.s4% eo... 5.0 gm ERE Oi. geo pede Aus te fe: 1.0 gm MesOue (sO ics ola tGeeee. 0.5 gm SSC Ee ee oe a: eer Meee ere 0.5 gm NCAT Meas eG is ls PE 15.0 gm 1000.0 ml The organic nutrients may be reduced to Distilled water....... one half or even one tenth of above concen- tration, in order to favor production of aerial mycelium in some cultures of actinomycetes. 16. Glycerol-peptone-beef agar: (li Genoa 4) eae da attest) ee 20.0 gm PeDtONG Yea Rae a Hae d-8 5.0 gm Beef extract. ........4.04. 3.0 ¢m S\N eee te ee ee 15.0 gm Distilled water............ 1000.0 ml pH 7.0 17. Tryptone-yeast agar: CEP DUONG peak ths ac aha ten acs 1.0 gm VGast:extlacbs cule cas aa Ys 1.0 gm 5 gm NTO ee roe gdp) Sys 5 MSE 17.0 gm Tap water... ..... 24's... 00020) mal 18. Glucose-yeast-ammonium agar: GIUCOSE ss 673 tors acitaay ote 10.0 gm WeAast Extract. ocd o.ss6 ees 1.0 gm GNP is ERP Oe e7) vonsn ce 1.0 gm | 50) MORE een ae en ee ee 1.0 gm MigSOi: (20 os c aee ese ees 0.2 gm PREIS ES Ne enter 5 veal a on Pee 15.0 gm Wistilled water.....:.:.... 1000°O. ks2e derkle 1.0 gm Casein digest (N-Z-Amine A). 2.0 gm CoG aie Oi. os eee 0.02 gm 20.0 gm Distilediwater?.....:¢. 2225) 10005 0eml Adjust to pH 7.3 This medium is often known as Hickey and Tresner’s agar. 34. Oatmeal agar: Rolkedvoaits 2). ah sina gee 20 to 65 gm ita waber. = x. a8 spat on 100020Gml Cook to thin gruel in double boiler, filter through several layers of cheesecloth, and make up to a liter while still hot. Add 18 to 20 gm agar. Adjust to pH 7.2 with NaOH. 34. Tomato paste-oatmeal agar: 20.0 gm 20.0 gm 500.0. ml Add these two ingredients to the 500 ml of Heinz baby oatmeal food... . Tomato Paste ss 14 26.25 4 oa Pap Wathen bets: St ts ht boiling tap water. Do not adjust pH. DiCarlo aera ees LosO vom ABAD Wallet. .2 cucu fete 500.0. ml Melt by steaming at 100°C for 15 to 20 min. Do not adjust pH. Mix the two solutions, steam at 100°C for 10 min, dispense, and sterilize for 15 min at 121°C. 35. Soil extract agar: IBGChOxtlaiGus. aren te sees 3.0 gm PepuGne'sso5 24 ts tan ae Ses 5.0 gm INOTS «oe eda een neh as ee 15.0 gm SOMNERCEACh. 62g bs ae was 1000.0. ml pH: 7.0 THE ACTINOMYCETES, Vol. II The soil extract is prepared by treating | kg garden soil with 2.5 liters of tap water for 1 hour in autoclave at 15 lb pressure. Filter hot. Add tale for clarification if necessary. 36. Carbon nutrition medium: Carbon source......... 10.0 gm CNIPD) 0 One eee ee eas 2.64 gm IPO ae eee 2.38 gm KOEI RO.) aa: ee: 5.65 gm MigS Occ (Hb Oe see 1.00 gm CusOmoko On. 2 eae 0.0064 gm RESO (Hs On ee cae 0.001 gm Mn@ls=4i Oo a8 See 0.0079 gm TmsSOie(H>oOs 2k Aer 0.0015 gm ANON Chae ae eel OP eet 15.0 gm Distilled water... .. 7 100020mnl Some carbon compounds may have to be sterilized separately either by filtration or by heating in aqueous solution. This me- dium was used by Pridham and Gottlieb (1948), and has found general application in the study of utilization by actinomycetes of different carbon sources (Fig. 66). 37. Medium for nitrate reduction: EDLONE: de oe ee eee eae 5.0 gm Mieativextractecc ft. ase 3.0 gm TRIN GOs. e258 2 ae, ae ee 1.0 gm Distilled water: 02° 2 2: . 000020 pH 7.0 38. Medium for hydrogen sulfide production: Peptone-iron agar (Difco)... 36.0 gm Yeast extracte 2+: oe ee 1.0 gm Distilled water 27% «026i. 1000.0 ml 39. Cellulose medium: Filter paper saturated with synthetic solution, free from other carbon sources. 40. Potato plug: Plugs of potatoes of desirable size and shape; distilled water added and steri- lized. The importance of the variety and health of potato has been discussed by Grein and Kuster (1955). 41. Milk or litmus milk: Skim milk powder is used. Litmus or brom-cresol may be added. APPENDICES Control (No Carbon) 1% Glucose 1% Sucrose 0.15% NaOAec c f ( = an = fay Streptomyces Sp. 10 Days at 28°C FIGURE 66. Growth responses of streptomycetes, on chemically defined medium, to various carbon sources (Courtesy of T. G. Pridham 42. Melanin formation medium: Use 4 to 5 ml portions in test tubes. Incubate Yeast extract . l.0gm_ at 27°C and read after 1 to 2 days and after L-Tyrosine. . 1.0gm 4 days. NaCl... 8.5e@m 43. Other media: Agar. >... 16.0 em A variety of other media are frequently Tap water... 1000.0 ml used, often depending upon the nature of the 304 THE ACTINOMYCETES, Vol. II organism and the problem. This is true par- ticularly of the pathogenic forms, of organ- isms not growing readily upon ordinary media, and of those used for special purposes, such as antibiotic production. Among the complex media may be listed certain egg media (Dorset’s), blood agar, blood serum, ‘carrot plug, and others (Waksman, 1919; Levine and Schoenlein, 1930; Pridham et al., 1956). References” Rules of Gen. AINSworRTH, G. C. anv Cowan, 8S. T. nomenclature for fungi and bacteria. J. Microbiol. 10: 465-474, 1954. Aso, K., Aral T., YANAGISAWA, F., AND NaKa- gima, M. Studies on the distribution of actino- mycetes and their antagonistic strains in Japan- ese soil. J. Antibiotics (Japan) 2: 240-248, 1948. Aumauist, E. Untersuchungen iiber einige Bak- teriengattungen mit Mycelien. Z. Hyg. Infek- tionskrankh. 8: 189-197, 1890. ANDERSON, L. E., Exuruicu, J., Sun, 8. H., ann BURKHOLDER, P. R. Strains of Streptomyces, the sources of azaserine, elaiomycin, griseo- viridin, and viridogrisein. Antibiotics & Chemo- therapy 6: 100-115, 1956. Aoki, M. Agglutinatorische Untersuchung von Aktinomyzeten. Z. Immunititsforsch. 86: 518— 524; 87: 196-199, 200-201; 88: 60-62, 1935-1936. Avery, R. J. AND BuaNnk, F. On the chemical com- position of the cell walls of the Actinomycetales and its relation to their systematic position. Can. J. Microbiol. 1: 140-148, 1954. Backus, E. J., Duacar, B. M., anp CAMPBELL, T. H. Variation in Streptomyces aureofaciens. Ann. N. Y. Acad. Sci. 60: 86-102, 1954. Baupaccr, E. La denomination ‘‘Actinomyces bovis”’ Harz doit etre supprimée comme ‘‘nomen dubium.’’ Bull. Sez. Ital. Soe. int. Microbiol. 8: 99-101, 102-105, 1936; 9: 138-146, 1937. Baupaccr, E. Contributo alla sistematica degli Attinomiceti V-VIII. Atti Ist. botan. Pavia (4) 11: 191-231, 1939. 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Actinobacillus, 198 annulatus, 174 ligniersi, 320, 322 aquatilis, 316 paulotrophus, 35 arborescens, 316 Actinobacteriuwm, 20, 297 aromaticus, 316 abscessus, 20, 322 asteroides, 35 cellulitis, 15, 20, 322 asteroides var. serratus, 316 tsraelit, 17, 20 atrodiastaticus, 113 liquefaciens, 20 atroolivaceus, 123, 217, 326 meyeri, 20, 322 atypica pseudoturberculosa, 317 propionici, 2 aurantiacus, 90 Actinococcus, 198 aurantiogriseus, 271, 326 Actinomyces, vil, 1, 2, 4, 5, 12-20, 77, 95,496, 297, aureoverticillatus, 292 315, 316, 322 aureus, 87, oe abikoensum var. spiralis, 166, 326 aurint, 237, ¢ acidophilus, 122 avadi, 317 acidoresistans, 316 baarnensis, 287, 317 acrimycint, 123, 229, 326 bacillaris, 218 acrimycint var. globosus, 123, 229, 326 badius, 287, 326 actinoides, 316 bahiensis, 317 actinomorphus, 316 baudetiz, 12, 13, 15 aerugineus, 316 beddardi, 122 africanus, 34 bellisari, 317 agrestis, 316 berardinist, 317 albidoflava, 316 berestneffi, 317 albidoflavus, 14, 120, 122, 123, 168, 197 bicolor, 131, 288, 317, 326 albidofuscus, 316 biverticillatus, 272, 326 albido-fuscus, 316 bobili, 87, 89 albidus var. invertens, 169, 326 nee ae res ole alboatrus, 87, 316 bostroemi, 317 alboflavus, 87, 120 bovis, 12, 13-15, 17, 20, 29, 78, 278 albopurpureus, 316 bovis albus, 317 alborubidus, 122, 326 bovis farcinicus, 317 albosporeus, 86 bovis luteoroseus, 317 albovinaceus, 326 bovis sulfureus, 317 albus, 6, 71, 87, 90, 118, 119, 120, 264, 320 bovis var. nigerianus, 317 albus-acidus, 316 brasiliensis, 285 albus asporogenes, 316 bronchialis, 317 albus chlamydosporus, 172, 316 bronchiticus, 317 albus var. acidus, 316 brumpti, 317 albus var. alfa, 316 brunt, 317 albus var. fungatus, 172 buccalis, 317 albus vulgaris, 172, 316 caeruleus, 91 allenbachi, 316 Californicus, 87 almquisti, 119, 122, 316 cameli, 317 alni, 197 caminiti, 317 americanus, 316 candidus, 91, 122, 223 anaerobicus, 316 candidus var. eke 122, 188, 326 anaerobies, 316 canis, 20, 317 347 348 INDEX OF canis familiarts, 317 caprae, 38 carneda, 317 crnews, 60 carougeaut, 317 catarrhalis, 3 cati, 317 caucasicus, 218 caviae, 317 cellulitis, 12, 15,8 cellulosae, 89, 192 cerebriformis, 317 cereus, 317 chalmersi, 317 christophersoni, 317 chromofuscus, 145, 326 chromogenes, 90, 144, 202, 208, 209, 235, 253, 273, 317 chromogenus. 6, 71, 87, 118, 119, 122, 144 cinereo-niger, 317 cinereonigeraromaticus, 317 cinnabarinus, 131, 326 cinnamonensis var. proteolyticus, 196, 326 circulatus, 91, 324 citrea, 317 citreus, 87, 196 citrotremeus, 317 cloacae, 317 coccocidus, arn coelicolor, 90, 282, 283 coelicolor var. pie 326 coelicolor var. flavus, 326 coeruleofuscus, 131, O88, 326 coeruleorubidus, 131, 288, 326 coerulescens, 131, 288, 326 coerulescens var. longisporus, 288, 326 coeruleus antibioticus, 324 colorata, 317 congolensis, 317 convolutus, 318 coremiales, 318 cremeus, 170, 326 cretaceus, 91, 318 cruoris, 318 crystallophagus, 318 cuniculr, 318 cyaneus, 90 cyanofuscatus, 197, 218, 326 cylindraceus, 318 cylindrosporus, 91 daghestanicus, 196, 326 dassonviller, 318 decussatus, 318 denitrificans, 318 dermatonomus, 318 diastaticus, 86 ORGANISMS diastaticus var. ardesicicus, 201 diastaticus var. venezuelae, 201 diastato-chromogenus, 87, 89 discofoliatus, 12, 15-16 dispar, 318 donnae, 318 dori, 318 equptt, 318 elaeagni, 318 elephantis primigeniz, 318 enteritidis, 318 equi, 318 erysipeloides, 318 erythreus, 122 erythrochromogenus, 87, 89 exfoliatus, 87, 122 farcinicus, 122 farinosus, 91, 188 fasciculus, 188 ferrugineus, 318 flava, 318 flaveolus var. rectus, 326 flavidovirens, 170, 326 flavidovirens var. fuscus, 170, 326 flavotricini, 235, 326 flavus, 87,90, 170, 179, 192, 197, 210, 213, 252, 318 flocculus, 122 fluorescens, 218, 318, 324 foulertoni, 318 fradiae, 90, 138 fradiae var. spiralis, 324, 326 Fradi, 87 freeri, 318 fulvisstmus, 89 fumanus, 196, 326 fumosus, 91 fusca, 319 fuscus, 319 gabritscheoskt, 319 gartent, 319 gedanensis, 122, 319 gelaticus, 122 genesil, 319 gibsoni, 319 glaucescens, 131, 218, 326 glaucescens var. badius, 218, 326 glaucus, 90 globisporus, 90, 218, 226 globisporus albus, 218 globisporus circulatus, 218 globisporus diastaticus, 218 globisporus flaveolus, 218 globisporus griseus, 218 globisporus lactis, 218 globisporus scabies, 218 globisporus streptomycini, 143, 218, 226 INDEX OF ORGANISMS 349 globisporus var. caucasicus, 326 globisporus var. flavofuscus, 326 globisporus vulgaris, 218 globosus, 91 gobitricint, 286, 326 goensis, 319 gonadiformis, 319 gougeroti, 119, 122 gracilis, 90 graminearus, 319 graminis, 319 griseoflavus, 89 griseoincarnatus, 227, 326 griseoloalbus, 170, 326 griseomycint, 123, 148, 326 griseorubens, 123, 217, 326 griseoruber, 131, 272, 326 griseorubiginosus, 131, 266, 326 griseorubiginosus var. spiralis, 266, 326 griseostramineus, 210, 326 griseo-viridis, 319 griseus, 87, 89, 90, 123, 133, 1385, 143, 218, 286 griseus variabilis, 123, 319 griseus zonatus, 123, 287 gristnus, 218 gruberi, 319 gqueguent, 319 guerrat, 319 guignardi, 319 gypsotdes, 319 halotricus, 319 halstedit, 87, 90 heimi, 319 heimat, 122 hobnesi, 319 hofmanni, 319 hominis, 14, 819 hvidhansent, 12, 16-17 incanescens, 285, 319 indicus, 319 innominatus, 319 interproximalis, 20, 319 invulnerabilis, 319 israeli, 14, 16 israeli var. indo-sinensis, 17 israelit, 12, 13, 16, 17-18, 19, 20, 31, 78 iverini, 326 japonica, 319 japonicus, 319 jollyt, 319 jucous, 325 keratolyticus, 319 kimbert, 122 krainskiz, 209, 319 krauset, 319 kurssanoviz, 231, 326 lacertae, 319 lanfranchii, 319 lasseret, 319 lateritius, 285, 326 lavendulae, 87 leishmani, 319 lepromatis, 319 levoris, 197, 218 levyi, 319 lieskez, 122, 320 liguire, 