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Full text of "The actinomycetes"

THE 
ACTINOMYCETES 

VOLUME II 




inand Colin I L828- 1898), who was the first to observe and describe an ac 
mycete (1875), under the name Streptothrix Foersteri. 



J I 



THE ACTINOMYCETES 

Vol. II 

CLASSIFICATION, IDENTIFICATION AND 
DESCRIPTIONS OF GENERA AND SPECIES 

by 
Selman A. Waksman 







It A LI I MORE 

I II E WILLI A M S & W I L K I IN S C O M P V N Y 

196 1 



THE ACTINOMYCETES 



Vol. 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 L922, Professor I). II. Bergey of the University of Pennsylvania wrote to 
me thai 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 thai volume of a section dealing with the actinomycetes. 

This group of organisms had occupied my attention for the previous several 
years, and alone ( L919) and with Roland Curtis ( 1916), 1 had described 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 tar published provided sufficient information for the 
accurate identification of most of the species recorded in the literature; (Id only 
four years previously, I had been warned by the dean of American cryptogamic 
botanists, Roland Thaxter, not to make further descripl ions of new species based 
solely or largely upon cultural and biochemical properties; and finally (c) I was 
not (wen certain at that time whether the actinomycetes should he included 
with the bacteria. 

I told Professor Bergey all this and suggested thai it would he better to wail 
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 
somewhal 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, "] 
will do it." The besl that I could accomplish a1 that time was to use cultural and 
biochemical characterist ics 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. 1 have not, however, always had the 
final word in organization of the material for all the various editions. Alone 
i I! 1 10), and together with Professor A. T. Ilenrici of the University of Minnesota 

(194:)), I proposed two systems for classificati f the act Lnomycetes, the second 

of w Inch consisted of a thorough revision of the group and its separal ion into four 
genera. The most significant change in this revision was the proposal, in L943, of 
the new generic name. Streptomyces. This second system has been the basis for 
the organization of the material in the lasl 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 a ug the bacteria and not among the 

fungi. Ample evidence of this belief has been presented in Volume I of this trea- 
tise. Unfortunately, the firsl reason for my hesitancy in L922, I believe, remains 
valid; the accuracy of the information available for species identification i> ,-iill 

v 



vi THE ACTINOMYCES ;s, 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 excepl by a few dedicated investigators. Recently. 
however, several important contributions (Hesseltine et ah, 1954; Flaig and 
Kutzner, 1954; Kutzner, 1956; Waksman, 1957; Ettlinger et al., 1958; Pridham, 
L959) 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 ;i 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 tit a certain "standard." Often, this 
standard has turned out to lie 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. Cordon and Dr. Hubert A. Lechevalier 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 consideral ion is given here to the various closely related genera thai 
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 
10 genera. 

A. Spores formed, bu1 not in sporangia. 

I. Vegetative mycelium fragmenting into bacillary or coccoid elements. 
Family I. Actinomycetaceae Buchanan. 

1. Anaerobic or microaerophilic, nonacid-fast. 

1 . ActinomyC( s I larz 

2. Aerobic, partially acid-fasl or nonacid-fast. 

2. Xocarrfia Trevisan 

11. Vegetative mycelium nonseptate, not fragmenting into bacillary or coc- 
coid elements. 

Family II. Streptomycetacecu 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 1 . Mesophilic forms, in pairs. 

."). Waksmania Lechevalier and Lechevalier 
b 1 . Thermophilic forms, in pairs or in chains, 
ti. Thermopolyspora Henssen 
•_'. Aerial mycelium not produced. 

a. Spores occurring singly on short sporophores. 
a 1 . Mesophilic forms. 

7. Micromonospora j )rskov 
b 1 . Thermophilic forms. 

s. Thermomonospora Henssen 

B. Spore.- occurring in sporangia. 
Family III. Actinoplanacecu Couch 

I. Aerial mycelium usually not formed, coiled conidial chain- lacking, spor- 
angiospores mot ile. 

!t. Actinoplam s Couch 
II. Aerial mycelium abundant, coiled conidial chains ;i- well ;i> sporangia 
formed in some species, sporangiospores nonmotile. 
10. stn ptosporangium Couch 



viii THE ACTINOMYCETES, Vol. II 

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 

Preface V 

Introductory vii 

1. The Specie- Concept in Relation to the Actinomycetes 1 

2. The Genus Actinomyces 12 

3. The ( renus Nocardia 21 

4. Characterization of Streptomyces Species 61 

.'). System.- of Classification and hlent ifical ion of ( Sroups and Specie,- of the 

( renus Streptomyces 82 

6. Series and Species of the Genus Streptomyces 11.") 

7. Classification of Streptomyces Species L52 

8. Description of Species of Streptomyces 165 

9. The Genus M icromonospora 293 

10. The Genus Waksmania I M icrobispora) 298 

I 1 . Thermophilic Actinomycetes 300 

12. Actinoplanaceae 310 

L3. Incompletely Described Species of Actinomycetes 315 

Appendix I. Color Designations for Describing Actinomycetes (Lin- 

denbein) 327 

Appendix [I. Certain Important Media for the Study of Actinomycetes 328 

References 335 

Index of Organisms 347 

General Index 360 




80932 



C h a p t e r I 



The Species Concept in Relation to 
the Actinomycetes 



Systematic Position <>(' the Actinomy- 

<<t< » 

In the preface to this volume, the state- 
ment was made thai "I am now certain of 
one thing, namely, thai 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 treatise." Nevertheless, 
-nine reiteration is warranted at this point. 

The taxonomic position of the actino- 
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 t hem 
with the bacteria. Their branching and man- 
ner of sporulation have suggested their rela- 
tionship to the fungi. Still other properties of 
actinomycetes seemed to warrant their con- 
sideration as a transition group between the 
bacteria and the fungi. 

Recenl 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 Coryni bactt num. 

2. Neither actinomycetes nor bacteria 
have heei i shown to contain i rue nuclei; they 
both contain only chromatin granules dis- 
tributed through the hyphae or the cells. 



:!. The diameter of actinomycete my- 
celium and spores is similar to that of bac- 
teria. Actinomycetes also, a- a rule, hick 
septa. 

}. Act inomycetes are subjeel to at tack 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 resistanl to those antibiotics, like the 
polyenes, that are active upon fungi hut not 
upon bacteria. 

(i. Chit in is absent from the cell substance 
of actinomycetes as well as from bacterial 
cells, but is present in fungus mycelium and 
spores. In their lack of cellulose, actino- 
mycetes are also similar to mosl 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- 
teria." Cummins and Harris (1958) went 
even further by suggesting that the order 
Actinomycetales be abolished altogether and 
that the families of the actinomycetes be 

included in the Eubocti Hall S. 

7. hike bacteria, but unlike mosl fungi, 
actinomycetes a- .-i rule are sensitive to 
an acid react ion of I he medium. 

8. The close relationship of the actino- 
mycetes to the bacteria is also evident from 
the work of ( 'ouch L95 I . who found that 
certain Micromonospora-like form- resemble 



THE ACTIXOMYCETES, 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 thai this genus may represent :i con- 
necting link between the bacteria and the 
lower fungi. 

The Generic Problem with Actinomv- 
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. Al- 
though occasional efforts had been made to 
separate the actinomycetes into several gen- 
era, such attempts usually failed to receive 
more than passing attention. The work of 
Waksman (1919), 0rskov (1923), Jensen 
(1931), and Erikson (1935) finally led Waks- 
man and Henrici to suggest, in 1943, the divi- 
sion of the actinomycetes into four genera. A 
new genus, Streptomyces, was proposed to in- 
clude those forms that are characterized by 
the production of an aerial mycelium with 
catenulate spores. Most of the important an- 
tibiotic-producing organisms subsequently 
have been found to belong to this genus. 

Unfortunately, this generic separation 
brought with it a number of new problems, 
which can be briefly summarized as follows: 

1. There is considerable overlapping 
among the different genera, notably between 
certain forms of Streptomyces that have lost 
the capacity to produce aerial mycelium and 
species of Nocardia, as brought out in a re- 
cent paper by Gordon and Smith (1955); 
there is also overlapping between certain 
nocardiae and mycobacteria. 

2. The formation by species of Strepto- 
myces and by certain forms of Nocardia of 
two different types of mycelium, substrate 
and aerial, and the influence of previous con- 
ditions of cultivation upon the growth and 



biochemical activities of these organisms 
served to confound the existing confusion. 

The nomenclatural status of the genera of 
Actinomycetales has recently been discussed 
by Lessel (1960). 

Lechevalier et al. (1961) described a new 
genus Micropolyspora (type species M. bre- 
vicatena), an organism that fragments like 
the members of the family Actinomycetaceae 
and sporulates like a member of the Strepto- 
mycetaceae, by forming chains of conidia on 
aerial hyphae; it also forms chains of conidia 
on the substrate mycelium. These authors 
suggested that the family Strcptomycetaceae 
be dropped and the family Actinomycetaceae 
be enlarged to include the genera Actinomy- 
ces, Micromonospora, Thermoactinomyces, 
Waksmania, Micropolyspora, Nocardia, and 
Stri ptomyces. 

What Is a Microbial Species? 

In the study of the taxonomy of any group 
of living organisms, including microorgan- 
isms, one is faced sooner or later with the 
problem of defining what is meant by a 
species. With microorganisms, in usual prac- 
tice, a microbial culture is designated by 
a name, sometimes qualified with a strain 
number; its morphological and cultural 
properties, and frequently its ecological and 
etiological characteristics, are described suf- 
ficiently so that anj'one who finds this or- 
ganism in nature will be able to recognize it 
from the description. If possible, the type 
form of the species is preserved in a type 
culture collection, to aid in the future identi- 
fication of the species. 

Unfortunately, microbial forms and types 
of organisms are not fixed in nature or even 
in culture. Some strains, even those closely 
related to the fixed type, may differ enough 
to raise a question as to their exact or specific 
identity. This frequently leads, often on the 
basis of only minor differences, to the crea- 
tion of new species that are given new epi- 
thets. This is particularly true of those mic- 



SPECIES CONCEPT !\ RELATION TO UTIN< >MYOETES 



r 'ganismSj like the actinomycetes, thai 

occur abundantly in nature; some of the 
newly isolated cultures may differ greatly 
from the fixed types. The difficulty of estab- 
lishing and recognizing •"species" under these 
conditions may become particularly perplex- 
ing. Raper (1954) was fully justified in say- 
ing, *Tt is almost axiomatic thai the ease 
with which a species of microorganism can be 
recognized tends to vary inversely with the 
number of isolates available for observation 
and examination." 

The concept of "species" first used during 
the seventeenth century gradually came to 
denote the fundamental units of a biological 
classification. These units came to be re- 
garded as fixed or static entities, created by 
nature, which can be grouped into higher 
categories, namely, genera, orders, and 
classes. A> the evolutionary theory was grad- 
ually accepted, especially with the develop- 
ment of modern genetics and cytology, the 
concept of "species" began to undergo a 
change. 

Ilucker and Pederson (1931) emphasized 
that the difficulty of dividing lower forms 
into well-defined species has led many to 
question whether these are natural groups 
and whether they can be considered to be 
similar to "species" among higher forms of 
life. The problem always arises: How much 
difference must exist between two cultures 
of bacteria before we are justified in regard- 
ing them as distinct species? 

Krassilnikov (1938) was very emphatic in 
stating that many investigators, without 
considering the rules of nomenclature pro- 
posed at international congresses, either de- 
Scribe the same forms under different names 
or combine various organisms into the same 
species. He said: "Even the concept of 
'species' is considered differently by various 
workers depending on their individual point 
of view, frequently considering a minor lack 
of correlation of a certain character as suffi- 
cient justification for creating a new species." 



.lust as in the case of many groups of true 
bacteria, one of the causes of the chaotic 
state of nomenclature of the actinomycetes 
is the lack of type cultures. It has actually 
been suggested (Skerman, L949) that even 
the available cultures be completely rede- 
scribed, priorities being based on existing 
names, and those names and descriptions for 
which no type cultures are available be dis- 
carded. 

In comparing the species concept among 
microbes with that of higher plants and 
animals, Cowan (1956) suggested that con- 
sideration be given to the following aspects: 
(a) whereas larger plants and animals have 
geographical distribution areas, few microbe- 
have such particular areas; (b) morphology 
is essential for the separation of species 
among algae, fungi, and protozoa, but it 
barely distinguishes higher ranks among bac- 
teria; (c) cytology is useful at the generic 
level, but "at the species level the bacteriol- 
ogist relies more on physiological than on 
morphological differences"; (d) interfertility 
is hardly to be considered as a species char- 
acter, since bacteria and actinomycetes re- 
produce asexually; (e) the introduction of 
certain characters in microbiology not uti- 
lized by botanists and zoologists adds satis- 
factory classification criteria; these include 
"nutritional requirements, metabolic and 
catabolic products, antigenic structure and 
pathogenicity." 

In discussing bacterial classification, 
Sneath (1957) came to the following con- 
clusions: (a i an ideal classification is one 
which has the greatest content of informa- 
tion; (b) over-all similarity is the basic con- 
cept of such ;in ideal classification, and i- 
measured in terms of the number of similar 
feature- possessed by two organism-: c 
every feature should have equal weight; (d) 
the division into taxonomic group- i- made 
upon correlated feat ures. 

To avoid the growing confusion from con- 
flicting ideas, (iihnour ( l'.lAS) suggested sep- 



THE ACTINOMYCETES, Vol. II 



aval ion of the concepts of "nomenclatural 
taxonomy" from those of "experimental tax- 
onomy." It is to be remembered that species 
are, after all, convenient "artificial creations 
of human imagination" rather than "real 
biological entities." Gilmour further sug- 
gested that "nomenclatural categories of 
genus, species, variety, etc." are excellently 
suited for the purpose of "a broad map of 
the diversity of living things." It would, 
therefore, be "a great advantage if they were 
not subject to continued attempts to bring 
them up to date and to redefine them in 
evolutionary terms." 

Speciation of Actinomycetes Other than 
Streptomycetes 

Krassilnikov (1938) wrote, "In spite of the 
most extensive literature, we have no definite 
idea concerning the natural systematics of 
the actinomycetes, nor a single opinion of 
their structure and development." The re- 
cently accumulated information leads us to 
conclude, however, that we need not be so 
pessimistic. 

According to Pridham (1959), there are 
now known more than 100 genera of actino- 
mycetes and well over 1500 subgeneric names 
and specific, or subspecific, epithets. Some of 
the descriptions of these forms are good, 
others lack essential details, and many are 
worthless. Morphological criteria are be- 
lieved to play an important role in separa- 
tion at the generic level (Fig. 1), with a 
gradual intergradation in complexity of re- 
productive units. The actinomycetes are 
looked upon as a heterogeneous group of 
organisms, ranging from the simple myeo- 
cocci and the seemingly more complex no- 
cardiae to the straight or flexuous strepto- 
mycetes and the verticillate forms, and from 
the relatively simple micromonosporae to 
forms such as Waksmania, Actinoplanes, and 
Streptosporangium (the latter two genera pos- 
sibly having some affinities with the chy- 
trids). Some of these organisms have definite 



affinities with true bacteria, others with both 
bacteria and microfungi, and still others with 
phycomycetous fungi. 

This heterogeneity is further emphasized 
by the facts that the actinomycetes contain 
forms that are anaerobic, microaerophilic, or 
aerobic; forms that fragment and those that 
do not; and forms that produce aerial my- 
celium and those that do not. Pridham sug- 
gested that some of the present concepts 
centered around the three genera Actino- 
myces, Nocardia, and Streptomyces be ac- 
cepted. Thus included in the Actinomyces 
would be the anaerobic to microaerophilic 
forms; in the Nocardia, the aerobic types 
that either form no aerial mycelium or pro- 
duce an aerial mycelium that generally has 
no catenulate spores; and in the Streptomy- 
ces, the aerobic forms that generally produce 
catenulate spores. 

Although time and again taxonomists have 
emphasized that an effective system of clas- 
sification should be based upon criteria that 
are expressed in consistently reproducible re- 
sults, this has hardly been applied, at least 
so far as our present knowledge is concerned, 
to the species characterization of actino- 
mycetes. Many "new species" have been de- 
scribed on the basis of a single difference — 
frequently a quantitative variable — from 
"old species." One often wonders what the 
composition of the medium, the conditions of 
growth, and the natural variability observed 
so frequently among duplicate cultures have 
to do with these distinguishing properties. 

The species concept among the actinomy- 
cetes must be considered as the continuity 
between different groups of organisms desig- 
nated as species, with various transitional 
forms bridging the gaps between species. The 
concept of natural classification applies to 
actinomycetes perhaps better than to many 
other bacterial groups: there are the chemi- 
cal approach (chemical composition, pres- 
ence of specific chemical compounds), the 
morphological approach (type of aerial my- 



SPECIES CONCEPT IN RELATION TO ACTTNOMYCETES 



Mycococcus 




Streptomyces 


j,f*w 


Jensema 


if 


Chainia'* 


^ik 


Polysepta 


ft" 


Streptoverticillium* 


4T 


Mycobacterium 


!W % 




7f^ 


Actinomyces 


U< 








A 




C 


Nocardia 


'\ 


Micromonospora 


s* 


Proactinomyces 


A 


Jhermoactinomyces 


**- 


Pseudonocardta 


^C 


Thermomonospora 

Microbispora t "~\ 
Tfiermopo/yspora "-> 
Waksmanta J 








Actmoplanes 


/*■»< 






Streptosporangium 


A,; 




B 




D 



Figure 1. Morphology of the various genera of the Actinomycetales. Of these, only Actinomyces in 
A. Nocardia in B, Streptomyces in C. Micromonospora, Thermoactinomyces, Waksmania, Actinoplanes, 
and Streptosporangium in D are recognized in this i realise as true actinomycetes; Nocardia and Proac- 
tinomyces are synonyms (Courtesy of T. G. Pridharo of the Northern Regional Laboratory, Agriculture 
Research Service, U. S. Department of Agriculture). 



THE ACTINOMYCETES, Vol. II 



celium, type of speculation, shape and sur- 
face of spore), and finally the ecological ap- 
proach (anaerobic versus aerobic, pathogenic 
versus nonpathogenic, thermophilic versus 
mesophilic). The idea of a physiological clas- 
sification includes formation of antibiotics 
and of enzymes, utilization of carbon com- 
pounds, and transformation of nitrogenous 
compounds, all of which can supply supple- 
mentary information. 

Speciation of Streptomycetes 

What has been said for the actinomycetes 
as a whole applies particularly to the large, 
heterogeneous, and variable group of organ- 
isms represented in nature by the aerial my- 
celium-producing strains, most of which are 
included at present in the genus Streptomy- 
ces. These organisms are found in the soil in 
the form of hundreds of thousands of spores 
and of bits of mycelium per gram. They are 
also found extensively in manures and in 
composts, in various fresh-water basins, in 
dust, and on food. They are almost entirely 
absent from peat bogs and the sea. 

The actinomycetes belonging to the genus 
Streptomyces have recently come to occupy 
an eminent place because many of them are 
important producers of antibiotics, vitamins, 
and enzymes. 

With the growing economic significance of 
members of the genus, the establishment for 
each species of certain characteristics which 
would be adequate to enable the investigator 
to recognize freshly isolated cultures in well 
defined specific terms becomes of great theo- 
retical and practical importance. 

Following the first descriptions of Cohn 
(1875), very few additional species of the 
aerial mycelium-producing actinomycetes 
were recognized until 1914. This was true in 
spite of the rapidly accumulating literature 
on the occurrence of such actinomycetes in 
the soil and in the causation of plant diseases. 
The common designations were limited 
largely to the names " Actinomyces albus" 



and "Actinomyces chromogenus," depending 
on the color of the aerial mycelium or the 
formation of soluble, dark pigments in com- 
plex organic media. 

Rossi-Doria (1891) was the first to de- 
scribe an organism, under the name Strepto- 
thrix alba, which was later designated as the 
type of the genus Streptomyces proposed by 
Waksman and Henrici in 1943. The most 
important characteristics of this species are 
its white aerial mycelium and the tendency 
for colonies to form concentric rings of this 
aerial mycelium. Rossi-Doria noted the abil- 
ity of his organisms to grow on numerous 
complex organic substrates. 

Thaxter (1891), who first described an im- 
portant economic species, the causative 
agent of potato scab (which he believed to 
be a fungus, Oospora), was highly critical of 
the efforts to describe "species" largely on 
the basis of cultural properties of the organ- 
isms. In this respect, the actinomycetes do 
not differ from any of the other groups of 
bacteria, where cultural properties and bio- 
chemical reactions have to supplement in- 
sufficient morphological information. Physi- 
ological activities and ecological properties, 
which are the expression of the response of 
organisms to their environment, are too 
numerous and often too variable among ac- 
tinomycetes to justify unlimited confidence. 

Krainsky (1914), Waksman and Curtis 
(1916), and Waksman (1919) emphasized 
the use of synthetic substrates, in addition 
to organic media. Carbon and nitrogen utili- 
zation tests were employed. Added attention 
was given to micromorphology. Many new 
species were described. Jensen (1930a, 1931) 
and Duche (1934) added various new species, 
the latter investigator stressing the use of 
various combinations of carbohydrates and 
nitrogenous compounds as media ingredients. 

One of the reasons for the limited recog- 
nition of species among the aerial mycelium- 
producing actinomycetes prior to 1914 was 
the fact that protein-rich media were 



SPECIES CONCEPT IN RKLATION TO ACTINoMYCKTKS 



employed for their cultivation. With t he in- 
troduction of synthetic media, it became 
definitely established that the aerial myce- 
lium-producing actinomycetes comprise a 
large number of tonus, differing greatly in 
their physiological and biochemical proper- 
ties, and to a lesser degree in their morphol- 
ogy. It was also recognized that, if a suffi- 
ciently large number of cultures was isolated 
and examined, many differences would he 
noted suggesting variability of the type spe- 
cies. The concept "species-croups," with one 
culture as the type species, was suggested. 
Waksman ( 1919) emphasized, therefore, ihat 
in spite of variation of individual biochemi- 
cal characteristics of the actinomycetes 
there are certain well defined properties, no- 
tably morphology, color of aerial mycelium, 
and formation of soluble pigments, that char- 
acterize these organisms, especially when 
grown on standard synthetic media and un- 
der carefully controlled conditions of tem- 
perature and aeration. 

It is easy to pick out a few cultures of 
actinomycetes (or streptomycetes) which 
possess characteristic properties that can be 
recognized as distinct species, and to discard 
all the others. This was actually done by 
Waksman and Curtis in their early (1915- 
1916) classification of actinomycetes, since 
they were faced with such a large number of 
freshly isolated cultures that it was impos- 
sible to consider more than a very small 
number of them. How many others have 
acted likewise it is dillicult to say. Should 
the various intermediate strains be consid- 
ered, one might be inclined to regard each 
as a different species, distinct from the 
others in at leasl one variable property, be 
it morphological, cultural, or biochemical. 
With the examination, in recent years, of 
many thousands of cultures of actinomy- 
cetes for their antibiotic properties, such an 
attitude was frequently reduced to an ab- 
surdity. There are those who contend thai 
the insistence on permanent characteristics. 



preferably a group of | hem, in describing new- 
species, would limit greatly our recognition 
of the growing economic importance of these 
organisms. Then there are those who reason 
thai not enough species of actinomycetes 
have so far been described, thus justifying 
random descripl ions of many freshly Isolated 
si rains as new species. 

Even synthetic media did not yield the 
final answer to the species problem of this 
group of organisms. Their cultural proper- 
ties, or growth characteristics in media of 
different chemical composition, properties 
that were at first greatly emphasized, were 
found to be extremely variable. Type cultures 
were shown to change their specific charac- 
teristics when grown in artificial media. 
Saltations and mutations came to play a 
highly important part in changing such prop- 
erties. When morphology was recognized at 
all, it was limited largely to observations on 
the curvature of the sporophores or to the 
size and shape of the spores. Drechsler ( I !> 1 !) I 
was the first to make a detailed study of the 
morphology of the actinomycetes thai pro- 
duce aerial mycelium. Unfortunately, he 
limited his study to a small number of cul- 
tures; this prevented him from establishing 
the existence of many specific types which 
could have been recognized on the basis not 
only of cultural but also of morphological 
properties. 

It must be regarded as a considerable step 
backward when Lieske (1921) completely 
disregarded the work of Krahisky (1914), 
Waksman and Curtis (1916), Conn (1917), 
and Waksman (1919). He believed ihat the 
classification of actinomycetes was impos- 
sible, since the properties observed were 
highly variable. His skeptical attitude to- 
ward the question of speciation of actino- 
mycetes was due largely to his use of complex 
media for t he growth of these organisms, and 
to a lack of sufficient appreciation of the 
significance of simple media tor their char- 
acterizat ion. 



s 



THE ACTINOMYCETES, Vol. II 



Burkholder et al. (1954) were led to con- 
clude that the species concepts formulated 
by an individual investigator depend a great 
deal upon the investigator's personal expe- 
rience, and whether he is a "splitter" or a 
"lumper." They suggested further that mi- 
crobial species should be characterized by 
multiple, readily recognizable, and reason- 
ably stable properties; the history of the 
cultures and the nature of the medium in 
which they are growing are of prime im- 
portance. 

With the genus Streptomyces gaining con- 
siderable economic importance, the creation 
of many new species based upon biochemical 
properties, notably formation of antibiotics, 
resulted in much confusion in the recognition 
of some of the species. The use of various 
mutagenic agents, such as irradiation, led to 
the formation of new forms or strains which 
are often markedly different in their nutrient 
requirements and biochemical activities from 
the mother cultures. 

According to this concept, in the classifi- 
cation of a group of living organisms, no 
single feature can be taken as the predomi- 
nant character. Only when this is combined 
with a group of other characters is one able 
to separate the group into subgroups, no- 
tably genera and species. In selecting a char- 
acter, no matter what its importance in the 
primary subdivision of a group of actino- 
mycetes, one may begin with color; or 
structure of aerial mycelium; or certain bio- 
chemical reactions, which may comprise 
proteolytic activities, utilization of carbo- 
hydrates, production of antibiotics, or phage 
sensitivity. The important thing is to select 
a group of properties to characterize each 
species, with fewer characters, perhaps only 
one, such as antibiotic production, charac- 
terizing varieties. One always encounters, of 
course, the intermediate forms between the 
species. Each investigator will have to decide 
upon the basis of the combination of charac- 
ters whether to place an unknown culture 



with one species or another. Thus the concept 
of species-group or section has come into 
being. As a further illustration one may take 
S. griseus and S. griseinus, two species be- 
longing to the S. griseus group; both are non- 
chromogenic; the color of the aerial myce- 
lium of both is similar; they are both similar 
morphologically; yet they are different from 
the standpoint of carbon utilization, phage 
sensitivity, and antibiotic production. 

Flaigand Kutzner (1954), Kutzner (1956), 
Baldacci (1959), and numerous others em- 
phasized both physiological and morphologi- 
cal criteria. Gause et al. (1957) emphasized 
the color of substrate and of aerial mycelium 
as well as morphology of sporulating hyphae. 
Numerous new species and varieties were 
described, although very few prior named 
species were discussed or placed into their 
system of classification. Many of these spe- 
cies and varieties are no doubt synonymous 
with previously described forms. 

With streptomycetes, the species are 
linked together so gradually that it is very 
difficult to say where one species ends and 
another begins. The creation of "sections," 
"groups," or "series" to occupy an inter- 
mediate place between genera and species 
may help in clarifying relationships, but it 
does not do away entirely with the poten- 
tial confusion in the creation of new species, 
especially when the relation of such species 
to those already established is not sufficient ly 
understood. This confusion has led some in- 
vestigators to question "whether the species 
concept is tenable in microbiology, and if it 
is not, what we are to substitute for it." It 
has even been suggested that the idea of 
static species must be abandoned in favor of 
something more elastic. 

Even now, after many additional data 
have accumulated concerning the morphol- 
ogy of the actinomycetes, and after these 
organisms have been separated into a num- 
ber of genera, there is still no general agree- 
ment concerning characterization of species. 



Sl'KCIKS CoNCKPT IN RELATION TO A.CTINOMYCETES 



Krassilnikov (1949) insisted that the shape of 
the spore, as seen in the lighl microscope, 
should be recognized as the major criterion 
for species differentiation. It is doubtful, 
however, whether Krassilnikov's various 
"longisporus" and "globisporus" types, with 
their many subtypes, can greatly facilitate 
the solution of the problem of species char- 
acterization. The cultural properties of these 
organisms still offer si >f the most im- 
portant criteria for species differentiation. 
There is also now available sufficienl addi- 
tional information concerning morphology, 
such as formation and branching of the 
sporophores, formation and nature of spores, 
and especially the spore surface as shown by 
the electron microscope, to make possible 
the use of these criteria not only for supple- 
mentary but often for major characteriza- 
tion of the species. 

Several factors have thus contributed to 
the confusion in establishing and recognizing 
species of act iiiomycetes : (a) lack of clearly 
defined morphological characters; (b) great 
variability of these organisms; (c I occurrence 
of numerous transition types; (d) ease of 
formation of mutants; (e) lack of sufficiently 
recognizable type species; (f) lack of empha- 
sis upon species-groups and upon type cul- 
tures; and (g) insufficient recognition of the 
formation of well defined chemical com- 
pounds which could be used as additional 
criteria for species characterizat ion. 

The suggestion that closely related spe- 
cies be placed in "species-groups" or "aggre- 
gate-species" has recently been gaining con- 
siderable attention. Such a unit should be 
characterized by various reproducible prop- 
erties under standard c litions of culture. 

Baldacci et al. I L953, L956) suggested that 
micromorphological criteria, namely, seg- 
mentation and branching of vegetative my- 
celium, presence or absence of -pores, and 
arrangement of sporophores, be used for 
generic classification. The genus Streptomy- 
ces was then divided, on the basis of pigmen- 



tat ion of i he vegetat ive and aerial mycelium, 
into a number of "-eric-." each of which 
was further subdivided into specie-. Gause 
d <il. (1957) made use of the "series" con- 
cept and created a number of groups based 
on the pigmentation of the aerial mycelium. 

When so many different cultures of ac- 
t iiiomycetes can be isolated easily from natu- 
ral substrates, it is but natural thai various 
intermediate type- should be found and that 
established species should tend to overlap 
one another. \i one were to isolate only a 
small number of cultures, it would be simple 
to recognize a few well defined species. But 
when hundreds of similar strains are found 
in nature and when many of them show only 
minor variations from one another, varia- 
tions which are not important enough to 
warrant creation of new species hut are 
nevertheless variations from the established 
type, the difficulties mount rapidly (Fig. 2). 

When study is based upon a single strain, 
a particular species may he described as hav- 
ing a yellow or yellowish aerial mycelium. 
Another strain may produce, on the same 
medium, an aerial mycelium only a shade 
different in color from the original type; 
this pigment may he designated as sulfur- 
yellow, cream-yellow, saffron-yellow, or even 
brownish, all other physiological and mor- 
phological properties being similar. Would 
one he just ilied in calling such a new strain a 
different species? The answer is definitely 
"no." One culture may produce a strong 
tyrosinase react ion, and another only a weak 
reaction, as indicated by pigmentation with 
potato, gelatin, and other protein media. 
( me would he inclined to accept these as 
mere quantitative variations a 1 Iowa hie for an 
established species. This must he recognized, 
since it is well known that had the test been 
repeated in another laboratory, where the 
medium mighl he slightly different in coin- 
position, the method of -1 erili/ai a f the 

medium different, or the age and origin of 
the inoculum different, these variations 



II) 



THE ACTINOMYCETES, Vol. II 




1 





Figure 2. Schematic representation of tuft and cluster formation by certain Streptomyces species 
(Reproduced from: Shinobu, R. Mem. Osaka Univ. Lib. Arts and Ed. B. Nat. Sci. 7, 1958). 



might have been sufficient to account for the 
minor differences in the color of mycelium 
or in the pigmentation of the medium. But 
what is one to do when the original culture 
is recorded as producing a yellow aerial my- 
celium on a given medium, whereas the new 
isolate gives a buff or brown mycelium? The 
answer would be that if all the other recog- 
nizable properties are the same or similar, 
this would be nothing more than a variant. 
Were one to plate out a single culture and 
pick a large number of colonies, similar varia- 
tions could no doubt be observed. 

Unfortunately, it has frequently been 
found much easier lo assign undue impor- 
tance to these variations and designate a 
freshly isolated culture as a new species. 
Some justification for this attitude has been 
found in the fact that the new culture may 
possess an important economic property, 
such as the production of a new antibiotic. 
1 1 is largely for this reason that within the 
last I.") years more "new" species have been 
created than in all the previous 7."> years 



since Ferdinand Cohn first described his 

Streptothrix. 

Requirements for Adequate Species De- 
scriptions 

In accordance with the rules of the Inter- 
national Code of Nomenclature of Bacteria 
and Viruses, certain procedures must be fol- 
lowed in describing bacterial species. These 
are summarized b^y Ainsworth and Cowan 
(1954) as follows: 

The name must be effectively published. 

The name must be validated by a concise de- 
scription of the diagnostic features of the new 
isolate. 

The etymology of the name should be ex- 
plained. 

No Latin diagnosis is required. 

When descriptions are reported in a language 
unfamiliar to the majority of workers, it is recom- 
mended that the authors simultaneously publish 
the diagnosis in a more familiar language. 

Subcultures of the type strains should be de- 
posited at one or more of the national culture 
collections. 

Unfortunately, these simple rules have 



SPIX'IKS CONCKPT IN RELATION TO A.CTINOMYCETES 



I I 



not always been adhered to. Numerous 
names of actinomycetes are reported in the 
literature with qo descriptions whatever. 
Some of the descriptions have been published 
partly in languages nol generally accessible, 
or in the form of patents, or even as news 
announcements in trade or popular journals. 
Although every effort has been made in 



this treatise to include all species that have 
been adequately described, numerous forms 
must be listed as "incompletely described" 
(Chapter 13). Various names are listed for 
which not even an inadequate description is 
available, the temptation to name a culture 
as a new organism, in order to claim the 
discovery, being too areal . 



Chapter 2 



The Genus Actinomyces 



The genus Actinomyces comprises anaero- 
bic or microaerophilic organisms. They are 
mostly pathogenic in nature. The pathogenic 
forms are nonacid-fast, nonproteolytic, and 
nondiastatic. These have been isolated from 
granules in the pus of morbid tissues of a 
human and animal disease known as actino- 
mycosis. They produce no filterable stages 
and show no serological reactions with other 
genera. 

There are also on record observations con- 
cerning the occurrence in various natural 
substrates of nonpathogenic, mesophilic, 
anaerobic actinomycetes that can with full 
justification be included in this genus. Al- 
though few of these have been sufficiently 
.studied, one such species is included. The 
saprophytic forms may be proteolytic, ac- 
tively fermentative, and may possess marked 
reducing properties. 

The natural relationship of this genus to 
the other genera of the actinomycetes, based 
primarily upon morphological and cytologi- 
cal studies, has recently been examined by 
Bisset (1959). 

< Jassilieatioii of the Genus Actinomyces 

I. Pathogenic tonus or forms isolated from 
pal hogenic specimens. 

1. Colonies soft, smooth, uniform, not adher 
ent to the medium. No aerial hvphae. 

a. Causative agent of certain animal dis- 
eases. 

I. Actinomyces bovis 
li. Isolated from human saliva and carious 
teeth. 

8. Actinomyces odontolyticus 

2. Colonies tougher in texture and warted in 



appearance, adherent to medium. Aeria 

hvphae rare. 

a. Hvphae gram-positive and stain faintly 
with hemotoxylin. Causative agent of 
certain human diseases known as actino- 
mycosis. 

6. Actinomyces israelii 
a 1 . Related form. 

4. Actinomyces discofoliatus 

1). Hvphae in pus granules stain with basic 
stains. Cause of actinomycosis in cats 
and dogs. 

2. Actinomyces baudetii 
II. Nonpathogenic forms. 

1. Occurs in human mouth. 

7. Actinomyces naeslundii 
a. Related form. 

3. Actinomyces cellulitis 

2. Occurs in ground waters. 

5. Actinomyces hvidhanseni 

According to Thompson (1950), there arc 
two distinct species of anaerobic organisms 
that should be included in the genus Actino- 
myces: A. hurls which is responsible for most 
cases of lumpy jaw in cattle, and .1. israelii 
which causes most of the typical infections 
in man. This separation of the genus agrees 
with the concepts of other investigators. 
One strain of A. israelii was recovered from 
a bovine source, and it was suggested that 
some bovine infections may be due to ^4. 
israelii. On the other hand, the work of Holm 
(1951) and Lentze (1948) indicates that a 
small number of human infections may be 
due to A. bar is. 

Cummins and Harris (1958) fully sup- 
ported the conclusions of Erikson (1940) 
and Thompson (1950) that bovine and hu- 
man strains of Actinomyces are distinct. On 



12 



thi: <;i:x us actixouycks 



13 



the basis of their chemical data, they sug- 
gested thai i here was very little justification 
for placing bovine strains even in the same 
genus with the si rains of A. israelii. Of the 
12 strains received as .1. bovis, two were 
identical with the human strains, two showed 
a cell wall pattern unlike anything hitherto 
recorded, i wo appeared to be corynebacteria, 
and the remaining six formed a homogene- 
ous group which seemed to he closely related 
to lactobacilli. If cell wall composition is to 
he considered as any guide to the classifica- 
tion of these strains, the criteria used for the 
identification of .1. bovis are insufficient and 
many of the investigators who identified the 
strain were not properly qualified to do so. 

Thompson and Lovestedl (1 ( .>.">1) isolated 
cultures from the months of 'J 1 patients. In 
addition to two positive cultures of .1. 
israelii, nine of the cultures comprised an 
organism which grew under both aerobic and 
anaerobic conditions. They considered the 
latter to lie a saprophyte found in the mouth, 
frequently confused with A. israelii. The 
name .1. naeslundii was proposed for these 
cultures. 

Howell et <d. (1959) made a comparison of 
200 strains of Actinomyces isolated from the 
oral cavity in the absence of actinomycosis, 
and 11 isolated from actinomycotic lesions. 
These strains were of two main types, one 
corresponding to the organisms described 
under the name .1 . naeslundii, and the other 
essentially identical to those isolated from 
lesions, which should be designated as .1. 
israelii. They recommended that .1. naes- 
lundii Thompson and Lovestedl be accepted 
as the proper name for the rapidly growing 
facultative type of Actinomyces. 

One may finally report the results of ;i 
comparative study (Pine et el.. I960) of II 
bovine strains of Actinomyces isolated from 
typical cases of lumpy jaw and 15 human 
-trains which had been identified as .1. 
israelii and .1. naeslundii. Of the bovine 
strains, one was a typical .1. israelii, whereas 



the remaining strains formed a homogeneous 
group <>f fast growing, catalase-negative 
diphtheroids which invariably failed to form 
a true mycelium in vitro; they were thus 
different from both .1. israelii and .1. naes- 
lundii. The last 10 -trains comprised the 
classical .1. bovis. They produced two kinds 

of colonies, depending on the medium: one 
smooth colony, identical to that of Coryne- 
bacterium acnes, and one rough similar to 
that of A. israelii but with no mycelium. 
They were anaerobes, forming acid from glu- 
cose but none from xylose, raffinose, or man- 
nitol; nitrates were not reduced and starch 
was rapidly hydrolyzed. They were less path- 
ogenic for animals than human strains, but 
induced lesions in which actinomycotic my- 
celial clumps were formed. The .1. israelii 
strains were also anaerobes; they formed 
acid from glucose, usually from xylose and 
mannitol, and less often from raffinose; ni- 
trates were sometimes reduced to nitrite-. 
and starch was poorly hydrolyzed if at all. 
A. naeslundii strains were facultative anaer- 
obes and formed acid from glucose and 
raffinose, but none from xylose or mannitol; 
nitrates were reduced to nitrites and starch 
was poorly hydrolyzed. Micromanipulative 
methods for the study of microaerophilic 
organisms have been examined by Erikson 
(1954); the catalase reaction of .1. bovis was 
reported by Suter ( L956). 

According to Emmons,* there is little 
value in presenting as valid all the following 
species until they h;ivel>een -I ndied carefully 

in pure culture. He suggested to accepl only 
.1. bovis, A. israelii, .1. baudetii, and .1. 
naeslundii. He went so far a.- to suggesl that 
the staining reactions of .1. baudetii are 
hardly sufficienl for its differentiation. 

Descriptions of Species of Actinomyces 

I. Actinomyces bovis Harz (Harz, ('. < >. 
In Bollinger, < >. Centr. med. Wiss. L5: 185, 

Personal communical ion. 



14 



THE ACTINOMYCETES, Vol. II 









chins mycelium; cul- 

X 1000 (Reproduced 



Figure 3. A. boms, br; 
tured from human tonsil 
from: Emmons, C. W. Puerto Rico J. Public 
Health Trop. Med. 11: 720, 1936). 



1877; Jahr. Munch. Thierarzeneisch 5: 125, 

1877). 

Actinomyces bovis was the first authentic 
actinomycete described as a causative agent 
of disease; it is natural, therefore, that it 
should have a number of synonyms. These 
are given here, without any guarantee that 
the list is complete. 

Synonyms: Discomyces bovis Rivolta, 
1878; Bacterium actinocladothrix Afanasiev, 
1888; Nocardia actinomyces de Toni and Tre- 
visan, 1889; Actinomyces hominis Bostroem, 
1890; Streptothrix actinomyces Rossi-Doria, 
1891; Cladoihrix bovis Mace, 1891; Oospora 
bovis Sauvageau and Radais, 1892; Actino- 
myces albidoflavus Rossi-Doria, 1891; Actino- 
myces sulphureus Gasperini, 1894; Nocardia 



bovis R. Blanchard, 1895; Streptothrix israeli 
Kruse, 1896; Cladoihrix actinomyces Mace, 
1897; Streptothrix actinomycotica Foulerton, 
L899; Discomyces bovis R. Blanchard, 1900; 
Streptothrix spitzi Lignieres, 1903; Sphaeroti- 
lus bovis Engler, 1907; Cohnistreptothrix 
israeli Pinoy, 1911; Actinomyces israeli Vuil- 
lemin, 1931. See also Baldacci (1937). 

Morphology: Grows in the form of sulfur- 
colored granules in the pus of cases of ac- 
tinomycosis. The radiating hyphae are cov- 
ered with extraneous material deposited by 
the host to form clubs. Organism is gram- 
positive, nonmotile, nonacid-fast. Colonies 
are dull white in color, only slightly ad- 
herent to the medium. No aerial hyphae. 
Mycelium undergoes fragmentation very 
rapidly into V- and Y-forms. Extensive 
branching is rare. Hyphae less than 1 p. in 
diameter (Fig. 3). 

Semisolid media: Growth excellent, espe- 
cially with paraffin seal. No soluble pigment 
produced. 

Gelatin: Growth scant, flaky. No lique- 
faction. 

Liquid media: Occasional turbidity with a 
light, flocculent growth. 

Egg or serum media: No proteolytic ac- 
tion. 

Milk: Turns acid; no coagulation and no 
peptonization. Sometimes there is no growth. 

Sugar utilization: Acid from glucose, su- 
crose, and maltose; no acid from salicin or 
mannitol. 

Temperature: Optimum 37°C. Does not 
grow at 22°C. Killed at 00°C. 

Oxygen requirement: Anaerobic to micro- 
aerophilic. Grows readily in an atmosphere 
of C0 2 . Bovine strains are more oxygen- 
tolerant on egg or serum media than strains 
of human origin. 

Viability: Pure cultures do not live more 
than 10 to 14 days. On Dorset's egg medium, 
they may survive in an ice chest for 3 to 4 
weeks. 

Habitat : ( )riginally found in lumpy jaw of 



THE GENUS ACTIN0M1 ( ES 



L5 



cm 1 1 le. Usually found in and aboul mouths of 
animals. 

Remarks: King and Meyer (1957) re- 
cently suggested thai in order to implement 
proper identification of .1. bovis, certain se- 
lected differentia] criteria, such as catalase 
test, litmus milk reactions, and the utiliza- 
tion of xylose, salicin, and raffinose, can be 
used. Slack and Moore (1960) suggested the 
use of fluorescent antibody formal ion for the 
further identification of this organism. 

2. Actinomyces baudetii Brion, 1942 (Brion, 
G. de. Rev. de Med. Voter. 91: 157, 1942; 
Brion, G. de, Goret, and Joubert. Proc. VI 
Congr. Intern. Patol. Comp., Madrid 1: 48, 
1952). 

Morphology: Granules from histological 
preparations show tangled, radiating hy- 
phae; ends of hyphae rounded and ovoid, 
forming a crown. Hyphae take basic stains. 
Mycelium composed of slender hyphae, 0.2 
to 0.4 fjL. Nonseptate. Ends swollen and 
rounded. Copious branching. In artificial 
media hyphae are frequently short, rarely 
exceeding 20 m m length. 

Agar colonies: Dull, whitish granules ad- 
hering slightly to the medium. 

Liquid media: A sediment of white gran- 
ule- i- produced. 

Gelatin: Xo liquefaction. 

Blood serum: In 1 to 5 days, surface cov- 
ered with white granules which are the size 
of a pill head. 

Serum media: Xo proteolytic action. 

Brain extract: Growth favored in some 
media. 

Indol: Production slight . 

Sugar utilization: Acid from glucose, su- 
crose, and starch. 

I Oxygen demand: Anaerobic to microaero- 
philic. 

I Optimum temperai me: 37 ( '. 

Pathogenicity: Pathogenic when inocu- 
lated into dogs, rabbits, and guinea pigs 
(forms subcutaneous 



Source: Isolated from various types of le- 
sions in cats and dogs. 

.'!. Actinomyces cellulitis (Linhard, 1949) 
now comb. (Linhard, J. Ann. inst. Pasteur 
76:478, 1949). 

Synonym: Actinobacterium cellulitis bin- 
hard. 

Morphology: Polymorphic rod-, showing 
primary, secondary, and sometimes tertiary 
branching. Length 5 to 7 n, diameter 0.6 n. 
Xonmot ile. < tram-positive. 

Agar media: Colonies lenticular. No gas. 

Glucose broth cultures: No turbidity. 

Abundant growth, settling to bottom. 

( telatin: No liquefaction. 

Milk: Unchanged. 

Serum: Serophilic, but can be adapted to 
serum-free media. 

Nitrate reduction : Positive. 

Oxygen demand: Anaerobic and micro- 
aerophilic. Colonies produced at 4 to 5-mm 
depth in agar media. 

Reduction: Does not reduce neutral red or 
safranin. 

Carbon utilization: Positive utilization of 
glucose, fructose, maltose, galactose, and 
sucrose. Produces volatile acids (propionic 
and formic). Production of gas may suggesl 
either a contaminated cull ure or the absence 
of an Actinomyces. 

Pathogenicity : Nonpathogenic. 

Habitat : ( >ral cavity of man. 

4. Actinomyces discofoliatus (Griiter, 
L932) Negroni (Negroni, P. Mycopathol. 1: 
81-87, L938 L939). 

Morphology: Deep colonies in semisolid 
glucose agar are whitish, lens-shaped, crossed 
or forming dihedral angles; margins of colo- 
nic- regular; consistency of colonic- slimy. 
Bacteria-like entities measuring 3 to 1 M to 
10 to 15 m by 0.8 fi, occurring as isolated 

element- or V- Or Y-shaped element-. ( '..in 
pact colonic- in hanging-drop cultures. The 
filaments have a tendency to dichotomous 



L6 



THE A(TIX(>.MV('KTi:S, Vol. II 




Figure 4. .1. israelii, grown : 
Institute of Pathology). 



Forces 



branching, with prevailing development of 
one branch. 

Glucose agar: Discoid, moist, and brilliant 
colonies; slightly elevated in the central pari 
with nearly regular margins. 

Gelatin: Xo liquefaction. 

Glucose broth: Slimy sediment and some- 
times a slight turbidity. The medium be- 
comes clear at the end of 8 to 10 days. 

Carbon sources: Acid but no gas from glu- 
cose, maltose, fructose, lactose, sucrose, and 
iiiulin; very little or no acid from mannitol. 

Starch: Not attacked. 

Sucrose: Not inverted. 

Nitrate reduction: Negative. 

H 2 S: Formed. 

Indol: Slight quantity produced. 

Fats: Slightly attacked. 

( Hive oil: Not attacked. 

Optimum temperature: 37°C. 

( >xygen demand: Facultatively anaerobic. 

Remarks: Vitality weak. Deep cultures in 
semisolid media die if held for longer than 8 
to 10 days at 37°C, or for longer than 30 
minutes at 60°C. Exposure for longer than 



a few minutes in dilute mineral acids kills 
the organism. The organism can be kept 
alive for 2 to 3 months if cultures are kept 
in an ice chest, in a dried state, or under 
vacuum. 

Habitat: Lachrymal concretions and hu- 
man actinomycotic lesions. 

5. Actinomyces hvidhanseni (Hvid-IIan- 
sen, 1951) nov. comb. (Hvid-Hansen, X. 
Acta Pathol. Microbiol. Scand. 29: 335-338, 
1951). 

Synonym: Actinomyces Israeli Hvid-Han- 
sen. 

Morphology: Gram-positive, nonacid-fast , 
nonmotile. Polymorphic, bent, and often 
branched rods. Obligately anaerobic. 

Meat liver agar: Colonies circular or ir- 
regular, often in the form of bodies bounded 
by four concave surfaces meeting in four 
acute vertices, of highly varying size and of 
a pale pink color. Surface colonies circular, 
convex, grayish-white or white; transparent 
S-colonies of a butyrous, viscous, but not 
mucous consistency. 

Meat liver broth: Diffuse growth at first, 



THE GENUS ACTINOMYCES 17 

followed rapidly by a fairly voluminous pale israeli Pinoy, 1913; Nocardia israeli Castel- 

pink, homogenous precipitate. lani and Chalmers, L913; Brevistreptothrix 

Thioglycollate medium: Growth either israeli Lignieres, L924; Proactinomyces israeli 

diffuse, netlike, or dispersed and granular. Jensen, L931; Cor ynebacterium israeli Lentze, 

Gelatin: Liquefied. L938; Actinomyces israeli var. indo-sinensis 

Milk: Coagulated in 24 to 48 hours and Reynes, 1947. 

peptonized in :! weeks. Morphology: Large, club-shaped forms 

Blood: All strains hemolyze human blood are seen in morbid tissues. Substrate myce- 

iiii solid media bul do not form a soluble lium consists of rapidly septating and spor- 

hemolysin. ulating hyphae. The branches may extend 

Sugar utilization: Galactose, fructose, and into the medium in long filaments or may 

glycerol vigorous; inulin, maltose, mannitol, exhibit fragmentation and characteristic an- 

saccharose, starch, dulcitol, and lactose gular branching. Hyphae occasionally sep- 

soniewhat less readily; xylose and arabinose tate, hut no definite spores are formed. 

not at all. Colonies exhibit a considerable degree of 

Reduction: Some strains form a little hy- polymorphism, but no stable variants have 

drogen sulfide. Sulfites and sulfates are not been established. Colonies are tougher in 

reduced. Nitrates reduced to nitrites and in texture than those of .1. bovis. Old colonies 

some cases to ammonia. Safranin, phenosaf- warted in appearance (Fig. 4). 

ranin, or neutral red not reduced. Most Gelatin : Growth scant, flaky . No liquefac- 

Strains decolorize methylene blue in 4 to 24 tion. 

hours; some do not . Liquid media: Growth in form of white 

Temperature: Optimum 37°C. Heating to compad colonies or granular sediment. Me- 

.">() lit) (' for L5 minutes injurious. dium shows no turbidity, usually remaining 

Remarks: All strains catalase-positive. All clear. No gas and no odor, 
produce ethyl alcohol, aldehyde, acetone. Pigments: No soluble or insoluble pig- 
ammonia. A faint indol reaction is found in ments. 

alkaline distillate. The presence of volatile Egg media: No proteolytic action. 

acids, tartaric acid, and lactic acid has been Milk: Becomes acid, but usually does 

demonstrated, but not succinic acid. Pro- not clot. No peptonization. Frequently no 

pionic acid and formic acid in ratios of from growth. 