320 lingualis, 320 Lipmanii, 87 liquefaciens, 320 listerz, 122 litmocidini, 326 londinensis, 320 longisporus, 90, 122, 172, 218, 320 longisporus-flavus, 90 longisporus-fungatus, 172 longisporus griseus, 172, 320 longisporus-ruber, 90, 271, 320 longissimus, 91, 212, 320 luridus, 212 luteolus, 320 luteo-roseus, 320 macrodipodidarum, 320 malachiticus, 130, 287, 326 malenconi, 122, 320 matruchoti, 320 melanocyclus, 90 melanogenes, 248, 320 melanoroseus, 320 melanosporeus, 243, 320 metchnikovt, 320 mexicanus, 37, 320 micetomae, 320 microflavus, 320 mihi, 320 mineaceus, 320 minimus, 320 minutissimus, 320 mirabilis, 122 mishagiensis, 320 mordoré, 321 mucosus, 320 multifidus, 320 muris, 49 muris-ratti, 320 musculorum, 320 mutabilis, 320, 326 myricae, 320 naeslundit, 12, 13, 18-19 necrophorus, 320 neddeni, 320 neschezadimenki, 320 300 INDEX OF ORGANISMS nicoller, 320 rectus, 91, 209, 281, 303 niger, 90, 247, 320 rectus brunneus, 281 niger aromaticus, 247, 320 reticult, 87, 91, 122 nigrescens, 231, 326 reticulus, 272 nigricans, 320 reticulus-ruber, 91, 272 nigrificans, 90, 247, 320 ribeyrot, 321 nitrogenes, 320 rivieret, 321 nocardit, 320 rodellae, 321 nodosus, 320 rogersti, 321 nondiastaticus, 320 rosaceus, 321 non-fluorescens, 320 rosenbachi, 321 ochraceus, 60, 320 roseochromogenes, 268 ochroleucus, 60, 275, 320 roseodiastaticus, 201 odontolyticus, 12, 19-20 roseoflavus, 133 odoratus, 320 roseofulvus, 270, 326 odorifer, 320 roseolilacinus, 286, 326 oidiosporus, 91 roseolus, 286, 321, 326 oligocarbophilus, 320 roseoviolaceus, 285, 326 olivaceoviridis, 130, 210, 326 roseoviridis, 286, 326 olivaceus, 89 roseus, 87, 89, 268 orangico-niger, 321 ruber, 90, 182, 204, 271, 321 orangicus, 321 ruber sterilis, 54 panginensis, 321 rubidaureus, 321 parvus, 87 rubiginosohelvolus, 143, 326 pelogenes, 321 rubiginosus, 128, 217, 326 penicillotdes, 321 rubrocyanodiastaticus, 118 phaeochromogenus, 89 rubrocyanodiastaticus var. impiger, 201 phagocidus, 321 rubrocyanodiastaticus var. piger, 201 phenotolerans, 90, 321 Rutgersensis, 86 pijpert, 321 sabrazés, 321 pinoyt, 321 saharae, 321 plurichromogenus, 321 salmonicolor, 90, 239 pluricolor, 90, 283, 321 salvati, 321 pluricolor diffundens, 259, 321 sanfelicer, 321 polychromogenus, 321 sanguinis, 321 ponceti, 321 sanninii, 119, 122 pranicolor, 131 saprophyticus, 122, 322 pretorianus, 321 saprophyticus var. chromogenes, 322 protea, 321 sartoryt, 322 prunicolor, 145, 326 scabies, 90, 197, 277 pseudonecrophorus, 321 scabies var. anglica, 322 pseudotuberculosae, 321 sendatensis, 322 pseudotuberculosis, 321 septicus, 322 pseudotuberculosus, 321 serratus, 322 pulmonalis, 321 setonit, 90 puntonit, 321 setonit flavus, 275 pur peo-chromogenus, 87 silberschmidtt, 322 purpureus, 321 somaliensis, 122, 322 purpurogenus, 87, 321 sommert, 322 putori, 49 spinae, 322 putridogenes, 321 sptnosporus, 322 putrificus, 321 spttzi, 322 pyocyaneus, 321 splenicus, 322 pyogenes, 321 spumalis, 90, 322 radiatus, 321 streptomycint, 148, 218, 226 raffinosus, 218 subtropicus, 220, 326 INDEX OF ORGANISMS dol sulfureus lacertae, 319 sulphureus, 14, 278 syringint, 286, 326 tarozzi1, 322 tenuis, 322 thermodiastaticus, 119 thermophilus, 119, 122, 300, 301 thermotolerans, 322 thibrerget, 322 thjottae, 322 thuilleri, 32 tossicus, 322 totschidlowskiz, 90, 322 toxicus, 218, 322 toxytricini, 235, 326 transvalensis, 322 tricolor, 198, 322 tyrosinaticus, 322 umbrinus, 200, 326 upcottit, 122 urethritidis, 322 urinarius, 322 valvulae, 322 valvularis, 322 valvulas desiiens bovis, 322 variabilis, 266, 326 vartabilis var. roseolus, 266, 326 venezuelae var. spiralis, 281, 326 Verne, 87 verrucosus, 322 verticillatus, 91 verticillatus viridans, 282 violacea, 285, 322 violaceochromogenes, 145 violaceorectus, 131, 285, 326 violaceus, 90, 251, 282, 283 violaceus-caesert, 86, 131, 251, 285 violaceus chromogenes, 285 violaceus cristallomicini, 325 violaceus-niger, 87, 90 violaceus-ruber, 87, 89, 282, 283 violaceus var. rubescens, 285, 326 violascens, 326 virgatus, 90 viridans, 90 viridis, 91, 120, 130, 136, 236, 287, 317 viridis sterilis, 287 virtdochromogenes, 90, 172, 306 virido-chromogenus, 87 virtdodiastaticus, 201 viridiflavus, 288 viridoviolaceus, 131, 285, 326 vulgaris, sae waksmanii, 282, 283, 322 willmorez, 12 xanthostromus, 275, 322 Actinomycetaceae, vit, 2 Actinomycetales, vit, 1, 2, 75, 316 Actinomycetes, incompletely described, 315-326 Actinoplanaceae, vit, 310-314 Actinoplanes, vi1, 4, 5, 81, 84, 310-313 philippinensis, 311-313 Anaeromyces bronchitica, 317 Asteroides, 323 liskeyi, 323 pseudocarneus, 323 Bacillus, 322, 323 actinoides, 322 bifidus, 323 coelicolor, 140 mycoides corallinus, 23, 40 ramosus, 323 Bacterium, 322, 323 actinocladothrix, 14, 322 Brevistreptothrix, 323 israeli, 17 Chainia, 5 Cladothriz, 315, 323 actinomyces, 14, 323 asteroides, 35 bovis, 14 dichotoma, 119, 122 foersteri, 318 tnvulnerabilis, 122 liquefaciens, 122 odorifera, 119, 122 rubra, 129 Cohnistreptothriz, 20, 323 americana, 316, 323 israeli, 14, 17 silberschmidti, 318 Corynebacterium, 1 acnes, 13 israeli, 17 Discomyces, 315, 322, 323 asteroides, 323 bovis, 14, 17 decussatus, 318 israeli, 17 lingualis, 319 pleuriticus, 322 pleuriticus canis familiaris, 322 Escherichia coli, 137, 174 Eubacteriales, 1 Flavobacterium, 323 salmonicolor, 323 Fungus sterilis, 153 Jensenia, 5 Lactobacillus, 1 Leptothrix oculorum, 318 Microbacterium mesentericum, 24 302 INDEX OF ORGANISMS Microbispora, 5, 84, 298-299 atlantica, 33, 36, 37 amethystogenes, 299 aurea, 317, 318 amethystogenes var. nonreducans, 299 autotrophica, 179 chromogenes, 299 babiensis, 239 diastatica, 299 berestneffi, 317 parva, 299 bicolor, 317 rosea, 298 bifida, 323 rosea var. nonnitritogenes, 299 blackwelliz, 28, 33, 37 Micrococcus pelletieri, 256 bovis, 14, 30, 323 Micromonospora, vit, 1, 2, 5, 84, 293-297, 300, 301, brasiliensis, 27, 29, 32, 37, 38, 58, 183 306, 309 brumpti, 239 bicolor, 