."> to 1 up to 20 to 1 for the six strains ex- Starch : Slight hydrolysis, 

amined. Oxygen requirement: Anaerobic. 

Habitat : ( i round water. Nitrate reduction: ( ienerally negative. 

Remarks: Kalakoutskii (1960) found an- Sugar utilization : Greater ability to utilize 

aerobic actinomycetes in natural waters and sugars than .1. bovis. Acid but no gas from 

in the air of apartments occupied by man, glucose, galactose, lactose, fructose, maltose, 

but not in the soil. raffinose, sucrose; no acid from inulin. 

(i. Actinomyces israelii (Kruse) Lachner- Hemolysis: Slight to marked. 

Sandoval, 1898 (Wolff, M. and Israel, J. Serological reactions: Pack oi serological 

Arch, pathol. Anat. 126: 11, 1891). affinity with A. bovis. 

Synonyms: Streptothrix israeli Kruse, Temperature: Optimum 37°C. Destroyed 

1896; Discomyces israeli Gedoelst, 1902; at 55-60°C in 30 minutes. 

Actinomyces bovis Wright, 1905; Discomy- Habitat: Dental caries, tonsils, and natu- 

ces l><>ris Brumpt, L906; Actinobacterium ral cavities of man and animals. Chief etio- 
israeli Sampietro, 1908; Cohnistreptothrix logical agent of human actinomycosis, de- 



IS 



THE ACTINOMYCETES, Vol. II 






■A. 



^ 



Figure 5. .4. israelii (Reproduced from: Pre- 
vot, A. R. 6th Intern. Congr. Microbiol., Symp. 
Actinomycetales, Rome, 1953, p. 45). 

scribed first by Wolff and Israel (1891) and 
later by Wright (1905). 

Remarks: Vitality weak. Cultures no 
longer viable after 8 to 10 days. Erikson and 
Porteous (1953) succeeded in obtaining good 
growth by continued subculture in a medium 
containing 99 parts of 1 per cent casein hy- 
drolyzate and 1 part of heart broth and 0.5 
per cent glucose. Antigenic structure of or- 
ganism has been recently studied by Kwa- 
pinski (19(30). 

According to Grootten (1934), the organ- 
ism is highly polymorphic. Rods varying in 
length are formed in young culture. They are 
straight or slightly curved, with round or 
oval extremities. Occasionally, long or even 
filamentous forms are found. Some of the 
filaments end in small spherical or pear- 
shaped swellings. It does not form spores. 
In agar tubes, it does not grow in the upper 
5- to 10-mm zone; below that zone, it forms 
a layer of 2 to 4 mm with numerous small 
colonies; in the deeper layers, the colonies 
are fewer, but may attain diameters of 2 to 3 
nun. \<> gas and no odor are produced. 
Liquid media remain clear. The organism is 
nonproteolytic; milk is not coagulated. Blood 



is rapidly hemolyzed. It does not grow on 
potato plugs, except poorly when glycerin- 
ated. It slowly attacks glucose, lactose, mal- 
tose, sucrose, and mannitol. It does not grow 
in glucose-gelatin medium. Animal infection 
is obtained by introducing a culture into the 
peritoneum of rabbits. 

Negroni (1954) described A. israelii in 
further detail. Deep colonies in semisolid 
glucose agar are globous, 1 to 2 mm in 
diameter, whitish, opaque, and with an ir- 
regular surface. Colonies are of a cheesy 
consistency and cannot be homogeneously 
suspended in water. On glucose or glycerol 
agar slants, the colonies are elevated, mam- 
milated, and whitish, with moist and bril- 
liant surface and irregular margins. Sub- 
merged mycelium is well developed. The 
colonies have a cheesy consistency and can 
easily be removed from the medium with a 
platinum loop (Fig. 5). 

According to Erikson and Porteous (1955), 
the conversion of a "rough" typical strain of 
A. israelii to a "smooth" soft form more 
tolerant of oxygen is a result of the physical 
trapping within the mycelium of a few alien 
facultative anaerobes, usually staphylococci. 

7. Actinomyces naeshmdii Thompson and 
Lovestedt, 1951 (Thompson, L. and Love- 
stedt, S. A. Proc. Staff Meet. Mayo Clinic 
26: 169, 1951). 

Morphology: Organism forms small, whit- 
ish, firm colonies. Mycelial branching, but no 
segmentation. Not acid-fast. 

Artificial media: Good growth. 

Hormone agar: Rough and smooth colo- 
nies, 1 to 2 mm in diameter, after 4 days. 
Surface of colonies varies from smooth to 
nodular to wrinkled. Consistency varies from 
butyraceous to tough and adherent. Colo- 
nies are opaque, with color varying from 
white to cream. 

Glucose brain broth: Growth rapid and 
abundant. Acid produced. 

Gelatin: Growth slow. Xo liquefaction. 

Starch: Not hydrolyzed. 



THE GENUS ACTINOMYCES 



1!) 



Milk: ( ii'owth scanl or absenl . 

Aerobiosis: ( rrows bo1 h under aerobic and 
anaerobic conditions, somewhal better aero- 
bically. 

Temperature: Optimum al 37°C; some 
growth al 32 ('. 

Pathogenicity: Nonpathogenic. 

Habitat : Human mouth. Considered to be 
a saprophyte found in the mouth and fre- 
quently confused with A. israelii. 

8. Actinomyces odontolyticus Batty, 1958 
(Hatty, 1. .1. Pathol. Bacterid. 75: 155 159, 
L958). 

Morphology: At first, the organism ap- 
pears in the form of shorl rods subdivided 
by one or two transverse septa. Later, these 
rods gradually elongate 1 until a septate sub- 
mycelium is produced. At the end of these 
filaments, globular "initial cells" are pro- 
duced which germinate to produce a non- 
septate secondary submycelium, which soon 
commences to break up. Finally, in a week 
to 10 days, small spores commence to form 
singly upon short side branches. The size of 
the mature spores varies greatly in different 
strains. 

Appearance of colonies: Colonies are usu- 
ally few in number with an initial appearance 
similar to those of a-hemolytic streptococci 
of comparable age. Later, they develop a 
dark red hemin-like pigment, easily dis- 
tinguishable. At and after this stage the 
colonies are exceedingly difficult to subcul- 
ture. After prolonged artificial culture the 
organisms can be submit ured at any stage. 
Attempts to isolate the organism upon horse 
serum agar or nutrient agar are usually un- 
successful, but after several subcultures a 
profuse growth of small convex nonpig- 
mented colonies is obtained on both these 
media. All strains grow equally well under 
aerobic and anaerobic conditions at all 
stages in their life cycle; in agar stab cultures 
a filiform growth is obtained throughout the 
line of inoculum. ( Irowth in peptone brol h is 
sparse, but in this medium enriched with 



« 




Figure c>. A. odontolyticus, various stages ol 
culture development (Reproduced from : Batty, I. 
J. Pathol. Bacterid. :.">: 155 159, L958 

yeast extract, a characteristic glutinous ropy 
sediment is produced which disperses to 
give an even turbidity (Fig. 6). 

\o strain produces catalase, oxidase, in- 
dole, hydrogen sulfide or acetylmet hylcar- 
binol; all are methyl red-negative and all fail 
to ferment fructose, maltose, trehalose, 
starch, inulin, dextrin, glycogen, xylose, 
rhamnose, glycerol, dulcitol, and salicin. A 
few strains ferment sucrose, galactose, arabi- 
uose, or mannitol, with the production of 
acid but no gas. About half the -trains pro- 
duce ammonia from peptone, acidify and 
coagulate litmus milk, and are tolerant of a 
concentration of 1 in !()()(» potassium tellu- 
rite. Some strains hydrolyze urea. None 
liquefy gelatin, Loeffler's medium, or coagu- 
lated egg medium. All reduce nitrate to 
nitrite within 18 hour-. 



20 



THE ACTINOMYCETES, Vol. II 



Habitat: Human saliva in deep dental 
caries. 

Remarks: This organism resembles .4. 
bovis in its life cycle. 

Incompletely Described Forms of Ac- 
tinomyces 

In addition to the above well described 
and readily recognizable forms belonging to 
the genus Actinomyces, numerous other an- 
aerobic organisms have been listed in the 
literature. Some of these organisms are no 
doubt strains of the well described forms and 
their names would be in synonymy. Others 
may represent distinct species. 

It is of particular interest to cite, in this 
connection, the ideas of Prevot (1957), who 



considered Actinomyces bovis as an aerobic 
organism and, therefore, the genus Actino- 
myces as an aerobic group. He suggested 
I ha I Actinomyces israelii represent the an- 
aerobic group, and the generic name Actino- 
bacterium Haas, 1906 (Syn. Cohnistrepto- 
thrix Pinoy, 1913) be given priority for desig- 
nating the anaerobic forms. Prevot (1957) 
divided the genus Actinobacterium into six 
species: (1) ^4. israelii, (2) A. meyeri, (3) A. 
abscessus, (4) .4. liquefaciens, (5) .4. cellu- 
litis, and (6) A. propionici. 

In addition to the above, numerous other 
anaerobic forms have been described, such 
as A. canis Levy, 1899; A. inter proximalis 
Fennel, 1918; and others. Some additional 
names will be found in Chapter 13. 



Chapter 3 



The Genus Nocardia 



Characterization of Genus 

The genus Nocardia represents a group 
of aerobic actinomycetes which includes both 
pathogens and saprophytes. The relation- 
ship of this genus to, and possible overlap- 
ping with, the genus Mycobacterium, on the 
one hand, and the genus Streptomyces, on the 
other, have already been discussed (Volume 
I i. Numerous cultures of nocardiae have 
been isolated from human and animal infec- 
tions, and claimed to be the causative agents 
of the particular disease. The fact, however, 
thai a culture of an organism has been iso- 
lated from a lesion of a man or an animal is 
no proof thai it is primarily responsible for 
the particular disease; it may actually be a 
secondary invader or a member of a mixed 
infection. Some specie- of Nocardia are def- 
initely associated, however, with certain 
diseases, or have at least been isolated from 
infected tissues. This gave origin to the term 
"nocardiosis," descriptive of these disease 
conditions. 

The colonies produced by nocardiae are 
cither smooth, or rough and much folded; 
they are either of a sofl or dough-like con- 
sistency, or compact and leathery, especially 
in early stages of growth. Many specie.- of 
Nocardia do not form any aerial mycelium; 
Mime give rise to a limited aerial mycelium 
which may structurally be similar to that of 
the substrate mycelium; still others may 
produce aerial hyphae and spores which may 
be indistinguishable from those of Strepto- 
myces and are thus responsible for various 



case- of overlapping between these two 
genera. 

Nocardias multiply by concentration 
and segmentation of the protoplasm within 
a filamentous cell, followed by dissolution oi 
the cell membrane. The fragmented portions 
of the mycelium usually develop into fresh 
mycelium under favorable conditions, either 
by germ tubes or by lateral budding. Strep- 
tomycetes produce true spores or conidia, the 
vegetative mycelium not segmenting spon- 
taneously into bacillary or coccoid forms, but 
remaining nonseptate and coherent even in 
old cultures, thus producing the characteris- 
tic tough textured, leathery growth. In no- 
cardiae, the aerial mycelium represent- an 
extension upward of the vegetative myce- 
lium; it does not exhibit any differentiated 
protoplasm and is sterile and abort ive. When 
a streptomycete has lost the capacity of 
producing aerial mycelium, a form analogous 
to thai of a nocardia may result, except for 
the structure of the mycelium and the 
capacity of the degenerated streptomycete 
to regain the lost capacity. It is occasionally. 
therefore, a matter of personal preference 
whether to place a freshly isolated culture in 
one genus or another. Some nocardiae are 
acid-fasl or partially acid-fast, and others 
are not . 

The mode of branching of the substrate 
mycelium (see Volume I, Chapter 5), the 
biochemical properties (proteolytic and sero- 
logical activities), and chemical nature of the 
cell walls of nocardiae appear to distinguish 

them from the streptomycetes. Boare and 



22 



THE ACTINOMYCETES, Vol. II 



Work (1957) have shown that these genera 
can be differentiated by the configuration of 
the diaminopimelic acid present in whole cell 
hydrolysates; streptomycete cell Avails con- 
tain the L-isomer, whereas nocardiae cell 
walls contain the DL-isomer. Cummins and 
Harris (1958) reported that the presence or 
absence of arabinose in the hydrolysates of 
the intact organisms can also be used to 
identify them; nocardiae cell walls contain 
arabinose, whereas streptomycete cell walls 
do not. The sensitivity of most Stnptomyces 
species, but not of nocardiae, to the action of 
lysozyme on their cell wall preparations, 
studied by Sohler, Romano, and Nickerson 
(Volume I, p. 159), provides further criteria 
for distinguishing between members of these 
two genera. Studies of infrared absorption as 
a taxonomic criterion (Riddle et al., 1956) 
has also been suggested.* 

In view of the overlapping between certain 
forms placed for convenience in either one 
genus <)]• the other, the separation of atypical 
strains of Nocardia or Streptomyces by mor- 
phology or fermentation tests alone may be 
difficult, as pointed out by Gordon and 
Mihm (1957). 

The genus Nocardia has been described in 
the last edition of Bergey's Manual as 
follows: 

"Slender filaments or rods, frequently 
swollen and occasionally branched, forming 
a mycelium which, after reaching a certain 
size, assumes the appearance of bacterium- 
like growths. Shorter rods and coccoid forms 
are found in older cultures. Conidia not 
formed. Stain readily, occasionally showing a 
sligh.1 degree of acid-fastness. Xonmotile.t 

* Personal communication from Dr. X. M. 
McClung. 

t The existence of motility among the nocardias 
was considered by Jensen (1953) as indisputable, 
and this really is not surprising in view of the 
numerous observations on motility in the closely 
related coryneform bacteria. The species in the 
order Actinomycetales cannot any longer be re- 
garded a,< constantly nonmotile (Fig. 14). 



Xo endospores. Aerobic. Gram-positive. The 
colonies are similar in gross appearance to 
those of the genus Mycobacterium. Paraffin. 
phenol and m-cresol are frequently utilized 
as a source of energy. 

"In their early stages of growth on culture 
media (liquid or solid), the structure of 
nocardias is similar to that of actinomycetes 
in that they form a typical mycelium; hy- 
phae branch abundantly, the branching 
being true. The diameters of the hyphae vary 
between 0.5 and 1 /x, usually 0.7 to 0.8 jx, 
according to the species. The mycelium is 
not septate. However, the further develop- 
ment of nocardias differs sharply from that 
of actinomycetes; the filaments soon form 
transverse walls and the whole mycelium 
breaks up into regularly cylindrical short 
cells, then into coccoid cells. On fresh culture 
media, the coccoid cells germinate into my- 
celia. The whole cycle in the development of 
nocardias continues for 2 to 7 days. Most 
frequently the coccoid cells are formed on 
the third to fifth day, but in certain species 
they can be found on the second day. 

"The multiplication of nocardias proceeds 
by lission and budding; occasionally they 
form special spores. Budding occurs often. 
The buds are formed on the lateral surface 
of the cells; when they have reached a cer- 
tain size, they fall off and develop into rod- 
shaped cells or filaments. The spores are 
formed by the breaking up of the cell plasm 
into separate portions usually forming 3 to 
5 spores; every portion becomes rounded, 
covered with a membrane and is t ransformed 
into a spore; the membrane of the mother 
cell dissolves and disappears. The spores 
germinate in the same way as those of ac- 
tinomyces. They form germ tubes which 
develop into a mycelium (Fig. 7). 

"The colonies of nocardias often have a 
paste-like or mealy consistency and can 
easily be taken up with a platinum loop; they 
spread on glass and occasionally render the 
broth turbid. The surface colonies are 



THE GENUS NOCARDIA 



23 



3K 




1 "<*aJ 



Figure 7. A', opaca: (a) grown for 4 days on ra-dodecane and mineral salts; gram stain, X 960; b 
same grown 3 days; X 3700; (c) same as (a) bu1 using fat stain. X L920; (d) two-day growth, X 12,500 
CReproducedfrom:Webley, D. M. J. Gen. Microbiol. 11:425, 1954). 



smooth, folded or wrinkled. Typical nocar- 
dias never form an aerial mycelium, but 
there are cull tire- whose colonies are covered 
with a thin coating of shorl 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 cull tires 
are colorless. The color of the culture serves 
as a stable character. The type species is 
A ocardia farcinica Trevisan.'"' 

Classification of Nocardia Species 

De Toni and Trevisan (1889) described 
five species of Nocardia: X. farcinica, N. 
actinomyces, N. foersteri, X. arborescens, and 
A'. /« rruginea. 

* Sec the work of < rordon and Mihm (1958). 



Jensen (1932a) found that a number of 
organisms previously described a- species of 
Mycobacterium actually belong, on account 
of definite mycelial growth in the initial 
stages of their life cycles, to the feints Nocar- 
dia* Mycobacterium agreste ( rray and Thorn- 
ion and B. mycoides corallinus Befferan were 
found to be similar to one another and were 
regarded as one species. A', corallina. The 
-.■one was true of .1/. salmonicolor den I )ooren 
de Jong, which was designated as N. sal- 
monicolor. Mycobacterium opacum den 
Dooren de Jong and .1/. crystallophagum 
Gray and Thornton proved to be identical 
and were named N. opaca. Mycobacterium 
erythropolis, a closely related form, was des- 

• I reneric name Proa< nomyces used. 



24 



THE ACTINOMYCETES, Vol. II 




Figure 8. JV. 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). 




Figure 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). 

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 Mycobacterium. 
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. 



A. Unstable mycelium (a-form), with 
short mycelium (if formed at all), 
bacterial (diffuse) growth in liquid 
media, bacteria-like colony. 

B. Stable mycelium (/3-form), producing 
long hyphae, colony growth in liquid 
media, and Streptomyces-like type of 
colony. 

Umbreit (1939) modified the system of 
Jensen as follows: 
I. Partially acid-fast, nonproteolytic, non- 
diastatic ; constantly utilize paraffin. 

1. a-Mycelium type: N. opaca* N. eryth- 
ropolis. 

2. /3-Mycelium type: 

a. Iled-colored : N. polychromogena 
N. asteroides. 

b. Yellow-colored: N . paraffinae. 

II. Nonacid-fast forms, constantly diastatic 

1. a-Mycelium type: 

a. Nonproteolytic: N. mesenterica. 

b. Proteolytic: N. actinomorpha. 

2. 0-Mycelium type: 

a. Yellow-colored: N. flavescens. 

b. Red to orange: N. maculata. 
Krassilnikov (1938) divided the genus 

Nocardia* into two groups: 
I. Well developed aerial mycelium, with 
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 cyl- 
indrical with sharp ends. No spirals or 
fruiting branches. This is the same as 
group /3 of Jensen. 

II. 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 Proaclinomyces used. 



THK GENUS N0CARD1 I 



25 



Table I 
Summary oj growth characteristics of t8 strains of Nocardia (McClung, 1949) 



Germina 

tinll 


Primary 
brani hing 


lit 


hr 


8 


16 


6 


L2 


9 


15 


It) 


36 


11 


11 


11 


13 


11 


12 


10 


12 


10 


14 


14 


30 


10 


28 


10 


16 


9 


11 


10 


12 


11 


15 


10 


15 


10 


13 


8 


13 



Set (Miliary 

branching 



I ragmen tat ion 



\ ■ 



Type 1 



I 



Type 3 



( rroup I 

\Y F 

.V. aquosus 

KI..I 

15 B 

N. erythropolis . . 

X . polychromogt nes 
(Ik. up II 

L3-20 

13-8 

N. ruber 

.Y. polychromogenes 

N. astt roides 

Group III 

21 3 

13-10 

L8-2 

13 15 

13 3 

20-6 



13 
12 
13 

11 
11 
11 

1!) 
is 
20 
120 
96 



40 50 

30 65 

Unknown 

1 'ills now n 



McClung (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. 
II. Extensive mycelial development, 
straight branches which do not over- 
lap. Colonial texture soft and pasty; 
pigment intracellular and insoluble. 
III. Extensive mycelial development, no 
fragmentation of hypliae, contorted and 
profusely produced branches which 
overlap. Colonial texture waxy or 
cartilaginous; generally both intracel- 
lular and soluble pigments are pro- 
duced ( Figs. 8, 9). 
The pattern in Nocardia fragmentation 
can he separated into three types. In Type 
1, ao acute angle is formed in a hypha pre- 
ceding division, which occurs at the apex of 



the bend; following division the new hypha] 
tips giow out parallel to each other. In Type 
2, division occurs in a straighl 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 
hypha) tip bends and continues to grow. 
Type 1 fragmentation is characteristic of 
Group I, and Type :! of ('.roup II. Type 2 
occurs in both groups, and Group III lack- 
fragmentation (Fig. 10). 

A summary of the growth characteristics 
of various strains of Nocardia belonging to 
these three groups is presented in Table l. 

In an attempt to find a group of depend- 
able properties for the separation of the 
genera Nocardia, Streptomyces, and Myco- 



26 



THE ACTINOMYCETES, Vol. II 




Figure 10. A 7 , 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, 1040). 

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, 
since the physiological reactions of the 
strains were the same as in the previous 
group of sporulating cultures; only five 
strains, or 4 per cent of the cultures possessed 
nocardia] 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 nt 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, 1050) 



Property 


N. asteroid.es 

(98 strains), 
positive 
strains 


N. brasiliensis 

(SO strains), 
positive 
strains 




i i 


% 


Decomposition of: 






Casein 





98 


Gelatin 


36 


100 


Tyrosine 





100 


Xanthine 








Hydrolysis of starch 


58 


56 


Acid from : 






Adonitol 








Arabinose 








Erythritol 


6 





( ralactose 


24 


92 


Glucose 


100 


96 


( Glycerol 


98 


98 


Inositol 


2 


100 


Lactose 








Maltose 


6 





Mannitol 





94 


Mannose 


19 


68 


a-Methyl-D-glucoside 








Raffinose 








Rhamnose 


33 





Sorbitol 








Xylose 








Nitrite from nitrate 


85 


92 


Growth at : 






50 °C 


24 





40°C 


00 


56 


35°C 


100 


100 


28°C 


100 


100 


10 °C 


12 


30 


Utilization of: 






Acetate 


100 


100 


Citrate 


33 


98 


Mai ate 


100 


100 


Propionate 


100 


100 


Pyruvate 


100 


100 


Succinate 


100 


98 


Benzoate 









THE GENUS NOCARDIA 



27 



Tabu: 3 
Comparativi properties of certain acid-fast nocardias (Suter, 1951) 






Growth on 

potato 



Aerial 
mycelium 



Growth at 
room temperature 



N. fastidiosa 
N. leishmanii 
X . caprat 
\ .'. pretoriana 
X. pulmonalis 
X . paraflinai 
N. transvalensis 
X . polychromogt nes 

X. minium 

X . coeliaca 

X. rubropertincta 

X . astt roidt s 

X. salmonicolor 

X. rubra 

X . farcinica 



+ 


+ 




+ 




+ 


+ 


+ 


+ 




+ 


+ 


+ 


+ 




+ 




+ 


+ 




+ 


+ 


+ 


+ 




+ 


+ 


+ 


+ 




+ 


+ 




- 


+ 


+ 


+ 


+ 


- 


+ 


+ 


+ 




- 


+ 


- 


+ 


+ 


- 




+ 


+ 


+ 


- 


- 


+ 


+ 


+ 


- 




+ 


+ 




- 




- 


+ 


+ 


- 


- 



Mycobacterium. In the case of the Strepto- 
WM/ces-designated cultures, 83 per cent pro- 
duced sporulating aerial hyphae, 9 per cent 
nonsporulating aerial hyphae, and 8 per cent 
tunned no aerial hyphae. The Nocardia- 
designated culture- 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 .Y. asteroides (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 SS 
per cent grew at 40°C. Eighty-six per cent 
reduced nitrate to nitrite, ">4 per cent hy- 
drolyzed starch, and 34 per cent decomposed 
gelatin. A Large number of cultures desig- 
nated as Nocardia (X. corallina, X. ery- 
thropolis, X. globerula, X. lutea, X. opaca, 
X. rhodnii, X. rubra) were tentatively as- 
signed by Gordon and Mihm to the myco- 
bacteria under M. rhodochrous (Overbeck) 
now comb. (Table 2). 

Of five species of aerohic act iiiomycetes 
associated with various mycetomas, Mariat 
(1957) recognized only N. asteroides and A'. 
brasiliensis a- nocardiae; Streptomyces >na- 



durae, S. pelletieri, and S. somaliensis were 
considered as streptomycetes, although 
.Mariat was not quite certain of their exact 
systematic position. 

Bojalil and Cerbon (19.59) divided the 
genus Nocardia 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 (', break- 
ing it down into amino acids and giving an 
alkaline reaction. A', brasiliensis belongs to 
this group. (2) Produces flaky growth easily 
dispersed through medium; poor growth on 
gelatin. A', 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 carbon and nitrogen sources, as 
well as in certain other physiological and 
biochemical properties, is reported in Table 
2. Some comparative properties of several 
nocardiae are given in Table '■>. 

Spalla (1958, 1959) criticized the various 
descriptions of Nocardia species on the basis 
of an insuliicient number of characters. He 
suggested that the following properties be 
used for characterization and classification 
of nocardiae: 



ft 
« -2 



H ^ 



JBSB 3Ul3BiBCl 

-SB-3SO0nj{) 


OOQO^ggo 


JESB |0J93X]0 




mnipaui 

3UIUlB-Z-\ 


oooort^o 



mmpoAui 
[Busy 



33UB5Sisai ppy 



I I I I I I I + 



I I I I ++ I I 



jo uoij-ejnS-eoj 



+ + + + 1 I I + 



uijBjaa 
jo sisXiOjpXH 



+ + + + + I I + 



ipJBJS 

jo sisX|OjpAH 



I I I I I I I + 



ajTjjjui 
jojioijjnpa-jj 



I I I I + I + + 



I I I I + I I I 



+ + + + I I 



+ + + + + I + 



JOJ33XIO I + + + + + + + + 



111 + 



I + 



111 + 



111 + 



asouiqiuv-/ + + + + I 1 I + 



I I I + + I + + 



+ + + + I + I + 



+ + + + 111 + 



+ + + + I I I + 



. N lO W >« ^ 

e !M OS <M -S s 

1 .s .2 .s ! -i s -s 

* r. 7i s3 •*» e ■* h 

&. £ $1 is gS -~ §5 

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£ £ £ ^ £ 



£ • 

'- % 

II 5 

of 

• - <a 

O OQ 

'•S 

o s 

I! 

'as a> 
>. ■- 

II & 

o 



28 



THE GENUS NOCARDIA 



20 



T Uil.K .") 

Serological relations of major pathogenic actinomycetes (Gonz&lez Ochoa and Vasquez Boyos, L953 

Pre< ipitin formation 



\ . brasilien X. asteroides S. madurat S. pelletieri S 

"o. 412 No. 1185 No. 1065 ensi No J- : 



sis \«. 417 No. 19 



.1. bovis No. 527 + + 

.V. brasiliensis No. 1 17 + + 

.V. brasiliensis No. 168 + + 

N. mexicana No. 414 + + 

.V. asteroides No. 18 + + 

N. asteroides No. 19 + + 

.V. asteroides No. 20 + + 

N. asteroides X<>. 652 + + 

.V. asteroides No. 69-4 + + 

N. gypsoides No. 9911 + + 

.V. carnea No. 616 + + 

N. leishmanii No. 1030 + + 

S. iniiihinu No. 412 — 

S. pelletieri No. 11S5 - 

S. africana No. 615 — 

*S. somaliensis No. 1065 — 

S. somaliensis No. 1064 — 

jS. somaliensis Xo. 1066 — 

5. paraguayensis No. 285 — 

5. aZ6us No. 693 

5. griseus No. 975 — 

N. lavendulae No. 9963 - 



1 . Color of growth on several synthetic and 

at least one organic medium. 
_\ Acid production from various carbon 

sources. 
;. Heat resistance. 

Size of terminal fragments. 



4. 

.">. St a 



■tie 



»lv 



d-fi 



ft. Diastase formation. 

7. ( 'iclatin liquefaction. 

S. Reduction of nitrate to nitrite. 

!). 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 .V. 
rugosa belonged to the same species as the 
parent, and that A', rugosa, X. asteroides, X . 
rubra, and A', blackwellii represent distind 
species, with a low degree of similarity. 



A summary of some of the properties of 
A", 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 A', gardneri and the 
other nocardiae may be noted, particularly 
in utilization of carbon sources, starch hy- 
drolysis, and formal ion of aerial mycelium. 
These differences account for the fact that 
A', 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 •">. Further information on the 
relation of Mycobacterium to Nocardia is 
found in the work of Ilaag (1927) and 
Cordon ( L937). 

of the various treatments of the genus 

Nocardia, the following three systems of 
classification of species are presented here: 



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 n in diameter. 

Nocardia minima 

2. Rapidly growing organisms; cells 1.0 to 
1.2 n in 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 cocci. 

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. 
a 1 . Growth in nutrient agar opaque, 

cream-colored; cocci in broth 
culture. 

Nocardia opaca 
b 1 . Growth on sugar-free nutrient 
agar watery; no cocci in broth 
culture. 

Nocardia erythropolis 

2. Substrate growth hard, yellow, with 
white aerial mycelium; sporophores 
divide into chains of acid-fast cocci. 

Nocardia paraffinae 
B. Nonacid-fasl organisms with weakly refractive 
cells; no distinct formation of cocci. Diastatic. 
I. Nonproteolytic; no aerial mycelium; 
marked production of cystites. 

Nocardia mesenterica 

II. Proteolytic organisms. 

1. Growth on nutrient agar with rapid 
formation <>f unbranched diphtheroid- 
like rods; no typical cystites; broth tur- 
bid. 

Nocardia actinomorpha 

* Designated by Jensen as Proactiiwmijc.es. 



2. ( '.rowth 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 Krassilnikov 

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 occa- 
sionally forming septae; the Iryphae 
break up into long rods, 15 to 30 /x; 
spherical bodies not formed. 

a. Saprophytes, found on dead sub- 
strates. 

Nocardia actinoides 
1). 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. 

a 1 . Grow on protein media. 

Nocardia actinomorpha 
b 1 . ( irow on paraffin. 

Nocardia paraffi // " < 

b. Parasites. 

Nocardia bo vis 
II. 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 

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 VOCARDIA 



:;i 



Organism: 

man and 



he bodies of 



mimals. 



B. Cultures pign 



Anaerobes, living in absence of 
oxygen. 

Nocardia ono< robicus 
Aerobes, <>r microaerophilic 

forms. 

a 2 . Strict aerobes. 

Nocardia lignieresi 
1)-. Facultative aerobes 
.1 ( 'ells nonacid fasl 

Nocardia israi h ' 
b 3 . Cells acid fast. 
Nocardia muris 
ited. 
I. Cultures pigmented violet or blue, the 

pigments diffusing into the substrate. 

1. Well developed substrate mycelium 
produced, hyphae forming occasional 
septae and breaking up into long rods, 
20 to 30 m; colonies form a faint aerial 
mycelium with straight sporophores ; 
spores cylindrical. 

Nocardia gabritschewski 

2. No aerial mycelium produced, hyphae 
or substrate mycelium forming fre- 
quent septae and breaking up into 
short rods and cocci. 

Nocardia cyam us 
II. 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. 

a 1 . Cultures not forming any soluble 
pigment in medium. 
Nocardia fructiferi 
b 1 . ( 'ultures producing a brown sub- 
stance. 

Nocardia polychromogenes 

b. Parasites living in bodies of man and 
animals. 

Nocardiafreeri 

2. Hyphae forming frequent septae and 
breaking up into shorl rod- and COCCi; 
no aerial mycelium produced. 

a. Saprophytes living on dead sub- 
<t rates. 

Nocardia ruber 

b. Parasites living in bodies of man 

and animals. 



* See Chapter 2 for description of Actino 
israelii. 




Figure 11. N. asteroides, strain 730, grown on 
oil e\t ract agar (Reproduced from: Gordon, R. E. 
ind Mihm, J. M. J. Bacterid. 7:>: 240, 1958). 

a 1 . Cells acid fast. 

Nocardia asU roides 
b 1 . Cells nonacid-fast. 

Nocardia variabilis 
HI. Cultures cit ron-vellow or bright yellow. 

1. Faint aerial mycelium with straight 
sporophores and cylindrical spores 
produced. 

a. Saprophj tes. 

Nocardia flavescens 

b. Parasites. 

Nocardia somalit nsis ' 

2. No aerial mycelium produced. 

a. Saprophytes. 

a 1 . Cultures yellow or bright yellow. 

Nocardia flaws 
b 1 . Cultures cit ron-vellow . 

Nocardia citreus 

b. Parasites. 

a 1 . Cells acid fast. 

Nocardia farcinica 

b 1 . ( 'ells nonacid fa-t . 

Nocardia putoriat 

IV. Cultures pigmented green. 

1. Saprophytes. 

Somalia I iriilis 

2. Parasites. 

Nocardia /"/"</< n< s 

V. ( 'lilt lires black. 

1 . Sapropl 

\ ocardia niger 

2. Parasites. 

Nocardia s< ndaiensis 

' Now recognized as a S 



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. 

a 1 . Pathogenic. 

a 2 . Substrate mycelium white, 
buff, or pale yellow. 

18. Nocardia farcinica 
b 2 . Substrate mycelium yellow to 
red. 

6. Nocardia asteroides 
b 1 . Not pathogenic. 

a 2 . Paraffin decomposed. 

42. Nocardia polychronio- 
genes 

b 2 . Cellulose decomposed. 

13. Nocardia cellulans 

b. Substrate mycelium white to pink. 
a 1 . Gelatin not liquefied. 

a 2 . Growth on nutrient agar 
opaque, cream-colored. 
38. Nocardia opaca 
b 2 . Growth on nutrient agar pink. 

10. Nocardia calcarea 

a 3 . Aerial mycelium on milk 
white. 

31. Nocardia leishmanii 
b 3 . Pellicle on milk pink. 

11. Nocardia caprae 

c 3 . Pellicle on milk yellow. 
9. Nocardia brasiliensis 
d 3 . Causing galls on blueberry 
plants. 

53. Nocardia vaccinii 
b 1 . 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 

3. Substrate growth cream colored, later 
becoming yellow. 

51. Nocardia serophila 

4. Substrate grow T th hard, orange-yellow. 

58. Nocardia variabilis 

II. Initial mycelium very short, rapidly divid- 
ing into rods and cocci. 

1. Growth pink. 

a. No cystites (swollen cells) formed. 
a 1 . No indigotin from indole. 

16. Nocardia corallina 
b 1 . Indigotin from indole. 

26. Nocardia globerula 

b. Cystites formed. 

49. Nocardia salmonicolor 

2. Growth coral -red. 

47. Nocardia rubropertincta 

3. Growth white, tan, or pink. 

a. No aerial mycelium. 
a 1 . Growth tan. 

15. Nocardia coeliaca 
b 1 . Growth white. 

28. Nocardia intracellular is 

b. Aerial mycelium produced. 

a 1 . Growth frequently pinkish. 

53. Nocardia transvalensis 
b 1 . Growth never pink. 

50. Nocardia sebivorans 

4. Produces no pigment, no growth on 
potato, coagulates milk. 

19. Nocardia fastidiosa 

B. Nonacid-fast organisms with weakly refrac- 
tive cells; no distinct formation of cocci; di- 
astatic. 
I. Nonproteolytic, although some give gela- 
tin liquefaction. 

1. Growth on agar pale cream. 

a. Gelatin not liquefied; starch hy- 

drolyzed. 

35. Nocardia mcsentcrica 
1). Gelatin liquefied; starch not hy- 

drolyzed. 

48. Nocardia rugosa 

2. ( irowth on agar whitish. 

4. Nocardia albicans 

3. Growth on agar yellow. 

20. Nocardia flava 

4. Growth on agar green. 

59. Nocardia viridis 

5. Growth on agar yellow-green. 

14. Nocardia citrea 



THE GENUS WCARDIA 



33 



6. Growth initially colorless, producing a 
yellow-green pigmenl in 2 to I days. 

54. Nocardia turbata 

7. Growth on agar dark brown and even 
black. 

a. No liquefacl ion of gelal in. 

37. Nocardia nigra 

b. Gelatin liquefied. 

20. Nocardia iron nsis 

s. Crowth consistency soft; aerial my- 
celium sparse. 

33. Nocardia lutea 
9. Growth consistency medium; aerial 
nix celium profuse. 

8. Nocardia blackvx llii 

10. Growth cream-colored to pink; aerial 
-pikes produced. 

52. Nocardia sumatrat 

11. < Irowt h grayish yellow. 

36. Nocardia maris 

12. Growth yellowish-orange. 

55. Nocardia uniformis 

13. Pigmenl on protein media deep brown. 

44. Nocardia rangoonensis 

14. Pigment on protein media light brown. 

12. Nocardia caviat 
II. Proteolytic, although some are only 
weakly proteolytic. 

1. Growth on nutrient agar with rapid 
formation of unbranched diphtheroid- 
like rods; no typical cystites; broth 
turbid. 

1. Nocardia actinomorpha 

2. Growth white, shiny or pale. 

a. Dough-like consistency; breaks up 
into short rods. 

3. Nocardia alba 

b. Membranous, myceloid growth. 

32. Nocardia listeri 

3. Growth on nutrienl agar with extensive 
mycelium; simple unbranched rods not 

formed; Cystites present. Broth clear. 

21 . Nocardia flavescens 

4. Growth cream-colored. 

a. Rapid liquefaction of gelatin. 

a 1 . No aerial mycelium. 

25. Nocardia gibsonii 
1)'. Aerial mycelium scant, white. 

56. A hi a at in upcottii 

b. Slow liquefaction of gelatin. 

17. Nocardia dicksonii 
.">. Growth rose-colored to brighl red or 
red-orange. 

24. Nocardia fructift ra 
6. Growth pink to red. 




Figure 12. N. rubra, election micrograph (Re- 
produced from: McClung, X. M. First Reg. Conf. 
Asia and Oceania, Tokyo, 1956). 

a. Gelatin not liquefied. 

2. Nocardia africana 

b. ( '.elat in 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 fordii 

9. Growth yellow to reddish-brown; solu- 
ble pigment brown to red. 

30. Nocardia kuroishi 

10. (Irowt h 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. 

3 l . A oca rdia man mi 

b. ( bowt h yellow-orange. 

7. Nocardia atlantica 

13. Produce nodules on root- of plants. 

5. Nocardia alni 

In addition to the species included in the 
above classification and described below, 
many more species of Nocardia have been 
recorded in the literature, either under this 
or under oilier generic names. Some are 
listed in ( !hapter L3, under the incompletely 
described forms. < Miters are synonyms. There 
is no question that some of the Streptomyces 
species described in Chapter 8 could just as 



:;i 



THE ACTIXOMYCETES, Vol. II 



well have been included among the Nucardia 
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 m- In older cul- 
tures, rods 2 to 3 fi 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: Growth dry, 
wrinkled, pink to orange. 

Egg medium: Growth raised, dry, smooth, 
salmon-buff. 

Gelatin: Colonies round, saucer-like, 
white, raised rim, edges burred. Liquefaci ion 
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. coeliaca in 
liquefaction of gelatin. No acid from glucose, 
lactose, sucrose, or glycerol. 

2. Nocardia africana Pijper and Pullinger, 

* For further details concerning some of the 
species, the last edition of the Bergey Manual 
should he consulted. 



1927 (Pijper, A. and Pullinger, B. D. J. 
Trop. Med. Hyg. 30: 153-156, 1927). 

Synonym: Actinomyces africanus (Pijper 
and Pullinger) Nannizzi Pollacci, 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. 

Egg medium: Colonies small, colorless, 
blister, partly confluent; becoming wrinkled, 
depressed into medium. Liquefaction slight. 

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 (Krassilnikov, 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-like 
consistency; shiny or pale. Substrate myce- 
lium breaks up into short rods 2.7 by 0.7 to 
0.8 n, later changing into a mass of coccus- 
like cells, 0.7 to 1 ij.. Many cells are swollen, 
others form side buds. Not acid-fast. No 
aerial mycelium. 

Synthetic agar: Inorganic salts used as 
sources of nitrogen; sugar, starch, or organic, 
acids utilized as sources of carbon. 

Nutrient agar: Good growth. No aerial 
mycelium. 



THE GENUS NOCARDIA 



35 



Gelatin: Growth good. Positive liquefac- 
tion. 

Milk: ( !oagula1 ion and peptonizal ion. 

Starch: Rapid hydrolysis. 

Cellulose: No growth. 

Paraffin: No growth. 

Nitrate reduction: Negative. 

Sucrose: I liveried. 

Source: Soil. 

Remarks: Several subspecies were also 
listed: N. chromogena, X. pauhtropha (Ac- 
tinobacillus paulotropkus Beijerinck, 1914), 
N. alba lactica, N. diastatica, X. hoffmanni. 

4. Nocardia albicans (Krassilnikov, L941) 
Waksman (Krassilnikov, X. A. Act inomyce- 
tales. [zvest. Akad. Xauk, SSSR, Moskau, 
L941). 

Morphology: Growth red, hyphae break- 
ing up into rod-shaped cells, 12 to 25 by 0.6 
to 0.7 m. "p to 50 ju 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. 

Xulrient agar: Growth good, smooth, 
shiny. 

( relatin: No liquefaction. 

Milk: No change. 

Starch: Hydrolyzed. 

( lellulose: No growth. 

Nutrienl broth: Growth poor, produces 
fainl turbidity, which settles on bottom and 
leaves a surface ring. No true mycelium. 
Cells rod-shaped 5 to 10 jtt, seldom 15 to 20 fi. 

Nitrate reduction: Negative. 

Sucrose: Inverted. 

Paraffin: Xot utilized. 

Source: Soil. 

Remarks: Glycerol used as a source of 
carbon, and nitrate as a source of nitrogen. 

5. Nocardia alni (Peklo emend, v. Plotho, 
1941) Waksman (von Plotho, 0. Arch. 
Mikrobiol. 12: 1 IS, 1041 I. 

Morphology: Mycelium contain- fatty 



globules; cells filiform, branching, disinte- 
grating into shorl rods and cocci. Aerial 
mycelium usually absent , bu1 may be formed 
on cultivation. Sporulating cultures form 
white, spherical to oval spore-. 

Agar media: Substrate growth compact, 
shiny, colorless or slightly brownish. 

Gelatin: Surface pellicle. Liquefaction 
positive. Soluble pigment brownish. 

Liquid media: Slimy surface film. 

Tyrosine: Utilized as source of nitrogen; 
color turns red-brown. 

Cellulose: Xot utilized. 

Carbon sources: Produces lactic acid from 
glucose and lactose. 

Optimum reaction for growth: pll 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. 

C). Nocardia asteroides (Eppinger, 1891) 
Blanchard, 1895 emend. Cordon and Mihm, 
L959 (Eppinger, H. L. Beitr. Pathol. Anat. 
9: 287, 1891; Blanchard, P.. In Bouchard. 
Traite Pathol. Gen. 2: 811, 1895; Cordon, 
R. E. and Mihm, J. M. .1. Gen. Microbiol. 
20: 129, 1959). 

Synonyms: Cladothrix asteroides Eppinger, 
1890; Streptothrix eppingeri Rossi-Doria, 
1891; Actinomyces asti roides < rasperini, L892; 
Oospora asteroides Sauvageau and Radais, 
L892; N. asteroides P. Blanchard, 1895. Ac- 
cording to Ochoa and Sandoval (1956), N. 
leishmanii Chalmers and Christopherson, 
and A', phenotolerans Werkam and Gammel 
are synonyms of A', asteroides. According to 
Cordon and Mihm (1959), N. caprae (Silber- 
schmidt) Waksman and Ilenrici. A', ep- 
pingeri, X. minima, and X. sylvodorifera are 
also synonyms. 

Morphology: Typical actinomycete 
growth, usually yellow to orange to orange- 
red. Mycelium -traight and line: it breaks up 
into small, coccoid forms and rod-. Some 
strains are acid-fasl : other- 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, scant, 
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. Some 
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 
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 Alihm, 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 m- 
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 slightly 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. 

Alginic acid: Decomposed. 

Carbon sources: Acid produced from arab- 
inose, xylose, rhamnose, raftinose, fructose, 
galactose, sucrose, gum arabic. No acid 
from lactose, dulcitol, mannitol, or sorbitol. 
Organic acids utilized: gluconic, lactic, malo- 
nic. Organic acids not utilized: acetic, 
butyric, citric, etc. 

Optimum temperature: 28-oO°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 NOCARDIA 



37 



Remarks: Another closely related culture 
lias been described as Proaetinomyces flavus 

(sec .V. marina I. 

8. Nocardia blackwellii (Erikson, L935) 
W'aksniaii and Henrici, L948 (Erikson, 1). 
Med. Research Council Spec. Repl . Ser. 203: 
32 33, L935). 

Morphology: Growth consisting of short, 
rod-like filaments, growing out into longer 
sparsely branching hyphae. Aerial mycelium 
short, straight; frequently Large, round or 
ovoid cells are interposed in the irregularly 
segmented chains of cells. 

Glycerol nitrate agar: Growth extensive, 
granular, irregular, thin, pinkish. 

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, colorless, minute, 
along line of inoculation. Later, abundant, 
colorless colonies to 10 mm below surface; 
larger pink-yellow surface colonies with 
white aerial mycelium. No liquefaction. 

Milk: Surface pellicle heavy, convoluted, 
bright yellow. No coagulation; no peptoniza- 
tion. Spalla states that milk is coagulated. 

( Jarbon utilization: See Table 4. 

Source: Hock joint of foal. 

Type culture: ATCC 6846; NCTC 630. 

!). Nocardia brasiliensis I Lindenberg, L909) 
Castellani and Chalmers (mend. Gordon and 
Mihm, 1959 (Lindenberg, A. Arch. Parasitol. 
13: 2(15-282, 1909; Castellani, A. and Chal- 
mers, A. .1. Manual of Tropical Medicine, 2d 
Ed. William Wood & Co., 1913, p. 816; 
Cordon, R. E. and Mihm, .1. M. .1. Gen. 
Microbiol. 20: 121), L959). 



Synonyms: Gonzalez Ochoa I 1945, 1953), 
Gonzalez Ochoa and Sandoval (1956), and 
Gordon and Mihm (1958) consider A', bra- 
siliensis a- the proper name for ihi- organ- 
ism. .1. mexicanus Boyd and Crutchfield, 
A', pretoriana Pijper and Pullinger, and A'. 
transvalensis Pijper and Pullinger, are con- 
sidered as synonyms. 

Morphology: Angularly branched fila- 
ments, bearing a few short straight aerial 
hyphae; later, growth becomes spreading 
and extensive. Aerial hyphae long and 
branching to short and gnarled; divide to 
form oval and cylindrical spores. Acid-fast- 
ness variable, from 100 per cent to none. 

Glycerol nitrate agar: Growth in form of 
piled up pink mass. Aerial mycelium very 
scant , white, at margin. 

Glucose nutrient agar: Colonies pale buff, 
umbilicated and piled up. 

Yeast-glucose agar: Highly mutable. 
Growth yellow to yellowish-orange, finely 
wrinkled. Some strains produce no aerial 
hyphae; other strains form mat of whitish 
aerial hyphae. A few strains form amber to 
brown soluble pigment. 

Potato: Colonies small, raised, pale pink; 
plug and liquid discolored. Later, growth 
dull buff, dry and convoluted at base, round 
and zonate at top of slant. Aerial mycelium 
white. 

Gelatin: A few colorless flak*-. No lique- 
faction 

Milk: Surface growth yellowish. Pale pink 
growth up the wall of the tube. Solid COagU- 
lum in 1 month; later, partly digested. 

Egg medium: Colonies few, round, color- 
less in :! days. Later, irregular, raised pink 
mass, warted appearance; moderate degree of 
liquefaction. 

Serum agar: Growth raised, convoluted, 
slighlly pinkish. 

Source: A case of mycetoma of the che>t 
wall in a South African native. 

Pathogenicity: To guinea pigs and humans. 

Remarks: According to Cordon and 



38 



THE ACTINOMYCETES, Vol. II 



Mihm, N. brasiliensis is distinguished from 
N. asteroides by positive decomposition of 
casein and tyrosine and by acid formation 
from inositol and mannitol. Additional char- 
acteristics of this species include the follow- 
ing: xanthine not decomposed; acid pro- 
duced from glucose and glycerol; no acid 
from arabinose, lactose, maltose, xylose, and 
other sugars; utilizes acetate, citrate, malate, 
propionate, pyruvate, and succinate, but not 
benzoate. 

According to Mariat (1958), A r . asteroides 
is characterized by a lack of proteolytic ac- 
tivity; utilization of urea, (NH 4 ) L >S04 and 
KNOs as sources of nitrogen, not of nitrite. 
Glucose, fructose, glycerol, and mannitol are 
utilized as sources of carbon, but not galac- 
tose, xylose, maltose, and starch, although 
paraffin is utilized. 

N. brasiliensis is characterized by gelatin 
hydrolysis; utilization of urea, (NH 4 )2S0 4 
and KNO3 as nitrogen sources; utilization 
of glycerol, glucose, fructose, galactose, man- 
nitol, xylose, and paraffin as carbon sources. 

Type culture: IMRU 850. 

10. Nocardia calcarea Metcalfe and 
Brown, 1957 (Metcalfe, G. and Brown, M. 
E. J. Gen. Microbiol. 17: 568-569, 1957). 

Morphology: Gram-positive and partially 
acid-fast. Mainly short rods (1.5 to 2.0 by 
1.0 n) together with unbranched aseptate 
filaments up to 10 m in length and occasional 
branched filaments. Some filaments show 
lemon-shaped swellings. After 4 days, short 
rods show snapping division typical of 
corynebacteria; abundant unbranched fila- 
ments (5.0 to 8.0 by 1.0 m) and a few branched 
filaments and cocci (1.0 /x) also present. 
Many rods show differentiation of a swollen 
spore-like strucl lire; these are usually formed 
terminally or subterminally, one per rod. In 
the filaments they are often formed in chains. 
Colonies after 14 days consist of short rods, 
cocci and rods with swollen cells. 

Agar media: Colonies circular, raised, soft, 
without aerial mycelium; pink or cream 
colored with distinct pink tinge when small. 



Sucrose agar: Filaments are rare and short 
rods and cocci are the predominant forms 
throughout. Occasional lemon-shaped cells 
are formed on all media. 

Glucose and mannitol agars: Very long 
branched filaments (10 to 25 m) present after 
2 days, often with terminal chains of swollen 
hyaline cells; these filaments usually frag- 
ment, but a few persist. 

Yeast extract-peptone agar: Cycle shorter 
than on previous media, most of the fila- 
ments having fragmented into rods and cocci 
after 3 days. 

Starch agar: Growth slight; no hydrolysis. 

Milk: Heavy growth, turned alkaline; no 
peptonization. 

Gelatin: Beaded growth at top of stab. 
Xo liquefaction. 

Nitrate reduction: Positive. 

Carbon utilization: Utilizes glucose, su- 
crose, and maltose; poor growth with lactose. 

Paraffin: Growth heavy in basal salts me- 
dium with ammonium salt and flakes of 
paraffin wax. 

11. Nocardia caprae (Silberschmidt, IS!)'.)) 
Waksman and Henrici, 1948 (Silberschmidt, 
W. Ann. inst. Pasteur 13: 841-853, 1899). 

Synonyms: This organism has been var- 
iously described as S. caprae (Price-Jones, 
1901). 0. caprae (Sartory, 1923), .4. caprae 
(Xannizzi, 1934). 

Morphology : Substrate growth forms thin, 
branching filaments, breaking up into rods. 
Aerial mycelium abundant on all media with 
tendency to form coherent spikes; mycelium 
not very polymorphous, but occasional 
thicker segments appeal'. Slightly acid-fast. 
Brownish soluble substance. 

Glucose -peptone -beef extract agar: 
Growth irregular, bright pink, tending to be 
heaped up. Later abundant masses, frosted 
over with thin, white aerial mycelium. 

Blood agar: Colonies minute, round, color- 
less, aggregated in broad pink zones. Aerial 
mycelium pale. Xo hemolysis. 

Egg medium: Colonies few, colorless, some 
Dink: aerial mycelium white. Pater, growth 



THE GENUS VOCARDIA 



::«.) 



becoming dull pink, 

while aerial myceliun 

Potato: Growth al 



;ular, with -cant raised, pale pink, confluent growth. Aerial 

mycelium white. Plug discolored. 
nt. Aerial myce- Gelatin: A few colorless flakes. No lique- 



li. 



pale pink. Growth becomes membra- faction. 



nous, considerably buckled. 

Gelatin: Growth extensive, dull, with 
small raised patches of pink aerial mycelium; 
later, ribbon-like, depressed. No liquefac- 
tion. 

Milk: Surface pellicle red. Solid coagulum; 
no peptoni/.at ion. 

Starch: No hydrolysis. 

Source: Lesions in goats. 

Pathogenicity: To rabbits, guinea pigs, 
and mice. 



Milk: Surface growth colorless. Aerial 
mycelium white. Coagulation positive. 

Nutrient broth: Surface pellicle cream- 
colored, wrinkled, extending up wall and 
breaking easily; moderate bottom growth, 
flaky. Medium discolored. 

Source: Infected guinea pigs from Su- 
matra. 

Remarks: Schneidau and Shaffer (1957) 

report that the organism is not acid-fast, 

grows at 46°C, utilizes paraffin, liquefies 

Remarks: According to Gordon and Mihm gelatin, hydrolyzes casein, liquefies starch, 

(1959) A', caprat is a synonym of A', aste- and shows positive hemolysis. 

routes; according to Schneidau and Shaffer 1; . Nocardia cellulans Metcalfe and 

I L957), however, the organism does not uti- 1WvM ,,,- 7 ( Metcalfej <;. and Browllj M 

li/e paraffin and does noi hemolyze blood, K ,, (>11 Microbiol. 17: 569 570. L957). 

as shown on p. (IS, Volume I. Morphology: Gram-positive and partially 

Type culture: IMRI 783. acid-fast. Branching aseptate filaments. 30 

12. Nocardia caviae Snijders, 1924 (Snij- i<> 40 ^ in length, often with swellings at in- 

ders, Geneesk. Tijdschr. Ned. Indie 64: 47. tervals; shorter filaments are less than 7.0 m 

7."), P.I24). in length. Fragmentation commences aboul 

Morphology: Growth consists of initial the fourth day, the number of shorl rods 

segmented hyphae, producing elements of (1.5 to 2.0 by 1.0 m) increasing rapidly. 

approximately even thickness, arranged in Hyaline spore-like structures are produced 

from the seventh day as slight terminal 
swellings on the filaments. After 28 days, the 
colonies consist of very short rods, cocci and 
spore-like cells. 

Agar media: Colonies raised, soft, without 
aerial mycelium; cream-colored on most 
media hut characteristically bright yellow on 
yeasl ex1 ract-peptone agar. 

Glucose agar: Filaments fragment less 
rapidly and are occasionally found after 28 
days. Numerous Y-forms .-ire found in older 

cultures. 

Cellulose tubes: After ii days there are 
long (20 to 30 n) branched and unb 



angular apposition; later, forms long, pro- 
fusely ramifying threads with strongly re- 
tractile protoplasm. Aerial mycelium straight 
and branching, the sporophores forming oc- 
casional coiled tips, divided into cylindrical 

-poll'-. 

Glucose agar: Growth piled up. convo- 
luted, cream-colored to pale pink. Aerial 
mycelium white. 

Glycerol agar: Growth scanty. 

Potato agar: Growth spreading, colorless. 
Aerial mycelium dense white. 

Egg medium: Growth heavily corrugated. 
pale pink, with submerged margin. Aerial filaments, many with terminal swelling 
mycelium dense while. After 3 weeks, color- Fragmentation is rapid and shorl rods and 
less transpired drops. cocci predominate during the stage of active 

Potato: Colonies -mall, colorless. Aerial cellulose decomposition, old cultures are 
mycelium white, powdery. Later, abundant, composed almos! entirely of cocci. 



40 



THE ACTINOMYCETES, Vol. II 



Milk: Acid and curd produced. 

Gelatin: Beaded growth at top of stab. 
No liquefaction. 

Starch agar: Starch not hydrolyzed. 

Nitrate reduction: Positive. 

Carbon utilization: Glucose, sucrose and 
maltose utilized; acid produced. 

Paraffin: Growth heavy with trace of 
yeast extract; no growth without yeast ex- 
tract . 

Type culture: ATCC 12,830. 

14. Nocardia citrea (Krassilnikov, 1938) 
Waksman and Henrici, 1948 (Krassilnikov, 
N. A. Bull. Acad. Sci. USSR No. 1: 139, 
1938). 

Morphology: Growth yellow to yellow- 
green, usually rough and folded, of a dough- 
like consistency. No soluble pigment. In 
young cultures, mycelium consists of very 
very fine threads 0.3 to 0.5 n in diameter. 
After several days the cells break up into 
short rods 0.5 by 1.5 to 5 n and into cocci 
0.3 to 0.5 n in diameter. Cells are the small- 
est of all the nocardias. Multiplies by fission 
and bud formation. No aerial mycelium. Not 
acid-fast. 

Synthetic medium: Growth and pigmenta- 
tion typical. 

Nutrient agar: Growth good. 

Gelatin: Liquefaction rapid. 

Milk: Coagulation and peptonization. 

Starch: Hydrolyzed rapidly. 

Sucrose inversion: Positive. 

Cellulose: No growth. 

Fat: Weak growth. 

Paraffin or wax: No growth. 

Nitrate reduction: Positive. 

Habitat: Soil and water. 

15. Nocardia coeliaca (Gray and Thornton, 
1928) Waksman and Henrici, 1948 (Gray, 
P., and Thornton, H. Centr. Bakteriol. 
Parasitenk. Abt, II, 73: 88, 1928). 

Morphology: Growth in form of short, 
curved, uneven-sided rods, 0.8 by 5 p; oc- 
casional filaments up to 10 to 12 p. long; fre- 



quently beaded, occasionally swollen or 
branched; coccoid forms 0.8 to 1.2 ji in 
diameter are common, especially in older 
cultures. Not acid-fast, or occasionally 
slightly acid-fast. 

Nutrient agar: Colonies less than 1 mm 
in diameter, round or irregular, raised, white, 
resinous, edge irregular, burred. Deep col- 
onies irregularly round or oval, edge slightly 
broken. Slant filiform, convex, white, rugose, 
resinous, edge undulate. 

Potato-glycerol agar: Growth dry, crum- 
pled, orange-colored, becoming brown. 

Gelatin: Surface colonies irregular, raised, 
white, rugose, dull edge entire. Deep colonies 
irregular, smooth or slightly broken. Stab 
convoluted, buff-white to yellowish, dull. 
Below surface the growth forms many ir- 
regular hollow lobes, giving a glistening ap- 
pearance, to a depth of 3 to 4 mm. 

Milk: Slightly alkaline after 5 to 7 days. 

Nutrient broth: Turbid. 

Nitrate reduction: None. 

Phenol: Utilized. 

Egg medium: Growth raised, smooth, 
moist, verrucose, buff -colored. 

Temperature: Optimum 22-25°C. 

Source: Soil. 

Remarks: No acid from glucose, lactose, 
sucrose, or glycerol. No chromogenesis. 
Hollow lobes produced in deep gelatin cul- 
tures. 

Type culture: ATCC 13181. 

16. Nocardia corallina (Bergey etal., 1923) 
Waksman and Henrici, 1948 (Hefferan, M. 
Centr. Bakteriol. Parasitenk. Abt. II, 11: 
459, 1904; Bergey et a/., Manual, 1st ed., 
1923, p. 93). 

Synonyms: Nocardia minima (Proactino- 
myces minimus Jensen). Bacillus mycoides 
corallinus Reader, 1926. 

Morphology: Growth pink to red to 
orange-yellow. Branching mycelium, gener- 
ally curved. In older cultures, hyphae de- 
generate generally into shorter rods and 
cocci. Not acid-fast . 



THE GENUS NOC IRD1 I 



II 



Nutrient agar: Colonies smooth, pink, 
shining; border lighter, edge filamentous or 
with arborescenl projections. As the colony 
grows, the cells in the interior break up into 

short rods and cocci which eventually form 
the mass of the colony. Cells on the outside 
remain filamentous, giving the colony a 
burr-like appearance, and often forming long 
arborescenl processes. No soluble pigment. 

Potato-glycerol agar: Growth filiform, 
raised, dry, wrinkled, yellowish-brown to 
coral red. 

Gelatin: Surface colonies round, convex, 
smooth, pink, shining, edge filamentous; 
deep colonies, burrs. No liquefaction. 

Milk: Reddish pellicle; milk becomes alka- 
line. 

Nutrient broth: Usually turbid. Pink 
scum. 

Paraffin and phenol: Utilized. 

Nitrate reduction: Positive. 

Starch: Not decomposed. 

Sucrose: Not inverted. 

Egg medium: Filiform, raised, dry, 
wrinkled, orange. 

Temperature: Optimum 22-25°C. 

Habitat : Soil. 

Remarks: Some strains produce acid from 
glycerol and glucose. No acid or gas from 
sucrose, maltose, or lactose. Phenol and nt- 
cresol are utilized. Some strains utilize 
naphthalene. Krassilnikov (1949) reports for 
his strains, good growth in high salt concen- 
trations. Schneidau and Shaffer I 1957 I reporl 
positive acid-fastness, positive hemolysis 
and urease formal ion. 

17. Nocardia dicksonii (Erikson, 1935) 
Waksman (Erikson, I). Med. Research 
Council Spec. Rept. Ser. 17: 203, 1935). 

Morphology: Growth consists of long fila- 
ments, sometimes wavy. Aerial mycelium 
straight . Spores cylindrical. 

Glycerol nitrate agar: Growth granular 

and wrinkled, cream-colored. Medium deeply 
discolored. 



Glucose-asparagine agar: Growth wrin- 
kled, colorless. 

Potato agar: ( '.row th abundant , colorless. 
Egg medium: Growth yellowish-brown. 
Starch agar: Strong hydrolysis. 
Gelatin: ( '.row th smooth, cream-colored on 
surface. Liquefaction limited. 
Milk: Coagulated, peptonized. 
I labitat : I nknown. 

18. Nocardia fun-mica (Nocard, 1888) 
Trevisan and De Tom, L889 'Nocard. M. 
E. Ann. inst. Pasteur, 2: 293, 1888; Trevi- 
san, V., I. generi e le specie delle Batleri- 
acee, Milan, 1889, p. ( .»). 

Morphology: Growth yellow, of doughy 
consistency. Markedly acid-fast. 

Nutrient agar: Colonies yellowish- white, 
irregular, refractive; mycelium filamentous. 

Potato: ( Irowth abundant , dull, crumpled, 
whitish-yellow. 

Gelatin: Colonies small, circular, trans- 
parent, glistening. No liquefaction. 

Milk: No coagulation; no peptonization. 

Starch: No hydrolysis. 

Nutrient broth: Clear, with granular sedi- 
ment, often with gray pellicle. 

Nitrate reduction: Negative. 

Temperature: Optimum .'>7°0. 

Pathogenicity: Pathogenic to certain 
domestic animals and guinea pigs. 

Source: Cases of cattle farcy. 

Type culture: IMP!' .'CIS. 



19. 
L. S. 

M< 
on al 
striki 



Nocardia fastidiosa Suter, 1951 (Suter, 
Mycologia 13: 658 676, 1951 I. 
irphology: The organism- were similar 
1 media studied, showing in general a 
ng pleomorphism with coccoid, bacil- 
and filamentous forms. Many of these 
clubbed; some bore a striking resem- 
•»• to spermatozoa. Other- were thick at 

•nd and tapered down to filamentous 
Filamentous forms were up to 25 m in 
h and. not considering clubs or swollen 
ons, measured 0.2 to 1.2 ^ in diameter. 
verage being about 0.8 to 1 (t. Stained 



42 



THE ACTINOMYCETES, Vol. II 



preparations never showed a richly branch- 
ing character, bu1 an alternate type of 
branching was fairly easy to demonstrate 
after about 7 days' incubation at 37°C. The 
coccoid forms were round, oval, or drop- 
shaped. Neither septa nor nuclei were seen. 
Spores were formed in short chains within 
mycelial strands and were of the same diame- 
ter as the mycelial strands. Similar spores 
were also found singly and extracellularly. 
The organisms taken from cultures were 
partially acid-fast . 

Growth on agar media: Colonies were slow 
growing, appearing after 2 to 3 days as tiny 
specks, which after 7 days' incubation finally 
achieved, but. never exceeded, a size of about 
1 mm in diameter. To the naked eye they 
appeared grayish- white, compact, and 
smooth, and under low-power magnification 
they appeared fluffy, raised, compact at the 
center, and irregular and stringy at the edge, 
due to the presence of radiating and tangled 
filaments. Zigzag arrangements of elements 
and branches, clubs, and curls were seen at 
the periphery. The colonies were adherent 
to the medium. The top surface was dry and 
could be scraped off with a stiff wire loop, 
but neither the whole colony nor any part 
of it could be removed intact. On blood agar 
after 7 days' incubation, the colonies viewed 
by transmitted light showed a characteristic 
dense reddish center and a clear outer zone, 
both areas being very sharply defined. 

Optimum temperature: 37°C. 

Oxygen requirements: The organism is a 
facultative anaerobe, growing equally well in 
the presence or absence of oxygen. 

Proteolytic activity: The organism is non- 
proteolytic. No odor of putrefaction was 
perceived in any of the cultures; gelatin was 
not liquefied; no growth occurred on serum 
plates or on coagulated human serum. 

Gelatin stab: No growth after 28 days at 
17-20°C. 

Potato: No growth at 37°C. 

( 'arbon sources: Acid formed from glucose; 



not from lactose, sucrose, maltose, or glyc- 
erol. 

Nitrate reduction: Negative. 

Habitat: Isolated from penile ulcer. 

Remarks: N. fastidiosa is different from 
previously described species of Nocardia in 
the following ways: It is very fastidious in 
its growth requirements. It does not grow 
in synthetic media to which carbohydrates 
have been added; it will not utilize paraffin; 
it will not grow on potato or carrot ; it will 
not grow on acid-maltose agar nor on acid- 
glucose agar; attempts to grow it on nutrient 
agar and on BHI agar have given variable 
results; its optimum temperature is 37°C. It 
is delicate and is relatively slow growing; it 
is never hardy or richly branching, and it 
does not produce a surface scum or a con- 
fluent or filiform growth. It is a facultative 
anaerobe, differing in this respect from all 
other Nocardias described with the excep- 
tion of N. farcinica and .V. rubropertincta. 

The author summarizes its distinctive 
properties as follows: It produces a fairly 
compact colony composed of tangled myce- 
lium and exhibits radiating, clubbed, 
branched, and curled elements at the per- 
iphery. Fragmentation of the mycelium and 
post-fission movement (zigzag arrangement) 
occur at the periphery of the colony. Arthro- 
spores are produced. Stained preparations re- 
veal partial fragmentation into bacillarv 
and coccoid forms. Mycelial forms and spores 
average slightly less than 1 ^ in diameter. 
Neither nuclei nor septa were observed. 
Branching is of an alternate type. It is par- 
tially acid-fasi . 

20. Nocardia fiava (Krassilnikov, 1938) 
Waksman and Henrici, 1948 (Krassilnikov, 
N. A. Bull. Acad. Sci. USSR No. 1: L39, 
1938). 

Not Proactinomyces flavus Humm and 
Shepard . 

Morphology: Cells at first filamentous, 
0.7 to 0.8 n in diameter; later, they break 
into long rods and then into cocci 0.7 m in 



THE GENUS VOC \.RD1 I 



i:; 



diameter. Some strains form chlamydo- 
spores. Numerous inflated cells of the bulbi- 
form or fusiform type. ( Jell mull iplical ion by 
fission, cross wall formation, rarely by bud- 
ding. \'oi acid-fasl . 

Synthetic agar: Colonics brighl yellow or 
golden. 

Nutrient agar: Growth dirty, lustrous, or 
rough and folded, of a dough-like consist- 
ency, yellow to straw-colored. No soluble 
pigment. 

( ielat in : No liquefad ion. 

Milk: Xo coagulation and no peptoniza- 
tion. 

Starch hydrolysis: Slight . 

Sucrose: Weak inversion. 

Cellulose: No growth. 

Paraffin and wax: Xo growth. 

Fat : Weak growth. 

Habitat: Soil. 

21. Nocardia flavescens (Jensen, L931) 
Waksman and Henrici, L948 (Jensen, H. 
Proc. Linnean Soc. X. S. Wales 56: 301, 
L931). 

Morphology: Substrate growth forms long, 
luanched, nonseptate hyphae, 0.4 to 0.6 fx. 
On nutrient agar and potato, septa are 
formed, mycelium fragmenting, partly re- 
sembling highly branched mycobacteria. 
Aerial mycelium consists of fairly long 
hyphae of the same thickness as the vegeta- 
tive hyphae, not very much branched, with- 
out spirals, often clinging together in wisps; 
hyphae break up into fragments of variable 
lengths, from 1.2 to 1 .5 up to 10 to 13 m, 
showing an irregular, granulated staining. 
Xot acid-fast. 

Nutrient agar: Substrate growth raised 
and much wrinkled, first dirty cream-col- 
ored, later dark yellowish-gray, of a soft, 
moist, curd-like consistency. Aerial myce- 
lium absent. Soluble pigment absent. 

Glucose agar: Substrate growth super- 
ficial, wrinkled, honey-yellow, of a hard 
and cartilaginous consistency. Aerial myce- 



lium thin, smooth, white. Soluble pigment 
yellow. 

Potato: Substrate growth much raised 
and wrinkled, first cream-colored, later 
yellowish-brown, soft and smeary. Xo aerial 
mycelium. Xo soluble pigment . 

( ielatin: Liquefad ion slow. 

Milk: Coagulation; slow peptonization 
with acid reaction. 

Starch: i Iydrolyzed. 

Cellulose: Xo growth. 

Paraffin: Xo growth. 

Sucrose: Inverted. 

Glucose broth: Rather scant growth. 
Granulated, yellowish sediment ; no surface 
growth. Broth clear. Xo pigment. Xo acidity. 

Nitrate: Slight or no reduction. 

Source: Soil. 

22. Nocardia fordii (Erikson, L935) Waks- 
man (Erikson, D. Med. Research Council 
Spec. Rept. Ser. 203: 15, 1935). 

Morphology: Substrate growth consists of 
filaments of medium length. Aerial myce- 
lium short, straight, sparse. Small oval 
spores on potato agar and starch agar. 

Glycerol nitrate agar: Growth thin, ex- 
tensive, golden brown, convoluted. 

Xutrient agar: Colonies small, creamy 
to golden, ring-shaped; later, heaped-up 
patches, becoming golden brown and con- 
voluted. 

Egg medium: Colonies minute, cream- 
colored, elevated, becoming golden brown, 
raised. 

Potato: ( rrowth yellowish in thin terminal 
portion, tending to be piled up. Aerial myce- 
lium scant, white, at top of slant. Later, 
growth abundant, golden brown, confluent, 
partly honeycombed, partly piled up. 

Gelatin: No visible growth, slighl soften- 
ing of gelatin; later partial liquefaction. 

Milk: Surface ring brownish. Coagulation 
positive. 

Starch: Xot hydrolyzed. 

Source: Human spleen in a case of acho- 
luric jaundice. 



44 



THE ACTLXOMYCETES, Vol. II 



23. Nocardia formica Harris and Wood- 
ruff, 195.3 (Harris, D. A. and Woodruff, H. 
B. Antibiotics Ann. 1953-1954, 609-614). 

Morphology: Mycelial development ex- 
tensive, with no fragmentation of hyphae. 
Ghost filaments and cytoplasmic condensa- 
tions produced. In submerged culture, 
straight and curved rods develop, exhibiting 
the Y- and V-forms. Rods are 0.9 to 1.1 by 
1 .3 to 6.0 n. Not acid-fast. 

Sucrose nitrate agar: Growth very faint 
or none at all. 

Glucose-asparagine agar: Growth fair. 
Aerial mycelium grayish-white. Sporulation 
poor. 

Nutrient agar: Growth fair. Aerial myce- 
lium none. 

Peptone-glucose agar: Growth tannish- 
colored. Aerial mycelium white to grayish, 
gradually covering surface. Reverse side 
dark brown. Soluble pigment brown. 

Egg medium: Growth excellent, buff-col- 
ored, convoluted, moist. No liquefaction. 
Medium not discolored. 

Starch agar: Starch hydrolyzed. 

Gelatin: Liquefaction rapid. Soluble pig- 
ment none. Growth settled on bottom of the 
tube. 

Potato: Very poor growth. 

Nitrate reduction: Positive. 

Casein: Hydrolyzed. 

Paraffin: Not utilized. 

Optimum temperature: 28°C; good growth 
at 37°C. 

Carbon utilization: No acid production in 
organic media from glucose, glycerol, lac- 
tose, maltose, and sucrose; acid produced in 
inorganic media from glucose, glycerol, 
lactose, maltose, but not from sucrose. 

Antagonistic properties: Produces an anti- 
biotic substance active against Trichomonas 
and swine influenza virus. 

Source: Isolated from an abandoned nest 
of African ants in an imported mahogany 
log. 

24. Nocardia fructifera (Krassilnikov, 



1941) Waksman (Krassilnikov, N. A. Ac- 
tinomycetales. Izvest. Akad. Nauk. SSSR, 
Moskau, 1941). 

Morphology: Growth not compact, mostly 
of dough-like consistency, smooth or rough. 
Hyphae breaking up into rods and in some 
cultures into cocci. Not acid-fast. Aerial 
mycelium well developed, whitish to rose- 
colored. Sporophores long, straight or weakly 
wavy, but not spiral-shaped. Spores cylin- 
drical, 1.5 by 0.7 id. 

Synthetic agar: Growth rose-colored to 
bright red. No soluble pigment. 

Nutrient agar: Aerial mycelium weakly 
developed or absent entirely. 

Gelatin: Liquefaction slow. 

Milk: Coagulation positive; peptoniza- 
tion weak. 

Sucrose: Inverted. 

Starch: Hydrolysis weak. 

Cellulose: Poor growth. 

Paraffin: No growth. 

Fats: Good growth. 

Source: Soil. 

Remarks: One strain was obtained as a 
mutant of another Nocardia; another strain 
was changed, after 8 months of cultivation, 
into a typical Streptomyces. This species is 
considered as a transition form between the 
two genera. 

25. Nocardia gibsonii (Erikson, 1935) 
Waksman (Erikson, D. Med. Research Coun- 
cil Spec. Pept. Ser. 203: 36, 1935). 

Morphology: Young growing mycelium 
branches profusely at short intervals, finally 
grows out into long, frequently wavy fila- 
ments. Property of producing aerial myce- 
lium apparently lost. 

Nutrient agar: Colonies small, cream-col- 
ored, depressed, partly confluent, growing 
into an extensive wrinkled surface layer. 

Glucose nutrient agar: Growth cream- 
colored, wrinkled, membranous. 

Potato agar: Growth wrinkled, glistening, 
membranous. 

Blood agar: Colonies small, discrete, yel- 



THE GENUS N0CARD1 



15 



lowish, irregularly wrinkled, clear hemolytic 

zone. 

Egg medium: Colonies small, round, 
smooth, colorless, with conically elevated 

cciilcrs. 

Potato: No growth. 

Gelatin: Dull white flakes sinking as me- 
dium liquefies. Liquefaction rapid. 

Milk: Coagulation positive; peptoniza- 
tion limited. 

Starch: Not hydrolyzed. 

Source 1 : Human spleen in a case of acholuric 
jaundice. Injected into a monkey and reiso- 
lated. 

Type culture- ATCC 6852. 

26. Nocardia globerula (Cray, 1928) Waks- 
maii and Ilenrici, 1948 (Gray, P. Proc. Roy. 
Soc. (London) B 102: 265, 1928). 

Morphology: Growth orange to orange- 
buff. It consists of curved rods and fila- 
ments, 1 by 2 to 9 ju, with many coccoid cells, 
especially in old cultures. Rods and filaments 
frequently irregularly swollen. Not acid-fast. 
Capsules may be present. 

Nutrient agar: Surface colonies irregu- 
larly round, 3 to 5 mm in diameter, convex, 
white, smooth, shining; edge undulate, erose. 
Deep colonies, lens-shaped. 

Gelatin: Surface colonies irregularly 
round, 1 to 2 mm in diameter, convex, light 
buff, smooth, shining. Stab: nailhead, irreg- 
ularly round, convex, pinkish-white, smooth, 
shining. 

Potato-glycerol agar: Growth filiform, 
moisl , smooth, pale pink. 

Milk: Alkaline. 

Nutrient and peptone broth: Turbid with 
viscous suspension. 

Nitrate reduction: None. 

Egg medium: Growth spreading, raised, 
moist , orange-colored. 

Indole agar: Blue crystals of indigotin 
formed. 

Temperature: Optimum 25-28°C. 

Phenol: Utilized. 

Source: Soil. 



Remarks: This organism resembles most 
closely A', corallina. It is distinguished by 
producing a more watery type of surface 
growth, more nearly entire deep colonic-, 
and more particularly by the production of 
indigotin from indole. No acid from glucose, 
lactose, maltose, sucrose, or glycerol. 

Type culture: ATCC 13,130. 

27. Nocardia hortonensis (Erikson, 1935) 
Waksman (Krikson, D. Med. Research 
Council Spec. Kept. Ser. 203: 22, 1935). 

Morphology: Substrate growth made up 
of very slowly developing unicellular myce- 
lium, composed of long slender straight 
branching filaments. Aerial mycelium very 
sparse, forming straight hyphae only once 
on potato. Not acid-fast . 

Glycerol nitrate agar: Colonies coiled, 
colorless, lustrous patches, isolated, with 
central depression. 

Nutrient agar: Growth very slow, as few 
smooth, cream-colored, coiled colonies. 

Glucose nutrient agar: Growth as coiled 
and heaped up cream-colored translucent 
masses. 

Potato agar: Colonies colorless, blister; 
later dull green heaped and coiled mass. 
Medium becomes slightly discolored. 

Potato: Colonies abundant, colorless, 
umbilicated, round, some coiled in raised 
masses; later, liberal olive-green growth. 
Aerial mycelium dense, velvety gray-green 
at top of slant . 

Gelatin: Colonies round, cream-colored on 
surface and a few millimeters below. No 
liquefaction. 

Milk: Surface growth green; peptoniza- 
tion positive. Color at first purple, later 
brown. 

Source: From pus containing typical 
actinomycetic granules from parotid abscess. 

28. Nocardia intracellularis Cutting and 
McCabe, MM!) (Cutting, J. T. and McCabe, 
A. B. Am. .1. Pathol. 23: I 17, L949). 

Morphology: Filaments branched, becom- 
ing fragmented, composed of bacillary ele- 



Hi 



THE ACTINOMYCETES, Vol. II 



ments in scries, 0.2 to 0.45 m in width. Liquid 
cultures give branched colonies. The hyphae 
do not form club-shaped tips, and lack 
chlamydospores. Not discolored when 
stained with fuchsin and treated with acid 
alcohol. 

Agar media: Colonies circular, raised, 
wet-shining, smooth, and nonmucoid. 

Potato agar: No growth. 

Gelatin: Growth poor. No liquefaction. 

Glycerol broth: White, mucoid masses 
formed at bottom of lube. Medium remains 
clear. 

Milk: Acid after 20 to 30 days. 

Starch: Not changed. 

Tyrosinase reaction: Absent. 

Cellulose: Not decomposed. 

Nitrate reduction: None. 

Oxygen requirement: Does not, develop 
in the absence of oxygen, but grows in an 
atmosphere having 10 per cent C0 2 . 

Paraffin: Used as the only source of car- 
bon. 

Temperature: Grows at 37.5°C and tol- 
erates well temperatures up to 40°C. 

Habitat: Observed in granuloma of in- 
fected lymph nodes and in the feces of a 
living patient whose death it ultimately 
caused. Observed also at autopsy in widely 
disseminated granulomatous lesions which 
it produced. 

Type culture: ATCC 13,209. 

2'.). Nocardia ivorensis Combes, Kauff- 
mann and Vazart, 1957 (Combes, R., Kauff- 
mann, J., and Vazart, B. Compt. rend. 224: 
821-824, 1582-1587, 1957). 

Agar media: Substrate growth character- 
ized by the black coloration of its coccoid 
bodies, by their elongation, by their resist- 
ance io dryness and to heat, by their cellulo- 
lytic properties, and by their production of 
an orange pigment on different media. Col- 
onies at first whitish, centers becoming light 
orange to brown, later black. Black circles 
formed successively around the central circle 
and finally becoming confluent. Later, sur- 



face of colony is uniformly black, shiny, and 
waxy; at the periphery, grayish, radiant 
outgrowths develop in the agar, forming a 
more or less regular fringe; on the surface of 
this fringe brown, rapidly darkening, con- 
centric zones appear. 

Milk: Reddish-orange surface film, and 
isolated colonies adhering to the walls of 
the tube. 

Potato: Growth orange, darkening in 
places, with the appearance of coccoid forms. 

Gelatin: Growth scant, slow; later the 
culture is orange in color. Liquefaction 
positive. 

Cellulose (filter paper or washed cotton), 
moistened with the synthetic medium: Light 
brown colonies appear in 2 days and turn 
dark at 6 days, being entirely composed of 
coccoid forms; later, colonies are entirely 
black. Cellulose progressively disintegrates. 

Paraffin : White colonies appear at 5 days. 
They remain small and rapidly form coccoid 
elements. 

Nitrate reduction: Positive. 

Remarks: Three isolated cultures differed 
from each other mainly in the rapidity with 
which they formed coccoid elements. Or- 
ganism closely related to N. nigra. 

Habitat: Colony of termites on ivory 
coasi of Africa. 

30. Nocardia kuroishi Uesaka, 1952 (Ue- 
saka, I. J. Antibiotics (Japan) 5: 75-79, 
1952). 

Morphology: Aerial mycelium abundant. 
Sporophores slightly curved at first, later 
turning around each other. Acid-fast. 

Glycerol nitrate agar: Growth thin, pale 
yellow. Aerial mycelium punctiform, white. 
Soluble pigment yellow. 

Nutrient agar: Colonies wrinkled, grayish- 
yellow. No aerial mycelium. Soluble pigment 
faint grayish-brown. 

Glucose nutrienl agar: Growth abundant, 
at first yellowish-brown, then reddish-brown. 
Aerial mycelium scant, white at margin of 



THE GENUS NOCARDIA 



17 



colonics. Soluble pigmenl red to wine- 
colored. 

Potato: Growth moderate, at first red or 
brownish red, later dark brown. Aerial myce- 
lium grayish white. Soluble pigmenl dark 
brown. 

Glucose broth: Red colonies forming 
pellicle. Abundant, flocculenl sediment. 
Soluble pigmenl dark brown. 

Gelatin: Growth yellowish-brown, sinking 
into medium. No aerial mycelium. No lique- 
faction. Soluble pigmenl yellowish-brown. 

Milk: No coagulation. Slow peptonization. 
Brown pigmenl . 

Starch: Hydrolyzed. 

Carbon source: Lactose well utilized. 

Nitrate reduction: None. 

Antagonistic properties: Produces an 
antibiotic neonocardin, active againsl vari- 
ous bacteria. 

Source: Soil. 

31. Nocardia leishmanii Chalmers and 
Christopherson, 1916 (Bin, ('. and Irish- 
man, W. B. J. Byg. 2: 120, L902; Chalmers, 

A. and Christopherson, I. Ann. Trop. Med. 
Parasitol. 10: 255, 1916). 

Morphology: Initial cells frequently swol- 
len, large and irregular, aggregated in shorl 
chains and then branching out into regular 
narrow filaments; later entire colonics as- 
teroid in appearance, very fine and close 
angular branching, with aerial hyphae situ- 
ated singly. Whitish-pink aerial mycelium 
generally abundant with irregularly cylin- 
drical conidia. Acid-fast. 

Glucose nutrient agar: Colonies rounded, 
elevated, red, with paler frosting of sparse 
aerial mycelium. No soluble pigment. 

Glycerol agar: Colonies small, round, pink, 
tending to be umbilicated and piled up. 
Aerial spikes stiff, white. 

Potato agar: Colonies minute, colorless, 
round. Aerial mycelium white, in patches. 

Egg medium: Growth colorless, confluent, 
studded with little wart-like projections 



hearing stiff aerial spikes; later pinkish. 
Aerial mycelium white. Medium discolored. 

Gelatin: Colonies small, pink. No lique- 
faction. 

Milk: Sin-face growth; aerial mycelium 
white turning pink. Coagulum solid, later 
partly peptonized. 

Pathogenicity: To rabbits, rat-, and 
guinea pig-. 

Source: Fatal case of lung disease and 
pericardii is in man. 

Remarks: According to Gonzalez Ochoa 
and Sandoval (1956), A', leishmanii is a 
synonym of A'. asU roidi zs. 

32. Nocardia listen (Erikson, L935) Waks- 
mann (Erikson, I). Med. Research Council 
Spec. Rept. Ser. 203: 23 24, L935). 

Morphology: Sporophores short and 
straight . Spores oval. 

Glycerol nitrate agar: Growth abundant, 
moist, cream-colored. Aerial mycelium pow- 
dery, white, with exuded drops. 

Calcium malate agar: Growth poor, in 
form of a biscuit-colored membrane. 

Nutrient agar: Growth smooth, moist, 
cream-colored, margin depressed, center 
elevated. 

Glucose nutrient agar: Growth cream- 
colored, glistening. 

Potato agar: Growth extensive, colorless, 
warted surface. Dirty pink coloration after 
2 weeks. Scant white aerial mycelium after 
1 months. 

Potato: ( irowth abundant . dull, brownish, 
wrinkled. Aerial mycelium white. 

Gelatin: Surface colonies round, white; 
after 45 days, confluent -kin. Liquefaction 
-light. 

Blood agar: Colonic- small, round, cream- 
colored, with smooth, translucenl surface. 

No hemolysis. 

Serum agar: Colonies -mall, irregular, 
moist, cream-colored, tending to be heaped 
up; later somewhat transparent. 

Milk: Coagulated. No change in reaction. 

Source: From human material. 



IS 



THE ACTINOMYCETES, Vol. II 



33. Nocardia lutea Christopherson and 
Archibald, 1918 (Christopherson, J. B. and 
Archibald, R. G. Lancet 2:847, 1918).* 

Morphology: Growth consists of irregular, 
spreading, polymorphous colonies, compris- 
ing swollen and segmented cells of all shapes 
and sizes with markedly granular contents. 
Later cells more monomorphous, the fila- 
ments being arranged in angular apposition. 

Glycerol nitrate agar: Growth in form of 
yellowish-pink, wrinkled membrane. 

Nutrient agar: Growth abundant, co- 
herent, moist, pink, membranous with round 
discrete colonies at margin. 

Glucose nutrient agar: Growth scant, 
reddish, smeary. 

Potato agar: Small filamentous colonies 
are formed; irregular angular branching. 
Aerial hyphae few, isolated, short, straight. 

Potato: Growth carrot-red, moist, thick, 
granular in bands, partly raised and with 
discrete round colonies. Aerial mycelium 
sparse, colorless, very thin at top of slant. 

Gelatin: Growth pale pink, wrinkled on 
wall of tube. Colorless punctiform and stel- 
late colonies in medium. No liquefaction. 

Milk: Growth orange-red on surface and 
at bottom. 

Egg medium: Growth poor, dull pink. 

Source: Actinomycosis of the lachrymal 
gland. 

Remarks: According to Erikson, various 
saprophytes, such as N. rubra and N. poly- 
chromogenes, are closely related. 

34. Nocardia marina (Krassilnikov, 1949) 
Waksman (Humm, H. J. and Shepard, K. 
S. Duke Univ. Marine Sta. Bull. 3: 76, 
1946; Krassilnikov, N. A., Guide to the 
identification of bacteria and aetinomycetes, 
Moscow, 1949). 

Synonyms: Proactinomyces flavus Humm 
and Shepard. Proactinomyces citreus marinae 
Krassilnikov. 

* Description after Erikson, D., Med. Res. 
Council Spec. Rept. Ser., 203: 30, 1935. 



Morphology: Growth smooth, bright 
yellow color, of a dough-like consistency. 
Hyphae long, filiform, branching, breaking 
down into short rods and cocci. Xo aerial 
mycelium. 

Synthetic and protein salt water media: 
Good growth. 

Gelatin liquefaction: Positive. 

Agar: Liquefied. 

Milk: No coagulation; peptonization posi- 
tive. 

Nitrate reduction: None. 

Starch: Hydrolyzed. 

Carbon sources: Acetic, lactic, and butyric 
acids utilized. Acid formed from various 
sugars. 

Temperat ure : 25-30°C . 

Habitat: Atlantic Ocean marine deposit. 

35. Nocardia mesenterica (Orla- Jensen, 
1919) Waksman and Henrici, 1948 (Orla- 
Jensen, S. The lactic acid bacteria, 1919, 
181 ; Jensen, H. L. Proc. Linnean Soc. N. S. 
Wales 57: 373, 1932). 

Morphology: Growth forms extensive my- 
celium composed of richly branching hyphae 
of a somewhat variable thickness, 0.4 to 0.8 
ll. No aerial mycelium. Later, hyphae divide 
into fragments of varying sizes and shapes, 
partly diphtheroid rods, but no real cocci. 
There is, particularly in complex organic 
media, a tendency to form large, swollen, 
fusiform to almost spherical cells, up to 3.5 
ix in diameter. These may stain intensely 
with carbol fuchsin. 

Glucose-asparagine agar: Growth fair, 
raised, granular, very pale yellow, glistening. 
Condensation water-clear. 

Glucose-peptone agar: Growth excellent, 
spreading. At first flat and smooth, pale 
straw-yellow, perfectly hard and cartilagin- 
ous, later raised and strongly folded, of a 
loose, curd-like consistency, bright lemon- 
yellow. 

Potato: Growth scant, restricted, soft, 
cream-colored smear. 

Gelatin: Growth finely arborescent. 



THE GENUS NOCARDIA I'.i 

cream-colored in the stab. Surface colony Oxygen: Grows aerobically. Grows also 

raised, folded, pale yellow. \'o liquefaction, under anaerobic conditions. 

Milk: Small cream-colored granules along Acid production: Acid produced in serum 
the tube. No proteolytic action. agar media, with glucose and salicin, some- 
Starch: Hydrolyzed. times with maltose and lacto.-e. 
Cellulose: Not utilized. Habitat: Parasite inhabiting nasopharynx 
Nutrient broth: Good growth; volumi- of rats. Isolated from body of patienl bitten 
nous, flaky, whitish sediment; broth clear. by a rat. 

Nitrate reduction: Negative. Remarks: Similar organisms by a variety 

Sucrose: Inverted. of names, such as .1. putorii, were also 

Source: Fermented beets. listed. Above description based on data of 

Remarks: Sodium nitrate, ammonium Topley and Wilson (1946). 

phosphate, and asparagine are utilized, al- 37. Nocardia nigra (Krassilnikov, 1941) 

though these are interior to peptone as Waksman (not A', nigra Castellani and 

sources of nitrogen. CI, aimer.- not Streptothrix nigra Rossi- 

36. Nocardia maris (Schottmuller, 1914) Doria.) (Krassilnikov, X. A. Actinomy- 

de Melloand Pais, L918 (de Mello and Pais, cetales. Izvest. Akad. Nauk. SSSR, Moskau, 

Arq. Hig. Pat. Exot. 6: IS:!, L918). 1!,ll >- 

Synonyms: Streptothrix muris-ratti Schott- Morphology: Growth rough, folded, shiny, 

miiller, L914. Streptobacillus moniliformis dough-like consistency. Cells thread-like, 

Levaditi, L925. Actinomyces muris Topley breaking up readily into rods 2 to 10 by 

and Wilson, l!)4(i. Proactinomyces muris () ~ m and cocci, 0.6 to 0.8 m- No aerial 

Krassilnikov, L941. mycelium. Gram-positive, not acid-fast. 

Morphology: Slender branching filaments, ^S ar media: Growth poor, at first color- 

0.4 to ().(i n in diameter, breaking up into less or brownish, gradually becoming darker, 

rods and cocci. Often cells form long chains later dark brown and even black. Pigmenl 

of bead-like cells, with terminal club-like " ()1 excreted in medium. Many cells are 

swellings. Nonacid-fast. swollen to :; M in diameter. 

Growth: None on ordinary media. Growth Potato: Growth good. 

occurs in presence of serum, ascitic fluid or Gelatin: No growth. No liquefaction, 

blood. Milk: No change. 

N . . • . x> ,, Cellulose: No growth. 

Nutrient agar: No growth. _ , 6 

,,, x - , Paraffin and wax : No growth. 

( ducose agar: No growth. ., , . , ,. 

,, . . . Nutrient broth: Small sediment produced, 

berum agar: Grayish-yellow, clear col- . f j- , 

• .Medium clear. 

,,m, ' s < (K2 t0 ° 3 mm m diameter, with ( , ;n . ))()ii ulilizall()n: niliz( , s glucose ;m( , 

smooth, glistening surface and entire edge. mannosej with formation of acid. 

Easily emulsifiable. Source: Seldom found in soil. 

Gelatin: No growth. Remarks: Culture rapidly loses its vi- 

Potato: No growth. ability on continued cultivation. 

Mllk: No effect - 38. Nocardia opaca (den Dooren de Jong, 

Nitrate reduction: None. 11)27) Waksman and Henrici, 1948 (den 

Blood agar: Like serum agar. No he- Dooren de .long. I.. E. Centr. Bakteriol. 

molysis. Parasitenk. Abt. II, 71: 216, L927; Jensen, 

Egg medium: Similar to growth on serum II. L. Proc. Linnean Soc. N. S. Wales 57: 

agar. No liquefaction. 369, 1932). 



50 



THE ACTINOMYCETES, Vol. II 




Figure 13. N. opaca (V. erythropolis), showing scheme of branching; glycerol nutrient agar, first 
sketch 10 hours incubation; others at hourly intervals (Reproduced from: McClung, N. M. Lloydia 12: 
153, 1949). 



Synonyms: Nocardia crystallophaga (Gray 
and Thornton); N. erythropolis (dray and 
Thornton); Proactinomyces opacus (Jensen). 

Morphology: Growth lustrous, rose-col- 
ored to red. Hyphae long, curved, irregular 
and branching, breaking up into rods and 
cocci. Not acid-fast. Gram-positive (Fig. 13). 

Potato-glycerol agar: Growth dry, rough, 
crumpled, pink to buff-colored. 

Gelatin: Colonies round, convex, whitish, 
smooth, shining, with edges slightly ar- 
borescent. Stab: convex, whitish, smooth, 
resinous, filiform, erose. No liquefaction. 

Egg medium: Growth spreading, smooth, 
moist, salmon-colored. 



Potato: Growth covered with tufts of 
aerial hyphae. 

Milk: Grayish pellicle. No coagulation, no 
peptonization. Reaction slightly alkaline. 

Nitrate reduction: Positive. 

Starch: Noi hydrolyzed. 

Sucrose inversion: Negative. 

Carbon sources: Sat mated, long chain 
aliphatic hydrocarbons are utilized as sources 
of energy. 

Temperature: Optimum 30°C. 

Source: Seldom found in soils. 

Remarks: Differs from X. corallina and 
N. polychromogenes in that the cells are 
much longer than those of the former and 



THE GENUS NOCARDIA 51 

much shorter than those of the latter. Erik- 40. Nocardia paraffinou (Jensen, L931) 

son (1949) added the following character- Waksman and Henrici, L948 (Jensen, II. 

istics: Soft cream to pink growth on nut ri- Proc. Linnean Soc. \. S. Wales .">(>: 362, 

enl agar media. On synthetic media, growth I!*.'!! I. 

colorless and thin, producing an initial myce- Morphology: Growth hard, firm, yellow- 

I iui ii, the hyphae dividing rapidly into short ish, consisting initially of an extensive myce- 

rods; addition of 0.01 per cent MnS0 4 stim- limn, with long, richly branching hyphae, 

ulated production of pale pink pigment. 0.4 to 0.5 ju thick. After 5 to 6 days, numer- 

Acid-fasl cell elements predominated during ous end branches swell to about double 

periods of maximum growth and tree air thickness, and divide into oval, spore-like 

supply. A study of the morphology of A', elements, 0.8 to 1.0 by 1.2 to 1.5 m- Division 

opaca grown on hydrocarbons and fatty starts at the tips of the swollen branches 

acids has been made by Webley (1955). and proceeds basipetally until most of the 

,_, .. , ... , hyphae appear divided. Primary septa have 

39. A ticiinha paniae (hnkson) Waksman • , , , , • ... 

,^ ., -r^ A,-, '-r, i ,, •. , ||()t been seen in the hyphae. 1 he spore-like 
(Enkson, I). Med. Research Council Spe~ 



Kept. Ser. 203: L935, L6 17). 

Morphology: Substrate growth made up 
of very small, round colonies; unicellular 
mycelium with slender, branching filaments. 
Aerial mycelium not visible on any medium 



elements are markedly acid-fast. Aerial my- 
celium white consisting of short, straight, 
not very much branched hyphae, 0.1 to 0.6 
m thick, which never show any different iation 
into spores (see also Krikson, 1949). 



, . , . ... " Sucrose nitrate agar: Growth very scant, 

but occasional isolated aerial branches. , . . . ., , . . .. * 

as thin colorless veil. Aerial nivcehuni trace. 



Glycerol nitrate agar: Growth poor; scant 
colorless patch. 

Calcium malate agar: Growth colorless to 
pink, spreading; later, brighl red mass, 
buckled and shining, colorless submerged 
margin. 



white. 

Glucose-asparagine agar: Growth fair, 
hat, growing into medium ; pale ocher-yellow 
to orange, with raised outgrowths on the 

surface. Aerial mycelium scant, white. 
Nutrient agar: Growth slow, somewhat 



Nutrient agar: Growth irregularly piled . . ,. . . 

. . , ' . . . raised, ocher-yellow, hard, smeary >.... 

ip. convoluted, colorless, easily detachable, , . . , ,. ' ,, , . 

. , " loose. Aerial mycelium scant, small white 



brownisl 

Glucose agar: Small colorless coiled mass 
later heaped up as green growth. 

Gelatin: Liquefaction rapid. 

Milk: Surface growth pale green. Coagula 
t ion and peptonizat ion. 

Potato agar: Growth as small elevated 



t ufts. No soluble pigment . 

Potato: Growth mycelium granulated, 
first pale yellow, later dee]) ocher-yellow to 
orange. Aerial mycelium scant, white. No 
soluble pigmenl . 

Gelat in: No liquefaction. 

Milk: No coagulation; no peptonization, 
convoluted, colorless mass with purple tinge r,. . M , , , 

1 March: No hydrolysis. 

111 center - Cellulose: Not decomposed. 

Egg medium: Colonies small, round, Paraffin: Readily utilized, 

tough, colorless; margin well embedded. Nitrate reduction: Negative. 

Later, colonies elevated, waned, darkened. Sucrose: No1 inverted, 

medium discolored and broken. Slighl de- Liquid media (milk, broth, synthetic solu- 

gree of liquefaction; medium dark brown. tions): Small, round granules of various 

Source: An ulcer of the abdominal wall of yellow t ange color.-, firm but can be 

a patient in India. crushed into a homogeneous smear. In old 



52 



THE ACTINOMYCETES, Vol. II 




Figtjke 14. Orskov's motile Nocardia (Reproduced by courtesy of N. M. McClung] 



broth cultures, a thick, hard, orange to 
brownish surface pellicle is formed. 
Habitat: Soil. 

41. Nocardia petroleophila Hirsch and 
Engel, 1950 (Hirsch, P. and Engel, H. Ber. 
deut. bot. Gesell. 69: 441-454, 1956). 

Morphology: Grows slowly, but abun- 
dantly on all mineral media, faster in a 
petroleum atmosphere. It grows on certain 
organic media, but does not produce any 
aerial mycelium. Substrate mycelium breaks 
up readily into rods; "involution cells" are 
formed abundantly. Mycelial threads are 
long, monopodially branched, 0.0 to 1.2 ju 
in diameter, and contain "metachromatic 
granules," readily stained with aqueous 
methylene blue. Aerial hyphae have the 
same diameter as the substrate hyphae, 
little branched. Aerial mycelium wets with 
difficulty. Xo aerial spores. Mycelial seg- 
ments 1.2 to 5.0 by 1.2 to 12.5 m- Gram-posi- 
tive; not acid-fast. 

Glucose-asparagine agar: Growth limited. 
Colonies 0.3 mm, white, yellow reverse. 
Aerial mycelium snow-white. Soluble pig- 
ment none. 



Calcium malate agar: Growth limited. 

Nutrient agar: Growth limited. Colonies 
whitish-yellow, 0.5 mm. Xo aerial mycelium. 

Starch agar: Growth limited. Aerial my- 
celium snow-white; reverse yellowish. Starch 
not hydrolyzed. 

Potato: Xo growth. 

Gelatin: Growth in form of microscopic 
colonies. Xo liquefaction. Xo pigmentation. 

Milk: Growth limited, yellowish, dry. 
Xo aerial mycelium. Xo coagulation; no pep- 
tonization. 

Temperature: Optimum 25-28°C. Re- 
sistant to drying. 

Salt concentration: Resistant to high 
concentration. 

Habitat: Soil. 

42. Nocardia polychromogenes (Vallee, 
1903) Waksman and Henrici, 1948 (Vallee, 
H. Ann. inst. Pasteur 17: 288-292, 1903; 
Jensen, II. Proc Linnean Soc. X.S. Wales 
56: 79, 363, 1931). 

Morphology: Growth bright red, coral- 
red to red-pink, of a doughy consistency, 
later becoming leathery. Aerial mycelium 
whitish with pink hue. Substrate growth 



THK GENUS NOCARDIA 



53 



forms long wavy filaments, 0. 1 to 0.5 by 
70 to LOO ft, extensively branched bu1 with- 
out septa. Older cultures consist entirely of 
rods, 4 to 10 ft, frequently in V-, Y-, or 
smaller coccoid forms. Gram-positive, not 
acid-fast, frequently showing hands and 
granules. 

Nutrienl agar: Growth scant, orange-red. 
No aerial mycelium. No soluble pigment. 

Glucose agar: Growth raised, flat, glisten- 
ing, rose-colored, later becoming folded and 
coral-red. 

Gelatin: Growth along stab thin, yellow- 
ish, with thin radiating filaments. Surface 
growth flat, wrinkled, red. No liquefaction. 

.Milk: Growth starts as small orange- 
colored surface granules, later forming a 
thick, soft, orange-colored sediment. No 
coagulation; no peptonization. 

Starch: Hydrolyzed. 

Paraffin: Utilized. 

Cellulose: Xo growth. 

Temperature: Optimum 22-25°C. 

Source: Blood of a horse; soil in France 
and Australia. 

Remarks: Differs from A'. r<>r<tllin<i in the 
formation of very long filaments and in fili- 
form growth in gelatin stabs. 

Type culture: IMRU 3409. 

4:>. Nocardia pulmonalis (Burnett, L909) 
Waksman and Henrici, 1948 (Burnett, 

S. II. Ann. Kept. \. Y. State Vet. Coll. 
1 909 1910, 107). 

Morphology: Mycelium acid-fast, espe- 
cially in early stages of growth; breaks up 
readily into oval-shaped cells. Growth 
lemon-yellow with white aerial mycelium. 
Consistency of colonies leathery. 

Peptone-beef extract agar: Growth moist, 
raised, in form of small, spherical colonies. 

Glucose-peptone-beef extract agar: 
( irowth dull, whitish, convoluted. 

Potato: Growth abundant, in form of 
small, translucent, round colonies, becoming 
lemon-yellow. Later, growth becomes con- 



voluted or folded with chalky white aerial 
mycelium. Color of plug brownish. 

Gelatin: Colonies small, whitish, spheri- 
cal; edges of colony becoming chalky white. 
Limited liquefad ion. 