294-295 bruni, 317 cabaelli, 294 buccalis, 317 chalcea, 293, 295, 301 calearea, 32, 38 coerulea, 294, 295 candida, 317 elongata, 294, 295-296 caprae, 27, 32, 35, 38, 39 fusca, 293, 294, 296, 301 carnea, 29, 317 gallica, 293, 296 carougeaut, 317 globosa, 293, 294, 296 caviae, 33, 39 monospora, 294 cellulans, 32, 39, 40 parva, 294, 296-297 chalmersi, 317 propionica, 294, 297 christophersoni, 317 vulgaris, 294, 300, 301, 309 chromogena, 35 Micropolyspora, 2 citrea, 32, 40 brevicatena, 2 citreus, 31 Mycobacterium, vit, 5, 21, 23-25, 67, 322, 323 coeliaca, 27, 32, 34, 40 agreste, 23 convoluta, 318 albuvialum, 323 corallina, 23, 27, 30, 32, 40, 41, 45, 50, 53-56 crystallophagum, 23 crateriformis, 36 erythropolis, 28 cruoris, 318 opacum, 23 crystallophaga, 50 paraffinicum, 322 cunicult, 57, 318, 323 rhodochrous, 27 cyanea, 132 salmonicolor, 23 cyaneus, 31 tuberculosis, 137, 175 cylindracea, 318 Mycococcus, 5 dassonvillet, 318 Nocardia, vi1, 1, 2, 4, 5, 23-60, 66, 73-75, 77, 78, 81, decussata, 318 83, 121, 129, 216, 236, 247, 285, 309 diastatica, 35 acidophilus, 324 dicksonit, 33, 41 actinotdes, 30 elaeagnit, 30 actinomorpha, 24, 30, 33, 34 enteritidis, 318 actinomyces, 14, 23, 323 eppingert, 35 africana, 38, 34, 60 equi, 318 agrestis, 54 erythropolis, 24, 25, 27, 30, 50, 328 alba, 33, 34-35 farcinica, 23, 27, 31, 32, 41, 42 alba lactica, 35 fastidiosa, 27, 32, 41, 42 albicans, 30, 32, 35 ferruginea, 23, 318 albida, 323 filiformis, 323 albosporea, 323 flava, 32, 42 albus, 30 flavescens, 24, 30, 31, 33, 43 alni. 30% 33,/ 30 flavus, 31 anaerobicus, 31 foerstert, 28 appendicis, 323 ford, 338, 43 aquosus, 25 formica, 33, 44 arborescens, 23 forstert, 318 asteroides, 25, 27-29, 31, 32, 35, 36, 47, 67, 272, foulertoni, 318 318, 021 freerz, 31 fructifera, 31, 33, 44 fusca, 319 gabritschewski, 31 gardneri, 28 gartent, 319 gedanensis, 30, 319 genesit, 256, 319 gibsonit, 33, 44, 45 globerula, 27, 32, 45 goensis, 319 grubert, 319 gypsoides, 29, 36 hoffmanni, 35 hortonensts, 38, 45 indica, 256 indica var. flava, 319 intracellularis, 32, 45, 46 israeli, 17, 31 tvorensis, 33, 46 krainskii, 323 krauset, 319 kurotishi, 33, 46, 47 lanfranchit, 319 lasserez, 319 letshmanit, 27, 29, 32, 35, 47 lignieresi, 31, 32 ; lingualis, 319 liquefaciens, 320 listeri, 33, 47 londinensis, 320 lurida, 324 lutea, 27, 33, 48 macrodipodidara, 320 maculata, 24 madurae, 239 marina, 33, 37, 48 mesenterica, 24, 30, 32, 48, 49 mexicana, 29, 320 minima, 27, 30, 35, 40, 54-55, 323 minutissima, 320 muris, 31, 33, 49 myricae, 30 narasinoensis, 324 nicoller, 320 niger, dl nigra, 33, 46, 49 nitrificans, 248 oligocarbophilus, 30 opaca, 23, 24, 27, 30, 32, 49-51 panginensis, 321 panjae, 33, 51 paraffinae, 24, 27, 30, 32, 51, 52 paulotropha, 35 petroleophila, 32, 52 phenotolerans, 35 pijpert, 321 INDEX OF ORGANISMS pinoyt, 321 plurichromogena, 321 pluricolor, 321, 32: polychromogena, 24 polychromogenes, 25, 27, 30-32, ponceti, 321 pretoriana, 27, 37, 321 pseudocarneus, 36 pseudotuberculosis, 321 pulmonalis, 27, 32, 53 putoriae, 31 pyogenes, 31 ramosa, 323 rangoonensis, 33, 53, 54 rhodnit, 27, 33, 54 repens, 323 rogerst, 321 rosenbachi, 318 ruber, 25, 31 rubra, 26-28 rubropertincta, 27, 32, 42, 55 rugosa, 28, 32, 55 salivae, 60 salmonicolor, 23, 27, 30, 32, 55, 36, 2 sanfelicer, 321 saprophytica, 323 saturnea, 60 sebivorans, 32, 56, 57 sendaiensis, 31 serophila, 32, 57 silberschmidti, 322 somaliensis, 31 splenica, 322 sumatrae, 33, 57, 58 sylvodorifera, 35, 323 tenuts, 322 thibierget, 322 thiryei, 321 thuilliert, 322 transvalensis, 27, 32, 37, 58 turbata, 33, 58 uniformis, 33, 58, 59 upcottit, 33, 59 vaccinit, 32, 59, 60 valvulae, 322 variabilis, 31, 32, 60 viridis, 31, 32, 60 Oidium lactis, 140 6, 315, 316 alpha, 122 Oospora, anaerobies, 316 asteroides, 35 bovis, 14 buccalis, 317 caprae, 38 chromogenes, 317 48, 50, 52, 53 8, 33, 48, 54, 55, 25: 3D 3 354 INDEX OF ORGANISMS cretacea, 318 cylindracea, 318 decussata, 318 doriae, 122, 318 forsteri, 318 hominis, 229 micetomae, 320 mordoré, 321 rosella, 321 rubea, 321 spitzt, 322 tenaz, 322 Polysepta, 5, 323 Proactinomyces, 5, 24, 323 actinoides, 324 aquosus, 323 asteroides var. crateriformis, 323 asteroides var. decolor, 323 atlanticus, 36 citreus marinae, 48 cyaneus, 323 cyaneus antibioticus, 323 flavus, 37, 42, 48 freeri, 318 israeli, 17 minimus, 40 murts, 49 opacus, 50 pseudomadurae, 323 restrictus, 323 Pseudonocardia, 5, 302, 309 thermophila, 308, 309 Serr atia, 323 corralina, 323 Sphaerotilus bovis, 14 Streptobacillus moniliformis, 49 Streptomyces, vi, 2, 4-6, 8-10, 21, 22, 24, 25, 84, 293, 300, 301, 302, 303, 306, 309, 311, 314, 315, 322, 333 Characterization of, 61-81 Deseription of, 84 Groups of, 82-114 albus, 92, 120 griseus, 92 lavendulae, 92 parvus, 92 reticuli, 92 viridochromogenes, 92 Identification of, 82-114 Sections of biverticillus, 97 biverticillus-spira, 97 monoverticillus, 97 monoverticillus-spira, 97 rectus-flexibilis, 97 retinaculum-apertum, 97 sptra, 97 Series of, 95, 96, 115-151 albidoflavus, 95, 124 albosporeus, 96 albus, 95, 96, 111, 117-123, 211, 217 antibioticus, 95, 118, 124 aureus, 95, 96, 124 azureus, 131 bostroemi, 95, 113 caeruleus, 95, 113 cas-gri, 144 chromogenes, 96 chromogenus, 144, 147 chrysomallus, 96 cinereo-ruber, 96 cinereus, 111, 117, 123, 148 cinnamomeus, 96, 112, 117, 149 circulatus, 96 coerulescens, 96 diastaticus, 95, 113 erythrochromogenes, 112, 117, 149 flavoviridis, 95 flavus, 95, 111, 117, 123-129 flavus-parvus, 128 fradiae, 95, 96, 111, 117, 131-133 fuscus, 96, 138 griseoincarnatus, 95 griseocarneus, 96 griseus, 95, 96, 111, 112, 117, 133-143, 229, 244, 260, 279 helvolus, 96, 148, 144 hygroscopicus, 112, 117, 124, 143, 144 intermedius, 95 lavendulae, 95, 112, 117, 144, 145-149 lavendulae-roseus, 96, 147 madurae, 95, 129 melanosporeus, 129 nigrescens, 96 phaeochromogenes, 110 reticuli, 96, 112, 117, 144, 149-151 rimosus, 95 roseochromogenes, 95 roseoflavus, 