Milk: Colonies on surface of the medium. 
Coagulation and gradual peptonization. 

Pathogenicity: Nonpathogenic for rabbits 
and guinea pig>. 

Source: Lungs of a cow. 

44. Nocardia rangoonensis (Erikson, 1935) 
Waksman and Henrici, 1948 (Erikson, I). 
Med. Research Council Spec. Rept. Ser. 
203: :!•'! :!4, 1 <):;.")). 

Morphology: (irowth consists of branch- 
ing hyphae which segment and present 
slipping and angular arrangement. Aerial 
hyphae few, short, straight, later developing 
into a profusely branching, long, waving 
aerial mycelium. Nol acid-fast. 

Glycerol nitrate agar: ( irowth dull, mealy, 
pink, wrinkled. Aerial mycelium scant. 
white. Medium slightly discolored. 

Nutrienl agar: Colonies round, lobate, 
umbilicated, raised, cream-colored to pale 
pink. Later, colonies colorless, medium dis- 
colored dark brown. 

Glucose nutrient agar: (irowth con- 
voluted, coherent, cream-colored; medium 
discolored. Later, growth wrinkled, biscuit- 
colored, colorless margin. Aerial mycelium 
on border, white. Soluble pigment dark 
brown. 

Potato agar: ( lolonies small, round, lemon- 
colored, partly confluent. Submerged growth 
greenish. Aerial mycelium white. Medium 
colored light brown. 

Egg medium: (irowth extensive, colorless. 
Aerial mycelium in center, pale pink. 

Gelatin: Colonies abundant, minute in 
medium; larger, cream-colored colonic- on 
surface. Aerial mycelium white. Brown pig- 
ment surrounding growth. No liquefaction. 

Milk: Surface ring yellow. Coagulation 
positive; peptonization partial. Soluble pig- 
ment dark brown. 



:.4 



THE ACTINOMYCOTIC Vol. II 



Source : Human pulmonary case of st repl o- 
thricosis. 

45. Nocardia rhodnii (Erikson, 1935) 
Waksman and Henrici, 1948 (Erikson, D. 
Med. Research Council Spec. Rept. Ser. 
203: 29, 1935). 

Morphology: Substrate growth made up 
of minute colonies, composed of hyphal seg- 
ments arranged in angular apposition. Aerial 
mycelium is short and straight. Later, 
growth becomes extensive and spreading, 
made up partly of long, branching filaments, 
and partly of short segments exhibiting 
slipping branching, each giving rise to 
aerial hyphae. Angular branching very 
marked, delicate, spreading, herringbone 
patterns being formed. 

Sucrose uitrate agar: Colonies minute, 
colorless, round. 

Glucose-asparagine agar: Growth abun- 
dant, coral-pink, convoluted, piled up. 

Glycerol agar: Growth made up of dull 
pink colonies, round and umbilicated, he- 
coming piled up and deeper coral-red. 

Potato agar: Growth abundant, pink, 
piled up and stiff. Aerial mycelium white at 
top of slant . 

Egg medium: Membrane salmon-pink, 
granular. 

Gelatin: Colonies pale pink, in form of 
surface pellicle and as sediment. Liquefac- 
tion rapid. 

Milk: Growth bright orange. Medium 
unchanged. 

Nutrient broth: Salmon-pink flakes in 
sediment and colonies on surface. Medium 
discolored. 

Source: From reduviid bug, Rhodnius 
prolixus. 

Type culture: [MRU 653. 

4(i. Nocardia rubra (Actinomyces ruber 
sterilis Krassilnikov, 1949) Waksman (Kras- 
silnikov, X. A. ( ruide to the identification of 
bacteria and actinomycetes. Moskau, 1949). 

Agar media : ( rrowth red, smooth, nodular, 



slightly lustrous with a gravel-like appear- 
ance. Xo aerial mycelium produced under 
laboratory conditions. .Most strains form no 
soluble pigment. Some produce a brownish 
substance. Slightly acid-fast (Fig. 12). 

Milk: Unchanged. 

Krassilnikov examined 25 different strains 
said to belong to this type, but differing 
from one another in intensity of color and in 
certain physiological properties. He believed 
that under certain conditions of growth these 
cultures would develop an aerial mycelium 
and proper sporulation. 

This group was divided by Krassilnikov 
into four subgroups: 

a. Flat, compact colonies, red to pink in 
color, pigment insoluble. Gelatin not lique- 
fied, milk unchanged or only peptonized; 
starch not decomposed; nitrate not reduced. 

b. Colonies raised, dry, crumbling at con- 
tact with loop; red to brownish-red in color. 
Ready growth on synthetic media. Gelatin 
liquefied slowly; milk coagulated slightly or 
only peptonized; nitrate reduced to nitrite; 
ready growth in paraffin and fats. 

c Colonies compact, growing deep into 
substrate; pink to light red in color; brown 
substance excreted into substrate. Gelatin 
slightly liquefied; milk peptonized by some 
strains, starch slightly decomposed; nitrates 
not reduced to nitrites. Some cultures grow 
slowly in cellulose. Do not grow on paraffin. 

d. Flat or nodular colonies, growing com- 
pactly into medium. Frequently develop 
coremia on the surface; these consist of 
thickly interwoven sterile hyphae. The cul- 
tures grow poorly on artificial media. Gelatin 
not liquefied or only slowly; milk not 
changed or only slightly peptonized; starch 
not decomposed. No growth on cellulose. 
Ready growth on fats, paraffin, and wax. 

Remarks: Krassilnikov considers this 
group to comprise transition forms between 
Streptomyces and Nocardia. N. corallina is 
believed to be a related form; a number of 
synonyms are listed such as N. agrestis, N. 



THE GENUS NOCARDIA 



55 



minima, and others. According to Schneidau 
and Shaffer (1957), this form docs no1 pro- 
duce urease, whereas N. corallina does. 

17. Nocardia rubropertincta (Hefferan, 
L904) Waksman and Henrici, L948 (Grass- 
berger, R. Munch, med. Wochschr. 1(>: 343, 
L899; Hefferan, M. Centr. Bakteriol. Parasi- 
tenk. Abt. II, 11: 460, L904). 

Morphology: Growth in form of small rods 
0.3 to 0.9 by L.5 to 3.0 m, showing angular 
arrangement; later, nearly coccoid, 0.6 by 
0.8 p. Tendency for branching on glycerol 
agar, bul branching does not occur com- 
monly, though granules of aerial mycelium 
are sometimes seen. No1 acid-fast or varia- 
ble. 

Xntrient agar: Colonies small, granular, 
becoming pink to red, depending on com- 
position of agar. 

Potato: Growth slow but excellent, inten- 
sive red, becoming dull orange. 

Gelatin: Colonies irregular with crenate 
margin and folded surface, coral-red. ( rrowth 
in stab at firsl thin, then granular to arbores- 
cenl with chromogenesis. No liquefaction. 

Milk: Surface scales thick, fragile, dull 
coral-red; also sediment. Milk becomes 
somewhat viscid after .'1 to 1 weeks. 

Nutrient broth: Faini uniform turbidity 
with salmon-pink pellicle, which is renewed 
on surface as it settles to form a red sedi- 
ment . 

Xit rate reduction: None. 

Carbon sources: Utilizes benzene, petro- 
leum, paraffin oil, and paraffin. 

Temperature: Grows well between 20 and 
37°C. 

Oxygen requirement: Aerobic to faculta- 
tively anaerobic. 

Source: Isolated from butter, soil, and 
contaminants of t uberculin flasks. 

Remarks: MycobacU ///////dike colonies 
with coral to vermillioii-red chromogenesis 
on various media. 



L959 (DiMarco, A. and Spalla, C. Lab. 
Ricerche Farmitalia, Milano, L959). 

Morphology: Hyphae short, 0.6 to 0.8 /x 
in diameter, wavy, later angular, radiating 
from a center. After 20 to 24 hours, they 
break up into rods 8 to 20 n long. No 
aerial mycelium. Nonacid-fast. 

Glucose-asparagine agar: Growth color- 
less, raised, moist , wrinkled. 

Glycerol agar: Abundant, lichenoid 
growth, dull cream. No consistency. Red- 
dish-brown soluble pigmenl after L5 days. 

Nutrient agar: Thick, cream-colored pel- 
licle, rough and folded. Dough-like con- 
sistency. After 15 days, brown soluble pig- 
menl . 

Potato agar: ( rrowth smooth, folded, with 
wrinkled and lichenoid portions. Colorless. 
Soft consistency. 

Milk: Coagulation; no peptonization. 

Gelatin: Liquefaction positive. 

Nitrate reduction: Negal ive. 

Starch: Nondiastat ic. 

Sugar utilization: See Table 1. 

Optimum temperature: 34 ( '. 

Remarks: Nonpathogenic. Produces vita- 
min B12 . 

Habitat : Cattle rumen. 

Type culture: [MRU 3760. 

This species was described further by 
Spalla ( l ( .).~) ( .)i as follows: It produces a color- 
less growth on glycerol, glucose, asparagine, 
and N-Z-amine agars. Acid is produced from 
glucose, galactose, ribose, rhamnose, l- 
arabinose, glycerol, d-mannitol, and adoni- 
tol; but not from d-mannose, sucrose, malt- 
ose, lactose, trehalose, raffinose, inulin, 
//-sorbitol, inositol, dulcitol, or salicin. Ni- 
trate is not reduced. Starch is not hydro- 
lyzed. Gelatin is liquefied. Milk is coagu- 
lated. The organism will resisl a temperature 
of 60°C for M 2 hours, but not 3 hours. It is 
gram-positive. The terminal fragments are 
1.56 ± 0.266 (i. No aerial mycelium is pro- 
duced. 



is. Nocardia rugosa DiMarco and Spalla, 49. Nocardia salmonicolor (den Dooren de 



56 



THE ACTINOMYCETES, Vol. II 



«*.;*'■' 










Figure 15. A r . salmonicolor , growing on a hanging microdrop of liquid paraffin (a) surrounded by 
sucrose nitrate salt solution; (b) same plus 1.5 per cent agar (Reproduced from: Webley, D. M. J. Gen. 
Microbiol. 8: 71, 1953). 



Jong, 1927) Waksman and Henrici, 1948 
(den Dooren de Jong. Centr. Bakteriol. 
Parasitenk. Abt. II, 71: 216, 1927). 

Morphology: Growth made up of short 
mycelium disintegrating into rods and cocci. 
Aerial mycelium sometimes stretching into 
quite long filaments, with small refractive 
granules. Many cells at the edge of the 
colonies show club- or pear-shaped swellings, 
up to 2.5 to 8.0 /x in width; many of these 
swollen cells later germinate with the forma- 
tion of two more slender sprouts. Acid- 
fastness is found among the earlier stages of 
growth, especially in some of the strains and 
on some media (Fig 15). 

Glucose-asparagine agar: Growth re- 
stricted, rather flat, edges Lobate, surface 
warty, glistening; at first pale orange, later 
ocher-yellow; consistency crumbly. 

Glucose-peptone-beef extract agar: 
Growth excellent, of a doughy consistency, 
spreading, flat, dense, edges lobate, surface 
folded, glistening, yellow, gradually chang- 
ing to salmon-pink and deep orange-red. 

Potato: Growth good, raised, warty, 
crumbly, glistening, at first buff, changing 
to orange, and finally to almost blood-red. 



Gelatin: Growth in stab scant, arbores- 
cent. Surface colonies small, wrinkled, 
orange. No liquefaction. 

Milk: Pellicle of small cream-colored 
granules, later a thick orange sediment. No 
coagulation and no peptonization, although 
milk appears slightly cleared, the reaction 
becoming alkaline. 

Starch: Not hydrolyzed. 

Sucrose: Not inverted, although readily 
utilized with sodium nitrate as a source of 
nitrogen. 

Paraffin: Readily utilized as a source of 
carbon. 

Cellulose: No growth. 

Phenol: Not utilized. 

Remarks: A detailed study of the acid- 
fast properties of this species has been made 
by Erikson (1949). It closely resembles N. 
corallina. 



50. Nocardia 
(Erikson, D. J. 
393, 1954). 

Morphology: 
acid-fast. Aei 
characteristic 
substrate and 



sebivorans Erikson, 
Pathol. Bacteriol. 68: 



rram-pos 



lve, pa 
white, 



1954 
387- 

•tially 
with 



ial mycelium 

nonwetting properties. Both 

aerial mycelium show spon- 



THE GENUS NOCARDIA 57 

taneous segmentation into shorter and of small colonies, white at first, later be- 
longer cells of coccoid or bacillary dimen- coming yellowish cream-colored, 
sions. Biochemical properties: [ndole negative, 

Agar media: Colonies firmly attached to H 2 S positive, neutral red no1 reduced. Sugars 

medium. No soluble pigment. No acid pro- like glucose, sucrose, lactose, galactose, and 

duced from glucose, mannitol, lactose, mannose not attached. 

sucrose, starch, raffinose, galactose, rham- Milk: Coagulation positive; peptonization 

nose, sorbitol, maltose, dulcitol, glycogen, or positive, 

glycerol. Coagulated serum : Nol liquefied. 

Sucrose nitrate agar: Growth fair; nitrate Nitrate reduction: Positive. 

utilized. Oxygen demand: Strict aerobe. 

Gelatin: No liquefaction. Optimum temperature: 35 37 C. 

Carbon utilization : Paraffin is good source Pathogenicity: Pathogenic to guinea pigs 

of energy, also n-dodecane. Cresols not and rabbits. 

utilized. Habitat: Isolated from urine of patient 

Temperature: Can withstand exposure to suspected of renal tuberculosis. 

( .K)°C for 10 minutes in a phosphate buffer -.> v ,„.,.>• , ro •• i 

1 ' . , oZ. Nocardia sumatrae (Smiders emend. 

suspension; denser suspensions withstood 3 Erikson) (V em<cuH gnijd { .^ >k 

minutes a, LOO ( (Enkson, 1955). Tij(ls( . h X(i(1 ll|(|]( , f)J . 4 - ? 

Habital : Pus ma case oi empyema. X() , Streptothrix cuniculi Schmorl , 801 

Pathogenicity: Rabbits and gumea pigs, H()1 Nocardia mniculi ,,,. M( , |1() „„,, 

slightly for mice. Morphology: Growth made up of large 

Remarks: Cells have avidity for oily sub- ram ii flT1 ,., M . • ■ • ... . 

• • swollen cells, giving rise to ramifying fila- 

stances (lipophilic). I nder unfavorable con- ,„«,,* . , , , . u i • ,• i \ ■ , 

1 f ments or to small chains oi short, thick seg- 

ditions oi growth, on the surface oi solid , lwl ,, t . „.i- l i. i i 

, fe , ' , , , ments which branch out into more regular 

paraffin large clubs and hexagonal cells are hypha(> Sometimes the , n ,^ llal . elementg 

pr ° duced f lkS °"'rvr L* are besei with SI)i ">- processes befOTe g™g 

lype culture: N( K 8595. ,., S(1 |() lypi( . a , ,„„„. branching filaments . 

51. Nocardia serophila (Sartory and Later the picture becomes more mono- 

Bailly, L947) emend. Waksman (Sartory, A. morphous, and short straight aerial hyphae 

and Bailly, C. Compt. rend. 221: 1533-1534, are borne, which presently exhibit irregular 

1 *.)47 i . segmentation. 

Morphology: Hyphae produce angular Glycerol nitrate agar: Colonies small, 

growth, much branched, occasionally curved, round, elevated, cream-colored, margins 

04 to 0.5 n in diameter. Nonmotile. Rarely depressed; becoming smooth, discrete yel- 

certain secondary branches are spiral shaped, lowish. 

Terminal and intracellular arthrospores. ( rlucose-asparagine agar: ( Jolonies minute. 
Gram-positive. Acid and alcohol resistant. colorless, becoming dull pink, partly con- 
Growth characteristics: Crows with dim- fluent and piled up. Aerial spikes lew, stiff, 
cully on ordinary solid or liquid media; pink- 
grows well on serum or blood media. Nutrient agar: Colonies small, round, 
Liquid peptone and serum media: Non- elevated, cream-colored, umbilicated and 
viscous, cream-colored growth, detaches radially wrinkled. 

from tube by agitation, medium remaining Egg medium: Growth scant, pinkish, 

clear. smeary. 

Coagulated serum media: Growth in form Potato: Growth coral-pink, dry, granular, 



58 



THE ACTINOMYCETES, Vol. II 



covered to a considerable extent with white 
aerial mycelium; piled up in center, discrete 
colonies at margin. 

Gelatin: Few flakes. No liquefaction. 

Milk: Heavy yellow growth attached to 
walls; solid coagulum in 1 month. 

Nutrient broth: Surface colonies cream- 
colored, scale-like; abundant, flocculent 
bottom growth. 

Source: Infected rabbits. 

Remarks: Description given after Erikson 
(id:;;,.. 

53. Nocardia transvalensis Pijper and 
Pullinger, 1927 (Pijper, A. and Pullinger, 
B. D. J. Trop. Med. Hyg. 30: 153, 1927). 

Morphology: Initial growth made up of 
unicellular hyphae, the central branch being 
frequently broader and showing dense 
granular retractile contents. Aerial mycelium 
white, forming straight hyphae, in some 
cases becoming clustered into irregular 
spikes. ( 'dlorless drops are exuded and a pink 
coloration is produced in the densest part of 
the growth on synthetic glycerol agar. 
Angular branching with division of substrate 
filaments. Aerial hyphae irregularly seg- 
mented. Acid-fast. 

Glycerol nitrate agar: Growth in form of 
small, pink coiled masses. Aerial mycelium 
thin, white. 

Nutrient agar: No growth. 

Glucose nutrient agar: Colonies raised, 
granular, pink. Aerial mycelium white. 

Potato: Growth dry, raised, convoluted, 
pink. Aerial mycelium white. 

Gelatin: Growth poor, in form of a few 
irregular, colorless flakes. No liquefaction. 

Milk: No change. 

Starch: Not hydrolyzed. 

Egg medium: Growth in form of small, 
irregularly raised, coiled, dull pink mass. 

Source: A case of mycetoma of the foot , in 
South Africa. 

Pathogenicity: To guinea pigs. 

Remarks: According to Gonzalez Ochoa 



and Sandoval (1956), A', transvalensis is a 
synonym of N. brasiliensis. 

54. Nocardia turbata Erikson, 1954 (Erik- 
son, D. J. Gen. Microbiol. 11: 198-208, 
1954). 

Morphology: Typical actinomycete, pro- 
ducing a fine mycelium composed of slender 
filaments, 0.1 /j. in diameter, which fragment 
into rods and coccoid cells. Under appro- 
priate conditions, many cells are motile. 
Nonacid-fast. 

Agar media: Growth good. Colonies small, 
0.1 to 2.0 n. Initially colorless, later pro- 
ducing a yellow-green pigment on nutrient 
agar. Pigment production favored by free 
air supply, suppressed by acid reaction. 

Broth cultures: Turbid when young; sedi- 
mentation of cells later, when pellicle and 
clarification of medium produced. 

Acid production: Positive with glucose, 
sucrose, maltose, lactose, galactose, xylose, 
arabinose, glycerol, starch; negative with 
mannitol, raffinose, rhamnose, sorbitol, 
dulcitol (using a casein hydrolysate me- 
dium). 

Oxygen demand: Aerobic. 

Optimum temperature: 20-30°C. 

Nitrate: Utilized. 

Gelatin: No hydrolysis, except in presence 
of peptone (slowly). 

Paraffin utilization: Negative. 

Habitat : Probably soil. 

55. Nocardia uniformis Marton and 
Szabo, 1959 (Mail on, M. and Szabo, I. 
Acta Microbiol. Acad. Sci. Hung. 6: 131- 
135, 1959). 

Morphology: The filaments of the sub- 
strate mycelium rapidly break up into rods 
and less frequently into coccoid bodies. The 
size of these forms is 0.7 to 1.1 m by 1.1 to 
4.0 /j.. In old cultures, swollen, club- or bol tie- 
shaped forms appear. The hyphae of the 
slightly developed aerial mycelium are 
straight or waxed, nonseptate, and contain 



THE GENUS NOCARDIA 



:,«) 



oval oidiospores. The mycelium is gram- 
positive and is not acid-fast . 

Agar media : The strains give aonbutyrous 
colonics growing into the agar, with moder- 
ately striated dull surface covered with 
slightly developed white powder-like aerial 
mycelium. The color of the colonics is a 
constanl yellowish-orange; it never turns 
red or yellow; no soluble pigment is pro- 
duced. In liquid synthetic media a surface 
pellicle resembling agar colonies is formed. 

( ielat in: No liquefact ion. 

Milk: Xo coagulation; no peptonization. 

Sugar inversion: None. 

Starch hydrolysis: None. 

Nitrate reduction: Rapid. 

Paraffin utilization: Slight or none. 

Optimum temperature: 14 :;7°C. 

Carlton utilization: Does not utilize man- 
nose, dextrin, inulin. 

Habital : Deep layers Bi horizon of saline 
soils. 

oil. Nocardia upcotiii (Erikson, L935) 
Waksman (Gibson, A. G. J. Pathol. Bac- 
teriol. 23: :;.">7, 1020; Erikson, I). Med. Re- 
search Council Spec. Rept. Ser. 203: 22-23, 
L935). 

Morphology: Growth forms Long, straight 
filaments, much interwoven and ramified. 
Aerial mycelium slight, transient, slightly 
acid-fast . 

Glycerol nitrate agar: Colonies small, 
round, cream-colored, glistening; heavy 
texture, margins submerged, hater, growth 
very much convoluted and raised, broad 
submerged margin ; medium becomes slighl ly 
reddish. 

Calcium malate agar: Growth limited, 
colorless, membranous, with undulating 
margin. 

Nutrient agar: Colonies smooth, shining, 
round, cream-colored; margin submerged. 
Aerial mycelium scant, white. Later, colonies 
are large with greenish tinge; very sparse 
aerial mycelium gradually disappears. 

Glucose nutrient agar: Colonies smooth, 



round, cream-colored; margin depressed, 

centers elevated, hollow on reverse side; 
hit ei- a coherent membranous growth, yellow- 
ish. 

Potato agar: Growth poor, in form of 
small, colorless, blister colonic-. Medium 
slightly discolored. 

Egg medium: Colonies round, flat, color- 
less, scale-like, some marked by concentric 
rings and slightly hollowed in center. Growth 
becomes yellow-brown. 

Blood agar: Colonies huge, drab, heavily 
textured. Xo aerial mycelium. Xo hemolysis. 

Gelatin: Growth abundant, flocculent, 
cream-colored on surface. Gradual liquefac- 
tion. 

Source: From the spleen in a case of 
acholuric jaundice. 

57. Nocardia vaccinii Demarec and Smith. 
L952 (Demarec, J. B. and Smith, X. 1!. 
Phytopathology 12: 249-252, 1952). 

Morphology: Growth in form of rods and 
filaments, 0.4 n to 0.S M in diameter; granular 
appearance when stained; eventually Wreak- 
ing up into bacillary forms. Pew cells acid- 
fast. Fat demonstrated by staining with 
Sudan black B. 

Sucrose nitrate agar: Growth scant, gray. 

Nutrient agar: Growth p \ slow, granu- 
lar, gray, sometimes pinkish in old cultures. 

Gelatin: Growth dry, ribbon-like. Xo 
liquefaction. 

Starch nut rient agar: ( rrowt h dry, ribbon- 
like, pinkish to orange. Bydrolysis of starch 
posit ive. 

Potato: Growth slow, spreading, raised, 
gray. 

Milk: Growth dry, raised, gray with 
orange spot-. Xo peptonization. 

Nit rate redud ion : Positive. 

Carbon utilization: With ammonia as the 
source of nit rogen, acid formed from glucose, 
sucrose, glycerol, and niaiinitol; reactions 
variable with arabinose and xylose; no 
growt h on lactose or sorbitol. 

Paraffin: Utilized. 



60 



THE ACTINOMYCETES, Vol. II 



Temperature: Growth best at 25-28°C; 
inhibited at 32°C; none or very scant at 
37°C. 

Antibiotic activity: None. 

Habitat : Causes formation of bud-prolifer- 
ating galls on blueberry plants. 

Type culture: ATCC 11,092. 

58. Nocardia variabilis (Cohn, 1913) Waks- 
man (Cohn, T. Centr. Bakteriol. Parasitenk. 
Grig. 70: 290-306, 1913). 

Morphology: Cells initially filamentous, 
breaking up into rods and cocci. Nonacid- 
fast . 

Agar media: Colonies round, smooth and 
lustrous, sometimes nodular; light brownish 
in color to orange-yellow. Colonies attached 
fast to the agar and partly removed with 
some effort. 

Gelatin: Growth orange-yellow. No lique- 
faction. 

Milk: Surface pellicle gradually becoming 
light orange. No coagulation; no peptoniza- 
tion. 

Broth: Colorless surface pellicle, readily 
dropping to bottom. Medium remains clear. 

Temperature: Optimum 37°C; good 
growth at 42°C; weak growth at 45°C. 

Potato: Growth thin, colorless, becoming 
in time yellow to orange-red; finally brown. 

Blood media: Xo hemolysis. 

Oxygen demand: Markedly aerobic. 

Habitat: Isolated from bladder of cystitis 
cases in man. Pathogenic to guinea pigs. 

Remarks: Said to be similar to .4. ochroleu- 
cus, A . ochraceus, and A. carneus of Neukirch 
(1902). According to Krassilnikov (1949), it 
is closely related to N. africana. 

59. Nocardia viridis (Krassilnikov, 1938) 
Waksman and Benrici, 1948 (Krassilnikov, 



N. A. Bull. Acad. Sci. USSR. Xo. 1: 139, 
1938; Guide to the identification of bacteria 
and actinomycetes. Moskau, 1949). 

Morphology: Growth dark green in color. 
Colonies of doughy consistency on certain 
media (wort agar, potato), and compact on 
others (nutrient). Pigment insoluble in 
medium and in organic solvents. On protein 
media, cells develop to form a thin mycelium 
without visible cross walls. Cells often 
branching, 0.7 to 0.8 m in diameter, with 
cross wall. After 5 to 7 days the cells break 
up into rods 5 to 15 fi long. Cocci not ob- 
served. Cells multiply by fission, seldom by 
budding. Xo aerial mycelium. Gram-posi- 
tive. Xot acid-fast. 

Nutrient agar: Growth compact. Thin 
mycelium produced. 

Potato: Growth rough, much folded. 

Gelatin liquefaction: Slow or none. 

Milk: Xo coagulation; no peptonization; 
some reports of positive peptonization. 

Starch: Xot hydrolyzed. Spalla (1939) re- 
ported positive hydrolysis. 

Sucrose: Xot inverted. 

Nitrate reduction: None. 

Paraffin and fats: Growth good; less on 
wax. 

Cellulose: Xo growth. 

Habitat: Soil. 

Davis and Freer (1960) described as a new 
species N. salivae, an aerobic actinomycete 
isolated regularly from the human mouth. 
Strains of this species are characterized by 
their saccharolytic power, which thus distin- 
guishes them from the typical soil nocardias 
(see also von Magnus, 1947; Howell et ah, 
1959). Birsch (1960) described N. saturnea, 
an aerobic organism occurring in dust and 
capable of utilizing petroleum. 



Chapter 4 



Characterization of Streptomyces Species 



Important Characters to be Considered 
for Recognition of Species and Va- 
rieties of Streptomyces 

In the identification and characterization 
of Sin pt'innin s species, the following char- 
acters should be considered: 

1. Morphological properties: 

(a I Structure of substrate 1 mycelium. 

(b) Nature and formation of aerial my- 
celium. 

(c) Struct ure and branching of sporo- 
phores. 

(d) Size and shape of spores. 
(e I Surface of spores. 

2. Cultural properties on various media: 
(a i ( rrowth characteristics. 

(b) Development of aerial mycelium. 

(c) Color of aerial and substrate my- 
celium. 

.'!. Biochemical properties: 

(a) Production of soluble pigments in 
organic and in inorganic media. 

(b) It ilizat ion of carbon sources. 

(c) Starch hydrolysis. 
id i Sucrose inversion. 

(e) Cellulose decomposition. 

(f) Proteolytic activities: liquefaction of 
gelatin, bl 1 serum, and casein; co- 
agulation and peptonization of milk. 

(g) Utilization of nitrogenous com- 
pounds. 

(h) Formation of oxidases: tyrosinase 
and laccase. 

(i) Reductases: nitrate reductase, sul- 
fate reductase. 



(j) Formation of antibiotics and vita- 
mins. 

(k) Formation of IFS in peptone-iron 
agar. 

4. Sensitivity to antibiotics: 

(a) Sensitivity to pure antibiotic prep- 
arations. 

(b) Phenomena of ••cross-resistance" 
and "cross-sensitivity" on artificial 
media. 

5. Sensitivity to phages. 

6. Serological reactions. 

7. Chemical composition. 

8. Ecological properties. 

9. Genetic relationships. 

10. Age of <ailt ure. 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. 

11. 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 i- believed to re- 
duce physiological changes t<> 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. 

In characterizing Streptomyces species, 
only certain media should be used and well - 



lit 



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 
calcium 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 
H2S production; (e) tyrosine medium for the 
tyrosinase read ion; 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 septal ion. The compactness of 
this substrate growth is responsible for the 
fact that liquid media are always clear, un- 
less the culture has been subject to phage 
or lytic action. 

NATURE AXD PROPERTIES OF AERIAL MYCE- 
LIUM 

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. 

h. 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 STREPTOMYCES SPECIES 



<•>:; 




Figure 1»>. Schematic representation of differenl types of spirals produced by various Streptomyces 
species; the spirals range from long to short , from compart to irregular I Reproduced from: Shinobu, R. 
Mem. Osaka Univ. Lib. Arts and Ed. B. Nat. Sci. 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 this, too, is influenced by the 
composition of the medium (Ettlinger ei "/., 
L958; Shinobu, 1958). Verticil formation is 
also an important characteristic of certain 
species; it can be simple or branching (pri- 
mary or secondary verticils), the branches 
being straighl or forming spirals; but this 
property as well is influenced to some extent 
by the composition of the medium. Al- 
though aocardiae may produce sporulating 
aerial filaments, these are never spiral- 
shaped (Fig. lf»). 

Waksman (MHO, L950) 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 
sporulal ion hyphae. 

I. Straighl sporulating hyphae, monopo- 



dia] branching, never producing regular 
spirals. 
II. 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. 
Krassilnikov (1941, I'M!)) divided the 
genus Actinomyces (largely forms included 
in Streptomyces) on the basis ( ,i' the following 
properties: I I I spiral forming versus straighl 
sporophores; (2) alternate distribution of 
sporophores on aerial mycelium versus verti- 
cil formation; (3) spherical versus oval 
-pore-; (4) colorless versus pigmented cul- 
tures; (5) white versus colored aerial my- 
celium; iii) saprophytes versus parasites. 

Aiso et al. (1948) divided the genus Strep- 
tomyces on the basis of the structure of the 
aerial mycelium into -ix types: 
I. Spirals n<>t formed. 



64 



THE ACTINOMYCETES, Vol. II 




f g 

Plate I. Diagrammatic representation of the morphology of the sporophores of Streptomyees (Re- 
produced by special permission from El i linger et al. Arch. Mikrobiol. 31: 336, 1958). 
a. Sporophores produce straight branching verticils on sterile aerial hyphae; S. reticuli type. 1). Sporo- 
phores produce open spirals as side branches on sterile aerial hyphae; S. purpurascens type. c. Sporo- 
phores produce on sterile aerial hyphae verticils with open, more or less regular spirals; S. nourst i type. 
d. Sporophores formed as side branches <>n sterile aerial hyphae, straight or slightly wavy; S. phaeo- 
chromogenes . e. Sporophores produced as verticils on sterile aerial h\ phae, with open, irregular spirals; 
,s'. echinatus. f. 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. 



rHAI!A('Tl.l!l/ATI<>\ OF STh'EPTOMYCES SPECIES 



«;;, 




Plate II. Diagrammatic representation of the morphology of the sporophores of Streptomyces Re 
produced by special permission from Ettlinger et al. Arch. Mikrobiol. .'{1:337. 1958). 
li. Sporophores produce numerous shorl , monopodia! 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, straighl main axis, monopodially branched with frequent, regular spirals; S fradiae. 1. 
Sporophores monopodially branched, straighl or slightly wavy; ,S. antibioticus . m. Sporophores mono 
podially branched along the whole axis with open, irregular spirals: S. erythraeus. a. Sporophores mono 
podially branched, with narrow . compad spiral.-; S. violaceoniger. o. Sporophores sympodially branched, 
in the form of trees with a long main axis; S. vii ido<n ties. p. Sporophores monopodially branched, stiff 
and straighl ; S. i< ru zuelae. 



68 



THE ACTINOMYCETES, Vol. TI 



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. 

III. 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) criticized 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 distinc- 
tion between 1 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 
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. II). 

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 mycelium 
(powdery, cottony, leathery). 

b. Branching, especially the formation 
and nature of verticils. 

c. 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 
il should be considered in connection with 
composition of the medium. The aerial my- 
celium was classified into the following three 
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 



(17 



case of natural overlapping between man- 
made concepts of genera and species or the 
improper labeling of cultures. Gordon and 
Mihm (1957) emphasized that il is easy to 
understand how a culture of A", asteroides 
that formed acid-fasl coccobacilli, rods, and 
short filaments and whose growth was 
heavy, finely to coarsely wrinkled, cream- 
colored to orange, and without noticeable 
aerial hyphae, could be mistaken for a Myco- 
bacterium. A culture of N. 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 
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), Ktt linger 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. (1!»4.">) were the firsl to use the 
electron microscope for study of spores of 
Streptomyces. This was followed by the work 
of Carvajal (1946); Kiister (l<):>:i); Flaig et 
al. (1952, 1955, L958); Baldacci and Grein 
(Hi.").)); Grein (1955); Vernon (l ( .).V>); 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- 
face.-; 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 group.-: (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 I here are Spores with smooth 
surface.-, with spines, with hairs, or with 
warty protuberances, and -pore- that are 
globose, long-ovoid, and cylindrical. 

On the basis <>f a system of classification 
that they had outlined, Baldacci and Grein 
( 1'.).").")) examined 50 strain- of -t reptomycetes 
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 1 ; 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 
slightly 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, L959) be- 
tween spore characteristics and sporophore 
morphology (Table 6). 

According to l'reobrajeitskava et al. ( 1!).")!), 
]'.)(')()), 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 lilac 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 <l al. 
(1960) also emphasized the importance of 
spore surface in classifying species of Sin />- 
tomyces; size and shape of spores of mosl 
species were considered of limited usefulness 
in taxonomic different iatioti. 

Lechevalier and Tikhonienko I 1959 re 
ported that the spores of S. viridochromo- 
genes were mostly elongated and those of S. 
violaceus, spherical. The spines formed by 



THE ACTINOMYCETES, Vol. II 

Table 6 

Morphology of sporophores and sport* of streptonujcetcs* ( Pridham, 1959) 



Specifii 

epithet 


NRRL 
No. 


Original strain no. 


Sporophore morphologyt 


Spore morphology from 
electron micrograph 


griseus 


B-1598 


Carvajal SL 842 


Straight to flexuons (RF) 


Smooth-walled 


bikiniensis 


B-1049 


Waksman 3515 


Straight to flexnons (RF) 


Smooth-walled 


canescus 


2419 


Com. Sol. 811.0 


Straight to nexnous (RF) 


Smoot h-walled 


r, in zuelae 


B-902 


Gottlieb 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- 
rant ) (RA) 


Spiny to hairy 


II III IIS 


B-1685 


Waksman (ATCC 

618) 


Spirals (aberrant ) (S) 


Smooth-walled 


hygroscopicus 


B-1865 


NRRL isolate 


Spirals (S) 


Spiny 


chartreusis 


2287 


Upjohn K-180 


Spirals (S) 


Spiny 



* Data taken from Carvajal, 1946; Vernon, 1955. All other data on spore morphology supplied by 
K. L. Jones. 

f Sporophore morphology determined according to the methods of Pridham el al., 1958. 




Figure 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 



IS!) 



si rains of both species differed cytologically. 
The spines of the first seemed to be pan of 
the cell wall, whereas the spines of the sec- 
ond seemed to l>e very superficial, appearing 
only on the envelope. It was concluded that 
spine formation is a stable characteristic of 
the spores. The shape of tlie spores varied 
with tlie composition of the medium. It was 
suggested that complex organic media he 
avoided for spore study. 

COLONY STRTN TURE 

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 he readily determined 
by superficial examination. Various other 
properties may he 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 
he of some help. Pridham* and others have 
noted the following very general correlations: 

1. Straight to Hexuous cultures generally 
are flat with a velvety, granular, or powdery 
texture. 

'_'. hoop cultures generally are flat with a 
velvety to slightly granular texture. 

:;. 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 he 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. 

' Persona] communication. 




Figure is. Sclerotium formation in Strepto- 
myces (Prepared by H. Lechevalier, Institute <>!' 
Microbiology). 



f 

• 




f\ 




4) 




ny.. 

- 




f • 




* 









Figure 1!). Sclerotium in species of Strepto 
myces, designated as new genus Chainia by Thir 
umalachar (Prepared by II. Lechevalier, Institute 
of Microbiology). 

4. Verticillate culture- generally are ele- 
vated and floccose. Aherrant verticillate cul- 
tures generally are flat with a velvety tex- 
ture. 



70 



THE ACTINOMYCETES, Vol. II 




Figure 20. Sclerotium in species of Slrepto- 
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 monopodia] branching, is also a 
constant, although a variable, property. No 
single property, however, is 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), bu1 not with Gattani 
(1957), who denied its significance (Figs. 
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-produc- 
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, it 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; it 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. 

Tin 1 formation of deep brown to black pig- 
ment- on organic media containing proteins 



CHARACTERIZATION OF STREPTOMYCES SPECIES 71 

and protein derivatives, notably the ami luced in tyrosine (or protein containing 

acid tyrosine, is an important species charac- tyrosine) media. The tyrosinase reaction 

teristic. Certain species may produce only was, therefore, considered to be the proper 

faint brown soluble pigments on organic one. 

media, as well as on synthetic media. Differ- Shinobu t L958) attached greal importance 

ent cultures, especially on continued cultiva- to the "tyrosinase reaction" in the species 

ion on artificial media, will show greal characterization of Streptomyces. Ettlinger 

variation in pigmenl production. et al. (1958) also recognized the difference 

Since about one third of all species of between melanin formation and the tyrosi- 

Streptomyces now recognized are melanin- nase reaction. In line with the idea- of 

positive (Waksman, 101!); Skinner, L938), Beijerinck, Waksman, and Ettlinger et al., 

and since this property has been utilized ex- recognition will be given here to melanin 

tensively in the classification of actinomy- formation rather than to the tyrosinase re- 

cetes, a knowledge of this reaction is of greal action. 

importance. Gasperini (1891) first utilized The formation of yellow, red, blue, green, 

this property in dividing the aerobic A ctino- and other soluble pigments is also highly 

/////rev into .1. chromogenus and .1. albus. It characteristic of the species growing on syn- 

was later recognized, however, that melanin thetic media. There is considerable variation 

production is characteristic of a large number in the intensity of these pigments, depending 

of species, including such important forms upon the strain of organism. In view of the 

as the plant pathogen S. scabies. fact that color standards are not always 

Beijerinck (1900, 1011, L913) designated available, Lindenbein (1952) suggested a 
as "melanin" the dark pigment produced by series of color designations which are simple 
.1. chromogenus from peptone, although this and convenient. This system in a modified 
organism did not always produce the pig- form is given in Appendix I. 
ment from tyrosine. He considered the pig- Pigment formation is considered by Kras- 
ment as a catabolic product of the organic silnikov (lOOO) as a constanl specific prop- 
nitrogen, erty, although the nature of the pigments 

Lehmann and Sano (1008) first suggested varies with the composition of the medium, 

the expression "tyrosinase reaction." They The color of t he aerial mycelium is not con- 

used for their studies a tyrosine-containing sidered as constant and is greatly influenced 

medium, melanin being known to be an oxi- by the composite f the medium (see also 

dation product of tyrosine. Waksman (1916, Conn and Conn, L941). 
L919, L920) expressed considerable doubt The variability in pigmentation of differ- 
that the production of a soluble dark pig- ent strains of S. aureofaciens was studied in 
ment on beef-peptone agar is due solely to detail by Duggar <t al. (1954) and Backus 
this reaction. Gelatin, containing no tyro- <lul. (1954), and is illustrated in Table 7. 
-inc. gives the characteristic pigmentation. 

Some species producing a typical dark pig- utilization of carbon soi ia es 

ment on the beef-peptone agar may fail to The ability of different species of actino- 

do so on synthetic media containing tyrosine, mycetes to utilize as sources of carbon and 

Skinner (1938) recognized a difference be- energy various organic substances, such as 

tween the dark pigmenl produced in peptone carbohydrates, alcohols, salts of organic 

media and not in lyrosine-cont aining syn- acids, fats, and amino compounds, can be of 

thetic media and the black pigment pro- considerable diagnostic value. These studies 





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CHARACTERIZATION OF STREPTOMYCES SPECIES 



73 



date from the early work of Waksman 
(1919), who employed a synthetic solution 
to which he added various carbon- or ni- 
trogen-containing compounds. Liquid sub- 
strata were employed and cultures were 
incubated under static conditions. The use 
of static liquid substrata was later found to 
give misleading results. In some cases, uni- 
form inoculum distribution is no1 achieved 
unless considerable care is taken. Numerous 
studies (Pridham and Gottlieb, L948; Bene- 
dict et al., L955; etc.) indicate that solid 
substrates and different basal media were 
later used (Table 8 . 

I lata et al. (1!).">:;i found a correlation be- 
tween the "roups and types of organisms 
established on the basis of carbon utilization 
and their antistreptomytic and antibac- 
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 lie combined with other criteria. 
None of the 12.") cultures they studied could 
use dulcitol, for example. The best carbon 
sources for characterizing Streptomyces spe- 
cies were found to he raflinose, /-xylose, <l- 
fructose, Z-arabinose, and d-mannitol. Gor- 
don and Mihm (1959) considered asa species 
characteristic the utilization of acetate, 
malate, propionate, pyruvate, and succinate. 

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 lac- 
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, niamhtol, and certain other carbon 

sources. 



T LB] i 8 

Carbon soura utilization by l '. natural 

variant strains of S. aureofaciens 

Backus et al., 1954 



\<> apparent 


Utilization by 
all variants 


Utilization variable with -train 


utilization by 
anj variant 




tive 


tive 


Sodium 


( Hucose 


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11 


1 


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Maltose 


cit rate ' 






( ll\ cine 


Starches 


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'1 


3 


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9 


3 


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9 


3 




( Hycerol 
Sodium 


Magne 
sium lac 


7 


5 




succi 


tate* 








Date* 


Xylose 


6 


6 




Inulin 









* Used ;it 0.4 per cent level, all others at 1 per 
■cut . 



PROTEOLYTIC ACTIVITIES 

Among the proteolytic activities of diag- 
nostic value in separating genera, liquefac- 
tion of gelai in, 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 lo 
produce brown to black pigments, provides 
significant criteria lor species characteriza- 
tion. 

In a study of 177 cultures of Streptomyces, 
Stapp (1953) found only one that did not 
liquefy gelatin. Detailed studies <>n proteo- 
lytic activities of actinomycetes are found 
in the work of Waksman | L919), Jensen 
(1930), Gordon and Smith l L955),and Kutz- 
ner (1956), as well as in Vol. 1. pp. L83 186. 
Waksman 1 1919) reported that of :;."> cultures 
tested, 33 liquefied gelatin more or less raj)- 



71 



THE ACTINOMYCETES, Vol. II 



idly; when these tests were repeated three 
times, considerable variation in the degree 
of liquefaction was obtained. Kutzner (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 occa- 
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 (Krassilnikov, I960) to be 
characteristic of almost all actinomycetes 
and to have, therefore, no taxonomic signifi- 
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 specie* 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 sub- 
strates show some degree of inhibition of 
growth of other microorganisms, when tested 
on suitable media, suggested the ability to 
form antibiotics to be of potential diagnostic 
value. It is a question whether one is a 
"lumper" 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 cyclo- 
heximide. Others may form various actino- 
mycins, grisein, streptocin, or candicidin. 

It has been suggested that because the 



CHARACTERIZATION OF STREPTOMYCES SPECIES 



75 



growth of homologous strains of an organism 

is less inhibited than thai 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 lias also 
been emphasized by Gause (1955). 

. 1 ctinophage 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. 



1. Actinophages vary greatly in their host 
ranges. 

a. Mosl actinophages which were 
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 in 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 \<H-<ir<li<i 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. 
('lair 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 investigator.- (Hoehn, 
1949; Chang, 1953, Rautenstein and Kofa- 
nova, L957; Cause et al, 1957; Mach, L958; 
Shirling, 1959a, b; Kutzner and Waksman, 
1959a; Kutzner, I960). 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 
"lumper" 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 



70 



THE ACTIXO.MVCKTES. Vol. II 



si rains 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 ;i single species (Waks- 
man, 1951)). There have been various reports 
concerning phages which are active upon 
st rcptomycin-producing strains, but do not 
at lack grisein-producers or nonantibiotic- 
forming cultures (Woodruff et al., 1 1)47 ; 
Waksman et al, 1947; Reilly et al, 1947; 
Waksman and Harris, 1949; Hoehn, 1949; 
Carvajal, 1953; Burkholder et 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, I960), S. lavendulae (Gause et al., 
1957; Shirling, 1959), and S. olivaceus (Kha- 
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. 

::. Actinophages vary greatly when toted 
againsl 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 10 7 to 10 9 par- 
t icles 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 (1900) 
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 containing XaCl hut gave high 
plaque counts when plated on the same me- 
dium without NaCI (with or without CaCl 2 ), 
while other phages showed higher activity 
on NaCI than on CaCl 2 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 NaCI and gave high 
plaque counts, but showed no activity 
against other strains on the same medium. 
These results suggest that a medium found 
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 SIM < II S 



77 



been suggested for species identification of 
actinomycetes. Aoki (I'.)::.") L936) was thus 
able to differentiate 1 km ween representatives 
of three genera, Actinomyces, Nocardia, and 
Streptomyces. By means of sonic vibrations, 
Ludwigand Butchinson (1949) prepared an- 
tigen suspensions satisfactory tor 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 Ilata (1953) and Bata et al. (1953). A 
purified antigen of a streptomycin-producing 
strain was found active againsl immune sera 
of the same strain, bu1 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 Boyos (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 :> and 4, com- 
prising species of Streptomyces. Slack it al. 
(1051), however, found that antisera pre- 
pared witli .1. bovis brought about low titer 
agglutination of Nocardia and of two species 
of Streptomyces. They concluded thai a close 
antigenic relationship exists between mem- 
bers of the genus Actinomyces and thai 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 */ '//., 1 959. 

( 'In mical < 'omposition 

A detailed study of the chemical composi- 
tion of cells of actinomycetes has been pre- 
sented in Volume I (pp. L58 L63 1. 

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. (19581 
have shown thai 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. 

f. Thermophilic forms. 

g. Inhabitants of acidic (pll .'! to 6.5) sub- 
strates. 

h. Inhabitants of neutral to alkaline sub- 
strates (pi 1 6.5 and above). 
The temperature at which an organism is 
grown 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 import ant 
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 pi I 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. israelii, and "nocardio- 
sis," caused by different species of Nocardia. 
We associate S. scabies with the "scab" of 
potato tubers, and S. ipomoeae with a dis- 
cvise 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 mutanl 
strains of S. coelicolor (most probably S. 
violaeeoruber) . It has been brough.1 out in 
Chapter 6 of Volume I thai true hybrids can 
be obtained by mating two different mutant 
strains of an act inomycete. Welsch (1958) 
suggested thai 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 si rain 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 




Figube 21. Formation of straighl sporophores by Streptomyces sp., X L500 (Courtesy of -Miss A. 
Dietz, Dept. of Microbiology, Upjohn Co., Kalamazoo, .Mich.). 



equivalenl to the original soil isolates, as 
determined by the methods of assessmenl 
used. 

The prior growth of the organism in soil 
media (sterile soil treated with a small 
amounl of CaC( h , it' acid, and with a half 
per cent of dried Mood) 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 i real ments. 

Shinobu l L958) suggested the following 
method for making a soil medium: Into a 
test tube of L.5 cm diameter place 7 g dried 
fertile soil; add L.5 ml of 1* per cent solution 



Si) 



THK ACTIXOMVCKTKS, 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 o 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 Eidige Technischen Hochschule 
(ETH) Zurich; Centraalbureau voor Schim- 
melcultures (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 is 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 
easier 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 new species also have been 
created to facilitate the obtaining of patents. 

Hesseltine et al. (1954) suggested that the 
following steps be taken in the taxonomie 
study of a Streptomyces species: 

1. Collection of strains on the basis of 
pigmentation of aerial mycelium. 

2. Study of the morphology of strains 
growing 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 STREPTOMYCES SPECIES 



SI 



.">. 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- 
biotics. 

6. Identification of new strains with 
known species, and preservation of cultures. 

Pridham* emphasized that recently lie has 
been placing principal emphasis on micro- 
morphology, secondary emphasis on chro- 
mogenicity (deep brown to black diffusible 
pigments), and tei't iary emphasis on color of 
aerial mycelium. 

In an effort to determine whether freshly 
isolated cultures can he identified on the 
basis of published descriptions and what con- 
ditions justify the creation of new species, 
several obvious comparisons were made 
(Waksman, li>o7). Certain strains that 
mighl he included in various important 
species or ^roup-species were critically ex- 
amined. The following conclusions were 
reached: 

At present, various morphological, cultural, 
and biochemical properties arc know a which make 
it possible t<> 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 he re- 
quired in order to establish the degree of varia- 
tion of ;i culture before it can lie recognized as a 
neu 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 lie briefly 
summarized as follow a : 

1. On I he basis of all the accumulated evi- 
dence, actinomycetes are shown to belong defi- 
nitely to t he bacteria. 

2. The position of the true act i nom vcet es in 

relation to related bacteria] forms, aotably the 
* Personal communical ion. 



mycobacteria and corynebacteria, musl be recog 
ni/i'il; this is true especially of certain aocardial 
types 

3. The generic interrelationships among the 

actii ycetes are highly significant. The separa 

tion <)f members of the genus Streptomyces from 
those of Nocardia is difficult, especially when one 
is dealing with nonsporulating forms of the first 
nml 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 he recognized. A combina- 
tion of morphological and cultural properties 
permits the establishment of species-groups. 
Some of these comprise a large number of forms 
with many variable characteristics. 

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 
cut sugars are ample illustrations of this. 

ti. Cognizance of the strains and varieties 
within each species musl finally he taken. This 
may he 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 ant ihiot ic. 

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 form- of life 
one is always faced witli the selection of 
variet ies resistant to disease, or giving higher 
yields, or having other desirable qualities, 
so one must select strain.- of actinomycetes 
on the hasis of resistance to phage or of pro- 
duct ion of higher yields of a given antibiotic 
or other metabolic product . 



Chapter 5 



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 (species-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 
foi classification purposes, well-defined 
media and standard conditions of cultiva- 
tion must be used. 



Actinomycetes are differentiated from the 
true bacteria by their filamentous growth 
and by their true branching. It is often 
difficult, 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 by the constant and 
marked nature of their aerial mycelium, 
whereas the nocardias are characterized 
largely by the transitory and undifferenti- 
ated nature of this mycelium. 

In establishing differences between no- 
cardias and streptomyces, one must con- 
sider the following factors: 

I. Nocardias usually have been consid- 
ered incapable of forming aerial mycelium 
that could be differentiated from the sub- 
strate mycelium. It also has usually been 
assumed that no spirals are ever formed 
from the mycelium. Recently, however, 
Cordon and Mihm (1958) have reported 
that certain nocardias are able to form aerial 
mycelium similar to thai of streptomyces 



82 



GROUPS AND SPECIES OF GENUS STREPTOMYCES 



v; 



and that spirals also may be formed. A 
streptomycete tonus 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 multiplies 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 segmenl spontaneously into 
bacillary or coccoid tonus, but remains non- 
septate and coherent even in old cultures, 
thus forming the characteristic tough tex- 
t ured, leathery growth. 

:!. 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 and that 
faculty of the degenerated culture to regain 
the lost capacity. 

4. Another difference between nocardia- 
and streptomycetes is the acid-fastness or 
partial acid-fastness of some of the formei 
when grown in certain media; the latter are 
never acid-fasl . 

As pointed out in Chapter I, Gordon and 
Smith (l!>.V)i proposed six distinctive char- 
acter- for the separation of the two genera. 
These criteria are: (a) colony structure; (b) 
casein hydrolysis; (c) 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- 




Figure 22. Tufi formal 
of t he Streptomyces tins* u. 



suits in five or six of the physiological tests 
belong to Streptomyces; strains with four 
to six negative reactions, to Nocardia. 

Although considered a- somewhat arbi- 
trary, these criteria allowed clear-cut generic 
separation of 07 per cent of 251 strains 
studied, regardless of their morphological 
variation. Nmety-six per cent of the strains 
received as Streptomyces were positive in 
five oi .-ix 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 
to be descendants of typical sporulating 
ones, also were positive in five or -ix of 
these tests. Two-thirds of the strains la- 
beled Nocardia gave negative results in four 
to six of the same tests. Of the remaining 
third of the strain- received a.- Nocardia, 
•J I had the same reactions as accepted 
strains of Streptomyces and were assumed to 



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 Micromonospora. Species of 
Thermoactinomyccs also produce an aerial 
mycelium, similar to that of species of 
Streptomyces, but they form single spores, 
similar to those of Micromonospora. 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 1 1. 

Among the numerous species belonging 
to the various genera of actinomycetes, 
those of the genus Streptomyces are by far 
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 m and is usually 
only 0.7 to 0.8 n- 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. 

* Bol h designations were published, in different 
journals, the same month and the same year. 
Priority lias 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., 
I960) to form spiny, warty, or hairy spores; 
the remainder were smooth-spored. All the 
blue- to blue-green-spored forms had spiny 
spores. White, yellow, 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 limited usefulness for 
taxonomic differentiation. 

The growth of Streptomyces "colonies'' 
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, eithei 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 SIM '.('IKS OF (HATS STREPT0M1 CES 



s;, 




Figure 23. Various strains of Streptomyces californicus (Reproduced from: Bi 
Bull. Torrev Botan. Club »2: 111, 1955). 



P. H. ei al. 



Classification Systems of tin- Genus largely those forms that arc now included 
Streptomyces in the genus Streptomyces. This is true, for 

example, of the fii.-t classification of San- 
Early Systems of Classification felice (1896), and the subsequenl ones of 

Krainsky (1914) and of Waksman and 

Although the earlier systems of classifica- Curtis ( 1916). Only the more comprehensive 

tion of actinomycetes were also supposed to and more recenl systems are presented 

be concerned with all the forms usually in- here. The earlier ones were given in Chapter 

eluded in this group, they represented 1 of Volume I. 



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 Rutgersi nsis 



2. Pigment formed on synthetic media. 

a. Pigment dark blue. 

Actinomyces violaceu&-( 'aeseri 

b. Pigment brown. 

Actinomyces diastaticus 
II. No spirals. 

1. No soluble pigment. 

a. Growth orange-red, aerial mycelium 
white. 

Actinomyces albosporeus 






J**^ 



f 




V 




« 



•K 



P 



Figure 24. Sporophores of Streptomyces sp. producing spirals, X 1500 (Courtesy of Miss A. Diet: 
Dept of Microbiology, Upjohn Co., Kalamazoo. Mich.). 



GROUPS AND SPECIES OF GENUS STREPTOMYCES 
red, aerial mycelium 



^7 



I). (Irowth rose c 

rosy. 

Actinomyces Fradii 

c. Growth a mixture of white and yel 
low. 

a 1 . No conidia. 

Actinomyc* s albo flavus 
b 1 . Abundanl conidia. 

a 2 . Conidia mil shaped, powdery, 
gray-yellow. 
Actinomyces tins* us 
b 2 . Conidia spherical ami oval, 
grow th compact . cit ron-yel- 
low. 
Actinomyces citreus 

d. Growth at firsl colorless, then brown 
to black. 

a 1 . Aerial mycelium white. 

Actinomyces alboatrus 
1)'. Aerial mycelium dark gray. 
Actinomyc* s Lipmanii 
2. Soluble pigment produced. 
a. Soluble pigment green. 

Actinomyc* s Verm 
1). Soluble pigment dark blue. 

Actinomyces violaceus niger 
B. Gelatin rapidly liquefied; brown pigment 

I. Spirals produced. 

a. Growth rose-colored; aerial mycelium 

rosy. 

Actinomyces ros* us 

b. Growth colorless; aerial mycelium 
golden brown. 

Actinomyc* s aureus 
c Growth slightly brown; aerial mycel 

ium white. 

Actinomyces Halstedii 
II. Xo spirals. 

1. No soluble pigmenl on synthetic media. 
a. Growth red to red orange; no aerial 

mycelium. 

Actinomyces Bobili 
1 1. Growth white; aerial mycelium white. 

a 1 . Aerial mycelium thin. rare, net 
like. 

Actinomyces reticuli 
b 1 . Aerial mycelium thick, white to 
gray. 

Acti mull yet s albus 

2. Soluble brown pigment produced on 
SJ n! het ic media. 

a. Aerial mycelium white, abundant. 

Actinomyces diastato chromo- 

iji n us 



b. Aerial mycelium white, produced 

late or not at all. 

1 1 tinomyc* s chromog* u us 

group 

c. < bow t h green ; aerial mycelium \\ lute. 

Actinomyces virido chromo- 

IJI II us 

('. Gelatin slowlj liquefied; oo soluble pigmenl 
1. Spiral- produced in aerial mycelium. 

1. Soluble led and blue pigment-. 

Actinomyces viola* * us 

2. No soluble pigment; substrate growth 
red. 

Actinomyces < 'alifornicus 
II No spirals produced in aerial mycelium. 

1 . Growth yellow; no soluble pigmenl 

Actinomyces parvus 

2. Growth tends to crack; soluble brown 

pigmenl . 

Actinomyc* s < xfoliatus 
1). Gelatin slowly liquefied; brown pigment pro- 
duced. 
I. Spirals produced; aerial mycelium laven- 
der. 

.1 eh limn i/ri s Inn ml ulm 
II. Xo spirals. 

1. Growth yellow; aerial mycelium gray. 

Actinomyces Jim us 

2. Growth colorless; aerial mycelium 

purplish while. 

Actinomyc* s purpurog* nus 
:!. Growth black; aerial mycelium scant. 
Actinomyces i ith rochromo- 
genus 

1. ( irowth purple; no aerial mycelium. 

Actinomyc* s purp* o chromo 

I It It us 

2. WAKSMAN SYSTEM (1919) 

This was a modification of the previous 
system and was based upon a study of 11 
species. An examination was made of the 
morphology of the aerial mycelium on two 
media; growth, aerial mycelium, and solu- 
ble pigmenl <>n 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 pigmenl produced on organic 
media. 




Figuke 25. One of the early studies on the structure of the aerial mycelium and the manner of sporu- 
lation of Streptomyces cultures (Reproduced from: Drechsler, ('. Botan. Gaz. <>7: 66-83, 147-168, 1919). 



GROUPS AM) SPECIES OF GENUS STREPTOMYCES 



I . Pigmenl deep brown. 
II. Pigmenl taint brown, golden yellow, 
or blue. 
B. No soluble pigmenl on organic media. 
I. Strongly proteolytic. 
1 1. Weakly proteolytic. 
The need for the recognition of species- 
groups was also emphasized: "All the cul- 
tures should he divided into groups, the 
representatives of which have common 
morphological, physiological and cultural 
characters. These species-groups may show 
slighl variations within the groups, when 
several representatives are compared, but 
all of them possess in common the main 
distinguishing characters of the species, and 
are distinctly different from any othei 
species-group." 

3. JENSEN'S SYSTEM ( 1930) 

Jensen modified the above system of 
Waksman, in describing '.to s1 rains of actino- 
mycetes, largely streptomycetes, isolated by 
him. These strains were divided into several 
species-groups. 

A. No pigment produced on protein media. 

1. Red or blue pigments in synthetic media; 
marked reduction of nitrates. 

Actinomya s violaa us ruber 
_'. No red or blue pigments. 

a. Typical golden pigmenl in all synthetic 
media. 

Actinomyces fulvissimus 
I). Pigmenl not typical; abundanl aerial 

mycelium. 

a 1 . Aerial mycelium on synthetic media 
dark slate gray; lemon or sulfur- 
yellow pigments sometimes formed. 
a 2 . Vegel at ive mycelium on synl he1 ic 
agar light colored. 

Actinomya s c< llulosat 
I)-. Vegel ative mycelium on synl hetic 
agar turning dark. 

Actinomyces olivaceus 
li 1 . Aerial mycelium greenish- or yellow 
ish gray; very rapid liquefaction of 
gelatin and blood serum, 
a-. Aerial mycelium greenish. 
. 1 ct\ imin ill i 8 grist iis 



!)'-. Aerial mycelium yellowish. 

. 1 1 1 1 iimn iji i s gi i,<, oflavus 
B. Typical brown pigmenl in protein media 
"chromogenus" species I. 

1 . Deep brown growth and pigment in all syn 

thetic media. 

Actinomyces i>lm< ochromo 

IJI n us 

2. Pigment in synl hetic media of ol her color or 

a. Aerial mycelium absent or in traces; 

typical red vegetative mycelium. 
Actinomyces bobili 
li. Aerial mycelium more or less abundant . 

a 1 . Typical red pigment in synthetic 

agar. 

.1 ctinomyces erythrochromo- 

l/l II us 

h 1 . Pigment not red. 

a'-'. Aerial mycelium rose to cinna- 
mon brown. 

Actinomyces r<>s< us 
!>-. Aerial mycelium abundant, char- 
acteristic lead gray; light brown 
pigment in synthetic media. 

Actinomyces diastatochromo- 
genus 

In a subsequent contribution, Jensen 
(1931) emphasized again that the term 
"species" as applied to actinomycetes 
should Ik 1 used in the sense of Waksman's 
"species-groups," ora "broad group of strains 
agreeing in ceitain outstanding morpho- 
logical and biological features"; otherwise, 
"every strain of actinomycetes isolated from 
a plating from an ordinary soil could then 
he raised to the rank of species." 

4. KRASSILNIKOV SYSTEM ( 19 1 1 I 

'litis system was based primarily upon 
the morphology of sporophores of the cul- 
tures and the shape of their spores, and 
secondarily upon the pigmentation of the 
culture-. 

A. Sporophores branching monopodially. 

I. Spiral shaped sporophores, produced on 
hyphae of aerial mycelium. 
1 . Spores spherical or oval , 

a. Cultures colorless, no1 producing any 

pigment at ion. 



90 



THE ACTINOMYCETES, Vol. II 



a 1 . Aerial mycelium white. 

a 2 . Saprophytes, Living on dead 
material. 

Actinomyces albus 
b 2 . Parasites, living on plants. 
Actinomyces totschidlowskii 
b 1 . Aerial mycelium dark gray. 

Actinomyces griseus 
c 1 . Aerial mycelium green. 
Actinomyces glaucus 

b. Cultures pigmented bine. 

a 1 . Pigment of the anthocyanin type, 
similar to litmus. 

Actinomyces coelicolor 
b 1 . Blue pigment not changing with 
acidity of medium. 

Actinomyces cyaneus 

c. Cultures violet, forming two basic 
pigments (red and blue), both dis- 
solved into the substrate. 

a 1 . Cultures not forming any fluores- 
cent substance in liquid media. 
Actinomyces violaceus 
b 1 . Cultures producing in synthetic 
media a fluorescent substance of 
blue-green color similar to pyo- 
cyanin. 

Actinomyces pluricolor 

d. Cultures black-violet, forming red 
and blue pigments, as well as a brown 
pigment of the type of melanin, which 
changes the violet color of 1 he cull ure 
to violet -black. 

Actinomyces violaceus -niger 

e. Cultures red-colored, producing pig- 
ments insoluble in water, of the lipo- 
actinochrome type; color of medium 
not changing with acidity. 

a 1 . Cultures not forming any brown 
or black pigments; they arc al 
ways red, sometimes with a 
brownish tinge, but not black. 
Actinomyces ruber 
b 1 . Cultures producing on synthetic 
media, in addition to pigments, a 
black or dark brown substance 
which gives the culture a red- 
brown to black color. 

Actinomyces melanocyclus 
!'. Cultures yellow, citron-yellow, or 
brownish-yellow. 
a'. Saprophytes. 

Actinomyces Jim- us 
b 1 . Living on plants. 

Actinomyces setonii 



g. Cultures orange. 
a 1 . Saprophytes. 

Actinomyces aurantiacus 
b 1 . Parasites. 

Actinomyces phenotole reins 
h. Cultures green or brownish-green. 

.1 ctinomyces viridochromo- 
genes 
i. Cultures black, producing a pigment 
of the melanin type. 

Actinomyces niger 
j. Cultures pigmented dark brown, but 
not Mack. 
a 1 . Saprophytes. 

Actinomyces chromogem s 
l) 1 . Plant parasites. 

Actinomyces gracilis 
2. Spores cylindrical or elongated. 

a. Cultures colorless. 

Actinomyces longisporus 

b. Cultures red, sporophores mostly 
st raight . 

a 1 . Saprophytes. 

Actinomyces longisporus- 

ruber 
I) 1 . Parasites. 

a 2 . Living in bodies of men and 
animals. 

Actinomyces spumalis 
b 2 . Living on plants. 

Actinomyces sal man i color 

c. Cultures orange. 

Actinomyces fradiae 

d. Cultures yellow. 
a 1 . Saprophytes. 

Actinomyces longisporus- 

flavus 
b 1 . Parasites living on plants. 
Actinomyces scabies 

e. Cultures citron-yellow. 

Actinomyces virgatus 

f. Cultures green. 

Actinomyces viridans 

g. Cultures brown or chocolate-colored. 

Actinomyces halstedii 
h. Cultures black! 

Actinomyces nigrificans 
II. Sporophores straight or wavy, but not 
spiral. 

1 , Spores produced by means of fragmenta- 
tion of plasma within cells, 
a. Spores spherical or oval. 
a 1 . Cultures colorless. 

Actinomyces globisporus 



GROUPS AM) SPECIES OF GENUS STREPTOMYCES 



b 1 . Cultures green. 
a 2 . Saprophytes. 

Actinomya s i iridis 
l) 2 . Planl parasites. 

Actinomya s cr< taceus 
c'. Cultures brown. 

Actinomyces globosus 
I). Spores cylindrical or elongated. 
a 1 . Cult ures colorless. 

Actinomyces candidxis 
b 1 . Cultures pigmented. 

Actinomya s cylindrosporus 
2. Spores produced by means of segmenta 

linn of aerial hypliae. 

a. Cultures colorless. 

Actinomyces farinosus 

b. ( 'ult ures pigmented red. 

Actinomyces oidiosporus 
Actinomyces rectus 

c. < lultures yellow orange. 

.1 ctinomyces longissimus 

d. ( lultures blue. 

Actinomya s cm ruh us 
o. Cultures brown. 

Actinomya s fumosus 
B. Sporophores produced in verticils. 
I. Sporophores s1 raighl . 

Actinomyces vi rticillatus 
1 1 . Sporophores spiral -shaped. 
I . Spores spherical, oval. 

Actinomyces reticuli 
Actinomyces reticulus ruin >■ 
•_'. Spores cylindrical . elongated. 

Actinomyces circulatus 



9] 

faint brown 



a. figmeni absenl 

only. 

1). Pigmenl blue. 

c Pigmenl at first green, becoming 
brown, etc. 
"_'. Soluble pigmenl on organic media 

brown. 
•'!. No soluble pigmenl produced in or- 

ganic media. 

a. Proteolyl i<- action strong. 

I). Proteolyl ic act ion limited. 

c. Proteolytic ael ion very weak. 

B. Saprophytes; thermophilic. 

1. Yellowish growth on potato; dia- 
static. 

2. Dark-colored abundant growth on 
potato; nondiastatic. 

3. Thermotoleranl cultures. 

C. Plant parasites oi cultures isolated from 

diseased plants or from soil in which 
diseased plants were grown. 

1. Isolated from potato scab or from 
soil in which scabby potatoes were 
grown. 

2. drown on or isolated from sweet 
potatoes. 

3. Isolated from scat) on mangels and 



)f the larger groups of the genus, notably 
o the Albus and Flavus groups, as will lie 
shown in ( Ihapter ('». 



sugar beets. 
Detailed consideration was given to some D. Isolates from animal tissues; in the 

animal body, hyphae often show clavate 
enlargements at the ends. 

1. Limited proteolytic action. 

2. St rone; proteolytic action. 
I-]. Produce only substrate growth and no 

aerial mycelium. 
The last system, like the earlier ones, may 
now be considered as of purely historic in- 
terest, lor the purpose of this treatise, it 
has been considerably modified. It has been 
greatly enlarged to include all newly <\c- 
scribed organisms belonging to the genus 



."). WAKSMAN AND HENRICJ SYSTEM (1943 

This system was used in the last two edi- 
tions of Bergey's Manual of Determinative 
Bacteriology I L948, L957). It may be listed 
here among the earlier systems. It was 
based primarily upon the ecology of the 

organisms, product] >f soluble pigments 

in organic and synthetic media, and proteo- 
lyl ic proper! ies. 



A. Saprophytes; psychophilic to mesophilic. Streptomyces. Some of the feature- of the 
I. Soluble pigmenl on organic media older system, notably those pertaining to 

other than brown, or faint brown. ecology, have been left out altogether. 



95 



THE ACTINOMYCETES. Vol. II 



Recent Systems of Classification 

Several systems for classifying species 
belonging to the genus Streptomyces have 
recently been proposed. Some of these have 
been selected for detailed examination. They 
aie based largely upon morphology, cultural 
and biochemical properties, or combina- 
tions of these. 

1. HESSELTIXE, BENEDICT, AND PRIDHAM 
SYSTEM (1954) 

In this system, emphasis was laid upon 
morphology as the basis for separation of 
the genus into five basic groups. After a 
study of hundreds of cultures on a variety 
of media, the conclusion was reached that 
the morphology of any one strain of Strepto- 
myces is essentially the same on any medium 
where sporulation occurs. The major groups 
were subdivided into a number of subgroups 
on the basis of cultural properties, pigmen- 
tation of spores, and other criteria. 

I. Sporophores not restricted in length, 
bearing fertile branches in verticils, 
with spores more or less strongly at- 
tached. 

1. Fertile branches in simple verticils, 
branches not ending in spirals. 

2. Fertile branches in simple verticils, 
branches ending in spirals. 

3. Fertile branches with compound 
verticils, branches not ending in 
spirals. 

II. Sporophores with branches all straight, 
never ending in spirals; verticils ab- 
sent. 

III. Sporophores predominantly in tufts, 
never verticillate; outline of branches 
flexuous and irregular. 

IV. Sporophores with branches ending in 
spirals, verticils being absent; sporo- 
phores either as long stalks bearing 
very short branches, or as short stalks 
bearing branches irregularly. 

1. Branches ending in open spirals with 
many turns. 



2. Branches ending in closed spirals- 
wit h few turns, thus appearing as 
tight knots. 
V. Sporophores with long and straight 
branches with spirals of large diameter 
at their ends; spiials usually with only 
a few turns, never verticillate. 
Xo strains were observed in which the 
sporophores were unbranched, except when 
they were growing under unfavorable con- 
ditions oi- where degenerated type cultures 
were studied. 

Seven major Streptomyces groups were 
thus created as indicated by the following 
key: 

I. Sporophores produce verticils; spores not 
readily separating; aerial mycelium white, 
pink, lavender, or tan. 

Group I. Streptomyces reticuli 

1. Verticil branches simple. 

a. Sporophores straight. 

b. Sporophores spiral-shaped. 

2. Verticil branches compound. 

a. Ultimate branches straight. 

b. Ultimate branches spiraled. 

II. Sporophores not producing any verticils; 
spores readily separate; color of aerial myce- 
lium often not pink, white, lavender, or tan. 

1. Spirals always formed; color of aerial my- 
celium blue, blue-green, or green. 

Group II. Streptomyces viridochro- 
mogenes 

2. Spirals may or may not be formed; color of 
aerial mycelium different . 

a. Spirals never formed, tufts often present ; 
color of aerial mycelium greenish-tan or 
tan. never white. 

Group III. Streptomyces griseus 
1). Spirals produced; color of aerial myce- 
lium lavender, red, pink, or nearly tan. 
Group IV. Streptomyces lavendulca 

3. Sporophores straight . 

a. Sixties white or nearly so. 

Group V. Streptomyces albus 

b. Aerial mycelium never white. 
a 1 . Aerial mycelium yellow. 

Group VI. Streptomyces parvus 
b 1 . Aerial mycelium gray, gray-brown, 
olive-gray, blackish-gray. 
Group VII. Gray-spored group. 

It was suggested that the last group could 



GROUPS AM) SPECIES OF GENUS STREPTOMYCES 



9.3 



be subdivided 



5is of spiral 



FLAIG WD KUTZNEB SYSTEM (1954, I960) 

Flaig and Kutzner (1954) and Kutzner 
ui 2000 Sin ptomyces cul- 
i were authentic species 
soil isolates. They charac- 
•es by several criteria in- 
laracterisl ics on complex 
dia, morphology of the 
physiological properl ies, 
vity against five test or- 



- L956) studied 

tares, 63 of \ 

and the rest t'l 

terized their <• 

eluding cultun 

and synthetic 

aerial myceliu 

and antibiotic 

ganisms. On the basis of these studies, a 

key was prepared. At first the material was 

divided into "groups" on the basis of the 

color of the aerial mycelium on oatmeal 

agar, six spore colors being recognized. The 

gray color group was further divided, on the 

basis of the color of the substrate mycelium 

and soluble pigmenl on this medium, thus 

resulting in the 10 groups shown in Table <). 

batei', however, Flaig and Kutzner (1960) 
reached the conclusion that the subdivision 
of the gray-spored color group may lead to 
difficulties when new isolates have to be 
placed in one of these groups, although this 
system proved to be quite useful iii studying 
many si rains at t he same t ime. 

Each group was further divided into sub- 
groups (altogether 382) on the basis of pig- 



menl formation on glucose-peptone agar, 
morphology of the aerial mycelium, anti- 
biotic activity againsl five tesl organisms, 
and cultural characteristics on several 
media. The shape of the spores (observed 
with electron microscope) was given for 17.~> 
of the 382 subgroups. Various authentic 
species were included in these subgroups 
according to their characteristic properties. 
'Hie following species were placed in the 
various groups. 

Group I: S. albus, S. griseus, S. chryso- 
mallus, S. coelicolor, S. cali- 
fornicus 
Group II: S. longispororuber, S. bobiliae, 
S. roseochromogenes, S. Vene- 
zuela?, S. phaeochromogenes 
Group III: S. lavendulae, S. xanthophaeus 
Group IV: S. flavogriseus, S. globisporus, 

S. flaveolus 
Group V: S. diastaticus, S. globosus 
Group VI : S. flavus 

Group VII: S. craterifer, S. griseolus, S. 
halstedii, S. hygroscopicus, S. 
aun ofacit us, S. violaceorubi r 
Group Mil: S. purpurascens 
Group IX: S. cyaneus, S. viridochromo- 

(/< nes, S. chartn usis 
('■roup X: N. hirsutus, S. prasinus, S. 
prasinopilosus 



Table 9 
Grouping of iht genus Streptomyces according In characteristic coloration (Flaig and Kutzner. I960 



Group 



Aerial myielium 



Substrate mycelium and soluble pigmenl 



T 


Yellow ish i<> \ ellov* graj 


II 


Lighl rose t<> reddish 


Hi 


( }ray-rose i lavender 


IV 


Lighl gray to gray 


V 


Gray 


VI 


White to graj (col fconj 


VII 


( rray (dusl y 


VIII 


Light gray or pink 


IX 


Blue 


X 


( Ireen 



Colorless, brownish, reddish, greenish 
Colorless, orange, greenish, brownish to 

dark brown, pink to dark red 
Colorless, orange, brownish to dark brown 
Yellowish-green to green 
I') row ii 

( lolorless, brownish to brow d 
Colorless, greenish gray, brownish gray, 

orange-brownish, red or blue violet 
\ Lolel 

Blue-purple, bluish-green, brownish 
Colorless, greenish, brownish to rose-red 



94 



THE ACTINOMYCETES, Vol. II 



3. SYSTEM OF YAMAGUCHI AND SABURI (1955) 

Yamaguchi and Saburi also used morpho- 
logical features as the primary basis for the 
separation of the genus Streptomyces into 
groups, and physiological characteristics 
for further separation. Although they were 
concerned primarily with species of Strepto- 
myces possessing antitrichomonal properties, 
their system may apply to the genus as a 
whole. 

I. Sporophores straight, tuft -forming tendency 
in the margin; no verticils or spirals. 

1. Aerial mycelium gray. 

a. Soluble pigment on protein media. 
Light purple, reddish-purple, purplish- 
brown, sometimes yellowish-brown. 

St reptom yces p u rpeojusc us 

b. Xo soluble brown pigment. 

Streptomyces fasciculus 

2. Aerial mycelium pale yellowish-green. 

a. Soluble pigment on protein media 
brown— ATCC Culture No. 3309. 

b. Soluble pigment brown. 

Streptomyces griseus 
II. Sporophores straight, verticils produced. 
1. Cottony aerial mycelium white, light tan, 
or pale pink. 

a. Soluble pigment on protein media 
brown. 

Streptomyces reticuli 
1). Xo soluble brown pigment on protein 
media. 

Streptomyces hack ijoensis 
III. Sporophores spiral-shaped. 

1. Predominantly closed spirals produced. 

a. Aerial mycelium gray. 

a 1 . Soluble brown pigment on protein 
media. 

a 2 . Growth colorless, yellowish- 
brown, brown, or deep brown. 
Streptomyces olivochromogenes 
b 2 . ( rrowth light purple to purplish- 
black. 

Streptom yces p u rp u reoch ro - 
mogenes 
b 1 . Xo soluble brown pigment. 

Streptomyces o urcofocicus 

b. Aerial mycelium pale pink. 

a 1 . Soluble pigment on protein media 
brown. 

Streptomyces lavendulae 
I) 1 . Xo soluble brown pigment. 

Streptomyces fradiat 



2. Predominantly open spirals or compact 
spirals produced. 

a. Aerial mycelium white. 

a 1 . Soluble pigment on protein media 
brown. 
a 2 . ( rrowth reddish. 

Streptomyces ruber 
b 1 . Xo soluble brown pigment. 
Streptomyces farinosus 

b. Aerial mycelium gray. 

a 1 . Soluble pigment on protein media 
brown. 

a 2 . Abundant compact spirals pro- 
duced on aerial hyphae. 
Streptomyces naganishii 
b 2 . Growth colorless, white, light 
yellow, or yellowish-brown. 
Streptomyces diastatochromo- 
genes 
c 2 . Growth colorless, reddish-or- 
ange, or reddish-purple. 
Str< ptomyces griseoruber 
b 1 . Xo soluble brown pigment. 
a'-'. Growth colorless to creamy. 

Streptomyces albus 
b 2 . Growth colorless, white, light 
yellow, or yellowish-brown. 
G 167 (resembling Strepto- 
myces cacaoi) 
c'-. Growth colorless, light yellow, 
or light pinkish-brown. 

Streptomyces albogriseolus 
d 2 . Growth purple to pink to red. 
Streptomyces calif orn if us 

c. Aerial mycelium gray, but on certain 
media moist with dark, glistening 
patches. 

Streptomyces hygroscopicus 
IX. Xo characteristic features of aerial hyphae. 

1. Very limited aerial mycelium production 
on various media. 

a. Soluble pigment on protein media dee]) 
brown. 

a 1 . Growth yellow to yellowish-brown. 
Streptomya s flavochromogi m s 

b. Soluble pigment faint yellowish-brown. 

Streptomyces thioluteus 

2. Aerial mycelium white. 

a. Growth colorless, light yellow, or red 
dish-orange. 

Streptomyces ruber 

4. BALDACC1 SYSTEM (1956, 1958, 1959) 

Following the example of Sanfelice and 
Waksman, Baldacci divided the genus 



GROUPS AND SPECIES OF GENUS STREPTOMYCES 95 

1 pink (white to 



Streptomyces into sections, based upon the 
color of the substrate mycelium. Each sec- 
tion was divided into series, on the basis of 
the coloi of the aerial mycelium. Each series 
was divided into species. 

The genus was characterized by the pres- 
ence or absence of spores, the arrangemenl 
of spores, and the ramification and breaking 



10. Aerial myceli 

pink). 

Series Rost oflavus 

11. Aerial mycelium yellow with gray 

spot.-. 

Scries Flavus 

12. Aerial mycelium grayish. 

Series .1 urt US 



up of the substrate mycelium. The species (b) Substrate mycelium yellow to green- 
were characterized by enzymatic reactions, yellow. 

antibiotic activity, and soluble pigments 13. Aerial mycelium gray-white, 

spreading through t ho substratum, depend- Series Flavoviridis 

ing on nutrition and pH. 14. Aerial mycelium white to lemon- 

AJthough Baldacci discussed the genus yellow, 

under the name "Actinomyces," he actually Series Virgatus 

meant Streptomyces, without recognizing it (c) Substrate mycelium yellow with green 

as such, since he made no mention of other and pinkish spots. 



species and othei genera. 

A. Actinomycetes cum sporophora solitaria 

vel congregata. 
Section 1. Substrate mycelium colorless; 

scant development on agar, showing a 
veiled cobweb-like appearance; limited 
spornlat ion. 

1 . Aerial mycelium white. 

Series Albus 

2. Aerial mycelium sea-green. 

Series Griseus 
'■\. Aerial mycelium green-azure. 

Series Viridis 
1. Aerial mycelium azure. 

Seiies ( 'aeruh us 
5. Aerial mycelium white-wine-lavender. 

Sei'ies Lavendulae 
(i. Aerial mycelium light pink. 

Set ies Ros( us 
7. Aerial mycelium gray. 

Series Diastaticus 



15. Aerial mycelium white to pink. 
Serio Madura* 

(d) Substrate mycelium brown to black. 
10. Aerial mycelium white to gray. 

Series Scabies 

17. Aerial mycelium red. 

Series Roseochromogent s 

18. Aerial mycelium yellow. 

Series Sulphureus 
10. Aerial mycelium gray. 
Series Antibioticus 

20. Aerial mycelium grayish-flesh-col- 
ored. 

Sei'ies Griseoincarnatus 

(e) Vegetative mycelium brown to green- 
brown. 

21 . Aerial mycelium gray. 

Series Intermedius 

(f) Vegetative mycelium brown. 

22. Aerial mycelium white to leather- 
brown. 

Set ies Rimosus 



Seci ion II. Substrate mycelium colored; 

development generally abundanl on agar (g) Vegetative mycelium orange, 
(creamy, lichenoid, etc.); delayed or par- 23. Aerial mycelium seashell pink 

tial sporulation. Series Fradiai 

(a) Substrate mycelium yellow to yellow- (hi Vegetative mycelium flesh-rose. 
brown. 21. Aerial mycelium white. 

'.). Aerial mycelium white. Series Bostroemi 

Serio Albidoflavus (i) Vegetative mycelium red. 



96 



THE ACTINOMYCETES, Vol. II 



2.j. Aerial mycelium white to pink. 
Series Albosporeus 

26. Aerial mycelium ash-gray. 

Series Cinereo-ruber 
(j) Vegetative mycelium violet-blue-red. 

27. White to gray aerial mycelium with 
different shades. 

Series Violaceus 
B. Actinomycetes cum sporophora opposita 

1 1 verticillata* 
Section I. Substrate mycelium colorless. 

1. Aerial mycelium white or whitish. 

Series Circulatus 

2. Aerial mycelium gray and pinkish. 

Series Griseocarneus 
Section II. Vegetative mycelium colored. 

(a) Substrate mycelium lemon-yellow- 
creamy-colored. 

3. Aerial mycelium cinnamon-colored. 

Series Cinnamoneus 

(b) Substrate mycelium brownish-yellow. 

4. Aerial mycelium gray. 

Series Reticuli 

(c) Substrate mycelium brown. 

5. Aerial mycelium greenish-gray. 

Series Verticillat us 

(d) Substrate mycelium red. 

6. Aerial mycelium pinkish-red. 

Series Rubrireticuli 

5. CAUSE ET AL. SYSTEM (1957) 

Gause et al. modified Baldacci's system, 
leaving out the sections and combining the 
pigmentation of the aerial mycelium with 
that of the substrate growth for series char- 
acterization. Each series was subdivided, on 
the basis of formation of a soluble pigment 
in a complex organic medium, or of the 
structure of the sporophores, or of the pig- 
mentation of a synthetic medium. These 
investigators, like Baldacci, adhered to the 
genus designation Actinomyces, without, 
however, considering the accumulated in- 
formation concerning all other genera. 

* These sections were placed by Baldacci in ;i 
separate genus "Strt ptoverticillium." 



Little consideration was given to previously 
named species. 

I. Aerial mycelium rose-purple, sub- 
strate mycelium colorless. 
Series Lavendulae-roseus 
II. Aerial mycelium rose-colored; sub- 
strate mycelium yellow. 
Series Fradiae 

III. Aerial mycelium rose-colored; sub- 
strate mycelium brown. 

Series Fuscus 

IV. Aerial mycelium light rose; substrate 
mycelium violet : 

Series Roseoviolaceus 
V. Aerial mycelium rose-colored; sub- 
strate mycelium red. 
Series Ruber 
VI. Aerial mycelium yellowish-green or 
cream-colored; substrate mycelium 
colored or colorless. 
Series Helvolus 
VII. Aerial mycelium white; substrate 
mycelium colorless. 
Series Alb us 
VIII. Aerial mycelium white; substrate 
mycelium red or brown. 
Series Albosporeus 
IX. Aerial mycelium blue or greenish- 
blue; substrate mycelium colorless or 
blue-colored. 

Series Coerulescens 
X. Aerial mycelium gray; substrate 
mycelium colorless. 
Series Griseus 
XI. Aerial mycelium gray, then black 
(result of autolysis); substrate my- 
celium colorless. 

Series Nigrescens 
XII. Aerial mycelium gray; substrate my- 
celium yellow or orange. 
Series Aureus 

XIII. Aerial mycelium gray; substrate my- 
celium yellow-brown. 

Series Chrysomallus 

XIV. Aerial mycelium gray; substrate 
mycelium brown-black. 

Series Chromogenes 



GROUPS AM) SPECIES 01 GEN1 S STREPTOMYCES 



'.'7 



XV. Aerial mycelium gray; substrate my- 
celium blue-violel or red-brown. 
Series Violaceus 

6. PRIDHAM, HESSELTINE, AM) BENEDICT 

system (1957, L958) 

Id their earlier system, these invesl igators 
divided the genus Streptomyces into seven 
groups. Each group was characterized by a 
distind morphology of the sporophores in 
mature cultures, and by a distind color of 
the aerial mycelium. This system subse- 
quently was revised. Morphological sections 
and color series were established and, on the 
basis of literature study and laboratory in- 
vestigations, many species and known anti- 
biotic-producing strains were cataloged. It 
was suggested thai evaluation of the compo- 
nent strains in the sections and series, by 
physiological tests, would allow the deter- 
mination of ranges of variation and a more 
logical approach to speciation in the genus. 
The placement by these investigators of 
strains in morphological sections, regardless 
dt' species designation, has suggested synon- 
ymy, as well as misident ilicat ion of many 
strains. 

The following bases were considered in 
justifying these subdivisions: 

1. The morphology of the sporophores of 
a particular si rain does not appreciably 
change on substrata that supporl optimal 
formation of aerial mycelium, sporophores, 
and spores. Morphological patterns exhibited 
by streptomycetes ate not subject to con- 
siderable variation, unless degeneration of a 
particular strain has occurred through im- 
proper maintenance. Morphological exami- 
Dations should be made after two weeks' 
incubation at 28 30°C on several appropriate 
media. 

2. The color of the sporulating aerial my- 
celium of a given st rain at maturity was said 
not to differ appreciably from medium to 
medium. Each morphological section of the 
genus can he further subdivided into color 
"series." Each color series can he subdivided, 



on the basis of physiological criteria, into 
"species." Additional delineation can then 
he w^vil to create "varieties" or "physiolog- 
ical forms," if need he. 

:'». The present concept of the genus 
Streptomyces is interpreted rather broadly. 
Some of the strains identified as members of 
the genus may in reality belong in other 
genera. 

The proposed section- were designated as 
follows: 

I. Rectus-flexibilis (MY). Sporophores 
straight, flexuous, or fascicled; mi 
spirals. Type species S. griseus. 
II. Retinaculum-apertum (HA). Sporo- 
phores in 1 he form of hooks, open loops, 
or greatly extended spirals. Type 
species S. fradiae. 

III. Spira (S). Sporophores either shorl and 
gnarled, or in the form of compad 
spirals or of extended long and open 
spirals. Type species S. viridochro- 
mogenes I fig. 26). 

IV. Monoverticillus (MV). Sporophores in 
the form of primary verticils attached 
to long, straight branches; no spirals. 

Y. Monoverticillus-spira (MV-S). Sporo- 
phores as primary verticils attached to 
long, straight branches; elements of 
verticils spiraled. 
\"i. Biverticillus (BV). Sporophores as com- 
pound verticils attached to long, 
straighl branches; no spirals. Type 
species S. cinnamomi us f. cinnamonu us. 
VII. Biverticillus-spira (BIV-S). Sporo- 
phores as compound verticils attached 
to long, straight branches; element- of 
secondary verticils spiraled. 
In addition lo the above sections, another 
section was set np lo include .-train- for 
which no micromorphological data were 
available. 

Each '•section" was subdivided into 
"series" based on the color of sporulating 
aerial mycelium at maturity. The proposed 
series were designated as follows: 
I. White. 



98 



THK ACTINOMYCKTKS, Vol. II 





-^^*^ 


: J 










1 








f 




r 1 


\ 



Figure 26. Spiral formation by Streptomyces 240 (Reproduced from: Naganishi, H. and Nomi, R. 
J. Fermentation Technol. 32: 492, 1954 i. 



2. Olive-buff (buff to tan to olive-buff). 

3. Yellow. 

4. Blue (blue to blue-green to green). 

5. Red (pink to red to lavender to Laven- 
der-gray) . 

6. Gray (light gray to mouse-gray to 
brown-gray to gray-brown). 

An additional "unknown" series was set 
up to include strains for which no color data 
were available. 

7. ETTLIXGER, CORBAZ, AND HUTTER SYSTEM 

(1958) 

Ettlinger et al. considered four major 
characters of Streptomyces that were stable 
and reliable enough to justify their system of 
classification. These characters were: (a) 
morphology of the spores, (b) color of aerial 
mycelium, (c) morphology of aerial myce- 
lium, and (d) formation of melanoid pig- 
ment. 

These investigators suggest ed combination 
of sections 4 and (i, and sections 5 and 7 of 
the Pridham et al. (1958) system, since they 
had never observed nonbranching verticils. 
They recognized a total of 15 morphological 
types distributed among the five sections. 



They also recognized the following color 
groups for the aerial mycelium: (1) niveus 
(snow-white), (2) griseus (yellowish- to 
greenish-gray), (3) azureus (sky-blue), (4) 
cinnamoneus (light carmine to brownish), 
(5) cinereus (ash-gray), ((>) prasinus (leek- 
green). 

They observed certain constant relation- 
ships among some of the four basic prop- 
erties. The griseus and cinnamoneus color 
groups were found to occur only in strains 
with smooth spores. The azureus and pra- 
sinus color groups occurred only in strains 
with spiny or hairy spores. The latter always 
were found associated with the occurrence 
of spirals. 

Other properties, such as soluble pigment 
on synthetic media, antibiotic activity, and 
pigmentation of substrate mycelium, were 
found to be variable. Gelatin liquefaction, 
milk coagulation, starch hydrolysis, and 
other physiological properties were not con- 
sidered of great value from a systematic 
point of view, since no true negative gelatin 
liquefaction or negative starch hydrolysis 
was ever detected. 

On the basis of the above properties, the 



following system of elassifieat 
posed for the genus Streptomyc 



GROUPS AND SPECIES OF GENUS STREPTOMYCES 

was pi-o- 



99 



A. Spores spiny or hairy. 
I. Aerial mycelium blue. 

1. Streptomyces \ irido 
chromogt nes 

1 1. Aerial mycelium not blue. 

1 . Aerial mycelium white. 

2. Strt ptomyct s />u r 
purasct ns 

2. Aerial mycelium no1 white. 

a. Aerial mycelium green. 

a 1 . Spores with short spines. 

3. Streptomyces pra- 

Sl II IIS 

I) 1 . Spores with longer spines or with 
ha if. 
a' J . Spores with s1 ill spines. 

4. Strt ptomyci s hirsu 

his 
b 2 . Spores with flexible hair. 

."). Streptomyces pra- 
sinopilosus 

b. Aerial mycelium gray. 

a'. Sporophores in verticils. 

a 2 . Sporophores in open spirals; 
no melanin formation. 

6. Streptomyces nour- 
sei* 

b 2 . Sporophores in closed spirals; 
melanin produced. 

7. Strt ptomyces echi- 

n il his* 
I) 1 . Sporophores not in verticils. 

a'-'. Sporophores in closed spirals. 
s. Streptomyces albo- 
griseolus 
l>'-\ Sporophores in open spirals. 
a 3 . Spirals irregular. 

!). Streptomyces mac- 
rosport us 
b 3 . Spirals regular. 
a'. Spores spiny. 

10. Streptomyces grist 
ojlariis 

I) 1 . Spores hairy. 
a 5 . Spirals w it h > ."> I urns; 
melanin posit ive. 

11. Streptomyces pilo 
sus 




The \erl i( 



te nat lire of 



"«■• 



Figure 27. Sporogenons coiled hyphae of Strep- 
tomyces T3110; taken from a gray area of a colony 
(Reproduced from: Duggar, B. M. et al. \nn. 
X. Y. Acad. Sci. 60: 71 -85, 1954). 

!>'. Spirals with <o turns, 
melanin-negal ive. 
12. Strt ptomyces flaveo- 

lus 
B. Spores smool h. 

I. Aerial mycelium yellowish- to greenish- 
gray. 

1. Melanin -negative. 

L3. Sin ptomyct s gri- 
seus 

2. Melanin positive. 

14. Streptomyces michi- 
garu nsis 

II. Aerial mycelium white. 

1 . Sporophores in verticils. 

15. Sin ptomyt t s rubri- 
reticuli 

2. No verticils produced. 

a. Sporophores form spirals. 

Hi. Sh i ptomyt ' s nive- 
orubt r 

b. Sporophores straighl or wavy. 
a 1 . Melanin negative. 

17. Sin ptomya sfulvis* 

si III IIS 

li 1 . Melanin posit ive. 

is. Streptomyces 
phaeochromogenes 



100 



THE ACTINOMYCETES, Vol. II 



III. Aerial mycelium light 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 si raighl . 

21. Streptomyces vene 
zuela( 

b. Sporophores in spirals. 

a 1 . Spirals at end of long, straight 
sporophores ; melanin-positive. 

22. Streptomyces laven - 
dulae 

b 1 . Spirals different; melanin-nega- 
tive. 



a'-'. Spirals closed. 

23. Streptomyces vio- 
laceoniger 
b 2 . Spirals open. 

a 3 . Spirals regular, usually 
>5 turns. 

21. Streptomyces fra- 
ilnii 
b 3 . Spirals irregular, usually 
<5 turns. 

25. Streptomyces ery- 
thraeus 

IV. Aerial mycelium ash-gray. 

1. Sporophores in verticils. 

26. Streptomyces reti- 
culi 

2. Sporophores no1 in verticils. 

a. Sporophores straighl or wavy. 



\ 



^"H* 




Figure 28. Sporogenous coiled hyphae of Streptomyces T 3 IK); taken from a blue sector of a colony 
(Reproduced from: Duggar, B. M. et al. Ann. X. V. Acad. Sci. 60: 71-85, 1954). 



GROUPS AM) SPECIES OF GENUS STREPTOMY( ES 



101 



a 1 . Sporophores sympodially 

branched. 

27. Si 1 1 jiinm ij, , s viri- 
dogeni s 

b 1 . Sporophores monopodially 

branched. 

a 2 . Sporophores as side branches 
of sterile hyphae. 

28. Sin ptomyct s ramu 

Ins IIS 

I)'-'. Sporophores different . 

a 3 . Melanin negative. 

29. Strt plum ii, i s oliva- 
Ct us 

b 3 . Melanin positive. 

30. Si 1 1 ptomyc< s anti- 
bioticus 

b. Sporophores in spirals. 
a 1 . Spirals closed. 

;;i . Sin ptomyct s hygro- 

sinpirus 

b 1 . S|iirals open. 

a 2 . Spirals irregular, usually >5 
Minis. 

32. Streptomyces nun o 
faciens 

I) 2 . Spirals regular, < 5 turns. 
a 3 . Melanin-negal ive. 

33. Shi ptomyct s par- 
r ii 1 1 us 

Ii 1 . Melanin-positive. 

34. Streptomyces gali- 

I ar IIS 

8. SHINOBU SYSTEM ( L958b) 

Shinobu proposed the following system 
for grouping of the species of the genus 
Sin ptomyces: 

Group 1. Monopodial branching, straight or wavy 
aerial mycelium; never producing spirals. 

Subgroup 1. Tyrosinase reaction: positive; ni- 
t rite product ion: positive. 
Sin ptomyces olivact us 
Subgroup 2. Tyrosinase reaction: positive; 
nit rite production : aegal ive. 

Si, , ptomyces phaeopurpurt us 
Subgroup 3. Tyrosinase reaction: negative; 
nit rite product ion : posit ive. 
Strt ptomyces sp. No. 2 
Subgroup I. Tyrosinase reaction: negative; 
nit rite product ion : negative. 
Sin ptomyces sp. No. 232 
Group II. Spiral formation; long or short . loose or 
compact, and open or closed spirals. 



Subgroup 5. Tyrosinase reaction: positive; 

nit rite product ion : posh ive. 

Shi ptomyct s ' iridochromo- 

IJI III s 

Subgroup 6. Tyrosinase reaction: positive; 

nit rite product ion : negal ive. 
Sin ptomyi i s -p. No. 2076. 
Subgroup 7. Tyrosinase reaction: negative; 
nitrite produd ion : positive. 
Sin ptomyces -\>. X<>. 236 
Subgroup 8. Tyrosinase reaction: negative; 
nit rite product ion : negative. 
Sin ptomyces scabit s 
Group III. Verticil formation; primary and 
secondary verticils; rarely one tertiary verticil 
(Fig. 29). 
Subgroup 9. Tyrosinase reaction: positive; 

nil rite product ion : positive. 

Sin filinii i/< i s In fnslii mi nsis 

Subgroup 10. Tyrosinase reaction: positive; 
nitrite product ion : negal ive. 

Sin ptomya s /"'< overticillatus 

Subgroup 11. Tyrosinase reaction: negative; 
nit rite production : posit ive. 

Si 1 1 ptomyct s nisi mi 1 1 Hillii 

Ins 

Subgroup !'-. Tyrosinase reaction: negative; 
nit rite product ion : negative. 

Sin ptomyct s nl 1 1 ,,, i i In illnlus 

( rroup IV. [ntermediate group of Nitella type and 

Anitella t vpe verticil. 
Subgroup 13. 

Streptomyct s spiroverticillatus 

Shinobu further emphasized thai in ili<' 
Idenl ilicat ion of species of Strt ptomya s o\ her 
characteristics should be considered. These 
are the following: 

1. Morphological properties: type of colony, 
shape of spiral, etc. 

2. Physiological properties: cellulase, man 
nase, and amylase reactions; utilization of carbon 

and nil rogen sources, etc. 

3. Cultural properties: growth of colony, pro 

duct ion of pigment , etc. 

9. FROMMEE SYSTEM I L959 

This system does not apply to iln' genus 
Streptomyces as a whole bul only to the 
ael Lnomycin-producing species. 

A. ( ihromogenic group. 

I. Spirals not forme. I on aerial mycelium. 
( Occasionally a few spirals are found. 



102 



THE ACTINOMYCOTIC, Vol. II 




w 4^ 









^4- 



^L 



> 



# 



±c 



Figure 29. Verticil formation (Nitella type) including both primary and second; 
produced from: Shinobu, R. Mem. Osaka Univ. Lib. Arts and Ed. B. Nat. Sci. 7, 1958) 



u 



*: 






■y verticils (Re 



1. Aerial mycelium on synthetic agar 
media gray. Tyrosinase-negative. 

Strepto m yces an t ib iotic us 

2. Aerial mycelium on synthetic agar 
media yellow. Tyrosinase-positive. 

Streptomyces michiganensis 
II. Numerous spirals produced on aerial my- 
celium. 

1. Yellow or yellow-green pigment pro 
duced on synthetic agar. 

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 
svni hetic agar mouse-gray. 

Streptomyces chrysomallus v. 
fumigatus 
II. Numerous spirals on aerial mycelium. 

1. Aerial mycelium on glycerol-glycine 
agar grayish-rose. Prad ically no growth 
on synt het LC agar. 

Streptomyces murinus 



2. Abundant growth on synthetic agar. 
Aerial mycelium on glycerol -glycine 
agar cream-colored. 

Streptomyces galbus v. achro- 

mogeru s 

10. MAYAMA SYSTEM (1959) 

Mayama (1959) concluded that morphol- 
ogy, serological reactions, and types of 
growth on liquid media arc the most impor- 
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- 
faciens, S. griseolus, S. albus, S. rimosus, and 
S. reticuli. 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 



103 




Figure 30. Spore formation in Streptomyces species (Reproduced from: Shinobu, H. Mem. Osaka 
Univ. Lib. Arts and Ed. B. Nat. Sci. 7. L958). 



terminal portion of the branching hyphae. Series 10. Monoverticillate, straighl to 
Series 1. Sporophores straighl to flexu- flexuous. 

ous. Scries 1 1 . Monoverticillate, spirals. 

Scries 2. Sporophores form open loops. Series 12. Biverticillate, straighl to flexu- 

Series 3. Sporophores form spirals. ous. 

Section 11. Aerial mycelium branches in tuft Series \:\. Biverticillate, spirals. 

formations. Sporophores produced at the Section V. 

terminal portion of the branching hyphae. Scries 11. No aerial mycelium. 

Scries 1. Sporophores straighl to flexu- 
ous. 

Series .">. Sporophores form open loops. 

Scries 6. Sporophores form spirals. 
Section III. Aerial mycelium forms long 

main stem. Sporophores produced al the 

terminal portion of side branches. Non- 
vert ieillate. 

Series 7. Sporophores straighl to flexu- 
ous. 



I I . \m\1I SYSTEM 

Finally a purely morphological system 

may be listed. \ i (1959) proposed a 

division of the genus Streptomyces into eighl 
morphological groups. He returned to an 

earlier concept of Drechslcr (1919) that the 
nature of the turn of the spirals, namely 
sinistrorse and dextrorse, is an important 
characteristic of Streptomyces species. lie 



Series 8. Sporophores form open loops, recognized, however, that some cultures may 

Series !). Sporophores form spirals. he rather indefinite in tin- respect. 

Section IV. Aerial mycelium forms long 

', ,, \ Serial hyphae somewhal flexuous or straight; 

main stem. Sporophores produced a1 , ' , .... , ,., 

lew long nypnae. I lie terminal filaments de 
terminal portion ot side branches. Ver- velop into spiral-shaped sporophores. 

I ieillate. 1 . Spirals sinisl rorse. 



104 



THE ACTINOMYCETES, Vol. II 







J 







~<r 



^ 



M 



^ 







1 






<H= 






10 11 12 13 

Plate III. Morphological groups in the genus Slreptomyces (Mayama, 1959) (For details, see text, 



pp. 102-103). 



Section I: series 1—3 
Section II: series 4 — 6 
Section III: series 7 — 9 
Section IV: series 10 — 13 



GROUPS AND SPECIES OF GENUS STREPTOMYCES 



L05 



a. Spirals long, extended to compact. 