95 roseoviolaceus, 96, 131 roseus, 95, 129 ruber, 96, 111, 117, 129 rubrireticuli, 96 scabies, 95, 112, 117, 144, 145 sulphureus, 95, 124 thermophilus, 112, 151, 152, 302-304 verticillatus, 96 violaceoruber, 111, 117, 130, 131 violaceus, 96, 97, 131 virgatus, 95 INDEX OF ORGANISMS 3099 viridis, 95, 111, 117, 130, 286 viridochromogenes, 112, 117, 149 Species of, 115-151, 152-164, 165-292 abikoensum, 163, 165, 166, 267 aburaviensis, 156, 166 achromogenes, 158, 166, 167, 173 acidomyceticus, 163, 167, 196, 235 acidophilus, 159, 167, 168 aerocolonigenes, 291 afghaniensis, 163, 168 africana, 29 africanus, 256 albicans, 324 albidoflavus, 157, 168, 169, 232, 235, 268 albidofuscus, 264, 324 albidus, 159, 169 albireticult, 105, 150, 151, 1638, 169 alboflavus, 124, 160, 169, 170, 232, 235, 254, 255, 265 albogriseolus, 94, 99, 105, 158, 170 alboniger, 163, 170, 171 albosporeus, 129, 133, 160, 171 alboviridis, 130, 157, 171, 172 albulus, 324 albus, 29, 68, 93, 94, 102, 105, 109, 111, 117-121, 123, 156, 169, 172, 174, 219, 254, 287 almquisti, 219 althioticus, 158, 172, 173 ambofaciens, 158, 173, 177 aminophilus, 324 annulatus, 156, 173, 174, 187 antibioticus, 66, 101, 102, 105, 108, 142, 161, 174, 186, 205, 279 antimycoticus, 160, 174, 175, 246 arabicus, 324 arenae, 156, 175 argenteolus, 156, 175, 176 armillatus, 125, 159, 176 aurantiacus, 158, 176, 177 aureofaciens, 71-73, 93, 94, 101, 102, 105, 124, 127, 128, 157, 173, 177, 216, 274 aureoverticillatus, 292 aureus, 124, 162, 177-179, 228, 250, 279 autotrophicus, 156, 179 bacillaris, 324 badius, 324 beddardii, 161, 179, 180 bellus, 162, 180 bikiniensis, 68, 105, 161, 174, 180, 181, 228 blastmyceticus, 161, 181, 182, 324 bobiliae, 98, 130, 149, 162, 176, 182, 194 bottropensis, 158, 182, 183, 204 brasiliensis, 160, 183 cacaot, 94, 159, 183, 184, 254, 264 caelestis, 156, 184, 218 caeruleus, 105, 181, 157, 184, 185 caespitosus, 163, 185 caiusiae, 161, 185, 186, 234 californicus, 85, 93, 94, 105, 140, 141, 160, 186- 187, 268 calvus, 123, 156, 187, 188 candidus, 160, 187, 188 canescus, 68, 158, 188-190, 197, 198 canus, 160, 190 carcinomyceticus, 324 carnosus, 162, 190, 275 casei, 302, 303 catenulae, 156, 190, 191 cavourensis, 161, 19] celluloflavus, 129, 158, 191, 192 cellostaticus, 324 cellulosae, 123, 157, 192, 248 chartreusis, 68, 93, 149, 162, 184, 192, 193, 288 chattanoogensis, 324 chibaensis, 158, 193, 324 chrestomyceticus, 324 chromogenus, 144 chrysomallus, 93, 102, 129, 141, 142, 158, 193, 194. chrysomallus var. fumigatus, 102, 194 cinereoruber, 130, 149, 162, 174, 194, 195 cinereoruber var. fructofermentans, 130, 194 cinnamomeus, 117, 149, 163, 195, 247 cinnamomeus f. azacoluta, 195 cinnamomeus f. cinnamomeus, 195 cinnamonensis, 68, 105, 148, 161, 195, 196, 243, 250, 324 circulatus, 147, 150, 163, 196 citreus, 124, 159, 196, 197 clavifer, 145, 157, 197, 275 coelicolor, 70, 76, 78, 93, 102, 105, 106, 131, 132, 140, 157, 169, 190, 197, 198, 212, 282-284 collinus, 163, 168, 198, 199 colombiensis, 324 coroniformis, 156, 199 craterifer, 93, 117, 128, 156, 197, 199 cyaneofuscatus, 131 cyaneus, 93, 131, 149, 163, 199 cyanoflavus, 131, 132, 157, 199, 200 cylindrosporus, 162, 200, 262 dassonvillez, 130 decaris, 212 diastaticus, 93, 158, 167, 200, 201, 202, 239 diastatochromogenes, 94, 161, 201, 202, 222 echimensis, 105, 151, 324 echinatus, 64, 99, 163, 202, 203 elasticus, 156, 203 endus, 144, 160, 203, 204 erythraeus, 66, 100, 105, 129, 158, 176, 204, 291 erythrochromogenes, 117, 129, 149, 162, 198, 204, 205, 225, 291 eurocidicus, 163, 205 eurythermus, 161, 174, 205 lr oo INDEX OF ORGANISMS exfoliatus, 159, 205, 206 farinosus, 94 fasciculatus, 324 fasciculus, 94 felleus, 158, 206, 252 fervens, 149, 163, 206, 207 filamentosus, 159, 207 filipinensis, 162, 207, 208, 212 fimbriatus, 145, 163, 208 fimicarius, 124, 158, 166, 167, 206, 208 flaveolus, 68, 93, 99, 105, 124, 125, 142, 157, 203, 208, 209 flavochromogenes, 94, 161, 182, 209, 215, 222, 250 flavofungini, 324 flavogriseus, 93, 129, 158, 209, 212, 224 flavoreticuli, 163, 210 flavovirens, 105, 129, 142, 158, 210, 236 flavoviridis, 237 flavus, 93, 117, 124, 127, 129, 159, 198, 210, 211, 213, 252, 254 flocculus, 158, 211, 264 floridae, 141, 186 fluorescens, 324 fordiz, 124 fradiae, 66, 75, 94, 100, 105, 117, 132, 133, 134, 147. 154. 160, 209,210; 211, 212, 213; 238, 268, 284, 324 fragilis, 158, 212 fulvissimus, 99, 102, 159, 213, 276 fumosus, 160, 213 fungicidicus, 156, 213, 214 fuscus, 133, 163, 214 galbus, 102, 161, 214, 215 galbus var. achromogenes, 102, 215 galilaeus, 101, 162, 215 galtiert, 145, 161, 215 ganmyceticus, 324 ganmycicus, 324 gardnert, 156, 215, 216 garyphalus, 162, 216 gedanensis, 159, 216, 217 gelaticus, 159, 217 glaucus, 159, 184, 217, 218 globisporus, 70, 93, 156, 218, 254 globisporus streptomycini, 145 globisporus tundramycint, 324 globosus, 93, 162, 218, 219 gougeroti, 159, 219 gracilis, 162, 219 graminofaciens, 324 griseinus, 8, 114, 140, 160, 219, 220, 226 griseobrunneus, 161, 220 griseocarneus, 105, 150, 151, 163, 220, 221, 243 griseochromogenes, 161, 221, 222 griseoflavus, 99, 124, 127, 157, 176, 208, 222, 237, 249, 277 griseolus, 93, 102, 105, 156, 222, 223, 248, 252, 262 griseoluteus, 105, 107, 158, 223 griseoplanus, 159, 223, 224, 324 griseoruber, 94, 162, 224, 225 griseoviridis, 130, 162, 225 griseoviridus, 225 griseus, 8, 29, 66, 68, 74-76, 83, 98, 94, 99, 105, 114, 117, 128, 135-141, 148, 159, 169, 171, 179, 188, 192, 194, 220, 222, 225, 226, 236, 253, 256, 269, 275 griseus var. farinosus, 324 griseus var. purpureus, 140, 186, 187 griseus var. spiralis, 324 grisinus, 324 hachijoensis, 94, 149, 163, 185, 226, 227 hachijoensis var. fuscatus, 227 halstedizi, 93, 105, 160, 227, 252, 269 hawaiiensis, 145, 161, 227, 228 hepaticus, 217, 324 herbaricolor, 292 hiroshimensis, 101, 105, 109, 150, 151, 164, 228, 229 hirsutus, 93, 99, 130, 156, 229 hominis, 159, 229 humidus, 156, 229, 230 hygroscopicus, 68, 74, 98, 94, 101, 105, 107, 117, 124, 144, 160, 204, 230, 231, 259, 264 hygroscopicus var. angustmyceticus, 231 hygroscopicus var. decoyicus, 231 hygroscopicus var. odoratus, 231 intermedius, 123, 157, 231, 232, 286 tpomoeae, 78, 161, 174, 232 kanamyceticus, 133, 159, 232 kentuckensis, 163, 232, 233 kimbert, 156, 233 kitasatoensis, 164, 185, 233, 234 kitasawaensis, 161, 234 lanatus, 102, 162, 234 lavendulae, 29, 64, 74, 76, 77, 93, 94, 100, 102, 105, 108, 117, 146-148, 163, 196, 234, 235, 281, 287 lavendulae var. brasilicus, 235 lavendulae var. japonicus, 235 levoris, 325 leydenematis, 325 lieskei, 158, 232, 235 lilacinus, 325 limosus, 144, 158, 235, 236 lipmanit, 105, 160, 236, 268 litmocidini, 131 loidensis, 162, 236, 237 longisporoflavus, 160, 237, 254 longispororuber, 93, 252 longisporus, 325 lucensis, 161, 237 INDEX OF ORGANISMS 307 luridus, 133, 160, 237, 238 luteochromogenes, 325 luteolutescens, 325 luteoverticillatus, 101, 163, 238 lydicus, 158, 238, 239 macrosporeus, 99, 156, 239 maculatus, 157, 239 madurae, 27, 29, 159, 239, 240 marginatus, 161, 240 marinolimosus, 158, 240 marinus, 156, 240, 241 mashuensis, 163, 241 massasporeus, 292 matensis, 163, 241, 242 mediocidicus, 163, 242 mediterranet, 325 melanochr omogenes, 325 melanocyclus, 129, 160, 242, 243 melanogenes, 129, 162, 243 melanosporeus, 129 melanosporus, 325 michiganensis, 99, 102, 162, 243 microflavus, 105, 124, 129, 159, 169, 237, 243, 244, 270 mirabilis, 161, 222, 244 mitakaensis, 160, 244, 245 murinus, 102, 157, 245 naganishi, 94, 160, 245, 246 narbonensis, 158, 246, 252 natalensis, 325 netropsis, 100, 105, 151, 164, 246, 247 niger, 158, 247 nigrifaciens, 161, 247, 248 nigrificans, 144 nitrificans, 160, 248 nitrosporeus, 105, 158, 188, 248 niveoruber, 99, 129, 157, 248, 249 niveus, 123, 156, 249 noboritoensis, 162, 242, 249, 250 nodosus, 160, 250 noursel, 64, 99, 158, 250, 251 novaecaesareae, 131, 157, 251 odorifer, 163, 251 otdiosporus, 160, 251, 252 olivaceus, 76, 101, 102, 105, 131, 132, 159, 206, Zi 223, 227,246, 252, 281 olivochromogenes, 94, 161, 222, 252, 253 olivoreticuli, 164, 253 olivoverticillatus, 101, 163, 253, 254 omiyaensis, 160, 254. orchidaceus, 325 orientalis, 156, 254, 255, 265 ostreogriseus, 292 paraguayensis, 29, 159, 255 parvullus, 64, 101, 105, 123, 142, 157, 215, 255 parvus, 105, 124, 126, 127, 142, 157, 255, 25 paucisporogenes, 325 pelletiert, 27, 29, 159, 256 pentaticus, 164, 256, 257 phaeochromogenes, 64, 93, 99, 105, 162, 167, Dad 24a 2005 2D0 phaeochromogenes var. chloromyceticus, 281 phaeofaciens, 228, 325 phaeopurpureus, 101, 162, 257, 258 phaeoviridis, 157, 258 phoenix, 325 pilosus, 99, 161, 258 platensis, 144, 156, 231, 258, 259 pleofaciens, 325 plicatus, 325 pluricolor, 131, 157, 259 pluricolorescens, 158, 259, 260, 325 poolensis, 162, 260 praecox, 159, 260 praefecundus, 162, 261 prastnoptlosus, 93, 99, 130, 156, 261 prasinus, 93, 99, 130, 156, 261 promycint, 3825 psammoticus, 292 pseudogriseolus, 159, 261, 262 puniceus, 141, 186 purpeofuscus, 94, 200 purpurascens, 64, 93, 99, 105, 106, 130, 161, 182, 194, 263 purpureochromogenes, 94, 129, 162, 166, 200, 234, 262, 263 pur pureofuscus, 162, 262 purpureus, 186 putrificus, 159, 263, 264 pyridomyceticus, 160, 264 racemochromogenus, 325 raffinosus, 325 rameus, 161, 264, 265 ramnait, 158, 265 ramulosus, 65, 101, 158, 265, 266 recifer, 325 n rectus, 302, 303 resistomycificus, 161, 222, 266 reticuli, 64, 94, 100, 102, 105, 111, 117, 150, 151, 164, 210, 234, 266, 267 reticuli var. latumctdicus, 267 reticuloruber, 150 rumosus, 102, 124-127, 157, 173, 176, 204, 267 rimosus f. paramomycinus, 267 rochei, 159, 212, 267, 268 roseochromogenes, 93, 105, 108, 129, 161, 196, 204, 268 roseocitreus, 162, 269 roseodiastaticus, 160, 269 roseoflavus, 129, 159, 269, 27( roseoverticillatus, 101, 164, 27( roseus, 133, 159, 270, 271 INDEX OF ORGANISMS ruber, 94, 102, 105, 117, 129, 159, 225, 252, 271 rubescens, 160, 271, 272 rubrireticult, 212 rutgersensis, rulgersensis 99, 150, 164, 2 21, 229, 257, 270, 105, 160, 231, 250, 272, 273 var. castelarense, sahachirot, 157, 273 sakatensis, 325 salmonicida, 105, 151, 325 sampsonit, 157, 273, 275 sayamaensis, 157, 273, 274 scabies, 71, 260, 274, 2 setonit. 158, 2 sindenensis, 78, 101, 105, 117, 75 275 geo elie 159 1276 spheroides, 156, 2 276, 27 spiralis, 158, 242, 275, 277 sprroverticillatus, 101, 164, subtropicus, 325 231, 325 144, 145, 161, 252, 7, 278 sulphureus, 105, 157, 278, 279 tanashiensis, tendae, 100, 163, 279 tenuis, 161, 279 termitum, 322 thermodiastaticus, 302, 303, 320 thermofuscus, 301, 802, 303 thermophilus, 301, 303 thermoviolaceus, 302, 304 thermoviolaceus apingens, 304 thermoviolaceus pingens, 304 thermovulgar 7s, 302, 304 105, 162, 250, 279 thioluteus, 94, 105, 151, 163, 279, 280 toxicus, 325 toyocaensis, tricolor, 130 tumuli, 156, 325 280 tyrosinaticus, 212 vendargus, 3 25 venezuelae, 66, 68, 93, 100, 105, 146, 147, 163, 280, 281 venezuelae var. verne, 105, 157, verticillatus, verticillus, 3: 252, 281 roseosport, 281 150, 151, 163, 238, 281, 282 verticilloviridans, 150 25 vinaceous, 105 vinaceus, 105, 141, vinaceus-drappus, 325 violaceochromogenes, 131 violaceoniger, 282 violaceoruber, 78, 93, 117, 130-132, 157, 184, 197, 198, 212, 251, 2 violaceus, 67 186, 260, 262 66, 100, 105, 130, 144, 157, 231, 59, 282-284. , 157, 184, 212, 284, 285 violaceus-niger, 282 virgatus, 157, 285 virginiae, 105, 147, 148, 163, 196, 235, 285, 286 viridans, 129, 157, 286 viridifaciens, 325 viridis, 117, 180, 160, 184, 286, 287, 290 viridochromogenes, 67, 93,99, 101, 105, 117, 180, 149, 150, 162, 193, 215, 287, 288, 289 viridoflavus, 164, 215, 288-290 viridogenes, 66, 101, 161, 290 viridosporus, 325 vulgaris, 325 wedmorensis, 159, 290 willmoret, 156, 291 xanthochromogenes, 325 canthophaeus, 93, 157, 265, 291, 292 zaomyceticus, 325 Streptomycetaceae, vil, 2 Streptosporangium, Vil, 5, 81, 84, 310, 