S coelicolor, S. albogriseolus, S. fla 
its, S. parvullus 
I). Spirals compact to compressed. None 
found. 
2. Spirals dexl rorse. 

a. Spirals Long, extended to compact. 

S. viridochromogeru s 

b. Spirals compacl to compressed. 

Stn ptomya s sp. No. L89 
B. Most aerial hyphae long, straigh.1 or slightlj 
flexuous. They do not sporulate, l>ut give rise 
to short side branches whose terminal filaments 
develop into spiral shaped sporophores. 
1 . Spirals sinisl rorse. 

,S\ purpuras, i ns 

'_'. Spirals dexl rorse. 

Various unidenl ified forms. 
('. Aerial hyphae irregularly flexuous or wavy; 
long hyphae absent. Terminal filaments form 
spirals. 

1 . Spirals sinisl rorse. 

S. sulphureus 

2. Curvature of spiral indefinite. 

S gi iseoluU us 
1). Aerial hyphae lout;, straight, or wavy. They 
give rise to short side branches, which develop 
into spore-bearing hyphae containing spirals. 
1 . Spirals sinist rorse. 

S. hygroseopicus , S. violaceoniger, S. 

albus 

E. Aerial hyphae in clusters. The terminal fila- 
ments develop into sporophores, both spiral 
ami Qonspiral forming. 

S. vinaceous, S. microflavus, S. fra 
diae, S. lavendulae, S. virginiae, S. 
cinnamom nsis, S. roseochromogt //< s, 
S. pliui ochromogt nt s 

F. Aerial hyphae branch in clusters. No spiral- or 

loop-. 

S. n in :ui Int. S. tanashiensis , S. 
bikiniensis, S. antibioticus, S. aureo 
faciens, S. olivaceus, S. nitrosporeus, 
S. griseus, S. lipmanii, S. rutgersensis , 
S. parvus, S. fiavovirens, S. californi 
i us, s. i iiiiin us, S. ruber, S. caeruU us 

(1. Aerial hyphae long, straight or slightly flexu 
ous. Verticillate. No spirals. 

S. ri tii uli. S. griseocarneus , S. echt 
mi nsts, S. hiroshimensis , S. salmoni 
cida, S. thioluteus, S. albireticuli , S. 
m tropsis 

H. Aerial hyphae somewhat flexuous or wavy. 
Long hyphae and spirals are not produced 

S. albus (atypical), S. halsledii, S. 



scabies, S. verne, S. griseolus, S. 
■ i us Plates IV and V 

L2. OTHER S"5 STEMS 

( )t her systems have been proposed tor t he 
classification of the genus Streptomyces. 
Some of these systems are modifications or 
supplementations of that presented in Ber 
gey's Manual (7tli ed., 1957), or modifica- 
tions of one of the other of those outlined in 
this chapter. 

One of these is the system outlined by 
Routien (1959). The various species in- 
cluded in the genus Streptomyces were 
divided into three major groups: (1) sapro- 
phytes; (2) plant parasites or cultures 
isolated from diseased plants of from soil in 
which diseased plants were grown; (3) cul- 
tures isolated from animal tissues. These 
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, etc.). 
The color of the substrate mycelium and the 
various biochemical properties were 1 hen 
used for further subdivisions. Morphology 
(spiral formation, shape of spores) played 
only a minor role in this system. See also 
Sakai, L959. 

Summary of thi Properties I 'sal in 

Subdividing thi d'< mis 

Streptomyces 

Evaluation of the above systems of clas- 
sification leads to 1 he conclusion that sporo- 
phore morphology has been given hist 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 firsl 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- 



100 



THE ACTINOMYCETES, Vol. II 



>& 1 




Plate 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 straighl ; spirals extended to compact (S. coelicolor). 

B. Aerial hyphae straight; spirals on side branches OS. purpurascens) . 

(See continuation, next plate). 



CROUPS AM) Sl'KCIKS OF OKM'S STItKI'TOMYCES 



107 













Plate IV. {Continued) 
('. Aerial byphae wavy; spirals on terminal filaments (S. griseoluteus) . 

I). Aerial byphae long, straighl or wavy; sporophores as side Inane In-; spirals produced (S. h yg oscopi 
cus). 



108 



THE ACTINOMYCETES, Vol. II 



if 




Plate V. Morphological types, according to Nomi (1959). a. represents a schematic presentation of 
each typo; 6. gives the actual photograph. 

E. Aerial hyphae in clusters, terminal filaments developing into sporophores, both spiral and nonspiral- 
forming (S. lavendulae, S. roseochromogenes) . 

F. Aerial hyphae in clusters; no spirals or loops (S. antibioticus) . 

(See continuation, next plate). 



(IROITS AM) SPECIES OF (il'.MS STREPTOM\ CES 



100 




Plate V. (Continued) 
<!. Aerial hyphae verl ici ll.-i t e ; do spirals (S. hiroshimensis) . 
H. Aerial hyphae flexuous i>r wavy; do loog hyphae aDd do spirals (S. albus). 




110 



THE ACTINOMYCETES, Vol. II 



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 Kutzner (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 et al. (1954), Pridham et al. 
(1958), Ettlinger 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 is neces- 
sary, therefore, to combine several prop- 
erties in order to bring out the characterist ics 
of the group or series, and especially those of 
the species. 

Proposal System of Classification of (he Genus 
Streptomyces into Groups or Series 

In presenting the following system, full 
cognizance is taken of the criticisms to be 
directed against it, especially thai the for- 
mation of the melanin pigmenl 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 edit ions 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 



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 believe 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 
most part, transferred to other genera 
(Chapter 11). The animal and plant isolates, 
including both pathogens and saprophytes, 
have been distributed throughout the genus, 
among the various series, where they logi- 
cally belong on the basis of their morphologi- 
cal, 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 



III 



the names of the type species within the 
particular series. (See also Table LO.) 

Genus Streptomyces Waksman and Hen- 
i'ici, containing L6 series. Type species S. 
albus (Rossi-Doria) Waksman and Henrici. 

A. Subgenus Streptomyces Waksman, with 
I I series. Type species Streptomyces (Strepto- 
myces) albus (Rossi-Doria) Waksman and 
Henrici. 

B. Subgenus Streptoverticillium Baldacci, 
with ■_' series. Type species Streptomyces 
(Streptoverticillium) reticuli (Waksman and 
( !uri is) 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 while to lighl gray aerial myce- 
lium, covering the whole of the substrate 
growth; concentric rings may he formed. It 
is melanin-negative. A fainl brownish pig- 
ment may he produced on organic media. 
Sporophores ace spiral-shaped, occasionally 
broom-shaped. The species within this series 
are usually strongly proteolytic, without 
formation of bad-smelling products. It may 
he argued that the type species S. albus is 
no longer available and that many species 
possess similar properties. This series and 
this species must he recognized historically, 

whatever the final decision of the type 
species to he adopted (Pridham and Lyons, 

1960). 

Series 2. ( 'vru n us. This s( 
terized by a gray aerial myc 
in color from lighl gray to 
smoke-gray to ash-gray to 
may he white at first, later 1 
shades of gray. Substrate g 
colorless or yellowish, t urnim 
frequent ly a soluble yellow ] 
duced. The sporophores are 
or spiral-shaped. 



Ties 


is cl 


nil 


ac- 


•eliu 




ing 


ing 


mill 


ise-g] 


ay 


or 


hlui 


sh-gr 


ay; 
arii 


it 


urni 


ing v 


HIS 


rowl 


h m 


ay 


be 


I grs 


iv to 


da 


rk. 


>ign 


lent l 


s P 


iro- 


eith 


IT -1 


rail 


Jn 



Series .",. hiatus. 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 hy a yellow or 
yellow-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 pigmenl is 
usually produced. Sporophores are long, 
straight , or spiral-shaped. 

Series 4. Ruber. This series is character- 
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- 
sionally a yellowish to brownish 
iv be formed. Sporophores are 
spiral-shaped. 

Viridis. This series is character 
green to dark green substrate 
I by a white to gray to lighl 



duced; occa 
pigment in; 
st raight or s 

Series 5. 
ized by a 
growth, an< 
green aerial mycelium. Usually there is no 
soluble pigment; occasionally a lighl green 
pigment is formed. Sporophores are straighl 
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 i- blue 
at an alkaline and red at an acid reaction. 
Sporophores form spirals. 

Series 7. Fnulitu . 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 pigmenl may occasion- 
ally be produced. Sporophore> are si raight 
or spiral-shaped. Specie- are strongly pro- 
teolyi ic and antagonist ic. 

Series 8. (iris, us. This series is character- 
ized by colorless substrate growth, becom- 
ing, in certain media, brown to almosl olive- 
black. Aerial mycelium is yellowish with a 
greenish tint, or greenish-gray or sea-green. 



Ill' 



THE ACTIXOMVCKTES, Vol. II 



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. 

II. Melanin-positive 

Series 10. Scabies. Aerial mycelium is 
white to gray to buff. Substrate growth is 
brown to black. Sporophores are straight 
or spiral -shaped. 

Series 11. Lavendulae. 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. Erythrochromogenes. Aerial my- 
celium is white with brownish shade. Sub- 
strate growth is brown to black. Sporo- 
phores produce spirals. 

Series L3. 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. Thermophilic. This series com- 
prises six species. These are listed in Chapter 
11. 

B. Sporophores produce verticils. 

Subgenus Streptoverticillium 

Melanin-negative 

Series 15. Cinnamomeus. This and the 
next series are largely characterized by the 
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 



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 
fad that identification is frequently based 
upon comparison with published descrip- 
tions rather than with type cultures has 
result ed 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 
distinct and chemically different from the 
typical melanoid or chromogenic pigments, 
e.g. 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 "series" suggested here are 
quite distinct from those proposed by Bal- 
dacci et <il. (1954). They proposed, for ex- 



GROUPS AND SPECIES OF GENUS STREPTOMYCES 



113 



ample, a "Bostroem" series, for which no 
true representative can be recognized at 
present. Their scries "Antibioticus" and 
"Caeruleus" cannol be accepted for other 
reasons thai need not be discussed further 
here. Certainly, the idea expressed by Bal- 
dacci ei al. in L955 thai "it is not possible 
to speak of a natural systemization of these 
microorganisms a1 the present state of 
knowledge . . . for the time being, one must 
liniii oneself to a classification aiming solely 
at diagnosis and nomenclature," represents 
a defeatist attitude. It is well illustrated by 
his creat ion of a series named "Diastal icus." 
Here were included pigmented and nonpig- 
mented organisms, chromogenic and non- 
chromogenic, with such fantastic names as 
.1. rubrocyanodiastaticus, and such varieties 

as atrodiastaticus. This is certainly a g I 

cause for confusion. 

Similar criticism can he applied to the 
grouping of the species proposed by Gause 
ei 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 sections into 
series, Gause ei al. (1!)")") 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 'M old and 71 new specie- 
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 he assigned to each individual, 
since it is stated that "the study of the 
structure, classification, ecology and dis- 
tribution of actinomycetes occupies the 
attention of large scientific collectives in a 
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 



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, b~> series 
were adopted. This fad 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 -ingle 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 (1021) was the classical "lumper," 
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, and 
certain others have brought the system of 
classifying this important group of organ- 
isms to undesirable extremes. 

In 1 he decision to classify the genus Strt />- 
tomyces into lb series, it is well understood 
that in time other series will be added; some 
<>f those presented here may eventually be 
split 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 production of 
different antibiotics can be combined with 

* Hottinger's, to which no reference is given. 



114 THE ACTINOMYCETES, Vol. II 

certain other distinct properties, such as raising of the latter to a series status. In 

pigmentation, morphology, and carbon utili- other cases, however, the mere formation of 

/at ion, to justify the creation of new species. a different antibiotic without other accom- 

This has actually been done for the separa- parrying differences hardly justifies, for the 

tion of S. griseinus from S. griseus and the present at least, the creation of new species. 



Chapter 6 



Series and Species of the Genus 
Streptomyces 



The genus Streptomyces was created in 
L943, 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 Mime of the thermophilic groups, there 
are certain important properties that may 
be said to characterize this genus, thus 
separating it, if not for any other reason 
than thai of convenience, from the other-. 

The major important characteristic prop- 
erties that distinguish the genus Streptomy- 
ces from the others can he briefly summar- 
ized as follows: 

1. A more or less branched, nonseptate, 
substrate or vegetative mycelium (stromal 
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. 

3. During growth in stationary liquid 
media, no turbidity is produced except on 
lysis; the masses of growth appear as clumps 
or compad masses. 

4. The surface colony gradually becomes 
covered with an aerial mycelium, though 
this occasionally may not occur. 

5. The aerial mycelium produce.- sporo- 
genous hyphae or fruiting bodies, which are 
straight, or in the form of tufts, or curved, 
spiral-shaped, or verticillate. 



(i. The sporophores carry chains <>l 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 spore- < u 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 comprise-, by tar. 
the largesl number of species of actinomy- 
cetes now known to occur in nature. The 
various specie- belonging to this genus differ 
greatly in their morphological, cultural, 
physiological, and biochemical properties. 
They include the majority of antibiotic- 
producing actinomycetes. The growing eco- 
nomic importance of these organisms has 
tended to increase the need tni- the separa- 
tion of the "-enus into groups, each of which 
would contain one or more species. This 
need ha- recently been further emphasized 
by the creation of numerous additional spe- 
cies. 

The color of the aerial and substrate my- 
celium, the morphology of sporophores, mid 
the formation of melanin pigments have 
been largely used for the separation of the 
genus Streptomyces into series and species. 
For the supplementary characterizati f 



115 



Ill) 



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 1 of the spiral-form- 
ing aerial hyphae as a basis for classification. 
It was also true of YVaksman'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. 
( 195o), as well as Ett linger et al. (1958), pro- 
posed use of the nature of the spore surface 
as a species characteristic; unfortunately, 
this property, depending ;is il 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 et 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 keen 
laid, taking full advantage of the knowledge 
now available, for dividing the genus Slr< pto- 
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 is presented here. Some of the series 
are described in greater detail than others. 



SERIES AND SPECIES OF GENUS STREPTOMYCES 



117 



( 'haracU ristic Propi rtit s 



This is due either to their longer historical I. Series llbus 
background or to their capacity to form 
important economic products, especially an- 
tibiotics. •■'■ Sporophores produce spirals; spores 

In characterizing each series, the follow- spherical to oval, 
ing properties have been given special con- b. Color of aerial mycelium white, 
sideration. ''• Melanin-negative. 

a. Morphological properties, notably d - N " soluble pigmenl produced (excepl 
structure of sporophores and spores. a t ' ;,i "' brown pigmenl on certain media). 

b. Color of aerial mycelium on synthetic e " Weakly proteolytic and weakly antago- 
,■ nistic. 

The Minis -eric- comprises a large num- 
ber of organisms, characterized by the pro- 
duction of a typical leathery and compact 

substrate growth, colorless on various 
e. Other characteristic properties, such ,• , • , ,• . • 

media. Aerial mycelium is snow-wnite to 

,n white in color, assuming various shades as 



c. Formation of soluble brown pigme 
i melanin i on protein media. 

d. Spore surface. 



as color of substrate growth, for 

soluble, nonmelanoid pigments, rate of , h(l ( . u , tm . (1 grows ()1(1(M . Th( , spor0 p hores 

proteolysis, or production of specific anti- are [ ong and , nnn spirals; the spores are 

biotics. spherical to ovoid. The various strain- grow 

A summary of the properties of the 1.1 we l] ()U both organic and synthetic media. 

series within the genus Streptomyces is given They vary greatly in their proteolytic and 

n Table 10. diastatic properties. As a rule, this group of 



Table 10 
Charactt ristic properties of various series of Streptomyces 



No. of 
series 



\anu- "f merit's 



Minis 

Flavus 
Uubi 
Viridis 
Violact mnl, i r 

F i ml in, 
(his, us 

Hygroscopicus 
Scabu s 
Lin , ndulcu 
Erythrochromo 

Ijl III s 

V 1 1 idoch riinm 

I /, II , s 
< 'ill 11,1111,1111, us 

Reticuli 



White 

White to gray 

Mouse gray 

Rose 

Gray to green 

Gray 

Pink i" rose 

< trass green 



gray 



Whit 
( }ray 
Lavender 
Yellowish 

Green t< 

green 
Pinkish 
White to graj 



dive 



( lolor of growth 



yel- 



nge 
dive 



( Iolorless 
Colorless I 

Low 
Yellow 
Red 
( rreen 

Red to blue 
Yellow to o 
Colorless to 

buff 
I )ark gray to black 
Brow n to black 
( Iolorless 
Orange 

Brownish to green 

yellowish 
( Iolorless 



Spiral 




forma- 


pecies 


tion 




+ 


S. albus 


+ - 


S. craU • in i 


+ 


S. flams 


+ - 


S. ruber 


+ - 


S. viridis 


+ 


S iolaceorubt r 


+ - 


S. frmliii, 


_ 


S. grist us 


+ 


S. hygroscopicus 


+ 


S. scabies 


+ - 


S. In'-, intuitu 


+ - 


S. erythrochi 




III III s 


+ 


S. viridochromo 






— 


S. cinnamomeus 


+ - 


S. ■ liculi 



118 



THE ACTINOMYCETES, Vol. II 



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 
genus Streptomyces, were classified (sec Bei- 
jerinck, 1900, for example) into two groups: 
(1) .1. albus (Streptothrix alba), comprising 
those forms that produce a white aerial 
mycelium and no soluble pigment; (2) .4. 
chromog<- tins (Streptothrix chromogena), in- 
cluding those forms that produce a black 
pigment on protein media. Duche appeared 
to follow this system as late as 1934, since 
he included in his monograph (Duche, 
1934) on the actinomycetes only those spe- 
cies that were said to belong to the .1. albus 
group. In view of the significance of the 
specific name "albus," representing the type 
culture 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 concerning this group made by 
Baldacci (1939), who is frequently quoted 
here almost verbatim. Baldacci recorded 



about 30 synonyms, some of which are 
listed in Table 1 1. (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 Foersteri, 
isolated from the air by Gasperini (1890). 
Rossi-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- 
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 .1. albus. Gasperini (1894) 
recognized the difference between .4. albus 
and A. chromogenus. The latter possessed 
chromogenic properties, the pigment diffus- 
ing into the substrate. 

Sanfelice (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 represent at ive 
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. lie 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 (1 ( .)14) isolated from garden soil 
a culture which he described as .1. albus. 
This culture produced a well developed 
growth, white at first, then becoming gray 
on certain media such as glucose agar and 



SERIES AM) SPECIES OF GENUS STREPTOMYCES 



119 



Table 11 
Comparativi characters of Streptomyces albus at 









Mj 1 elium 




Proteolytic 


Spe< ific name 


Author 


Nat uri" and dimen- 
sions of spore 






Chronv 

1 n,'eneM> 


action 






Substrate 


Aerial 




Gelatin 


Milk 


Cladothrii a 


\l:„. 


+ 




White 


+ 






Streptothrix Foersteri 


( iasperini 


Oval 1.0-1.5) 


( 'olorless to yellow 

ish 


White 


~ 


+ 




Streptothrix a. 2 


Urn quist 


+ 


White 


_ 


+ 




a n. 3 


Almquisl 


+ 




White 


- 


- 




Si 1 /.iVM, t.r a//xi 


Rossi -Dorm 


+ 


1 !oloi less to black 


White 


— 




— 


Arl i iimiinri g nl/ms 


Krainsky 


Oval (1.0) 


( 'l)llP| 1.--S 


\\ hite less often 
gray 


- 


+ 




A . albus 


Waksman and 
( Jurtis 


Sphi 1 ical, o\ a! 1 1 .2 -1.6 
by 1.1-1.4) 


\\ hite or gray 


( nam 01 gray 


— 


+ 


+ 


.1 . nl him 


Jensen 


Rectangular (0.4-0.5 

bj 2 I 


Cream or yellow- 
ocher 


White 


+ 






A. albus 


Duehe 


+ 


While yellow 


White 


— 




— 


A. chroma-genus 


Gasperini 


Oval 1 •"> 


< Ichei tO black 


White 


+ 






( ladi Ihrii 


Rullmann 


+ (1.0) 




W hite or chalk 
white 


Weak 


+ 




A thermophilus 


Beresl new 


+ 




White 








A . tin rmophilus 


Gilberl 


+ (0.5-0.6) 


( traj yellow 


Gray 




+ 


+ 


A . I hi rmodiastaticus 


Bergej 


Oval 


Colorless 


White 




+ 


— 


. 1 . sil inn in 1 


Ciferri 


Round 


( !oloi less 


White or ivory 

white 


+ 


Weak 




A . almquisti 


Duche 


+ 


Yellowish 


- 


- 






1 gougeroti 


D.lehe 


+ 


Greenish 


White 


- 






Stl • ptoth 1.1 il> limn n 


Scheele and 


+ 


Colorless 


White 




Weak 




sis I 


Petruschky 














si 1 ptoth 1.1 Candida 


Petruschky 


+ 


( lolorless 


White 




+ 


+ 


Sir, ptothrix lathridii 


Petruschky 


+ 


Colorless 


White 




+ 


+ 


Cladothrii invul 


Vcosta and ( 1 


+ 




White 


- 


+ 




in ruhilix 


Rossi 















gelatin. The aerial mycelium was produced 
readily, the medium remaining colorless. 

The spores were oval, 1 ju in size. ( lela- 
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 .1. albus. It was similar to thai of Krain- 
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 .'_' to 1.6 by 
1.1 to 1.4 ft. 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 or 
yellow-ochre. The culture was said to pro- 
duce cylindrical spores, 0.1 to 0.5 by 2.4 ju. 

Duche" (1934) created an excessively 
broad "A. albus group." He was severely 
criticized by Baldacci (1939), who said thai 
"if it is meant by 'albus group' those species 
that produce white aerial mycelium in cul- 
ture, they are many more in numbers than 
those described by Duche\ These are also 

the 'viridis' species and those thai -how 
analogy with the 'flavus' form-." Baldacci 
further emphasized thai A. albus is a well 
characterized species thai doe- not permit 
confusion with others, while the "albus 
group" of Duche comprised 18 species, of 
which 15 were new one-; among these were 



120 



THE ACTINOMYCETES, Vol. II 



such forms as A. viridis, A. albidoflavus, and 
.4. alboflavus, which definitely belong to 
other groups. 

In his morphological study of the actino- 
mycetes, Duche recognized five types of 
sporulation, none of which was used for 
systematic purposes. The relationship of 
aerial hyphae, spirals, and spores was noi 
sufficiently emphasized. Duche 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" colonies, 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, 
"L'espece de Waksman and Curtis semble 
posseder toutes les proprietes de celle de 
Krainsky. . . . Notre espeee resemble aux 
deux precedentes." Baldacci concluded that 
the work of Duche, after trying to prove the 
diversity of the various interpretations in 
the literature, had not attained its purpose 
of establishing what the species A. 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 Krainsky 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 is at 
present as definite as any other. It has been 
recently studied intensively by Duche ( L934) 
and by Baldacci (1939). It is colorless, with 
white aerial mycelium, forming ovoidal 



spores in coiled chains on lateral branches 
of the aerial hyphae. It is proteolytic, lique- 
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 (inability 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 
ovoidal spores; by aerial mycelium colors 
generally interpreted as eretaceus (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 1 m 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 



121 



intersection, but it can follow in other di- 
rections or assume a wavy appearance. This 
mycelium originates direct ly from the germi- 
nating spores and can be rather abundant. 
It corresponds to the firs! 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 1.1 n); it is subhyaline 
with a tendency to assume yellowish colora- 
t ion. 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 ().(i to 0.7 by 1.2 
/i. 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. (dims. The 
difficulty in reaching an agreement con- 
cerning the shape and measurement of the 
spores is noi due, as claimed by Duche, to 
their small size, bu1 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 



attached to the glas> at their smaller sur- 
face, ;uid thus appear round. Spiral.- are 
abundant . 

The degeneration of the hyphae can be 
observed in the old substrate mycelium: 
zones of protoplasm with empty spaces 
simulating arthrospores can be seen. This 
phenomenon led earlier investigators, includ- 
ing Gasperini, to make unjustified generali- 
zations. This is also evident in the claim- 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 witli 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 
0rskoY and by Jensen. The "arthrospores 
irregulars" of Duche and others bring out 
very clearly this type of degeneration, which 
was erroneously interpreted as a type of 
sporulation. 

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; <>n some media, such as 
nulrient agar, it assumes a grayish tint. 
With age, growth of the culture becomes 
opaque or even yellowish, comparable to 
the color of the substrate, as can he ob- 
served in the cultures where the aerial mj 
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, independenl 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 

Krassilnikov (1949) included 19 species 
in the Albus series. He used a combination 
of different criteria for their separation and 
identification. For 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, etc. Krassilnikov also 
considered, for identification purposes, the 
shades of white in the aerial mycelium, tem- 



perature relation, proteolytic and antago- 
nistic properties, the secretion of a brown 
substance, growth in acid media, production 
of ammonia and HjS, decomposition of 
rubber, and formation of coremia. He in- 
cluded in this group various thermophilic 
and thermotolerant organisms. 

Cause 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: ^4. candidus, 
A. candidus var. alboroseus, and A. 
albidoflavus. 

b. Medium colored brown: A. longisporus 
and A. mirabilis. 

c. Medium colored brownish: A. albo- 
rubidus. 

The above characterization fails to recog- 
nize some of the fundamental cultural prop- 
ert ies 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 
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- 
quisti, A. beddardi, A. chromogenus, A. 
erythreus, A. exfoliatus, A. farcinicus, A. 
flocculus, A. gedanensis, A. gelaticus, A. 
gougeroti, A. heimii, A. kimberi, A. lieskei, 
A. listerii, A. malenconi, A. reticuli, A. soma- 
liensis, A. sanninii, A. saprophytic us, A. 
thermophilus, A. upcottii, A. willmorei; Clad- 
othrix dichotoma, C. liquefaciens, ('. invul- 
nerabilis, C, odorifera; Oospora doriae, 0. 
alpha; Streptolhri.v alba, Str. Candida, Str. 



IERIES AND SPECIES OF GENUS STREPTOMYCES 



123 



dassonvillei, Str. foersteri, Str. graminearum, 
Str. ieucea, Str. lathridii, 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 sufficienl to men- 
tion S. albus, S. calvus, and S. niv< us. 

II. Series Cinereus 

( 'haracteristic Properties 

a. Sporophores straighl or spiral-shaped. 

h. Color of aerial mycelium white to 
gray; occasionally dark humid stains or 
gu1 tation drops. 

c. Growth usually colorless, occasionally 
yellow to tan. 

d. Melanin-negative. 

When members of tins series form an 
aerial mycelium, the color is characteristi- 
cally gray. Although it may be white at 
first, it changes to various shades of gray, 
ranging from lighl 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 huff-colored; it 
is either opaque or somewhat slimy; the 
reverse is 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 greal number of incom- 
pletely described forms, as shown in Chapter 
12. 

A variety of antibiotics have been found 
to he produced by members of this series. 



Som, ( 'haracU ristic Specii s 

The following species may he tentatively 
included in this series: S. craterifer, S. inter- 
medius, S, parvullus, and S. c< lluhscu . 

< )ne may also include in this series various 
organisms described by Kxassilnikov I L949), 
including his emendation of A. griseus 
Krainsky (not S. griseus Waksman), .1. 
griseus variabilis, and .1. griseus zonatus. 
Tin- is also true of some of the forms de- 
scribed by Cause et <il. (1957) under such 
names as .1. rubiginosus, .1. griseomycini, 
.1. iverini, A. acrimycini, A. acrimycini var. 
globosus, .1. atroolivaceus, and .1. griseoru- 
bens. 



II. Sei 



Flai 



Characteristic Propertit 'S 

a. Sporophores Long, spiral-shaped; spores 
spherical to oval. 

1). Aerial mycelium white to .may to 
mouse-gray ; color of growth yellow to golden 
yellow. 

c Melanin-negative. 

d. Yellowish-green to golden soluble pig- 
ment may he excreted into the medium. 

e. Strongly proteolytic and antagonistic. 
The Flams series includes a large number 

of organisms, widely distributed in nature. 
The members of the series vary greatly in 
some of their cultural, biochemical, and 
morphological properties when grown on 
artificial media. This group has been recog- 
nized since 1891, when Rossi-Doria de- 
scribed a cult ure under the name of Sh; pto- 
thrix albido-flava. Another similar culture 
was soon described by Gasperini (1892) as 
Actinomyces albido-flavus. Sanfelice (1904 
designated the second of his three groups as 
Str. flava, comprising organisms isolated 
from the air. Caminiti (1907) was inclined 
to include in this group various pigmented 
forms, such as Str. citrea and Str. chromo- 
iji /hi. 

Numerous organisms belonging to the 



124 



THE ACTINOMYCETES, Vol. II 



Flavus series have been isolated from soil, 
dust, and other natural substrates by Krain- 
sky, Waksman and Curtis, and others. They 
have been designated by a variety of names, 
such as S. alboflavus, S. aureus, S. citreus, 
S. griseoflavus, and S. flaveolus. 

The Flavus series is characterized by 
cream-colored to yellow or golden yellow 
growth on most artificial media. The aerial 
mycelium is usually white to gray to mouse- 
gray. The sporophores are long, usually 
spiral-shaped. The spores are spherical, us- 
ually 0.7 n in diameter. No brown pigment 
is produced on protein media. A yellowish- 
green to golden pigment is often formed in 
synthetic and organic media. The various 
species in this group are strongly proteolytic 
and diastatic. Sucrose is inverted. Nitrate 
is reduced. Many of the strains are strongly 
antagonistic and are able to form active 
antibiotics, some of which have found ex- 
tensive application as chemotherapeutic 
agents. 

Krassilnikov (1949) recognized 13 distinct 
species as belonging to the Flavus series. 
Baldacci (1939), however, subdivided the 
actinomycetes with the characteristics of 
the Flavus series (various species producing 
yellow or golden growth) into a number of 
series: "aureus" "albidoflavus," "sulphur- 
eus" ''antibiotic us," and u hygroscopieus.' n 
Baldacci et al. (1954) included in the "Au- 
reus" series such species as S. aureus, S. 
aureofaciens, S. citreus, S. jimicarius, S. 
flavus, S. flaveolus, S. fordii, S. griseoflavus, 
S. hi/ijroscopicus, S. microflavus, and S. par- 
vus. 

A culture of an organism isolated by 
Takahashi (1953) in Japan was identified 
by him as S. flaveolus Waksman. To validate 
this identification, his description of this 
culture is presented in Table 12 alongside 
Waksman's description of the original type 
culture. These data show that, in spite of 
minor variations in color characterization, 
quantitative differences in gelatin liquefac- 



tion and nitrate reduction, and even in 
differences in antibiotic production, the 
identification of the species appears to be 
correct. 

The same is true of the characterization of 
S. parvus. The original culture of this organ- 
ism, which was used as the basis for its 
description in Bergey's Manual, has died out 
in the collection. Benedict, of the Northern 
Regional Research Laboratory of the U. S. 
Department of Agriculture, isolated from a 
sample of soil collected in West Africa a 
culture which he identified as S. parvus. A 
comparison was made of the culture origi- 
nally isolated and described by Krainsky 
(1914), the culture isolated by Waksman 
and Curtis (1916) and reported in Bergey's 
Manual, and the new culture of Benedict 
(Table 13). The results point definitely to 
the identity of the three cultures, thus prov- 
ing again that accurate identification of 
some species can be made by comparing 
freshly isolated cultures with written de- 
scriptions of type isolates. 

Finally, a comparison was made (Waks- 
man, 1957) of two published descriptions of 
S. flavus and S. griseoflavus, together with 
recent descriptions of two cultures that 
have been raised to the status of new species, 
namely, S. aureofaciens and S. rimosus, both 
important producers of antibiotics. The re- 
sults, presented in Table 14, show that S. 
aureofaciens and S. rimosus are sufficiently 
different from S. flavus and S. griseoflavus 
to warrant the creation of new species. S. 
aureofaciens is characterized by a deep gray 
aerial mycelium, by a lack of or limited 
spiral formation, by limited proteolytic ac- 
tivity upon gelatin and milk, and by poor 
growth on nutrient agar. It was concluded 
that these properties differentiated this 
culture sharply from the two older cultures. 
S. rimosus is characterized by pool- growth 
on synthetic agar and by the formation of 
abundant spirals in its aerial mycelium. 
These properties, together with certain 



SERIES AND SPECIES OF GENUS STREPTOM1 CES 



1 25 



Table 12 
Identification of Streptomyces Qaveolus (Waksman, L919; Takahashi, L953; Waksman, L957) 



Chanu Irri-Ii. - 



Morphology 

Sporophorea 

Spores 
Synt betic agar 

Subst rale growth 

Aerial mycelium 
Soluble pigmenl 

Calcium malate- 

\H,('l agar 

Subsl rale growth 

Aerial mycelium 

Soluble pigmenl 

Nutrient agar 
Sulist rate growth 

Aerial mycelium 
Soluble pigmenl 

( relatin 
Substrate grow ih 
Aerial mycelium 
Soluble pigment 
Liquefaction 

Potato 

Subsl rale grow I li 
Aerial mycelium 
Color of plug 

( Hucose broth 
Substrate growth 
Aerial mycelium 
Soluble pigmenl 

Milk 

Nitrate reduction 
Antibiotic production 



Numerous spirals on all media 

Oval to elliptical 

Lighl sulfur yellow turning cad 

mium-yellow 
White w it h ash gray patches 
Empire-yellow 



( 'ream colored 

Mouse gray, with white margin 

None 

Wrinkled, white 

Abundant , white 
None 

Abundant yellowish pellicle 

White 

( rolden to faint brown 

Rapid 

Wrinkled, cream colored 

White 

Faint brown 

Thin, yellow pellicle 

White 

( iolden 

Rapid coagulation and peptoniza- 
tion 
Si rong 

Produces act inoinvcin 



Numerous spirals on synthetic media 

Spherical or oval, 0.8 by 1.2 M 

Antimony yellow to chamois colored 

White, later smoke gray 
Buff yellow 



Pale olive buff to yellow ocher 
Vinaceous-buff to light mouse-gray 
None or faint yellowish 

Colorless to whitish, reverse cinnamon 
buff 

White 

Golden yellow 

Wrinkled, yellow 

White 

Faint yellowish brown 

Medium 

Wrinkled, golden yellow to orange 
White to seashell pink 

Faint brownish 

Colonial buff to honey-yellow 

White to smoke-gray 
Yellowish (golden yellow 
Rapid coagulation and strong peptoni 
zation 

Posit ive 
Produces fiaveolin 



other morphological and cultural differences A culture described as S. armillatus 

between this culture and the two older cul- (Mancy-Courtillel et al., 1954) appeared, on 

tares, justified creation of a separate species, the basis of the description, to be sufficiently 

especially because of the ability of S. rimo- close to S. rimosus to throw doubt upon its 

SUS to produce an important new antibiotic. distinct identity. Like the lat tor, it produced 

In view of the greal variability of these spirals in its aerial mycelium; on synthetic 

organisms and the temptation to establish agar it formed very poor growth without any 

separate species on the basis of minor differ- aerial mycelium and without pigmentation; 

ences in pigmentation, any attempi to on nutrient agar, it produced yellow-gray 

create such new species must be considered growth with poorly developed white aerial 

critically. mycelium and no soluble pigment ; on potato 



126 



THE ACTINOMYCETES, Vol. II 



Table 13 
Characterization of Streptomyces parvus (Waksman, 1957) 



Characteristics 


Krainsky 


Bergey's 6th Edition 


New culture received from N. R. R. L 
No. 3686 


Sul >s t rate 




Golden yellow to brick-red 


Bright yellow 


growth 




depending on composi- 
tion of medium 




Aerial myce- 


White to gray to rose-yel- 


Poorly developed, rose- 


Long, straight hyphae; no 


lium 


low depending on nitro- 


white; sporophores pro- 


spirals 




gen source 


duce spirals 




Spores 


More or less oval, 1.6 m in 


Spherical to oval, 0.9-1.3 


Short, oval 




size 


by 1.2-1.8 ix 




Synthetic 


Colonies small, yellow in 


Colonies small, yellow, 


Thin, yellow growth; thin 


agar 


color, with light colored 


with aerial mycelium 


white to yellow aerial my- 




aerial mycelium* 


light yellow 


celium; bright yellow sol- 
uble pigment 


Nutrient 


Yellow growth. Aerial my- 




Yellow growth; abundant 


agar 


celium appears late 




white with grayish tinged 
aerial mycelium; bright 
yellow soluble pigment 


Glucose as- 






Yellow growth; white to 


paragine 






gray aerial mycelium, 


agar 






golden soluble pigment 


Glucose agar 


Aerial mycelium light yel- 


Colonies small, yellow, 






low; appears late 


with aerial mycelium 
light yellow 




Gelatin 


Colonies flat or concave, 


Colonies yellow; liquefac- 


Cream colored growth drop- 




yellow in color; gelatin 


tion medium 


ping to bottom; good 




slowly liquefied 




liquefaction; bright yel- 
low soluble pigment 


Potato 


Colonies yellow, aerial my- 




Abundant wrinkled brown- 




celium white 




ish-yellow growth; abun- 
dant sulfur-yellow aerial 
mycelium; no soluble pig- 
ment 


Cellulose 


White surface growth 


Growth good 




Remarks 


Produces diastase; reduces 


Produces actinomycin; re- 






nitrate slowly; strongly 


duces nitrate slightly 






proteolytic 







* Calcium malate agai 



plug, it produced yellow-gray growth with a 
faint brownish soluble pigment; on gelatin, 
it formed a surface growth with white aerial 
mycelium, with a yellowish or brownish 
soluble pigment, and good liquefaction of the 
gelatin; on milk, it produced good grayish 
growth. These characteristics, together with 
the ability to produce oxytetrac/ycline, defi- 
nitely placed the culture in the S. rimosus 
species. Emphasis was laid upon the tact it 



formed flat colonies, hardly folded and not 
cracked like those of S. rimosus; it showed 
concentric circles in the aerial mycelium, a 
variable property. It did not form nitrite 
from nitrate, and it did not hydrolyze starch; 
these two properties were hardly sufficient, 
however, to justify the recognition of S. 
arm Hint its as a new species. 

Among the various members of the Flavus 
series, the actinomycin-prodncers occupy an 



SERIES AND SPECIES OF GENES STHEPTOMYCES 



127 



Table 14 
Characteristics of Streptomyces flavus and allied si oti \\ ii -man, 1957) 



Characteristics 


S. flavus (Bergey) 


5. griseoflavus (Bergey) 


S. aureofaciens (Duggar) 


S. rimosus (Sobin el al.)* 


Morphology 










Sporophoree 


Straight, much branched, 
no spirals 


Straight, no spirals 


Straight, flexuous; no spi- 
rals as a rule; occasional 
loose spirals 


Numerous spirals 


Spores 


Oval 




Spherical to oval, 1.5 n 

long 


Short, cylindrical, 0.6-0.7 
by 0.8-1.4/1 


Synthetic agar 










Substrate 


Yellow to sulfur-yellow 


Reddish brown to orange 


Heavy cream -colored, be- 


Submerged, colorless 


growth 






coming yellowish lirnu n 




Aerial myce- 


Straw-yellow 


White 


\\ bite, t inning mouse-gray 


None 


lium 






to brownish-gray 




Nutrient agar 










Substrate 


( Ire .nil colored, lichenoid 


Cream-colored 


Good, light brownish 


Cream-colored to brownish 


growth 










Serial myce- 


White to light gray 


White 


None 


None or white to gray- 


lium 








white 


Soluble pig- 


None 


N'iiiic 


None 


Faint yellowish or none 


ment 










i ielatin 










Substrate 


Yellowish 


Cream-colored to brown- 




Moderate 


growth 




ish-white 






Aerial 


None 


White 




White 


Hum 










Liquefaction 


Positive 


Slow 


None 


Medium to good 


Soluble pig- 


Faint yellow ish 


Faint yellowish 






ment 










Potato 










Substrate 


Lichenoid, brownish to 


Lichenoid, brownish to 


Wrinkled, orange-yellow 


Wrinkled, ochroid 


growth 


greenish-olive 


reddish-brown 






Aerial myce- 


White to gray 


H bite to gray 




Whitish tO drab 


< !olor of plug 


Brownish or none 


None 


Unchanged 


Yellow ish-brown pigment 
in none 


Yeast-glucose 










agar 










Substrate 


Rapid, lichenoid, brown- 


Cream-colored to brown- 


Heavy, cream-colored 


( iood, yellowish 


growth 


ish 


ish 






Serial myce 


\\ hite, later grayish 


White to grayish 


White to deep gray or dark 


Pallid drab 


hum 






gray 




Soluble pig- 


Yellow 


Yellowish 


None 


Yellowish 


ment 










Milk 


Coagulation and peptoni- 


No coagulation, rapid pep 


No coagulation, no pep 


No peptonization 




zation 


ionization 


tonization 




Production of 


An antibacterial agent 




i Ihlortel racycline 


Oxytet racycline and rimo- 


antibiotics 


formed 






cidin 



• \n earlier description of this culture on synt hetie agai was inronecth 
COSe in place of sucrose, which explains the difference between t he previo 



labelled; the medium was made up at that time with glu- 
ts and present obser\ a1 ions. 



interesting place. A culture (No. 3491) be- were included in the S. parvus subgroup, 
longing to S. flavus or to S. parvus was iso- Another culture (No. 3686), designated as 
lated in our laboratories in 1948 and found S. parvus, did not form any spirals. Still 
capable of producing actinomycin. It was another culture (No. 3687) produced only 
aonchromogenic and formed a straw-colored limited curling of the aerial mycelium and 
to yellow aerial mycelium. This culture was mighl l>e considered an intermediate lie- 
found to belong to the S. flavus subgroup; tween the two subgroups. It was suggested, 
other cultures (Nos. 3677, 3679, and 3680) therefore (Waksman and Gregory, 1954), 



128 



THE ACTINOMYCETES, Vol. II 







'«V 




Figure 31. Variation in morphology of spore-bearing hyphae in *S'. aureofaciens: (left) natural vari- 
ant A 377; (center) natural variant AB 374; (right) induced mutant A 377-2G55 (Reproduced from: 
Backus, E. J. et al. Ann. N. Y. Acad. Sci. 60: 101, 1954). 



that the whole series be designated as Flavus- 
parrus. Considerable variation was found 
among the members of this series. 

Morphological Characters 

Hyphae: (a) short, gnarled, and in clus- 
ters, with short oval spores; or (b) long, 
straight with spherical spores; or (c) long 
with long corkscrew spirals and spherical 
spores (Fig. 31). 

I'hysiological Characters 

Sucrose nitrate agar: Growth cream- col- 
ored, yellow to brownish to orange; reverse 
yellow to orange. Aerial mycelium cream- 
colored, straw-colored to citron-yellow, 
straw-yellow, grayish-yellow to bluish-gray 
to white, or absent. Soluble pigment light 
yellow to brownish. 

Glucose-asparagine agar: White to cream- 
colored growth, sometimes turning orange. 
Aerial mycelium white to gray. Soluble pig- 
ment none, or brownish to yellow. 

Nutrient agar: Growth cream-colored to 
yellowish to brownish. Aerial mycelium 
white, cream-colored to gray, or absent. 
Soluble pigment yellow to almost none. 

Gelatin: Cream-colored to yellow to 



orange-yellow ring on surface. Aerial myce- 
lium cream-colored, straw-green to gray, or 
absent. Soluble pigment brownish to yellow, 
or absent. Liquefaction varies from slow to 
rapid. 

Potato: Growth abundant, lichenoid, 
cream-colored to brownish to orange. Aerial 
mycelium white, cream-colored, gray to 
yellow. Usually no soluble pigment; occa- 
sional yellowish-orange pigment. 

Milk: Surface growth abundant or thin 
gray to black ring. Aerial mycelium white to 
gray or absent. Milk not coagulated but 
peptonized, the rapidity depending on ex- 
tent of growth. 

Antagonistic properties: Some members of 
the group produce highly important anti- 
biotics, such as the tetracyclines, that have 
found extensive application in chemotherapy 
and in food preservation (Kochi et al. ,1952). 

A careful study of the literature reveals 
the fact that a large number of species found 
in nature belong to this series. Some of them 
have been well recognized and described. 
Various others may be added, but many 
have been only insufficiently described. On 
the basis of the recognized information, the 
S. flavus series may be said to include the 



SERIES AM) SPECIES OF GENUS STREPTOMYCES 



12!) 



following species: S. Jliirus, S. Jhirorin US, S. 

flavogriseus, S. chrysomallus, S. celluloflavus, 
and S. viridans. 

IV. Series Ruber 

( 'haracU ristic Propertu s 

a. Sporophores straight or spiral- 
shaped. 

h. Substrate growth pink, red to red- 
orange t<» purple-red; pigment insoluble. 
Aerial mycelium thin, rose-white. 

c. Melanin-negative. 

The Ruber series comprises a large, highly 
heterogeneous group of organisms. Members 
of this series have been known since 1888, 
when Mace described an organism under 
the name of Cladothrix rubra. Numerous 
other cultures under different names were 
later placed in this group. 

The Ruber series is characterized by a 
bright red, red-orange, or rose-red substrate 
growth, the color depending on the composi- 
tion of the medium and on conditions of 
cultivation. The cultures may show con- 
siderable variation in color of the substrate 
mycelium, from purple-red to light rose. The 
pigment is usually not excreted into the 
medium, unless the latter contains fatty sub- 
stances in which the pigment is soluble. The 
aerial mycelium is not well developed; it is 
usually produced on synthetic media as a 
thin, rose-white cover, or it is formed only 
in isolated sectors or spots. Tin 1 sporophores 
are straighl or spiral-shaped; the spores are 
spherical to oval, 0.7 to 0.8 by 0.8 to 1.0 M . 

The members of the Ruber series are not 
very strongly proteolytic or diastatic. Su- 
crose is readily inverted. Some of the species 
belonging to this series are active producers 
of anl ibiotics. 

Baldacci did nol list a Ruber series, but 
one designated as "roseus," which is close 
enough to be considered similar to it. An- 
other was designated as " m< ianospOTi US," 



which is also close to the u ruber" series. 
Gause et al. (1957) divided the series into 

three subgroups on the basis of structure of 
the sporophores, namely, spiral-shaped, 
straight, and tuft-forming; the lasl appar- 
ently includes verticil-forming types. 

Some of these organism- may be con- 
sidered as forms intermediate in transition 
to the true chromogenic type-. Among the 
forms closely related to this series, one may 
include, for example, S. melanocyclus, S. 
melanosporeus, S. melanogenes, and possibly 
also S. erythrochromogenes, S. roseochromo- 
genes, and S. purpureochromogenes. The 
Ruber series is also related to the Fradiae 
and Flavus series, notably through such spe- 
cies as S. roseoflavus and possibly S. micro- 
flavus. 

Certain forms that may be considered as 
species of Nocardia are frequently included 
in this and in the next series. Somet imes even 
a new series is created for them, as was done 
by Baldacci (1942) for "madurae." 

S. albosporeus may be considered as a 
subgroup of the Ruber series. It is charac- 
terized by the formation of a rose-colored or 
red to brown substrate growth and a white 
aerial mycelium. Cultures belonging to this 
subgroup are characterized by strong pro- 
teolytic activity and by weak diastatic ac- 
tion. The sporophores are straight, with 
some close spirals. The first representatives 
of this subgroup were isolated by Krain.-kv 
in I'M 1 and by Waksman and ( 'urtis in L916. 

The separation of members of this series 
on the basis of carbon utilization has been 
suggested by Zahner and Kt t linger I 1957 1, as 
shown in Table 15. 

Although a large number of species found 
in the literature may be included in the 
Ruber series, only a few have been suffi- 
ciently described. It is sufficient to mention 
S. ruber, S. niveoruber, S. albospon us, and S. 

i r yi h nil US. 



130 



THE ACTINOMYCETES, Vol. II 



Table 15 
Utilization of carbon sources by a group of closely related 
Streptomyces species (Corbaz et al., 1957) 



S. purpurascens 


+ 


+ 


+ 


+ 


+ 


+ 


(+) 


+ 


(-) 


+ 


+ 


S. bobiliae 


+ 


+ 


+ 


+ 


+ 


+ 


+ 


(-) 


(-) 


(+) 


+ 


S. cinereoruber 


+ 


- 


(-) 


- 


+ 


- 


- 


- 


(-) 


- 


+ 


S. cinereoruber var. fructofer men- 


+ 


(+) 


+ 


(+) 


+ 


- 


(-) 


+ 


+ 


+ 


+ 


tans 

























+ = good growth; (+) = weak growth, questionable carbon utilization; (— ) = very weak growth; 
= no growth. 



V. Series I iridis 

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. 

c. 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. dassonvillei. 

The following organisms may be included 
in the Viridis series: S. viridis, S. prasinus, 
S. hirsutus and »S r . prasinopilous. 

Several forms described by Gause et <d. 
(1957) could be included in this series, 
notably A. malachiticus and A. olivaceoviri- 
dis. 

VI. Series Violuceoruber 

Characteristic Properties 

a. Sporophores produce spirals. Spores 
spherical to oval. Surface of spores smooth. 

b. Substrate growth colorless, becoming 



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 Hi, 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- 
cause 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 ^violaceus" 
"violaceoruber," "violaceoniger," "tricolor" 
and "pluricolor.'" The species capable of 
producing blue pigments are divided here 
into two distinct subgroups: S. violaceoruber 
and S. violaceoniger. 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- 
ganic media. 



SERIES AM) SPECIES OF GENUS STREPTOMYCES 



i:;i 



Table 16 
Streptomyces species, producing a blut pigment (Kutzner and Waksman, 1959) 



Organism 


Color of aerial my< elium 


Spirals 


Spore surface 


Melanin 
pigment 


Author 


S. caeruleus 


Light grayish blue 


_ 




_ 


Baldacci, 1944 


S. coelicolor 


( rrayish yellow 


- 


Smooth 


- 


Miiller, 1908 


S. cyaneofuscatus 


( treenish graj ish yellow 


- 




+ 


( rause - / al., L957 


S. cyaneus 


Bluish gray to blue 


+ 


Spiny 


+ 


Krassilnikov, 1949 


S. cyanoflavus 


Greenish-brow nish gray 


- 




+ 


Funaki et al., 1958 


S. litmocidini 


Gray, sometimes with 
brownish tinge 


— ; seldom 

+ 






( rause i i al., L957 


S. nnvaccacsarcac 


White with purple tinge 


+ 




- 


Waksman and Cur- 


(= A. violaa us 


due to substrate myce 


+ 






tis, 1916; Waks 


1 at sari I 


limn 








man, 1919 


S. pluricolor 


Whitish graj 


+ 






Berestnew, 1897; 
Krassilnikov j 
L949 


S. tricolor 


Light brown to light gray 


+ 






Wollenweber, L920 


S. violaceoruber 


Ash-gray 


+ 


Si 1 h 




Waksman and Cur- 
tis, 1916; Waks- 
man, L919 


S. olivaceus 


Ash-gray 


- 


Smooth 


- 


Corbaz ei al., 1957 


Streptomyces sp. No. 


Mouse-gray 






+ 


Kurosawa, 1951 


169 













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 scries as 
"violaceus," which he did qo1 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 scries "rioliici us" a variety of other 
forms that logically belong to this scries. 
Ettlinger ei al. ( 1958) designated as azun us 
the light blue pigmented forms. 

This series is not known for 1 lie product ion 
of any important antibiotics, although coeli- 
colorin has been reported for cultures of S. 
violaceorvher and chartreusin for S. char- 
In usis. 

The following species may be included in 
this series: S. violact oriibi r, S. novaecaesan ok , 
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 (iause et al. ( l ( .).">7l also belong 
here. These include .1. coeruhscens, A. glau- 
cescens, .1. coerukorubidus, A. tricolor, .1. 
coeruleofuscus, A. violaceorectus, A. prani- 
color, A. litmocidini, A. viridoviolaceus, A. 
griseorubiginosus, A. griseoruber, A. cinna- 
barinus, and others. 