311, 313, 314 album, 314 amethystogenes, 314 roseum, 312, 313, 314 viridialbum, 314 vulgare, 314 Streptothrix, 10, 144, 315, 316 actinomyces, 14, 316 actinomycotica, 14, 316 alba, 6, 118, 119, 120, 122 albido, 316 albido-flava, 128 alpha, 316 aquatilis, 316 aurea, 317 beta, 317 bovis communis, 317 candida, 119, 122, 187, 317 caprae, 38, 317 chromogena, 118, 128, 144 cinereonigeraromaticus, 317 citrea, 123 coelicolor, 140, 197 cunicult, 57, 318 dassonvillei, 122, 123 eppingert, 35, 318 erythrea, 318 farcinica, 318 flava, 1 Foersterz, 118, 119, 318 foersteri, 123 forsteri, 318 freerz, 318 gedanensis, 119 gelatinosus, 319 graminearum, 123 hominis, 229, 319 humifica, 319 INDEX OF ORGANISMS 309 israeli, 14, 17, 319 violacea, 131 lathridizi, 119, 123, 319 zopfi, 322 leucea, 123, 319 Streptoverticillium, 5, 96, 111, 150 leucea saprophytica, 319 Thermoactinomyces, vit, 2, 5, 84, 301, 302, 306-309 melanotica, 320 glaucus, 3807 murts-rattt, 49 monosporus, 307, 309 necrophora, 320 thalpophilus, 307, 308 nigra, 49, 320 thermophilus, 307, 308 nigrescens, 320 viridis, 300, 307, 308, 309 oidioformis, 320 vulgaris, 307, 309 orangica, 321 Thermomonospora, vit, 5, 302, 304, 305, 306, 307 paulotrophus, 321 curvata, 304, 305 putoriz, 321 fusca, 804, 305 pyogenes, 123, 321 lineata, 304, 305, 306 rattt, 321 Thermophilic actinomycetes, 300-309 rubea, 321 Thermopolyspora, vii, 5, 302, 305, 306 rubra, 321 bispora, 306 sanninit, 322 polyspora, 306 spirilloides, 322 Waksmania, 2, 5, 84, 298, 299 taraxeri-cepapt, 322 rosea, 298, 299 tartart, 322 See also Microbispora GENERAL INDEX Abikoviromycin, 166, 272 Acetomyein, 265 Achromoviromyein, 167 Acidomyein, 320 Actinoidin, 324 Actinolysin, 324 Actinomyces classification of, 12 description of, 12-19 genus of, 12-20 incompletely described forms of, 20, 316-323 species of, 13-20 Actinomycetin, 172 Actinomycin, 174, 192, 194, 209, 210, 211, 215, 234, 243, 245, 255, 324 Actinomycin-producing organisms, 141, 142 Actinomycosis, 78 Actinophage sensitivity of Streptomyces, 141, 142 Actithiazie acid, 196, 286 Aerial mycelium, 62-69 Aggregate-species, 9 Alazopeptin, 224, 324 Albomyein, 325 Allomyein, 825 Althiomyein, 173 Amicetin, 324, 325 Aminocidin, 324 Amphomyein, 190 Amphotericin, 250 Amylocyanin, 140 Anaerobie actinomycetes, =p 75-76, 12-20 Angolamyein, 205 Anisomyein, 223 Antagonisms between strains, 147 Antibacterial properties, 147 Antibioties 1968, 324 C and D, 325 produced by 323-325 production of, as species characteristic, 75 incompletely deseribed species, sensitivity of actinomycetes to, 74, 75 X, 324 Antibiotic-producing actinomycetes, 324-325 Antitrichomonal activity, 225, 262 Antitumor agent, 325 substances, 324 Arthrospores, 121 Asecosin, 190 Aspartoein, 324 Aureothricin, 192, 200, 28 Azaserine, 213 Bacteria, relationship to actinomycetes, v, 1 Bennett’s agar, 331, 332 Bergey’s Manual of Determinative Bacteriology, v 326 Biochemical properties of Streptomyces, 61 Biomyein, 325 Blasticidin, 221 Blastmyein, 182, 324 Blood agar, 334 B-mycin, 183 Borrelidin, 268 Bryamyein, 228 , Cacaomycetin, 183 Caerulomyein, 184 Camphomyein, 325 Candicidin, 227 Candidin, 290 Candimycin, 324 Carbomyein, 227, 239, 279 Carbon nutrition medium, 332 Carbon sources for actinomycetes, 71-73, 143 Carcinomycin, 324 Carrot plug, 334 Caryomycin, 207 Carzinocidin, 234, 324 Carzinophilin, 273 Catenulin, 191 Celesticetin, 184 Cell wall, composition of, 1, 22 Celloeidin, 193, 324 Cellostatin, 324 Cellulose medium, 332 Chartreusin, 193 Chemical composition of actinomycetes, Chitin, | Chlamydospores, 298 Chloramphenicol, 254, 281 Chlortetracycline, 177, 274, 325 Cinerubin, 182, 249 Cinnamyein, 195 Cladomyein, 325 Classification systems of genus Streptomyces, 82- 114 Baldacei, 94-96 Belin ger et al., | ~J 98-101 360 GENERAL INDEX Flaig and Kutzner, 93 Frommer, 101-102 Gause et al., 96-97 Hesseltine ef al., 92, 93 Jensen, 89 KrassilInikov, 89-91 Mayama, 102, 103 Nomi, 108-105 Pridham et al., 97, 98 Proposed new system for series, 110-114 Routien’s outline, 105 Shinobu, 101 Waksman, 87-89 Waksman and Curtis, 86, 87 Waksman and Henrici, 91 Yamaguchi and Saburi, 94 Cluster formation, 10, 66 Cohn, F., 0 Colimyein (probably neomycin), 324 Colony structure of streptomycetes, 69, 70 Color designations, 327, 328 Congocidin, 173 Conn’s medium, 329 Coremia formation, 68, 298 Croceomycin, 324 Crystallomycin, 325 Cyanomycin, 200 Cycloheximide, 140, 194, 226 Cycloserine, 325 Czapek’s agar, 328 Dextrin-casein digest agar, 332 Dorset’s medium, 334 Echinomycin, 208 Ecology of actinomycetes, 77, 78 Egg-albumin agar, 331 Ehrlichin, 235 Elaiomyein, 217 Elaiophylin, 325 Kmerson’s agar, 331 Endomycein, 172, 204 Erythromycin, 204, 205 Etamycin, 225 Etruscomycin, 237 Eurocidin, 169, 205 Fairy rings, 298 Fermicidin, 22: Fervenulin, 207 Filipin, 208 Flavensomycin, 191 Flavofungin, 324 Flavomycin, 270 Fluorin, 324 Fradicin, 212 Fungichromin, 192 Fungicidin, 214 Ganmycin, 3824 Gelatin agar medium, 330 Gelatin media, 330 Genera of actinomycetes, 2, 82-84 Genetics of actinomycetes, 78 Geomyein, 291 Glucose agars glucose-ammonium salt agar, 329 -asparagine agar, 328 -casein digest-yeast-beef agar, 331 -peptone agar, 329 -tyrosine agar, 329 -yeast-ammonium agar, 330 -yeast extract agar, 331 -yeast extract-beef-peptone agar, 331 -yeast-malt agar, 332 Glycerol agars glycerol-ammonium salt agar, 329 -asparaginate agar, 328 -Camalate agar, 329 -glycine agar, 329 -peptone-beef agar, 330 -starch-glutamate agar, 331 -urea agar, 329 -yeast-malt agar, 640 Granaticin, 252 Grisamine, 222 Grisein, 220 Griseoflavin, 222 Griseolutein, 223 Griseomycin, 223 CGriseoviridin, 225 Grisin, 324 Groups of Streptomyces, 93 Hehomyein, 620 Helixin, 175 Hickey and Tresner’s agar, 332 Homomycin, 250 Hydrogen sulfide medium, 332 Hydroxystreptomycin F221 Hygromycein, 231, 250 Incompletely described species, 11, 215-226 Kanamycin, 232 Leucomycin, 234 Levorin, 325 