VII. Series Fradiae 

( 'haracteristic Properties 

a. Sporophores usually straight ; occasional 
loops and spirals. 

b. Substrate growth yellow-orange to or- 
ange. Aerial mycelium seashell-pink, espec- 
ially on potato agar and on glucose-aspara- 
gine agar. 

C. Melanin-negat ive. 

This represents ;i fairly large group of or- 
ganisms, widely distributed in nature. S. 
fradia* was first isolated and described by 



L32 



THE ACTINOMYCETES, Vol. II 



Bin, 



Table 17 
■pigmented substances produced by actinomycetes (Kutzner and Waksman, 1959) 



Preparation 


Organism 


Melting point 


Solubility 


Author 


Amylocyanin 


S. coelicolor 


°C 


In water and in dimethylforma- 
mide; insoluble in other sol- 
vents 


Muller, 1908 


Litmocidin 


Nocardia cyarn a 


144-1 4G 


Slightly soluble in water at an 
acid reaction and extracted 
from it by ethanol, ether, or 
amylacetate 


Gause, 1946; 
Brazhnikova, 

1946 


Coelicolorin 


S. coelicolor 


142-140 


Very soluble in acetone, ethyl- 
acetate, or chloroform; solu- 
ble in ethanol, methanol, 
benzene, or ether; insoluble 
in petroleum ether 


Kominami, 1949; 
Hatsuta, 1949 


Cyanomycin 


S. cyanoflavus 


128 


Extracted from water at an 
alkaline reaction by chloro- 
form or methylenechloride 


Funaki el al., 
1958 


Granatacin 


S. olivaceus 


204-20G 


Extracted from water at an 
acid reaction by acetone; 
soluble in ethylacetate, and 
dimethylsulf oxide; insoluble 
in petroleum ether 


Corbaz el al., 
1957 


Actinorhodin 


S. coelicolor 


270 


Soluble in pyridine, piperi- 


Brockmann et al., 






(decomp) 


dine, or phenol; weakly sol- 
uble in dioxane or acetone; 
insoluble in ether, CSo , 
CCU , or petroleum ether 


1947, 1950, 1955 


Streptocyanin 


Streptomyces sp. 


290-300 


Soluble in acetone, dioxane, or 


Tonolo et al.. 






(decomp) 


pyridine 


1954 


Anthocyanin 


S. violaceoruber 




Extracted with hot or cold 
water and dilute alcohol 


Kriss, 1936 


Anthocyanin 


S. coelicolor 




Extracted with hot or cold 
water and dilute alcohol 


Kriss, 1937 


Anthocyanin 


S. violaceoruber 






Frampton and 
Taylor, 1938 


Bydroactino- 


Streptomyces sp., 




Soluble in water 


Kriss, 1936 


chrome 


producing violet 
growth and pig- 
ment 








Lipoactino- 






Insoluble in water 


Kriss, 1936 


chrome 











Waksman and Curtis (1916). Of the two 
neomycin-producing cultures isolated by 
Waksman and Lechevalier in 1949, one 
formed no spirals and thus agreed with the 
original description of the organism; the sec- 
ond produced some spirals of the closed type. 
Differences were also observed in the shade 



of color of the aerial mycelium on synthetic 
media. 

Some of the strains of S. fradiae were 
found capable of producing certain antibi- 
otics, notably members of the neomycin com- 
plex, as well as the antifungal agent fradicin. 
Several other species reported in the litera- 



SERIES AND SPECIES OF GENUS STREPT0M1 ( SI 



i:;;; 



ture appear to be related to the Fradicu 
series. Baldacci ei al. I L953) at firsl did nol 
recognize this organism as representing a 
distincl series, and apparently considered i1 
as a member within the "roseus" series. 
Later, however, Baldacci and Comaschi 
( 1956) gave it scries characteristics. 

Gause ei al. (1957) divided the Fradicu 
series, on the t>asis of spiral formation, into 
two subgroups: one, spiral-forming, com- 
prising .1. roseoflavus; the other, nonspiral- 
forming, comprising .1 . fradiat proper; other 
species and varieties were included in both 
subgroups. Most of the members of a new 
series, designated as "fuscus," could also he 
considered as members of the Fradiae series. 

Waksman and Scotti (1958) divided the 
Fradiae series into three subgroups. These 
were described briefly as follows: 

i. Substrate growth on synthetic media 
thin, smooth, colorless, almost entirely lim- 
ited to the surface of the medium; occasion- 
ally colored orange-yellow. Aerial mycelium 
light pink, seashell-pink, or salmon-colored. 
Some strains produced little if any growth 
on synthetic media. Bes1 sporulat ion took 
place on potato agar and on glucose-aspara- 
gine agar. On organic media, growth was 
smooth to wrinkled, yellowish or orange- 
yellow to orange-brown; aerial mycelium, if 
present, was white to seashell-pink. On cer- 
tain media, a soluble, pink to salmon-colored 
pigment was produced. Morphologically, all 
strains formed a straight aerial mycelium; 
some cultures, however, were able to form 
hooks and loops, and even occasional spirals, 
on certain media. These strains were con- 
sidered as representing typical S. fradiae 
proper. 

I la. On synthetic media, substrate growth 
thin, colorless, limited almost entirely to the 
surface of the medium; aerial mycelium 
white. On organic media, growth cream- 
colored to yellowish; aerial mycelium thin, 
white to grayish-white. On yeast-glucose 
agar, growth orange to brownish to greenish; 



aerial mycelium white. Abundant spirals 
were found in the aerial mycelium. 

tlb. < >n synthetic media, growth very 
poor. < >n organic media, growth generally 
pool'; growth best on yeast-glucose agar. 
Xo aerial mycelium was formed. 

A detailed characterizati if subgroup I 

is given in Table is. Among the other species 
apparently closely related to this section is 
S. kanamyceticus (Okami and Qme- 
zawa l. 

The following species may lie included in 
the Fradiae series: S. fradiae, S. luridus, S. 
albosporeus, perhaps also S. roseus and S. 
fuscus. 

A III. Series Griseus 

< 'haracteristic I 'mix rtu s 

a. Sporophores straight, produced in tufts. 
Spores oval; surface smooth. 

b. Growth colorless to olive-buff. Aerial 
mycelium water-green to grass-green to gray. 

c. Melanin-negative. 

d. Strong proteolytic activities. Produce :i 
variety of antibiotics. 

An organism, under the name of A . grist us, 
was first isolated and described by Krainsky 
iu 1914. Its substrate growth on artificial 
media was colorless; only a small amount of 
yellowish soluble pigment was produced. The 
aerial mycelium was of a green-gray color 
on both organic and synthetic media. When 
the concentration of nitrogen in the medium 
was increased to 0.005 per cent, the aerial 
mycelium became white. The culture was 
only weakly proteolytic. 

Soon afterward, in 1915, Waksman and 
Curtis isolated several cultures of what ap- 
peared to be the same organism, the com- 
parison with krainsky'- description being 
based primarily on the color of the aerial 
mycelium. Since this work was done during 
the years of World War I, Krainsky's 
original strain could not be obtained for com- 
parative studies. The new culture was des- 



134 



THE ACTINOMYCETES, Vol. II 



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SERIES AND SPECIES OF GENUS STREPTOMl CES 



135 



ignated as .1. griseus Krainsky, although 

certain marked differences were observed 
between the 1 WO isolates. 

Since no type culture of Krainsky's organ- 
ism was available for comparison to any 
investigator, all the subsequent descriptions 
were based upon the Waksman and Curtis 
culture, which was distributed to all collec- 
tions in the world. 

In 1919, Waksman amended the descrip- 
tion of Krainsky, as follows: "This organism 
was isolated numerous times from the soil. 
The name .1. griseus was used before by 
Krainsky so that the description of the latter 
is itself an amendment. Although this or- 
ganism was originally identified with the 
organism described by Krainsky under the 
same name (from description only, wiihont 
any actual comparison of cultures), this 
identification should be, therefore, corrected. 
The culture described here possesses a very 
strong proteolytic power, while Krainsky 
stated that his culture was not strong pro- 
teolytically." 

The differences between the two cultures 
can be briefly summarized as follows: .1. 
griseus Krainsky produced a greenish-gray 
to dark gray aerial mycelium, with a green- 
ish-yellow soluble pigment in older cultures; 
growth on potato was grayish, with white- 
gray aerial mycelium; Krainsky never stud- 
ied the morphology of his organism, except 
for the shape (oval) and size of the spores. 
.1. griseus Waksman and Curtis produced a 
water-green to yellowish-green aerial myce- 
lium; the sporophores were straight and were 
formed in tuft-like masses; growth on potato 
was yellowish, wrinkled, and without any 
soluble pigment. 

■n spite i >f these differences, Waksman 
hesitated at first to change the name of the 
culture which he and Curtis first isolated. 
This hesitation was due partly to the faci 
that the organism was found to undergo 
considerable variation upon continued cul- 
tivation on artificial media. The substrate. 



the temperature of incubation, the length of 
the incubation period, the amount and na- 
ture of inoculum, all tended to exert an in- 
fluence upon the morphological and cultural 
characteristics of the organism. At one time 
milk was clotted at 37°C in 2 days and then 
peptonized; at another time, under the same 
conditions, clotting of the milk required ."> 
to (I days; at still another time, the milk 
in some tubes was not clotted at all but was 
rapidly peptonized. There were other recog- 
nizable changes or variations. Drechsler, 
studying the morphology of the Waksman 
and Curtis cult tire, found that the aerial 
mycelium showed proliferation of fertile 
branches at moderately close intervals along 
the axial hyphae, thus suggesting tuft for- 
mation. This phenomenon alone would have 
definitely indicated that the culture should 
have been identified as a distinct species. 

In August 1943, in the laboratories of the 
Department of Microbiology of the New 
Jersey Agricultural Experiment Station, a 
culture was isolated which produced the 
highly important antibiotic designated as 
streptomycin. Upon careful examination, 
this culture was found to be similar to the 
Actinomyces griseus described by Waksman 
and Curtis in 1916. Since, in the meantime, 
Waksman and Henrici had proposed thai the 
generic name for the sporulating forms of 
actinomycetes be changed from Actinomyces 
to Streptomyces, the organism was named 
Streptomyces griseus. This name has been 
universally recognized, since 1944, as the 
official one for the streptomycin-producing 
organism, and has been so designated in nu- 
merous oilier collections throughout the 
world. A detailed description of this species 
was published in 1948 (Waksman, Reilly 
and Harris). 

Baldacci et al. ( 1954 I subjected the Grisi us 

series to a detailed study. They recognized 
that this representative species had come to 
the fore as a result of the important role 
that it played in the production of antibi- 



136 



THE ACTINOMYCETES, Vol. II 



otics. They emphasized that although first 
listed by Krainsky in 1 ( .)14, S. griseus was 
amended and described in detail by Waks- 
man in L919. They further added: 

"If we examine the characteristics given 
by Krainsky we are led to link this species 
with A. viridis. This conclusion appears still 
more logical when we study the coloured 
taldes prepared by Krainsky. However, in 
view of the impossibility of comparing 
Krainsky's original strain and the difficulties 
that would arise if one did not accept Waks- 
man's amendment for a species so generally 
studied in laboratories, it appears advisable 
to take as definite the characteristics speci- 
fied by the American author and given in 
Bergey's Manual .... Numerous strains have 
been isolated by us and compared, with 
satisfactory results, with Waksman's strains. 
. . .There is a considerable body of literature 
dealing with this species which has a faculty 
for mutation." 

Baldacci and Comaschi later (1956) wrote: 

"The examination of Krainsky 's descrip- 
tion and colored pictures would suggest that 
this species belongs to .1. viridis Lombardo 
Pellegrino (1903). The comparison between 
Krainsky's and Waksman's descriptions 
gives evidence — as even Waksman has par- 
tially pointed out — to the difference of 
proteolytic activity and. according to our 
opinion, the very important difference of the 
color of the sporulating colonies which are 
greenish in Krainsky's description. If we ac- 
cept Waksman's correction of the species 
and compare his descriptions with our 
strains, we find a perfect identity. Since it is 
impossible to compare the original strain of 
Krainsky with the others, the acceptance of 
the correction proposed by Waksman offers 
the advantage of maintaining the name 
"griseus" for an actinomycete so largely 
spread oul and studied in laboratories, so 
thai we agree with it according to this mean- 
ing." 

Many other cultures of S. griseus have 



since been isolated from soils, river muds, 
animal excreta, water, dust, and other nat- 
ural substrates, No1 all of them were found 
capable, however, of producing streptomy- 
cin; the majority of these cultures were 
either inactive or produced other antibiot ics, 
such as cycloheximide, grisein, streptocin, 
actinomycin, and candicidin. Some of the 
cultures yielded a mixture of streptomycin 
with other antibiotics. The ability to form 
streptomycin was at first considered as a 
strain, rather than a species, characteristic; 
later, however, it was decided (Waksman, 
1959) to raise S. griseus to the status of a 
series and the streptomycin-producing 
strains to a species status, Streptomyces 
griseus, Waksman and Henrici. 

Several procedures were developed for the 
isolation from natural substrates and for the 
identification of streptomycin-producing 
strains of S. griseus. These methods were 
based on certain physiological properties of 
the organisms and on the nature and ac- 
tivities of the streptomycin formed by them: 

1. Tolerance to fairly high concentrations 
of streptomycin in the medium. When a 
soil or other natural material was plated 
out on a medium containing 50 mg of strep- 
tomycin per liter, the great majority of bac- 
teria and actinomycetes failed to develop on 
the plate. Most of the actinomycete colonies 
were found to be of the S. griseus type. 

2. Ability of certain resistant strains of 
test bacteria to grow in the presence of 
streptomycin. 

:;. Sensitivity to a specific actinophage. 
When cultures of S. griseus are tested for 
their sensitivity to a specific actinophage 
which is active only upon the streptomycin- 
producing species, the inactive forms or those 
producing other antibiotics can be easily 
eliminated. 

4. Utilization of streptomycin-dependent 
strains of bacteria in testing for strep- 
tomycin. When a culture of S. griseus or of 
another organism suspected of producing 



SER] 



AND SPECIES OF GENUS STREPTOMYCES 



L37 



streptomycin was finally selected and grown 
in a liquid medium, the streptomycin-like 
nature of the antibiotic could be established 
by adding the culture filtrate to a nutrient 
broth and inoculating the latter with a strep- 
tomycin-dependenl strain of Escherichia coli 
or of some other bacterium. Growth of the 
bacterium definitely established the fact that 
the unknown antibiotic was streptomycin. 

5. Cross-streaking the unknown cultures 
on a suitable agar medium toward known 
streptomycin-producing cultures. The latter 
exerted only a slight inhibiting effect upon 
the unknown streptomycin-producers. 

Usually some soil or other material is 
plated on ordinary agar media favorable to 
the development of actinomycetes; colonies 
were picked and tested. The S. griseus col- 
onies could easily he recognized by the pale 
green to grayish-green shade of their aerial 
mycelium. A suitable agar medium can also 
he seeded with living cells of a nonpatho- 
genic strain of Mycobacterium tuberculosis 
and various dilutions of soil used for plating 
purposes, due plates are first incubated at 
28-30°C for 2 or :; days, to enable the acti- 
nomycetes to develop. This is followed by 
further incubation of the plates at 37°C for 
the development of the test bacterium. Col- 
onies thai have the capacity of inhibiting 
growth of the bacterium are found to be 
surrounded by clear zones. 

The antibiotic potency of an active culture 
of S. griseus was found to be fairly constant, 
in spite of the ability of the culture to give 
rise to inactive variants. Highly active 
strains tend to retain their relatively superior 
streptomycin-producing potency, whereas 
poor strains usually remain weak producers 
of this antibiotic. For the commercial pro- 
duction of streptomycin, however, it is es- 
sential to select continuously the mosl ac- 
tive strains. 

Since the streptomycin-producing culture 
isolated in L943 was found to be identical 
with the one described by Waksman and 



Curtis, it must be considered in the light of 
that description. The same is tine of die S. 
griseus strains isolated later and found to 
be able to produce grisein, candicidin, vio- 
niycin, and actinomycin. There are certain 
differences in the cultural and biochemical 
properties of the various strains belonging 
to the Grisi us series, especially in theirability 
to produce various antibiotic- and in their 
sensitivity to different phage-. This justifies 
the separation of the group into several dis- 
tinct species. 

The morphological and cultural properties 
of certain cultures belonging to the Griseus 
series are given in Table 19. 

Characterization 

The Griseus series is characterized by cer- 
tain morphological and cultural properties 
that make possible its identification and 
ready distinction from other groups belong- 
ing to the genus Streptomyces. As more and 
more cultures of S. griseus were isolated, it 
became recognized that this is a large series 
of organisms, the members of which vary 
greatly in their physiological properties and 
in their ability to produce various antibiot- 
ics. 

Waksman and Curtis described S. griseus 
as producing on sucrose nitrate agar a thin. 
spreading growth, developing dee]) into the 
medium, at first colorless, then turning olive- 
buff. This color may be lost on successive 
transfers. The aerial mycelium is thick, pow- 
dery, water-green in color. No soluble pig- 
ment was observed; the reverse of the gro\* th 
became brownish in 24 days. ( )n gelatin. 
at 18°C, »S'. griseus produced a greenish-yel- 
low or cream-colored growth developing deep 
into the substrate; the aerial mycelium was 
white-gray with a greenish tinge. There was 
no soluble pigment; liquefaction of the gela- 
tin was rapid. The culture was capable of 
utilizing a variety of carbohydrates, includ- 
ing pentoses, hexoses, sugar alcohol-, and 
organic acids. It was also able to obtain its 



138 



THE ACTINOMYCETES, Vol. II 



Table 19 
Morphological and physiological properties of certain strains and one mutant of Streptomyces griseus 







Synthetic agar 


Glycerol agar 


Gelatin* 


Potato 


Antibi- 


Strain of 


Morphology 














otic 
proper- 
ties 


organism 


Growth 


Aerial 
mycelium 


Growth 


Aerial 
mycelium 


Growth 


Aerial 
mycelium 


1915 W and C 


Sporophores 


Cream-col- 


Powdery, 


Cream -col- 


White to 


Cream-col- 


White to 


ot 


(N. J. strain) 


long, formed 


ored 


water- 


ored 


greenish- 


ored or 


greenish- 






in tufts, no 




green 




yellow 


greenish- 


yellow 






spirals 




color 






yellow 






1915 W and C 


Sporophores 


Cream-col- 


Powdery, 


Cream-col- 


White to 


Greenish- 


Grayish 


++ 


(Holland 


long, formed 


ored, turn- 


water- 


ored 


greenish- 


yellow or 






strain) 


in tufts, no 
spirals 


ing olive- 
buff 


green 
color 




yellow 


cream- 
colored 






1943 Strepto- 


Tufts, no 


Cream-col- 


Powdery, 


Cream to 


Cream-col- 


Greenish 


Greenish 


++ + + 


mycin pro- 


spirals 


ored, turning 


water- 


olive- 


ored 


yellow or 


tinge 




ducer 




olive-buff 


green 
color 


green 




cream- 
colored 






Grisein pro- 


Tufts, no 


Cream-col- 


Light gray 


Cream -col- 


Cream-col- 


Greenish- 


Grayish 


+ + 


ducer 


spirals 


ored 


to green- 
ish 


ored 


ored to 

greenish 


yellow 






Rhodomycetin 


Tufts, no 


Vinaceous 


White to 


Carmine- 


Gray- 


+ + + 


Gray 


++ 


producer 


spirals 




gray 


red 











* Brownish pigment produced by some strains. 
t Mutant of this culture produced an antibiotic 



nitrogen from a variety of compounds, in- 
cluding both inorganic and organic forms. 
In studies of streptomycin-producing 
strains, Carvajal (1946) characterized the 
morphology and life cycle of S. griseus in 
greater detail. The substrate mycelium when 
young is well branched, typically in a mon- 
opodial form. Transverse septa are formed 
in virtually all cases in the delimitation of 
the reproductive cells. Reproduction occurs 
by means of unicellular asexual spores and 
conidia, which are exogenously borne in 
chains on the aerial mycelium. The spores 
are of various shapes: barrel, oval, bean, 
spherical, and cylindrical. Differences in 
shape and size are found often, even 
among the spores of the same chain. Mature 
aerial spores often show small fragments of 
transparent film adhering to the outside wall. 
The spores germinate at one end or at both 
ends, usually from the points at which they 
are attached to the adjacent spores or to the 
hypha. Hyphal fusions and germ tube fusions 
also can be observed. Carvajal reported that 
he had demonstrated a nucleus in the germ 
lubes of S. yrixcKs in the young mycelium 



and in the developing spores. The nuclei 
were said to be well distributed throughout 
the cytoplasm of the mycelium; the spores 
may be uninucleate or multinucleate. 

Gottlieb and Anderson (1947) studied the 
course of spore germination and of develop- 
ment of the mycelium in submerged cultures 
of S. griseus. The exact time of spore germi- 
nation was difficult to determine, only an 
elongation of the spores being observed. 
After 6 hours, the mycelium was found to 
consist of some small hyphae and of longer 
branched hyphae which tended to develop 
into masses of mycelium consisting of a 
dense solid center and a periphery of 
branched radiating hyphae. Within '24 to 
30 hours, the entire body of the medium 
was filled with these mycelial clumps. The 
culture appeared viscous at this stage. After 
48 hours, the mycelium began to fragment, 
and spores were produced. At 84 hours, def- 
inite lysis of the mycelium took place; the 
dense central core of the masses of growth 
disintegrated into granular pieces. 

Measurement of viscosity and weight of 
mycelium revealed an increase which reached 



SERIES AND SPECIES OF GEMS STh'l-l'Ti >\l ) I ES 



139 



a maximum al 24 to •">() hours, followed by 
a decrease up to about 96 hours; a gradual 
Leveling of growth then took place. 

Growth of S. griseus in stationary cultures 
teaches a maximum iu 10 days, whereas 
maximum growth in submerged cultures is 
usually attained in 3 to 5 days. This is fol- 
lowed by lysis of the mycelium. Growth of 
the organism is accompanied by a gradual 
rise in pll value of the culture and in the 
ammonia and amino nitrogen content; the 
total nitrogen in the mycelium tends to be 
higher during the active stages of growth. 
The production and accumulation of strep- 
tomycin parallel the growth of the organism, 
reaching a maximum when lysis just sets in; 
this is followed by a decrease when the rate 
of lysis readies a maximum. 

Metabolism 

The metabolic changes of S. griseus in a 
glucose-peptone-meat extract medium have 
been found by Dulaney and Perlman (1947) 
to fall into two phases. During the first 
phase, the organism grows rapidly and forms 
extensive mycelium; this is accompanied by 
a reduction in the quantity of soluble con- 
stituents in the medium, namely, the nitro- 
gen, the inorganic phosphate, and the avail- 
able carbohydrate; the quantity of lactic 
acid present is first increased and then uti- 
lized to some extent ; the oxygen demand is 
high, and the Q02 values may reach L50; 
little streptomycin is produced; the soluble 
carbon content of the medium during the 
growth phase rapidly falls as the glucose is 
utilized; about 50 per cent of the carbon 
appears to be unavailable to the organism 
during the first stage; the nitrogen content 
of the mycelium varies with age. During the 
second or autolytic phase of growth con- 
siderable lysis sets in; streptomycin is pro- 
duced actively, and the pll of the medium 
rises; the quantity of mycelium is decreased 
as a resull of lysis; the lactic acid content 
remains more or less constant, as does the 



soluble carbon content of the medium; the 
oxygen demand slowly decreases; the am- 
monia nitrogen, soluble nitrogen, and inor- 
ganic phosphate contents of the medium 
rise rather markedly, paralleling die autoly- 
sis of the cells. 

Ammonium compounds, bu1 not nitrates 
are favorable sources of nitrogen for growth 
and streptomycin production. S. griseus 
rapidly assimilates phosphate in a phos- 
phorus-poor medium. An excess of phos- 
phorus lias a depressive elTeel both upon 
growth of the organism and upon strepto- 
mycin production. 

The supplementary addition of amino ac- 
ids or of more complex organic compounds 
has been found to stimulate production of 
streptomycin. Eiser and McFarlane (1948) 
found that, of the amino acids, histidine is 
essential for both mycelial growth and strep- 
tomycin production; inositol also increased 
the yield of both; valine favored the latter. 
and aspartic or glutamic acid the former. 
If the salt concentration is low, most of the 
streptomycin will be found in the mycelium, 
thus suggesting that streptomycin is a prod- 
uct of intracellular synthesis. Woodruff and 

linger ( 1948) reported that yields as high as 
1 g of streptomycin per liter are produced 
by S. griseus in media containing proline 
as the only source of nil rogen. 

The ability of S. griseus to form an en- 
zyme (mannosidostreptomycinase) which de- 
composes mannosidostreptomycin into strep- 
tomycin and mannose has been recently 
demonstrated. This enzyme is not produced 
by other actinomycetes or fungi (Volume I. 
p. 1ST). 

On a dry basis, the mycelium of S. griseus 
contains about 16 per cent ether-soluble 
material and about 37 per cent cold water- 
soluble substances. Little study has been 
made of the specific chemical composition 
of these and other tract ions. 

Stokes and Gunness < L946) grew S. griseus 
in stationary cultures in a nutrienl medium 



140 



THE ACTIXOMYCKTES, Vol. II 



containing 0.5 per cent meat extract and 1 
per cent glucose. The cell material was dried 
and then hydrolyzed by acid or alkali. The 
amino acid composition of this material, on 
a percentage basis of the dry material, was 
as follows: total nitrogen, 9.14; histidine, 
0.84; arginine, 2.90; lysine, 2.13; leucine, 
3.73; isoleucine, 1.49; valine, 3.40; methio- 
nine, 0.55; threonine, 2.33; phenylalanine, 
L.67; tryptophan, 0.62. 

In addition to the two forms of strepto- 
mycin, S. griseus produces several other 
antibiotics. Ether extracts from the myce- 
lium of the organism yield a substance 
designated as streptocin, which is active 
against gram-posit ive bacteria but not 
against gram-negative forms. Another anti- 
biotic, designated as cycloheximide, can be 
isolated by extracting the crude submerged 
culture with chloroform, evaporating the 
extract, and dissolving the residue in meth- 
anol. Cycloheximide is not active againsl 
bacteria but has strong antifungal prop- 
erties; it is particularly active against yeasts. 

Varieties and Mutants 

The Griseus series represents a large' 
widely distributed, and variable group of 
organisms. It has long been recognized 
(Waksman, 1959) that this series should be 
divided into several species. The formation 
of different antibiotics by the various specie- 
offers an excellent supplementary basis for 
such subdivision. The many cultures isolated 
and studied in detail can thus be classified 
into live distinct species. 

1. Streptomyces griseus Waksman and 
Henrici. This comprises strains of *S. griseus 
which produce streptomycin; they also pro- 
duce cycloheximide. 

2. Streptomyces griseinus Waksman. 
St rains of S. griseus which produce grisein or 
grisein-like substances. These strains are as a 
rule resistant to actinophage. Benedict and 
Lindenfelser (1951) demonstrated that a 
majority of streptomycin-producing strains 



of S. griseus form a green soluble pigment 
in calcium malate medium and a yellow 
pigment in calcium succinate medium; on 
the other hand, the grisein-producing strains 
of this organism do not form any green or 
yellow pigments in these media, although 
they show the typical greenish pigmentation 
of the aerial mycelium. 

3. Streptomyces coelieolor (Muller) emend. 
Kutzner and Waksman. This species com- 
prises strains which produce the antifungal 
agent candicidin but no antibacterial sub- 
stance. The first organsim belonging to this 
species was isolated by Muller (1908), and 
designated Streptothrix coelieolor. It produced 
a blue pigment similar to that formed by a 
diphtheroid organism which he called Bacil- 
lus coelieolor. The culture was a typical 
Streptomyces and formed concentric rings in 
its aerial mycelium. It developed well at 
room temperature and at 36°C. It grew on 
gelatin, with gradual liquefaction but with- 
out pigmentation. On agar media containing 
5 to 10 per cent dextrin, but not in glycerol 
media, a brown pigment was formed. The 
culture formed no aerial mycelium on ordi- 
nary agar media, unless serum, glycogen, 
dextrin, or starch was added. When glucose, 
sucrose, arabinose, or other sugars were used, 
no aerial mycelium was formed. 

Muller emphasized two important activ- 
ities of S. coelieolor: It possessed antagonistic 
properties, and it was active against Oiclium 
lactis. Muller was thus one of the first to 
demonstrate activities that were to make the 
whole group of actinomycetes famous. Mul- 
ler also studied the pigment extensively; he 
called it amylocyanin; it is produced best on 
potato media when grown at 30°C, but not 
at 36°C. 

4. Streptomyces californicus Waksman and 
Curtis. This species comprises strains which 
produce viomycin, active against gram-posi- 
tive bacteria only. Burkholder et al. (1955) 
classified these organisms as strains of S. 
griseus var. purpureas. Waksman (1958) 



SERIES AND SPECIES OF l JEN US STREPTOM\ < I S 



111 



Tab: i 20 
son of cultural characteristics of four strains of Streptomyces California - at media 

Burkholdei ■ I 



Agar medium 


Myc ilium 


S. floridae 


fornicus 


S. f>u ni i eu s 




u 1475 


1 tlycerol as- 
paragine 


Substrate 
Aerial 


.'. eto Light 
gray to slighl 

pin pie 

While 


light 
gray to purple 

White 


Purple 
White to light 


light 
gray to slighl 
purple 
W bite to light 


w bite to light yel 

lOW 

W hlte tO light 


i tryp 
tone 


Substrate 

\ei III 


Black with sbght 
purple tinge 

White to slighl 
gray pink 


Gray to slighl 

black 
Slight growth, 

white 


graj ■: 
( (raj to black 

i i ii nk 
to light gray- 


gray-green 
( iray to black 

:', pink 
to slight light 


green 
Light graj yellow 


Starch sj d 
thetic 


Substrate 

\ri ial 


Purple 
White to light 


Light gray to slighl 
purple 

White 


green 
Purple 

Wbitt i" light 


gray-green 
Whiten, i 

to slight purple 
Light gray-green 


Light yellow-gray 
green 


' lalcium malate 


Subst rate 


gray-green 
While 1(1 light 
yellow 


White to light 
yellow 


gray-green 
White 


White 


i Iraj j ellon to 

slight light 

brown 




Aerial 


White 


White 


Light gray-green 


White 


White to slight 


Nutrient 


Substrate 

Aerial 


White to slighl 
light yellow 

White 


While to slight 
light yellow 

White 


White to light 
yellow 

White 


White to light 

gray yellow 

White to slight 

light gray-green 


gray green 
Light graj yellow 

White to slight 
gray-green 



suggested thai they be raised to the status 
of a species within the S. griseus section. A 
further study of this species points to its 
identity to S. californicus, which lias pri- 
ority in species designation. 

.">. Streptomyces chrysomallus (Lindenbein) 
Waksman. These comprise strains which 
produce actinomyein. Welsch et al. (1957) 
studied 51 cultures of Streptomyces for their 
susceptibility to seven actinophages; 13 
of the strains produced actinomyein and 
eight represented nonact inomycin-producing 
strains of S. griseus. Certain actinomycin- 
producing organisms, including the Linden- 
bein culture of S. chrysomallus and a culture 
of S. parvus, were considered to belong to 
the S. griseus section. A del ailed study of the 
actinomycin-producing organisms has re- 
cently been made by Solovieva and Die- 
lova (1960). 

Various other organisms belonging to the 
S. griseus section are able to form at least two 
other antibiotics. One of these, cyclohex- 
imide, is active only againsl fungi, and 
another, streptocin, is active againsl certain 



protozoan-like organisms. No detailed s1 udy 
has as yet been made of these strains in an 
effort to raise them to specie- status. 

There are also those strains of S. griseus 
that produce no antibiotic at all, at leasl as 
far as one is able to detect by available 
methods. 

The streptomycin-producing strains of S. 
griseus give rise readily to mutants. Two 
such mutants have been reported: One was 
a colorless form, producing no aerial myce- 
lium, forming no streptomycin, and sensitive 
to this antibiotic; Dulaney <l al. I L949) re- 
ported, however, on a colorless mutanl that 
produced streptomycin. The other was a 
pigmented mutant, forming pink to vina- 
ceous-colored subsl rate growth and an aerial 
mycelium typical of S. griseus; 1 his mutant 
formed no streptomycin but gave rise to 
another antibiotic (rhodomycetin), which 
was not active against gram-negative bac- 
teria. According to Kutzner I I960), this 
strain shows much similarity, on the basis of 
phage sensitivity and other properties, to 
S. californicus. A detailed study of degenera- 



142 



THE ACTINOMYCETES, Vol. II 



Table 21 

Classification of actinomycin-producing organisms based upon utilization of rhamnose and 
raffinose (Ettlinger et al., 1956) 









Utilization of: 


Actinomycin 


Species 


Reference or origin of strain 
















Rhamnose 


Raffinose 


+ 


S. flavovirens 


Pridham and Gottlieb 


+ 


+ 


+ 


S. flaveolus 


Kurosawa 


+ 


+ 


+ 


S. antibioticus 


Pridham and Gottlieb 


+ 


_ 


+ 


S. antibioticus 


Burkholder et al. 


+ 


- 


X 


S. antibioticus 


orig. (Waksman) 


+ 


- 


X 


S. antibioticus 


NRRL (Raper) 


+ 


- 


c 


S. chrysomallus 


orig. (Lindenbein) 


+ 


- 


- 


S. flaveolus 


orig. (Waksman) 


+ 


- 


I 


S. parvullus 


orig. (Waksman) 


+ 


- 


X 


S. parvus 


NRRL (Benedict) 


+ 


- 


X 




3 ETH strains 


+ 


- 


c 




8 ETH strains 


+ 


- 


X 




ETH 9001 


- 


- 



Table 22 

Grouping of actinomycin-producing organisms (Ettlinger el al., 1956) 



Group 


Melanoid 
pigment 


Spore color 


Actinomycin 
complex 


Authentic strains 


ETH strains 


I 


+ 


cinereus 


X 


S. antibioticus (orig. and 
NRRL) 


3 


II 


+ 


griseus 


X 




1 


III 


- 


cinereus 


/I 

\x 


S. parvullus (orig.) 


1 

2 








c 


S. chrysomallus (orig.) 


17 


IV 




griseus 


|x 


S. parvus (NRRL) 


5 



tion and regeneration of S. griseus was made 
by Williams and McCoy (1953). 

Different varieties of S. griseus vary 
greatly in their cross-resistance and in their 
sensitivity to actinophages. 

It must further he noted that high-yielding 
streptomycin strains can he obtained by 
irradiation, by growth in media containing 
increasing concentrations of streptomycin, 
and by strain selection. 

Okami (1950a) examined 47 strains be- 
longing to the Griseus series. Five of these 
were grisein-forming strains and four were 



pink pigmented forms. The streptomycin- 
producing strains grew in maltose-containing 
media with NaN0 3 as a source of nitrogen, 
but not in glucose, glycerol, or sucrose media. 
The grisein strains grew in media containing 
any of the four carbon compounds. The pink 
strains grew only in glycerol media (Table 
23). The utilization of the carbohydrate was 
found to depend largely on the nitrogen 
source. In the presence of ammonium sulfate, 
the above differences disappeared. All strains 
utilized xylose, but not raffinose or rham- 
nose. Sensitivity to phage was said to be 



SERIES AND SPECIES OF GENUS STREPTOMYCES 



l i:; 



strain specific, bu1 doI characteristic of 
streptomycin production. The use of strep 
tomycin-resistanl and si reptomycin-de- 
pendenl cultures of bacteria as test organ- 
isms for the differentiation of the various 
strains of S. griseus was considered as supple- 
mentary to the foregoing differentiation 
methods. 

It is of particular interest, in this con- 
nection, to draw attention to the contusion 
that lias arisen in some cases from Krassil- 
nikov's attempt to change the name of the 
streptomycin-producing organism. Just as 
Waksman did previously, Krassilnikov came 
to the conclusion that there is a difference 
between the Krainsky and the Waksman 
and Curtis cultures of .1. griseus. Although 
he, likewise, had no opportunity to compare 
Krainsky's original isolate with tin 1 strep- 
tomycin-producing organism, he attempted 
to draw conclusions on the basis of cultures 
that he isolated himself, and proposed that 
the name of the streptomycin-producing 
organism he changed to Actinomyces globi- 
sporus streptomycini (later changed to A. 
sin ploini/rini). This suggested change was 
most unfortunate for several reasons: (a) ;i 
well described specific name, namely Strep- 
tomyces griseus Waksman and Henrici, was 
set aside merely for the sake of priority of ;i 
name of a culture (A. griseus Krainsky) 
which no one had ever seen and which was 
not available in any culture collection; (b) a 
name of an organism that had become rec- 
ognized throughout the world because of its 
important physiological and biochemical 
properties, and especially because of its 
capacity to produce a highly important 
chemical substance, streptomycin, was 
changed to a trinomial merely because of the 
existence of an insufficiently described vari- 
ety of an unknown culture. 

'Hie confusion thus became compounded 
by this attempl to change the name of the 
streptomycin-producing organism. We find, 
in addition to the two names suggested by 



Table 23 
ation of carbon sources In/ various strains of 
Streptomyces griseus with nitrate as soura oj 
nilrogi u (( >kami, L950 

Carbonsource Streptomycin (iris.-in Pink-pig- 

strain strain nu-nt strain 



( Hucose 
' .1\ cerol 
Sucrose 
Maltose 



Krassilnikov, the incorrect name- Actino- 
myces griseus Waksman listed by Koreuiako 
and Nikitina (Shorin, L957), Streptomyces 
griseus Krassilnikov by Znamenskaia et al. 
(1957), Actinomyces griseomycini by Gause 
et al. (1957), and finally Streptomyces glo- 
bisporus streptomycini by Severin and Gor- 
skaia (1957). This confusion was fortunately 
limited to the literature published in Rus- 
sian. 

For the reasons presented here, Krassil- 
nikov's modifications of the name N. grist us, 
with all the subsequent confusing names, 
cannot be accepted. In fact, the actual 
culture, A. griseus (Krainsky) emend. 
Krassilnikov (1949), belongs rather to the 
Cinereus series. 

The logical name for the streptomycin- 
producing species remains Streptomyces 
griseus Waksman and Henrici. 

. 1 tl<l it /Dual Organisms 

Numerous other organisms belonging to 
the Griseus series have been described as 
species and ;is varieties. Some of the descrip- 
tions are incomplete; and it is, therefore, 
rather difficult to give them an exact posi- 
tion. This is true, for example, of the cultures 
described by Gause et al. (1957) under the 
name .1. rubiginosohelvolus and some of the 
other species placed in the series Helvolus. 
See also llarada. L959. 

IV Series Hygroscopicus 

This series comprises organisms that form 
a white to gray aerial mycelium, with a 



144 



THE ACTINOMYCETES, Vol. II 



tendency to become dark gray; frequently 
black patches are produced in the mycelium, 
the whole often becoming black. The sub- 
strate growth is dark gray with a tendency to 
become moist, slimy, and finally changing 
to black. The species are melanin-negative, 
although on synthetic agar a brown to black 
soluble pigment may be produced. 

Morphologically the species give rise to 
spiral-shaped sporophores. This series com- 
prises a number of species, some of which 
are listed: S. endus, S. hygroscopic us, S. 
limosus, S. nigrificans, S. platensis, and S. 
violaceoniger. 

X. Series Scabies 

Characteristic Properties 

a. Sporophores produce spirals. 

b. Aerial mycelium light gray to dark 
gray. 

c. Melanin-positive. 

d. Some species are able to cause diseases 
of plants, notably seal) of potatoes. 

The melanin-producing capacity of certain 
actinomycetes, or their ability to form solu- 
ble brown to black pigments when grown in 
protein-containing media, was first recog- 
nized as a diagnostic characteristic by lvossi- 
Doria and Gasperini in 1891. Numerous 
cultures found capable of producing such 
pigments were isolated from different sub- 
strates, and designated as Streptothrix chro- 
mogena, Actinomyces chromogerms, or .1. 
chromogenes. Gradually it came to be recog- 
nized that all these isolates represented not 
a single species but a large number of organ- 
isms, differing greatly in their morphological, 
physiological, and biochemical properties. 

This was definitely established in 1900 by 
Beijerinck, who isolated two types of actino- 
mycetes (" Streptothrix") represented abun- 
dantly in nature. "One of these that I have 
Learned to recognize in the form of numerous 
varieties, 1 will designate as Sir. chromogena 
Gasperini, since I believe that one such 



variety was available to the author of this 
name. The other species I designate as Str. 
alba." The first was characterized by the 
formation of a brown pigment in meat ex- 
tract-gelatin media. 

Neukirch (1902) demonstrated the pres- 
ence in nature of two chromogenic types of 
actinomycetes. Krainsky (1914) described 
four chromogenic species, whereas Waksman 
and Curtis (1916) demonstrated the occur- 
rence in soil of various other chromogenic 
types, "each with such well defined charac- 
ters as to make it almost impossible to 
classify them as one species." 

One of the most important series among 
the chromogenic actinomycetes is S. scabies, 
which at one time was designated as S. 
chromogenus. In addition to the members of 
the Chromogenus series as such, numerous 
other species now included in other groups 
also possess chromogenic properties, espe- 
cially members of the Lavendulae and Re- 
ticuli series. 

The soluble pigment produced by various 
organisms when grown on protein-containing 
media was found to belong to the melanin 
type. It frequently involved the tyrosinase 
reaction. The intensity of the pigment varies 
with the organism and with the medium. 
The formation of the melanin pigment is 
usually determined by growing the organ- 
isms on tyrosine-containing media. 

Baldacci et al. (1953) did not recognize a 
"Chromogenus" series as such, although they 
listed one under "Cas-Gri." Gause et al. 
(1957) listed two series, one a "Chromogen us" 
proper, and the other "Helvolus," in which 
both pigment-producing and nonpigment- 
producing types are included. 

The organisms belonging to S. scabies were 
at first believed to be primarily associated 
with scabbiness in white potatoes, sugar 
beets, and mangels. Only the typical chro- 
mogens were at first included in this series. 
They formed a brown to dark brown to black 
growth, a gray aerial mycelium, and a brown 



SERIES AM) SPECIES OF GENUS STBEPTOMYCES 



145 



in black soluble pigmenl on organic media. 
It musi be conceded a1 mice thai not all 
organisms isolated from scabby potatoes or 
beets are able to produce a soluble brown 
pigmenl and certainly not all are capable of 
causing scabbiness in potatoes. 

Millard and Burr (1926) isolated a num- 
ber of cultures from scabby potatoes and 
beets. They proposed a key for the identifica- 
tion of the presumably potato-disease-pro- 
ducing actinomycetes (see Volume I, Chap- 
ter IS). '1'he medium -elected tin- this 
purpose (glycerol nitrate solution) could 
hardly be considered the most desirable sub- 
strate for bringing out the proper characters 
for a system of classification. Some of these 
cultures, notably S. clavifer and S. fimbri- 
atus, definitely belong to the Scabies series. 

Baldacci and Spalla I 1950) suggested that 
the strain of S. scabies isolated by Millard 
and Bun- be designated as S. scabies var. 
anglica. It is distinguished from the North 
American species first described by Thaxter 
as having a "gray substrate growth, a gray 
aerial mycelium and a yellow soluble pig- 
ment." 

The possibility that different strains or 
races of S. scabies were responsible for the 
infection of potatoes and mangels has been 
fully recognized. No definite correlation has 
been found, however, between pathogenicity 
and cultural and other properties of the or- 
ganism, although variants may differ from 
the parent culture in pathogenicity. High 
nitrogen contenl of the medium appeared to 
inhibit production of aerial mycelium in the 
parasitic strains but not in the saprophytes, 
of the 20 isolates tested by Schaal I MM 1 1 on 

three different media, six did not produce 
any spirals but 14 did. These spirals were of 
both sinistrorse and dextrorse types. 

Taylor and Decker I L947), in a study of 
14i] isolates obtained from -cabby potatoes, 
beets, and radishes, used the following 
criteria for their classification: acid-fastness; 
starch hydrolysis: formation of dark brown 



surface ring on milk; acidification of milk; 
reduction of nitrate to nitrite; utilization of 
certain sugars, organic acid-, and paraffin; 
gelatin liquefaction; pigment formation 
from tyrosine; and maximum growth tem- 
perature. The only true correlation between 
specific cultural properties and the ability to 
produce typical lesion- of potato scab was 
obtained in the production of a dark brown 
ring of surface growth on milk. 

The following species may be tentatively 
included in the Scabies series: S. scabies, S. 
hawaiiensis, and S. <j<ilti<ri. 

A number of other organisms isolated from 
potato tubers or directly from the soil were 
found capable of causing scab of potatoes 
and must be included in this series. This is 
true, for example, of .1. violaceochromogenes 
described by Krassilnikov (1040), and of .1. 
chromofuscus and .1. prunicolor of Gause <t 
al. (1057). Other closely related forms have 
been described, although pathogenicity tests 
were not made. 

A number of forms that apparently have 
nothing to do with scab formation, but have 
the characteristic properties of the series may 
also be included. 

\l. Series Lavendulae 

Charactt ristic Properties 

a. Sporophores straight or spiral-forming; 
spores oval, smooth surface. 

b. Aerial mycelium colored lavender to 
pale blue. 

c. Melanin-positive. 

This is one of the true chromogenic series 
of the genus Streptomyces. Organisms be- 
longing to the Lavendulae series are widely 
distributed in the soil and are represented 
there by a large number of species and varie- 
ties. Many of them are strongly antagonistic 
and are capable of forming various important 
antibiotics, such as streptothricin. Wood- 
ruff and McDaniel | 1958) reported that 90 
per cent of all the antibiotic- produced by 



1 If. 



THE ACTINOMYCETES, Vol. II 



actinomycetes are streptothricins ; here be- 
long various related compounds, such as 
streptin, streptolin, actinorubin, and anti- 
biotic 136. 

The most important species belonging to 
this series are S. lavendulae and S. venezuelae, 
organisms producing streptothriein and 
chloramphenicol respectively. 

S. lavendulae comprises organisms ex- 
tremely variable in nature. Many of them 
give rise, on cultivation, to different variants 
or mutants. Some of these variants produce 
a blue diffusible pigment on glucose-peptone 
agar; others form a brown pigment. The sub- 
strate mycelium of the blue pigment-forming 
variants is pale blue, with scattered, small 
pinpoint areas of deep blue. Upon complete 
sporulation, the substrate growth becomes 
covered with the characteristic lavender- 
colored aerial mycelium; occasional sunken 
areas have a slightly bluish tinge; the reverse 
of the substrate growth is cream-colored 
except for the small blue spots. Other vari- 
ants produce a colorless to cream-colored 
substrate growth free of any blue pigment 
whatsoever; a brown diffusible pigment ap- 
pears later, and the growth becomes covered 
with thick lavender-colored mycelium. The 
two types of variants are stable in nature. 
Some variants may lose the capacity to 
produce aerial mycelium. 

S. venezuelae, as well, gives rise to a num- 
ber of variants. Two strains were isolated 
and found to be similar to S. lavendulae in 
their cultural and physiological properties, 
although they differed in their ability to 
utilize various carbohydrates. Streptomyces 
venezuelae utilizes arabinose, rhamnose, 
xylose, lactose, and fructose; S. lavendulae 
has either no effect or only a limited effect 
upon these carbohydrates. The former also 
differs from the latter in its sensitivity to 
actinophage and in various serological re- 
actions. 

Streptomyces venezuelae was described as 
having a thin-walled substrate mycelium, 
colorless, hyaline, monopodially branched. 



the hyphae varying in diameter from 0.9 to 
1.8 n and the branches growing to about 150 
ix in length. The aerial mycelium appears 
lavender under the microscope, thick-walled, 
generally not much branched, straight or 
slightly and irregularly curved, not forming 
spirals, individual hyphae arising frequently 
from the primary mycelium at the surface 
of the substrate. The color of the colonies, 
when viewed on agar without magnification, 
is gray to light tan or pink, but not lavender. 
The upper portions of the aerial hyphae 
divide into chains of spores. These are oval 
to oblong 0.4 to 0.9 by 0.7 to 1.6 M - Individ- 
ual spores are colorless at maturity, but in 
mass appear tan to gray when viewed with- 
out magnification. 

Okami (1956) made a comparative study 
of the organisms commonly included in the 
Lavendulae series on the basis of the color of 
the aerial mycelium and certain other char- 
acteristics. He found that eight cultures, 
notably the streptothricin-producing forms, 
possessed the following properties which he 
considered as standard for the series. 

a. Aerial mycelium pink-lavender color 
when grown on yeast extract-glucose 
agar. 

b. Brown pigment when grown on yeast 
extract -glucose agar. 

c. Very sensitive to chlortetracycline and 
chloramphenicol; relatively sensitive to 
streptomycin; relatively resistant to 
neomycin; and resistant to strepto- 
thriein. 

d. Utilize: glucose, galactose, maltose, 
mannose. Do not utilize: arabinose, 
fructose, lactose, mannitol, raffmose, 
rhamnose, sucrose, xylose. 

e. They show certain growth-inhibiting 
effects (Tables 24 and 25). 

On continued cultivation for 40 years on 
artificial media, the original 1915 isolate of 
S. lavendulae (No. 3330) lost many of its 
characteristic propert ies : 

1. It no longer produced any aerial my- 
celium. 



SERIES AND SPECIES OF GENUS STREPTOMYCES 



117 



2. It did qo1 form any dark brown pig- 
menl . 

3. It was now able to utilize fructose, 
mannitol, rhamnose, xylose. 

1. It showed no or very weak antagonistic 
action. 

Od the basis of the above properties, 
Okami divided the S. lavendulcu series into 
10 subgroups ( Table 26). 

Table 24 

Antibacterial activity of different strains of 

Streptomyces lavendulae (Okami, 1050 i 



S. lavendulae 


Inhibition 


zone, test 


bacteria 


strain no. 


E. coli 


Staph, aureus 


B. cereus 




mm 




mm 


mm 


3330 













3440 8 


17 




16 


3 


3440-14 


6 




6 


2 


3516 


18 




17 


2 


3516 W 


10 




1(1 


2 


3530 


16 




16 


2 


3531 







(i 





3532 


27 




23 


18 


3542 


18 




17 


11 


3543 


11 




is 


6 


3568 













3445 













3465 


2 




4 


5 


3544 


11 




'J 


3 


3555 


33 




27 


21 



Baldacci considered S. lavendulae as a 
distinct group. Krassilnikov, however, 
looked upon these organisms as members of 
the "chromogt nus" group. Gause et al. 
created a series under the name "lavt minim - 
roseus" and subdivided it into three sub- 
groups, based upon the formation of a solu- 
ble pigmenl in organic media: (1) The first 
included cultures that formed no soluble 
pigment, such as S. virginiae; (2) The second 
included S. venezuelae, S. lavendulae, and S. 
circulatus, as well as a variety of others, all 
of which formed a brown to black pigmenl 
in organic media; (3) The third produced a 
yellow pigment. As the requirements for the 
group S. lavendulae presented above indicate, 
neither the first nor the third of these sub- 
groups belong to this group. 

Sanchez-Marroquin (1958) found that 
three related species of Streptomyces produc- 
ing a pink to lavender aerial mycelium on 
synthetic media could be distinguished from 
one another as follows: (a) S. fradim pro- 
duces spirals on short branches; (b) S. 
lavendulae forms spirals at the tip of long 
straight branches; (<•) S. venezuelat forms no 
spirals, but only straight sporophores; the 
first is nonchromogenic and the lasl two 
are chromogenic. The Lavendulm series was 



T \ki.k 25 
Effect of composition of medium on reciprocal antagonism between different strains of 
Streptomyces lavendulae (Okami, 1956) 





Inhibiti 


o„ Z one 


of S. 


lavendulae 3516 


Inhibi 


tion zone 


of V 


lavendulai 


Strain number 




Agar media* 






Agar media 














Soya 


YI>\ 








Soya 






















ASP 




Starch 






ASP 










mm 


mm 




mm 


mm 


mm 


mm 




mm 


mm 


3330 


4.0 


5.0 




7.0 


4.0 


3.0 







2.0 


3.0 


3440-8 


17.0 


15. 