Longisporin, 325 Luteomycin, 179, 279 Mangel scab, 280 Masumoto agar, 329 361 362 Matamycin, 180, 242 Media composition of, 62, 328-334 standard, 80 Mediocidin, 242 Melanin formation, 71, 333 -negative species, 111, 112 -positive species, 112 Mesophilic streptomycetes, 111, 112 Metabolism of Streptomyces griseus, 139, 140 Microbial species, concept of, 2-4 Micromonosporin, 296 Mikamyein, 245 Milk medium, 332 Miramyein, 244 Mitomycin, 185 Monilin, 325 Monomyein, 324 Morphology of streptomycetes, 5, 7, 61, 62-69, 116 Mutants of Streptomyces griseus, 140-143 Mycomycin, 324 Natural classification, 4 Necrotin, 325 Neomycin, 170, 212, 232, 266, 270 Nitrate reduction medium, 332 Nitrogen sources, 74 Nitrosporin, 248 Nocardia, 21-60, 323 acid-fast forms, 24, 27 biochemical properties of, 26-28 characterization of, 21-23 classification of, 28-34 colonies of, 24 description of, 27-29, 34-60 growth characteristics of, 24, 25 morphology of, 21-28 motility, 22 multiplication of, 21 serological properties, 29 Noecardorubin, 324 Noeardiosis, 21 Nomenclatural taxonomy, 4 Novobiocin, 216, 222, 249, 277 Nuelei, 4 Nucleoeidin, 187 Nutrient agar, 330 Nystatin, 251 Oatmeal agar, 332 Olivacein, 252 Oxytetracycline, 176, 223, 259, 267, 325 Pathogenic actinomycetes, 29 Pentamyein, 257 GENERAL INDEX Peptone-beef extract agar, 330 -beef-salt agar, 330 Phaeofacin, 325 Phagomyein, 223 Phthiomyein, 325 Physiological classification, 6 Picromycein, 206, 246 Pigments, nature of, 70, 71 Pimaricin, 325 Pleomyein, 825 Plotho’s agar, 329 Pluramyein, 260, 325 Pneumocin, 325 Polyenes, 179, 198 Potato glucose agar, 331 -peptone-glycerol agar, 331 Potato plug, 332 seab, 273, 275, 277 **Pox,:” 260 Proactinomycin, 216, 248 Progesterone, 176 Proteolytic activities of Streptomyces, 73, 74 Puromyein, 171 Pyridomycin, 264, 324 Racemomycin, 325 Raisnomyein, 2383 Ramnacin, 265 Reducing properties, 74 Resistomycin, 266 Rhodoeidin, 325 Rhodomyein, 194, 263 Rifomyein, 325 Rimocidin, 267 Ristocetin, 324 Roseocitrin, 269 Roseomycin, 268 Ruticin, 27: Sabouraud’s agar, 329 Sarkomyein, 205 Sclerotia formation, 69, 70 Sections, 9, 97, 98 Rectus-flexibilis, 97 Retinaculum-apertum, 97 Spira, 97 Monoverticillus, 97 Monoverticillus-spira, 97 Biverticillus, 97 Biverticillus-spira, 97 Series concept, 9 Series of Streptomyces, 115-151 Serological reactions, 76, 77 Sistomycosin, 825 Soil-extract agar, 332 Speciation of streptomycetes, 6-10 Species concept, I-11 descriptions, requirements for, LO, 11 groups, 7, 9, 66 Spiral types, 63-66 Spiramyein, 173 Spores of Streptomyces, 67-69 Starch agar, 330 -casein agar, 330 -peptone-beef agar, 331 Steroids, 266 Streptin, 272 Streptogramin, 324 Streptolin, 324 Streptomyces characterization of, 61-81, 153-155 classification of, 85-105, 155-164 description of, 10, 80, 81, 84, 165-292 generic name, 2 morphological groups, 104 series of, 115-151 species concept of, 2-4 species of, 84, 152-164 Streptomycetes actinophage sensitivity, 75, 76 antibiotic sensitivity, 74, 75 biochemical properties, 70-74 earbon utilization, 71-73 chemical composition, 77 colony structure, 69, 70 cultural properties, 70-74 ecology, 77, 78 genera of, 82-84 genetics, 78 growth response, 333 nitrogen utilization, 74 pigment formation, 70, 71 proteolytic properties, 73, 74 reducing properties, 74 serological reactions, 76, 77 standard media, 80 type cultures, 78-80 Streptomycin -dependent strains, 136 GENERAL INDEX -producing strains, 136, 141 production, 142, 143, 181, 226, 241, 252, 2 Streptonivicin, 249 Streptothricin, 169, 235, 244, 324 Streptovaricin, 276 Substrate mycelium, 62 Sucrose-nitrate agar, 328 Synthetic media, 7 Systematic position of actinomycetes, 1 Taitomycin, 168 Taxonomy of actinomycetes, 4 Tennecetin, 324 Tetracycline, 325 >) »- Thermophilic actinomycetes, 112, 300-309 Thiozolidone, 196 Tomato paste-oatmeal agar, 332 Toyocamyein, 325 Trichomycin, 227 Trichonin, 272 Tryptone-yeast agar, 330 Tuft formation, 10 Tumor-inhibiting substance, 243 Type cultures, 10, 61, 78-80 Tyrosinase reaction, 9, 71 Tyrosine-casein-nitrate agar, 329, 330 Tundramyein, 324 Ushinsky’s medium, 328 . >? Valinomycin, 213 Vancomycin, 254 Variations, cultural, 72 Varieties of Streptomyces griseus, 140-148 Vengicide, 325 65 363 Verticil-forming species of Streptomyces, 64, 65 Viomyein, 140, 186, 187, 253 Viridogrisein, 225 Virocidin, 210 Vitamin Bie , 824 Xanthicin, 325 Xanthomycin, 262, 273 Zaomycin, 325 ny Pewee lere sg see wal itieieines “- ftee iebeitiee * eaSeeAleL eet die ty Pie ermialelale eu, viel tiene clereleses ee fw hee, Pyelrleve oe tite Th oF * ie the rit Pitielere ge tee . wale rislststatstensiat ele PP eee pdiereouae fie aie aietey Fee wee ttelele aie tele “(eitpates tee ste beehete Op Oyepehelebece thee seeds heveyebecel ‘ ohare “hebejereie ole be yeraieiel jt treba le erle ye an fi behebeiele Titi tas eile etieiae herein eleiains cecaleyelaeny be wi ecey wit Bier elacager@heltlere etre ihe eerele Perea ayes e te SFiW elec acelepebepe ere sth eat yes card eine, itpthe irenpenes sriebraeitie oheeieye Treteiniele ei thetey PiPiaeiaje st errielems ithejeievegeie rat be elev, titheiere saeleus 4 1. te eltian x) eitue 19 10beheyey: te letete ie iebe ane bes “* uw fine a ieee ee WOi@isleveiaisisleresese . ttle Chale 4 veh WO aiteele ’ ripititieie ehe 4.1 ear Nett el epee TPR Bere hepa rey ehetelere ef 19 2 aitiasege 3 . * Bieceleue #.0\2 0 0, 0\4 SSTON Citieieltigeeie . “ite vie * eleleie strike PP ALALea teat aye ‘ i” shee leet Pielorel> Fee mee, aiesesd eet a thei cane “ thew alee PP mae lerelererey ttle fr 8ee tae " te? . eh leie eeres “OF Bie tele 1¢ eye eis Fite yeterereicia terse . tee . eerie tiaielele se * Hiejereres . * iM ithe " eke tid ea Haste Relics baeae eels feta haere *iei siete tsisheleters ey WS itietebaiereie a Lal esereheiess . tte ea, ieietel: te. stehelered O18) Lai. 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