20.0 


15.0 
















3440-14 


17.(1 


l.(i 




1S.0 


10.0 


o 













3510 





0.5 







(1 
















3516-W 





2.0 




3.0 


1.0 










(1 





3530 





1.0 




1.0 


1.0 
















3531 


12.(1 


1.0 




5.0 


8.0 


o 







II 





3532 


16.0 


10.0 




2.0 


12.0 


7.0 







10.0 


10.0 



VGA = yeast extract-glucose; ASP = asparagine -glucose. 



148 THE ACTINOMYCETES, Vol. II 

Table 26 
Classification of the lavendulae series into subgroups (Okami, 1956) 

Subgroup Culture No. Characteristics 

A Standard S. lavendulae 

8440-8 a. Aerial mycelium with pink-lavender color on YDA 

3440-14 1). Brown pigment on YDA 

3516 c. Very sensitive to chlortetracycline and chloramphenicol, relatively 

3516-W sensitive to streptomycin, relatively resistant to neomycin, resistant to 

3530 streptothricin 

3542 (1. Utilizes: galactose, maltose, mannose 

3544 Does not utilize arabinose, fructose, lactose, mannitol, raffinose, rham- 

3555 nose, sucrose, xylose 

e. Shows certain inhibiting effects 

B 3330 a. No aerial mycelium 

b. No dark brown pigment 

d. Utilizes fructose, rhamnose, mannitol, xylose 

e. Antagonistic action none or very weak 

C 3531 c. Relatively resistant to chloramphenicol 

D 3532 c. Relatively resistant to chlortetracycline 

e. Antagonistic action none or weak 

E 3543 b. No dark brown pigment 

c. Relatively resistant to chloramphenicol 

d. Utilizes arabinose. fructose, lactose, mannitol, raffinose, rhamnose, su- 
crose, xylose 

e. Antagonistic action none or weak 

F 3568 d. Utilizes arabinose 

e. Antagonistic action none or weak 

G 3465 b. No dark brown pigment 

e. Antagonistic action none or weak 

H 3555 c. Relatively sensitive to streptothricin VI 

d. Utilizes fructose 

e. Antagonistic action none or weak 

I 3625 c. Resistant to chloramphenicol 

3534 d. Utilizes arabinose, rhamnose, xylose 

3534-A e. Antagonistic action none or weak 

J 3651* d. Does not utilize mannose 

3C.52* 

* S. virginiae 

said to include not only S. lavendulae but also sufficient to cite the work of Kuchayeva 

S. cinnamonensis and S. virginiae. (1958). She collected 22 cultures, freshly 

To indicate the confusion in classifying isolated or obtained from different labora- 

members of the Lavendulae series, it is tories and believed to belong to this group. 



SERIES AND SPECIES OF GENUS STREPTOMYCES 1 »!) 

Eighl of the cultures produced straighl to black. On organic media, soluble pigmenl 

sporophores and II formed spiral-shaped is brown to deep brown to olive or purple or 

sporophores. The color of the aerial myce- black. 

limn of the substrate growth varied from The sporophores form spirals. 

yellow to reddish-brown. Some produced a This series is widely represented in nature 

melanin pigmenl and others did not, thus by a number of species, namely: S. virido- 

automatically excluding the lasl as members chromogenes, S. chartrensis, and S. cyaneus. 
of the Lavi ndulcu series. They varied greatly 

in their antagonistic properties, some in- XIV. Series Cinnamomeus 
hibiting the growth of all bacteria and fungi 

tested, and others having no effect either on Characteristic Properties 

gram-negative bacteria or on certain fungi. a. Sporophores produced in verticils. 

The following antibiotics were listed a- b. Aerial mycelium white, yellow, or 

product- of the Lavendulat series, thus sug- pinkish. 

gesting the possible specific differences in c. Substrate growth yellowish to pinkish. 

the series: streptothricin, streptin, ant ismeg- d. Melanin-negative, 

matis factor, antibiotic L36, lavendulin. This series is characterized by the forma- 

actinorubin, pleocidin, ehrlichin, actithiazic tion of verticil-bearing sporophores. The 

acid, antibiotic MD 2428, and grasseriomy- verticils are both primary and secondary. 

cin. Spirals usually are not produced, although 

occasionally some spirals are formed. The 

XII. Series Erythrochr onto genes species within this series are melanin-nega- 



tive, although a purplish pigmenl may be 
produced on certain organic media. The 



This is a large melanin-positive series oi 

organisms. The aerial mycelium is usually 

. •, ,, li • i 'i 111/ aerial mycelium is white to pinkish to cin- 

white with a yellowish or brownish shade to " . , _, ' 

• ., iii • i i i •. • namon-colored. Ihe substrate growth is 

gray with a bluish or greenish shade; it is ,, • , , 

,.' ,,., . , ,,,, , . yellowish to brown to pmkisl 

often reddish to brown. Ihe substrate ..„ . .... 



The species included in this series can be 
listed here: S. hachijoensis, S. fervens, and 
S. cinnamorm us. 



growth is colorless to orange to red or even 
brown to black. Certain species placed in 
this group often produce a greenish-yellow 
or reddish-brown to almost black soluble 
pigmenl on synthetic media, d he sporo- 
phores are straight or produce spirals. Characteristic Pro-pi Hi 

This melanin-positive series comprises a 
large number of species, such as represented 



\A . Series Reticuli 



Sporophores produced in verticils 
Tt or spiral-shaped, on the primary oi 



by the following: S. erythrochr omoaenes, S. 

,'.,. . a . ' secondary aerial hyphae. 

booiliae. and 6. cinereoruber . , . . . .: 

o. Aerial mycelium white to era\ 



XIII. Series \ iridochromoiU'iies 



c. Melanin-positive. 

This is one of the two series within the 
This melanin-positive series of organisms genus Streptomyces which are differentiated 
is characterized by an ash-gray to greenish from the other members of the genus pri- 
to olive-green to bluish aerial mycelium, marily by their morphology. Species within 
The substrate growth is cream-colored to tin- series are characterized by the radial 
brown to greenish to black. Soluble pigmenl arrangement of the sporophores, whereby 
on inorganic media is yellowish to greenish three or more branches originate from ;| 



L50 



THE ACTINOMYCETES, Vol. II 



node, thus forming a "verticil," frequently 
referred to as a "whorl." Very often the 
sporophores may be branched toward the 
end of the sporulating-hyphae, giving the 
appearance of a "broom shaped branch," 
or may give rise to the formation of a 
"cluster," which is to be distinguished from 
a typical verticil of sporophores. The verti- 
cils may be primary or secondary in nature. 
Among the variations of a verticil is the 
formation of tufts, when straight branches 
are grouped together on the aerial hyphae. 

The formation and nature of the verticils 
may be changed with the composition of the 
medium, a phenomenon first reported by 
Waksman and Curtis (1916) and more 
recently by Nakazawa (1955) and Shinobu 
(1955a). Both primary and secondary ver- 
ticils may be formed in the same culture. 

Baldacci (1953) did not recognize this im- 
portant series at all. He gave series recog- 
nition to one of its members, S. rubrireticuli, 
merely on the basis of its pigmentation. 
Gause et al. (1957), following this example, 
included in the "ruber" series one "tuft- 
forming" organism, also on the basis of its 
pigmentation. Later, however, Baldacci 
(1958) suggested separation of the verticil- 
producing organisms into a separate genus, 
Streptoverticillium, as shown elsewhere 
(Chapter 4, Volume I). 

Various investigators have used morphol- 
ogy as a basis for series separation of the 
genus Streptomyces. It is sufficient to list 
here three of them. 

Shinobu (1955a) proposed division of the 
genus as follows: 

1. Monopodial branching. This section has 
been divided on the basis of spiral forma- 
tion. 

2. Verticil formation: 

a. Nitella type. Typical radial branches 
almost equal in length. No spirals 
formed. S. reticuli is given as a typical 
representative. 

b. Anitella type. Radial branches differ 



from each other. Spirals formed with- 
out proper radial symmetry. S. virido- 
chromogenes is representative. 
The S. reticuli subgroup was further di- 
vided into: 

A. Verticils only primary. Sporophores straight. 

Streptomyces verticillatus 
Streptomyces hiroshimensis 

B. Verticils both primary and secondary. Sporo- 
phores form spirals. 

I. Colorless to brownish growth on synthetic 
media. 

1. Good growth on protein media. Spores 
spherical, oval. 

Streptom yces reticuli 
Streptomyces albi reticuli 

2. Poor growth on protein media. Spores 
cylindrical. 

Streptomyces circulatus 
II. Growth on synthetic media pink to red. 
Streptomyces reticuloruber 
Streptomyces griseocarm sus 
III. Growth on synthetic media greenish. 
Streptomyces verticilloviridans 

Solovieva et al. (1957) made a study of 
cultures belonging to the S. reticuli series 
and isolated from Pamir soils. These cultures 
were divided into two subgroups: 
I. S. verticillatus, with straight sporophores 
(primary and secondary). 
II. S. reticuli, with spiral-shaped sporo- 
phores. 

These subgroups showed very little dif- 
ference in their physiological and biochem- 
ical properties. Only one strain, S. rubri- 
reticuli, showed some differences (weaker 
gelat in liquefaction, strong nitrate reduction, 
weak growth on cellulose). From an anti- 
biotic point of view, however, there was a 
marked difference; members of the first sub- 
group showed strong antifungal activity, 
whereas subgroup 11 gave weaker activity 
or none. 

Pridham et al. (1958) divided the verticil- 
forming series into four morphological sub- 
groups: 

1. Monoverticillate, no spirals. 
'2. Monoverticillate, with spirals. 



SERIES AMI SPECIES OF GENUS STREPTOMYCES 



i:,i 



:;. Biverticillate, qo spirals. 

4. Biverticillate, with spirals. 

The following species were included in this 
series: N. reticuli, S. netropsis, S. thioluteus, 

5. griseocameus, and S. verticiUatus. 
Unfortunately, some cultures show both 

straight and spiral-shaped sporophores. 
N'omi (1960) criticized the above system; 
he could not accept the spiral-producing 
tonus among the true verticil types. He con- 
sidered the group to consist of the typical 
"biverticils" comprising both primary and 
secondary elements; the atypical '•mono- 
verticils" comprising primary verticils, 
mixed verticils, and sometimes more com- 
pound poly verticils. He included the follow- 
ing species: S. reticuli, S. hiroshimensis, S. 
albireticuli, S. echimensis, S. griseocarnens, 
S. thioluU us, s. salmonicida, and S. netropsis. 



Series Tfu 
ter 11, ana 
mycetes. 



mophilus is discussed in Chap- 

ng the thermophilic actino- 



Other Possible Series 

In addition to these L6 series of the genus 
Sin ptomyces, other series could he suggested, 
based either on the color of the aerial myce- 
lium or of the substrate growth; ecological 
or physiological criteria have also been pro- 
posed tor series characterization. Certain 
groups, such as the thermophilic forms be- 
longing to the j^enus Streptomyces are in- 
cluded in a special series (Thermophilus) , 
and are discussed elsewhere (Chapter 10), 
since they are definitely related, because of 
their close ecological and physiological re- 
lationships, to the other thermophilic genera. 



Chapter 7 



Classification of Streptomyces Species 



The difficulties encountered by an inex- 
perienced investigator in identifying a 
freshly isolated culture of a Streptomyces with 
previously described species have been 
brought out in the preceding three chapters. 
No wonder an inexperienced worker soon 
becomes discouraged and takes the easy path 
of creating a new species or variety for 
every newly isolated culture. This is es- 
pecially true if such a culture produces an 
antibiotic not previously described or an 
apparently different form of a known anti- 
biotic that he is anxious to patent or on 
which he wishes to establish priority. Among 
the significant factors that have contributed 
to this rash of "new" species are: 

1. Inadequate description of previously 
described species with which comparisons 
are made 

2. Overlapping of the morphological and 
cultural characteristics of strains or species 
previously described. 

3. Variations in composition of the media 
used in describing species. 

4. Failure to recognize natural variability 
of different strains that might be included 
in a single species. 

5. Idiosyncrasies of the particular inves- 
tigator, and his tendency to be either a 
"lumper" or a "splitter." 

In spite of these discouraging aspects of 
the problem of identification of particular 
organisms, classification and characteriza- 
tion of Streptomyces species have recently 
made considerable progress. Though various 
criticisms have been directed toward it, the 



system of classification of actinomycetes 
used in the seven editions of Bergey's Man- 
ual still appears to be the most logical and 
most workable, except for certain modifica- 
tions that are now desirable. In every new 
edition, advantage has been taken of the 
accumulated information to modify this sys- 
tem of classification, especially of the genus 
Streptomyces. In this treatise, a complete re- 
arrangement has been made in classifying 
the species included in the genus Strepto- 
myces, as compared with the last (seventh) 
edition of Bergey's Manual. 

The thermophilic species of Streptomyces 
have been placed in a separate series Thermo- 
philus and transferred to Chapter 11, in 
which all the thermophilic actinomycetes 
are included. Species of Streptomyces iso- 
lated from animal and plant infections, es- 
pecially those for which pathogenicity has 
not been established, have been distributed 
throughout the genus, thus removing the 
need for a major separation of these species 
into saprophytic versus parasitic forms. 
These properties are now given only sec- 
ondary consideration in characterizing spe- 
cies within the genus. It has further been 
recognized and emphasized, time and again, 
that it is most desirable to utilize morpho- 
logical properties in defining and charac- 
terizing species of Streptomyces, though in 
some instances this could not be done with 
any degree of assurance. 

New genera have been created, once cer- 
tain well-defined morphological and physio- 
logical properties suggested its advisability. 



I 52 



CLASSIFICATION OF STREPTOMYCES SPECIES 



153 



Whether this practice should be extended 
and recognil ion thus be given to the capacity 
to form sclerotia or to the ability to form 
verticils by certain Streptomyces species, 
thus placing them in separate genera, re- 
mains to be determined. The author's sug- 
gestion of many years ago thai the structure 
of the sporophores (straight versus spiral- 
forming, closed versus open spirals, tuft- and 
verticil-producing) be used in characterizing 
certain species or species-groups is gaining 
wider recognil ion, though some investiga- 
tors do not consider this a sufficiently con- 
stanl property for the major subdivision of 
the genus and suggest thai it lie left for 
secondary characterization. The shape and 
size of the spores appear to he less signifi- 
cant properties, although the surface* of the 
spores, as delected by the electron micro- 
scope, has been gaining approval. 

Among the most annoying characteristics 
of the genus Streptomyces are: (a) the loss of 
capacity by certain species to produce aerial 
mycelium, and (b) the overlapping property 
on the part of certain species of Nocardia to 
produce an aerial mycelium thai cannot he 
differentiated from that of Streptomyces. It is 
true, however, that cultures of Streptomyces, 
even if they have lost the capacity to pro- 
duce aerial mycelium, can still he recognized 
by the si ructure of their substrate mycelium 
and by certain cultural and physiological 
properties. The latter include the nature of 
their soluble pigments, their ability to 
liquefy gelatin, hydrolyze starch, invert 
sucrose, and coagulate and peptonize milk. 
Some strains that have lost the capacity to 
produce aerial mycelium may regain this 
property if they are grown in sterile soil or 
in special soil media, or are subjected to 
other special treatments. 

The growth of cultures of Streptomyces 
that have lost the capacity to produce aerial 
mycelium is often colorless, though some- 
times pigmented; it is smooth or lichenoid, 
leathery, compact, with a shiny surface. 



Some produce a soluble brown pigment. 
Some are able to form antibiotics. On the 
assumption thai such cultures, because they 
do not form aerial spores, should be con- 
sidered as sterile, Krassilnikov designates 
them as trinomials with a third component 
of the name "sterilis." This is analogous to 
Fungus sterilis among the fungi. Certain 
such species are included in the presenl 
classification; others may be considered as 
typical nocardias and have been transferred 
to that genus. 

Characterization of Streptomyces Spe- 
cies 

Among the properties to receive major 
consideration in describing individual spe- 
cies are the following: 

1. Morphological properties: These include 
formation and nature of substrate (vegeta- 
tive) growth and of aerial mycelium, manner 
of sporulation (spiral formation, verticil 
formation), nature and surface of spores. 

2. Cultural characteristics. These com- 
prise color and color changes of substrate 
growth and of aerial mycelium, and forma- 
tion of soluble pigments on synthetic and 
organic media. The most significant of these 
pigments are the melanins or melanoids pro 
duced in media containing tyrosine or pro- 
teins and peptones. The brown to black 
pigments produced in such media by certain 
species of Streptomyces are said to designate 
melanin-positive as opposed to melanin- 
negative reactions, or tyrosinase-positive 
versus tyrosinase-negative reactions. In view 
of the fact, however, that lew of the older 
investigators tested this read ion in tyrosine- 
free media, it is more desirable to use the 
designations chromogenic (melanin + ) or 
Qonchromogenic (melanin — ). In this case, 
chromogenicity refers specifically to the 
formation of brown to black pigments on 
protein-containing media. 

:;. Physiological and biocht mien/ prop, rtu s. 
These include: proteolytic activities, such as 



154 



THE A.CTINOMYCETES, Vol. II 



gelatin liquefaction, coagulation and pep- 
tonization of milk, and hemolysis of blood; 
utilization of carbon compounds; antagonis- 
tic properties and formation of antibiotics; 
effect of temperature, aeration, and reaction 
upon growth; formation of specific enzymes, 
such as oxidase, lipase, invertase, diastase, 
mannase and protease; reduction of nitrate, 
and formation of H 2 S. There is no sharp line 
of demarcation between cultural and physio- 
logical properties, on the one hand, and be- 
tween physiological and biochemical activi- 
ties, on the other. 

4. Ecology. The ability of the organism to 
cause animal or plant diseases, and its occur- 
rence in a natural environment are important 
characteristics. 

5. Supplementary characteristics. Addi- 
tional characteristics that may be utilized 
for descriptive purposes include: (a) sero- 
logical reactions, (b) phage sensitivity, and 
(c) sensitivity to specific antibiotics. 

In describing the various species, one has 
to depend frequently upon the information 
supplied in published reports, since type 
cultures often are not available. Where the 
desired information is lacking or where the 
description, for various reasons, is inade- 
quate, the species may be placed in the list 
of incompletely described forms. 

Recently there has been a tendency to 
overlook earlier described species, and to 
emphasize and often give new names to 
newly isolated cultures. One cannot con- 
demn this tendency too strongly. While, in 
most cases, it is difficult to establish synon- 
ymy because of a lack of type cultures or a 
possible change in such cultures upon pro- 
longed cultivation on artificial media, every 
effort must be made to give credit to the 
earlier investigator. One has no patience, 
therefore, with those attempts to set aside, 
willfully or unwillfully, older descriptions 
or to consider such organisms as varieties of 
newly isolated and newly named cultures. 

When n culture is freshly isolated, a study 



should be made both of its position in a 
particular group in the genus and of its 
classification as a species. Only by a com- 
bination of properties described under both 
can one determine the identity of the new 
culture. Obviously, no conclusions should be 
drawn that such a culture represents a new 
species merely on the basis of certain super- 
ficial observations, such as a delay in coag- 
ulation and peptonization of milk or in 
liquefaction of gelatin, or because of a differ- 
ence in the intensity of coloration of the 
potato plug, or in the shade of pigmentation 
of growth on a particular medium, or even 
in the degree of curvature of the sporophores. 
Any attempt to create new species on the 
b;ois of such minor variations must be con- 
sidered both unscientific and confusing. The 
tests must be repeated again and again to 
confirm the recorded observations. 

The need for a knowledge of the exact 
composition of the media used for descrip- 
tive purposes can hardly be overemphasized. 
Some species show their most characteristic 
property upon only one particular medium, 
and unless such a medium is used, the prop- 
erties of a new isolate can easily be over- 
looked. S. fradiae, for example, shows the 
characteristic color (seashell-pink) of its 
aerial mycelium upon potato-starch agar. 
Certain synthetic agar media, notably 
nitrate-sucrose, glucose-asparagine, and cal- 
cium malate, are among those which alone 
bring out characteristic properties of certain 
other species. Growth on potato is quite 
characteristic, although the variety of the 
potato, the manner of crop fertilization, and 
other factors may influence the nature of the 
growth of the organism and pigment formed. 

In view of the great interest at the present 
time in the screening programs for antibi- 
otics, when literally many thousands of cul- 
tures are being isolated and tested, there is 
naturally a tendency on the part of some 
investigators to consider the ability of a 
particular culture to form a specific anti- 



CLASSIFICATION OF STREPTOMYCES SPECIES 



I .v. 



property 
ither thai 



biotic as its major characterise 
Hut this capacity is often a s1 fain 
a specie.- characteristic. In view of the mu- 
tational possibilities of such cultures and of 
the marked effeet of composition of medium 
and environmental factors upon the qualita- 
tive nature and quantitative yield of the 
antibiotic, one must consider such a prop- 
erty, at best, as only a secondary character- 
istic and avoid assigning to it an important 
role in creating new species. 

Shinobu (1958b) emphasized again that 
only synthetic media should he used for the 
study of the sporulat ion of the aerial myce- 
lium, notably spiral formation, in Strepto- 
myces. In mosl species, the curvature of the 
spiral is sinistrorse (counterclockwise); in a 
few, dextrorse (clockwise). The diameter of 
the spiral varies from 1.5 to 8.0 n and is not 
a characteristic property, although it is fixed 
for some species. As pointed out previously 
(Chapter 4), three morphological groups 
were recognized: (1) Those forming straight 
or wavy aerial mycelium, (2) Spiral-forming 
types, and (3) Verticil-forming types. 

Other criteria have been suggested. Some 
of these may be utilized for supplementary 
information in describing species and vari- 
eties. A Subcommittee on Act inomycetes of 
the Society of American Bacteriologists 
(Gottlieb, L960) gave careful consideration 
to the various criteria used in describing and 
characterizing species of Streptomyces. They 
came to the conclusion that morphology is 
to be considered as one of the more impor- 
tant criteria. Color of the aerial mycelium 
and carbon utilization are also important. 
Supplementary characteristics are provided 
by the production of IIS, reduction of 
nitrate, and gelatin liquefaction. Such cri- 
teria as color of substrate growth and nitro- 
gen utilization were not considered of suffi- 
cient significance in describing new species. 

Many species, including a number of 
newly isolated forms, have been placed in 
Chapter b"» as incompletely described. ( Mhers 



have been listed as synonyms. Care ha- been 
taken to avoid the creation of many new 
varieties, unless it ha- been fully established 
that such varieties have a sound morpho- 
logical or cultural basis. Whether these 
varieties should be raised to the status of 
species remains to be determined by further 
study. Any such attempt would automat- 
ically lead to the temptation to create new 
species out of mutants, which are unfortu- 
nately altogether too common, whether 
naturally occurring or artificially created. 
for the time being, they may still be con- 
sidered as varieties. 

Classification of (ienns Strt>i>(<ntiyces 

In proposing the present system of clas- 
sification of the genus Streptomyces, the 
following properties have been given the 
greatest consideration: 

1. Morphology of sporulating bodies; size. 
shape, and surface of spores. 

2. Color of aerial mycelium and of sub- 
strate growth. 

3. Color of growth. 

4. Formation of soluble chromogenic or 
melanoid pigments in proteinaceous media. 
This property is used, together with micro- 
morphology, for the major subdivisions of 
the genus. 

o. Formation of soluble pigments in syn- 
thetic media. 

('). Certain biochemical properties, notably 
proteolysis, starch hydrolysis, nitrate reduc- 
tion, formation of II_S, utilization of carbon 
sources, and formation of specific anti- 
biotics. 

This system is a modification of the one 
originally used by Waksman and Curtis in 
L916, variously changed in subsequent years 
by Waksman and by Jensen, and used in 
modified forms in the various edition- of 
Bergey's Manual of Determinative Bac- 
teriology. 

A. Sporophores straight, wavy, or spiral shaped; 
no vert icils. 



156 



THK ACTIXOMYCETKS. Vol. II 



I. Proteinaceous media are not pigmented dec]) 

brown or black; melanin-negative Soluble 

pigment on various media is faint brown, 

pink, red, purple, yellow, blue, or absent. 

1. Soluble pigment only faint yellow or 

faint brown. 

a. Aerial mycelium white, abundant. 
a 1 . Sporophores produce spirals. 

a 2 . Occurs in soil and in certain 
other natural substrates. 
15. Streptomyces albus 
b 2 . Occurs in the sea. 

148. Streptomyces marinus 
b 1 . Sporophores straight. 

a 2 . Sporophores produce broom- 
shaped clusters. 
102. Streptomyces globispo- 
rus 
b 2 . No clusters produced. 
a 3 . Nonproteolytic. 

27. Streptomyces autotro- 
ph/ ClIS 

b 3 . Weakly proteolytic. 

179. Streptomyces orientalis 
c 3 . Strongly proteolytic. 

129. Streptomyces kimberi 

b. Aerial mycelium, white, scant. 

250. Streptomyces willmorei 

c. Aerial mycelium white to light gray. 
a 1 . Sporophores produce compact 

spirals. 

a 2 . Growth colorless. 

18. Streptomyces annular 
tus 
b 2 . Growth colorless to yellow- 
ish. 

92. Streptomyces fungici 
die us 
c 2 . Growth brown to orange- 
brown. 

21. Streptomyces arenat 
d 2 . No growth on sucrose nitrate 

agar. 

22. Streptomyces argenteo- 
lus 

I) 1 . Sporophores form loose spirals. 

40. Streptomyces calvus 
c 1 . Sporophores straight or wavy, 
a'-. Growth on sucrose nitrate 
agar yellowish-brown. 

2. Streptomyces abura- 
Vtensis 
!)-'. ( irowt h gray to greenish. 

60. Streptomyces coroni 
fur in is 



c 2 . Growth yellowish-white; 

utilizes paraffin and rubber. 
68. Streptomyces elasticus 

d. Aerial mycelium white to mouse- 
gray; spores bluish-gray. 

a 1 . Sporophores produce compact 
spirals. 

227. Streptomyces spheroi- 
des 
b 1 . Sporophores in clusters; a few 
compact spirals. 

45. Streptomyces catenulae 
c 1 . Sporophores produce open cork- 
screw spirals. 
a 2 . Soluble pigment yellow. 

a 3 . Growth on synthetic 
media cream-colored. 
167. Streptomyces niveus 
b 3 . Growth on synthetic me- 
dia yellow. 
143. Streptomyces macro- 
sporeus 
b 2 . Soluble pigment tan to 
brown. 

35. Streptomyces caelestis 
d 1 . Sporophores straight. 

111. Streptomyces griseolus 

e. Aerial mycelium white to gray, 
covered with dark humid stains or 
guttation drops. 

a 1 . Sporophores form spirals. 

a 2 . Growth buff to olive-colored. 
189. Streptomyces platensis 
b 2 . Growth colorless. 

123. Streptomyces humidus 
b 1 . Sporophores straight. 

a 2 . Growth on potato cream- 
colored. 

61. Streptomyces crate rife r 
b 2 . Growth on potato slimy to 
black. 
235. Streptomyces tumuli 

f. Aerial mycelium green. 
a 1 . Growth green. 

196. Streptomyces prasinus 
b'. Growth colorless. 

121. Streptomyces hirsutus 
c 1 . Growth red. 

195. Streptomyces prasino- 
pilosus 

g. Aerial mycelium limited, produced 
late; white with tinge of gray. 

97. Streptomyces gardneri 
2. Soluble pigment blue or purple. 



CLASSIFICATION OF STREPTOMYCES SPECIES 



L57 



a. Aerial mycelium white. 

63. Streptomyces cyanofla 
vus 

b. Aerial mycelium while to gray. 

a 1 . Soluble pigmenl produced only 
Oil potato and certain other 
media; pigmenl changes to red 
in an acid and to green in an 
alkaline reaction. 

58. Stri ptomyci s coi licolor 

h 1 . Pigmenl produced on all media, 
red in an acid and blue in an 
alkaline react ion. 

240. Streptomyces violaceo 
ruber 

C 1 . Pigmenl at first yellow red. 

changing to blue or bluish-green. 
190. Streptomyces pluri 
color 
d 1 . Pigmenl purple. 

171. Streptomyces novai <in 
san at 
e 1 Soluble pigmenl bluish to black. 
239. Streptomyces violaceo 
niger 
C. Aerial m\ celium blue. 
a 1 . No spirals formed. 

36. Streptomyces cat rult us 
b 1 . ( )pen spirals. 

241. Streptomyces violaceus 
3. Pigmenl at first green, becoming 

a. Aerial mycelium usually absent. 

237. Streptomyces vt rnt 

b. Aerial mycelium white. 

221. Streptomyces sum/) 
sonii 

c. Aerial mycelium brownish-white to 
brownish-gray. 

L87. Streptomyces phaeovi- 
ridis 

d. Aerial mycelium dark gray, olive 
colored, or grayish-green; sporo 
phores produce spirals. 

244. Streptomyces viridans 
1. Pigmenl yellow to golden yellow. 
a. Growth green to greenish-yellow. 
a 1 . Aerial mycelium weakly de 
veloped ; white or pale yellow . 

212. Streptomyces virgatus 
b 1 . Aerial mycelium gray to dark 
gray. 

125. Strt ptomyci s inU rmt 

ill us 

c 1 . Aerial mycelium scant , white. 



144. Sin ptomyces macula- 
tus 
d 1 . Aerial mycelium cinnamon- 
colored. 

L58. Streptomyces murinus 
b. < irnwt h green to dark green 

1 1. Streptomyces albovin 
dis 
C. < irou t h sulfur yellow. 

a 1 . Aerial mycelium white to pink 

ish. 

81 Si n ptomyci s flavt <>l us 
b 1 . Aerial mycelium light yellow . 
182. Sin ptomyces parvus 
c 1 . Aerial mycelium white to gray 
to reddish gray. 

251. Streptomyces xantho 
phaeus 
d 1 . Aerial mycelium light gray. 

is. Streptomyces cellulosae 
e 1 . Aerial mycelium ash gray. 

L81. Streptomyces parvullus 
f. Aerial mycelium yellowish green 
to sulfur-yellow. 

230. Streptomyces sulpha 

nus 

d. Growth carmine red, reddish brown 
to orange colored to cinnamon dralt. 
a 1 . Aerial mycelium chalk-white. 

166. Sin ptomyces niveoru 
her 
b 1 . Aerial mycelium white to gray. 
a 2 . No aerial mycelium on po- 
tato. 
208. Streptomyces rimosus 
b 2 . Aerial mycelium on potato 
white to gray to black. 
25. Streptomyces aureofa 

I'll IIS 

Closely related form. 

222. Streptomyces saya- 
maensis 

c-. Aerial mycelium on potato 

olive Lull 

57. Streptomyct s > 

c 1 . Aerial mycelium grayish brown. 

220. Streptomyces saha- 

chiroi 

d 1 . Aerial mycelium yellowish gray. 

1 10. Strt ptomyci s griseofla 

rus 

e. ( Irowth cream colored to brown, 
a'. Rapid liquefaction of gelatin. 

7 Sti ■ ptom \ji i s albidofla- 

rus 



158 



THE ACTINOMYCETES, Vol. II 



b 1 . Gelatin slowly liquefied. 

50. Streptomyces chibaen- 
s is 

5. Soluble pigment yellowish to yellow- 
green. 

a. Aerial mycelium white. 

51. Streptomyces chryso- 
mallus 

b. Aerial mycelium white to yellow. 
a 1 . Growth yellowish to green. 

134. Streptomyces lieskt i 
b 1 . Growth yellow, becoming black. 

135. Streptomyces limosus 

c. Aerial mycelium gray. 

a 1 . Growth on sucrose nitrate agar 
yellowish-green. 

85. Streptomyces fiavovi- 
rens 
b 1 . Growth on sucrose nitrate agar 
yellow. 

47. Streptomyces cellulo- 
flavus 

6. Soluble pigment yellowish-brown to 
reddish-brown. 

a. Growth cream-colored. 

a 1 . Sporophores flexible and hooked. 
112. Streptomyces griseolu- 
teus 
b 1 . Sporophores produced in clus- 
ters. 

165. Streptomyces nitrospo- 
reus 

b. Growth has rosy tinge. 

204. Streptomyces ramnaii 

c. Growth yellowish. 

17. Streptomyces ambofa 
dens 

d. Growth has reddish tone. 

191. Streptomyces pluricolor- 

i seeus 

e. Growth becoming red. 

a 1 . Aerial mycelium white. 

70. Streptomyces eryth- 
raeus 
b 1 . Aerial mycelium white to gray 
with greenish tinge. 

205. Streptomyces ramulo- 
sus 

c 1 . Aerial mycelium mouse-gray to 
drab. 

83. Streptomyces flavogri- 
seus 
d 1 . Aerial mycelium white to gray 
to olive-buff. 

224. Streptomyces setonii 



7. Soluble pigment in synthetic media 
brown. 

a. Growth coral-red. 

24. Streptomyces aurantia- 
cus 
1). ( iiowth yellow. 

32. Streptomyces bottro- 
perisis 

c. Growth yellowish-brown 

3. Streptomyces achromo- 
genes 

d. Growth brown to purplish. 

16. Streptomyces althioti- 
cus 

e. Growth black. 

162. Streptomyces niger. 

8. Soluble pigment on potato plug brown 
to brownish-red to reddish-purple. 

a. Growth on potato greenish-colored. 
a 1 . Spirals formed. 

65. Streptomyces diastati- 
cus 
b 1 . No spirals. 

142. Streptomyces lyi/icus 

b. Growth on potato gray; no spirals 
formed. 

42. Streptomyces canescus 

c. Growth on potato yellowish-colored. 
a 1 . Aerial mycelium white. 

87. Streptomyces flocculus 
I) 1 . Aerial mycelium gray to yellow- 
ish. 
a 2 . Growth cream-colored. 

80. Streptomyces fimica- 
rius 
b 2 . Growth yellow brown. 

75. Streptomyces felleus 

d. Growth on potato yellow turning 
white. 

147. Streptomyces marinoli- 

))IOSUS 

e. Growth on potato pink to reddish- 
purple. 

a 1 . Sporophores produce spirals. 
a 2 . Aerial mycelium cinnamon 
to drab-gray. 

170. Streptomyces noursei 
I) 2 . Aerial mycelium gray. 

11. Streptomyces albogri- 
seolus 
I) 1 . Sporophores both straight and 
spiral-forming. 

228. Streptomyces spiralis 
c 1 . Growth on various media yellow- 
orange to brown. 

89 Streptomyces fragilis 



CLASSIFICATION OF STREPTOMYCES SPECIES 
synthetic agar 



I.V.i 



9. Soluble pigmenl 
brown to black. 

a. Growth on potato gray to brown. 

7 t. Stn ptomya s < xfolia 
tus 

b. Growth on potato greenish to black. 

100. Streptomyces gelaticus 

c. Aerial mycelium pigmented green. 

101 . Streptomyces glaucus 
10. No soluble pigment on synthetic media. 

a. ( rrowth yellowish brown. 

160. Streptomyces narbo 

III IIS IS 

h. Growth yellowish to pink to black. 
a 1 . Aerial mycelium abundant . gray. 
23. Streptomyces armilla- 
tus 
h 1 . Aerial mycelium white, with 
pinkish to orange t inge on cer 
tain media. 

77. Streptomyces filamen 

Ins ns 

<•'. Aerial mycelium white to yellow . 
127. Stn ptomyces kanamy 
<i tints 
(I 1 . Aerial mycelium white to pink: . 
1 1"). Streptomyces madurae 
eh Aerial mycelium scant, white 

183. Streptomyces pelletieri 
f. Aerial mycelium while gray to 
black. 

225. Streptomyces somalien 
sis 

g 1 . Aerial mycelium black. 

isi). Streptomya s paraguay 

I IIS IS 

c. Growth yellowish to orange. 

a 1 . Aerial mycelium white to rose- 
colored. 

213. Streptomyces niscofla- 
vus 
b 1 . Aerial mycelium white. 

201. Streptomyces putrificus 
C 1 . Aerial mycelium scant, white to 
grayish-brown. 

'.to. Streptomyces fulvissi 

III IIS 

d 1 . Aerial mycelium orange to pale 
|iink. 

226. Streptomyces spectabi 

Us 
e'. Aerial mycelium yellowish to 
gray. 

Mi. Stn ptnui ijcrs Jlarns 
P. Aerial mycelium has olive tinge. 
122. Streptomyces hominis 



g 1 . Aerial mycelium -■ 
vellow. 



155. Stn ptomyces microfla 
li \erial mycelium white to orange 

colored. 

216. Streptomyces ruin r 
i'. Aerial mycelium gray to mouse 
gray. 

34. Si r< ptomyces cacaoi 

d. ( irowt h yellowish green to cil run 

yellow; aerial mycelium white to 
yellow to pinkish. 

56. Sin ptomya s citrew 

e. Growth colorless to cream colored. 

a 1 . Aerial mycelium scant . white. 
a 2 . Good growl h on milk. 

10 1. Streptomyces gougt ro 
tii 
!•'-. Xo growl h on milk. 

113. Streptomyces griseo 

jjlll II us 

b 1 . Aerial mycelium white to olive 
hull'. 

193. Streptomyces praecox 
c'. Aerial mycelium white, 
a'-'. Acid-sensitive. 

8. Streptomyces albidus 
h-'. Acid-resistant. 

5. Streptomyces acidophi 

his 

d 1 . Aerial mycelium white to gray. 
a-. Sporophores straight . 

240. Streptomyces wedmor 

I lis is 

1)'-. Sporophores produce spirals. 
1!)7. Streptomyces p$< udo 
griseolus 
e 1 . Aerial mycelium sandy lavender 

to dark gray. 

200. Streptomyces rochet 
I' 1 . Aerial mycelium rose-colored. 
215. Streptomyces roseus 
I'. Growth black. 

99. Streptomyces g< dam n- 

s is 

v.. < b'ow t h yellow to olive ocher. 

171. Sin ptomya s oh ■ a 
h. Growth colorless to yellowish to 

olive buff. Aerial mycelium water 

green. 

a 1 . ( ireen and yellow pigmenl - on 

malate and succinate media. 
116. Stn ptomyces griseus 



1(30 



THE ACTINOMYCKTES, Vol. II 



b 1 . No green and yellow pigments 
on malate and succinate media. 
106. Streptomyces griseinus 
i. Growth red or purple. 

39. Streptomyces californi- 
cus 
j. Growth colorless to black. 

a 1 . Aerial mycelium white to brown- 
ish-gray. 

202. Streptomyces pyrido- 
myceticus 
b 1 . Aerial mycelium on synthetic 
media dull gray. 

157. Streptomyces mita 
kacnsis 
k. Growth dark brown. 

a 1 . Sporophores produce spirals. 
a 2 . Aerial mycelium white to 
gray. 

a 3 . Growth on potato has 
green tinge. 
US. Streptomyces halstedii 
b 3 . No green tinge on potato. 
219. Streptomyces rutgers- 
ensis 
b 2 . Aerial mycelium olive-gray. 
169. Streptomyces nodosus 
b 1 . Sporophores straight. 

a 2 . Aerial mycelium gray-white. 
91. Streptomyces fumosus 
b 2 . Aerial mycelium dark gray. 
136. Streptomyces lipmanii 
1. Growth on synthetic media rose to 
gray. 

212. Streptomyces roseodia- 
staticus 
m. Growth cream-colored to yellow or 
yellow-orange. 

a 1 . Aerial mycelium on certain me- 
dia white, moist with dark, 
glistening patches. 

124. Streptomyces hygrosco 
pie us 
b 1 . Aerial mycelium white-yellow 
to brownish-yellow. 

138. Streptomyces longis 
poroflavus 
c 1 . Aerial mycelium white. 

a 2 . Aerial mycelium present on 
protein media. 

H . Streptomyces candidus 
I) 2 . Aerial mycelium absent on 
protein media. 
178. Streptomyces omiyaen- 



d 1 . Aerial mycelium powdery white, 
with yellow tinge. 
a 2 . Little spiral formation. 

10. Streptoniyces albofla- 
vus 
b 2 . Abundant spiral formation. 
43. Streptomyces canus 
e 1 . Aerial mycelium gray. 

20. Streptomyces antimy- 
coticus 
n. Growth colorless to pinkish to 
brown. 

159. Streptomyces naga- 
nishii 
o. Growth orange or red. 

a 1 . Growth yellowish to orange; 
aerial mycelium seashell-pink. 
a 2 . Produces antibacterial (neo- 
mycin) and antifungal (fra- 
dicin) antibiotics. 

88. Streptomyces frail me 
I) 2 . Produces antiviral (luridin) 
agent . 
140. Streptomyces luridus 
I) 1 . Growth rose to red; aerial my- 
celium white. 

a 2 . Growth yellow to red; weak 
proteolysis. 

13. Streptomyces albospor- 
eus 
b 2 . Growth pale pinkish-buff; 
strong proteolysis. 

33. Streptomyces brasilien- 
sis 
c 1 . Growth pale rose to red; aerial 
mycelium weakly developed, 
velvety, rose-white. 

173. Streptomi/ccs oidio- 
sporus 
d 1 . Growth red; aerial mycelium 
black. 

152. Streptomyces melano- 
cyclus 
p. Growth colorless, turning dark. 

69. Streptomyces endus 
q. Growth becoming salmon-pink; 
acid sensitive. 

217. Streptomyces rubescens 
r. Growth green to dark green; aerial 
mycelium whitish to grayish. 

245. Streptomyces viridis 
s. Growth on blood agar brick-red. 

164. Streptomyces nitrifi- 



CLASSIFICATION OF STREPTOMYCES SPECIES 



nil 



II. Proteinaceous media arc pigmented deep 
In-own to black; melanin-positive. 

1 . ( rrowl h colorless on synt het ic media 

a. Aerial mycelium thin, rose-colored. 
a 1 . Spirals produced. 

210. Streptomyces roseo- 

rliininiiij, ,n S 
b 1 . No s])irals formed. 

."ii Sin ptomyct s i innamon 
ensis 
1). Aerial mycelium white with pale 
pink or pale gray tinge. 

131. Streptomyces kitasa 
waensis 

c. Aerial mycelium gray to brown to 
reddish. 

a'. Growth on organic media green 
ish to black. 

175. Streptomyces olivochro- 
mogenes 
h'. Growth dark brown. 

206. Streptomyces resisto 
mycificus 

c 1 . ( Irowt h cream colored. 

163. Streptomyces nigrifa- 

d. Aerial mycelium cot tony, dark 
brown. 

66. Streptomyces diastato- 
chromogenes 

e. Aerial mycelium pale yellow to gray. 

30. Streptomyces blastmy 
ceticus 
2. Growth on synthetic media yellow. 

a. Aerial mycelium white. 

203. Streptomyces rameus 

b. Aerial mycelium white to gray. 

82. Streptomyces flavochro 
mogenes 
C. Aerial mycelium white to yellow. 

Hi Streptomyces cavouren 

S is 

d. Aerial mycelium ash-gray. 

188. Sin ptomyces pilosus 

e. Aerial mycelium mouse-gray to 
green-gray. 

9 1 Streptomyct s galbus 

f. Aerial mycelium hazel brown. 

13!). Streptomyces lucensis 

g. Aerial mycelium olive buff. 

a 1 . Soluble pigment green to olive 
to black. 

233. Streptomyces tenuis 



b 1 . Soluble pigmenl cream-colored 
to golden brown. 

146. Streptomyces margino. 

Ins 

h. Aerial mycelium white wit h patches 

of bluish green on standi media. 

126. Streptomyces ipomoeat 
'■'• ( iiou t h white to gray. 

a. Sporophores produce spirals. 
a 1 . Causes potato scab. 

223. Streptomyces scabies 

b 1 . I )oes not cause potato scab. 
a-. ( rrowl h on potato gray. 

1 lit. Streptomyces ha nun 
ensis 

b'-'. ( irowth on potato orange 
red. 

96. Streptomyces galtieri 

b. Sporophores st raight . 

a 1 . Aerial mycelium while to gray. 
29. Streptomyces bikinien- 

b 1 . Aerial mycelium white, cottony 
156. Streptomyces mi nihil is 

c. Sporophores tend to be straight; 
spirals less marked. 

28. Streptomyces beddardit 
4. (irowth cream- to brown-colored. 

a. Sporophores in (dusters. 

a 1 . Aerial mycelium on nutrient 
agar gray to yellow ish green 

19. Streptomyces antibioti- 
cits 
b 1 . Aerial mycelium on nut rient 
agar ash-gray. 

73. Streptomyces euryther- 
mus 
c 1 . Aerial mycelium on nutrient 
agar white. 

38 sin ptomyces caiusim 

b. Sporophores not in (dusters. 

a 1 . Aerial mycelium white to gray. 
109. Streptomyces griseo- 
rhroiiioiicncs 
b 1 . Aerial mycelium olive gray. 

248. Streptomyces virido- 
genes 
C 1 . Aerial mycelium olive buff to 
water green. 

107. Streptomyces 

hr il II in us 
."). ( irow t Ii red to reddish orange. 

a. Aerial mycelium white. 

200. Streptomyces purpur- 

nsn ,is 



162 



THK ACTIXOMYCKTES. Vol. II 



I). Aerial mycelium white to gray. 

114. Streptomyces griseo- 
ruber 

c. Aerial mycelium gray. 

a 1 . No soluble pigmenl on synthetic 
media. 

95. Streptomyces galilaeus 
I) 1 . Soluble pigment on synthetic 
media light carmine. 

52. Streptomyces cinereo- 
ruber 

d. Aerial mycelium scant; ability to 
produce such mycelium easily lost . 

31. Streptomyces bobiliat 

e. Aerial mycelium pink, with bluish- 
green spores. 

28a. Streptomyces lull us 

6. Growth white to cream-colored. 

98. Streptomyces gpirypha- 
lus 

7. Growth buff to dark brown. 

a. Aerial mycelium gray to dark olive. 

105. Streptomyces gracilis 

b. Aerial mycelium dark gray. 

103. Streptomyces globostts 

c. Aerial mycelium white. 

64. Streptomyces cylindro- 
sporus 

d. Aerial mycelium tan to light brown. 

115. Streptomyces griseovi- 
ridis 

8. Growth on synthetic agar dark green 
to olive-buff. 

a. Aerial mycelium white to light green 
to blue. 

246. Streptomyces virido- 
chromogenes 
1). Aerial mycelium thin, white. 

211. Streptomyces roseocit- 

c. Aerial mycelium pale gray to blue- 
gray. 

49. Streptomyces chartreu 

9. Growth dark brown to black. 

a. Growth on potato orange to orange- 
red. 

a 1 . No aerial mycelium on potato. 
200. Streptomyces purpur- 
eochromogenes 
b 1 . Aerial mycelium scant to none; 
light brownish-gray. 

ISO. Sin plum arcs phaeo 
purpureus 
c 1 . Aerial mycelium on potato 
abundant , gray. 



198. Streptomyces purpur- 
eofuscus 
d 1 . Aerial mycelium on potato pow- 
dery white. 

132. Streptomyces lanatus 
b. Growth on potato brown to black. 
Aerial mycelium on synthetic agar 
white to brownish. 
a 1 . Aerial mycelium abundant. 
a 2 . Spirals formed. 

185. Streptomyces phaeo- 
chromogenes 
b 2 . No spirals. 

153. Streptomyces melano- 
genes 

b 1 . Aerial mycelium on synthetic 
agar slight. 

168. Streptomyces nobori- 
toensis 

10. Growth on synthetic media colorless 
to light orange. 

a. Aerial mycelium gray to cinnamon- 
drab. 

26. Streptomyces minus 

b. Aerial mycelium white to gray. 

192. Streptomyces poolensis 

c. Aerial mycelium olive-buff. 

194. Streptomyces praefe- 
cundus 

11. Growth on synthetic agar whitish- 
yellow to grayish-yellow. 

a. Soluble pigment light yellow. 
a 1 . Aerial mycelium white-gray. 

a'-. Aerial mycelium on potato 
gray. 
231. Streptomyces tana- 
shiensis 
b 2 . No aerial mycelium on po- 
tato. 

78. Streptomyces filipinen- 
sis 
I) 1 . Aerial mycelium olive-colored. 
137. Streptomyces loidensis 

b. Soluble pigment brown to reddish 
brown. 

154. Streptomyces michigan- 
ensis 

12. ( irowth on synthetic agar gray to olive- 
gray. 

44. Streptomyces carnosus 

13. Growth on synthetic media red to 
purple. 

a. Aerial mycelium white to gray. 

71. Streptomyces erythro- 
chromogenes 

b. Aerial mycelium green. 



CLASSIFICATION OF STREPTOMYCES SPECIES 



L63 



6 Stn ptomyct s afghan 
it nsis 
c Aerial mycelium greenish to yellow 
1. Stn ptom yet s acidomy- 
ceticus 
d. Aerial mycelium chalk-white. 

59. Streptomyces collinus 

11. Growth colorless to cream -colored. 

a. Aerial mycelium cottony white 
lavender to vinaceous lavender. 

133. Streptomyces lavendu 

In, 

b. Aerial mycelium grayish pink to 
lavender. ' 

243. Streptomyces virginiat 
C Aerial mycelium white to cream- 
colored. 

172. Streptomyces odorifer 

15. Growth yellow to brown. Aerial myce- 
lium Light tan to pink. 

236. Streptomyces venezuelat 

16. Growth gray to black. 

79. Streptomyces fimbria- 
te 

17. Growth colorless to stone red. 

93. Stn ptomyces fuscus 
lv ( Irowth blue. 

62. Streptomyces cyant us 
B. Sporophores in aerial mycelium form verticils. 
I. Melanin-negative. 
1 . ( irowth yellowish. 

a. Aerial mycelium white to pinkish. 

117. Streptomyces hachi 
joensis 

b. Aerial mycelium greenish yellow. 

:-!7 . Stn ptomyces cat spito 

SUS 

c. Aerial mycelium gray. 

12s. Streptomyces kentuck 



(irowth pink to red 



7ti. Stn ptomyces fervt ns 
'■',. (Irowth yellowish to green to brown; 
aerial mycelium while. 

1 19. Streptomyces mashuen 

4. (irowth colorless on synthetic media; 

aerial mycelium white to lighl cinna 
mon. 

53. Stn ptomyces cinnamo 
metis 

o. (irowth colorless; aerial mycelium 

white. 

55. Stn ptomyct s circula- 
te 



6 Growth colorless to gray; aerial myce- 
lium white to gray. 

150. Streptomyces matensis 
1 1. Melanin posit ive. 

1. Sporophores do not produce any spi- 
rals. 

a < .row I h w bite to cream colored. 

108. Sin ptomyces </< ist <■ 
car m us 

b. ( irowth colorless to yellowish. 
a 1 . Strong proteolytic properties 

a 2 . Aerial mycelium on agar 
media absent or white 
patches. 

151. Streptomyces medioci 
dii us 

1)-. Aerial mycelium on agar 
media white, yellowish to 
gray. 

8 1 Stn ptomya s flavort ti 
cult 

1)'. Weak proteolyt ic act ion. 

a 2 . Aerial mycelium white to 
yellowish 

72. Streptomyces eurocidi- 
cus 
1)-'. Aerial mycelium white to 
pale olive-buff. 

12. Streptomyct s alboniger 

c. ( '.row t h yellowish-brown. 
a 1 . Aerial mycelium white. 

1. Streptomyces abikoen- 

sum 
1)'. Aerial mycelium white with 
yellowish tinge. 

2:54. Streptomyces thioluteus 

d. (irowth dark gray to gray green. 
a 1 . Strongly proteolyt ic. 

238. Streptomyces vt i ticilla 

I us 
h 1 . Weakly proteolytic. 

177. Stn ptomyces olivovt r- 
ticillatus 

e. ( irow t h brow n. 

111. Streptomyces luU 
lie i I In I us 

2. Sporophores produce spirals. 

a. Aerial mycelium none or limited. 
232. Streptomyces /< ndat 
1). Aerial mycelium j ellow to ash 
gray. 

(17. Streptomyces echinatus 
C. Aerial mycelium white. 

9, Streptomyces albireti- 
culi 



Ill I 



THK ACTINOMYCETKS, Vol. II 



d. Aerial mycelium pale vinaceous. 

161. Streptomyces netropsis 

3. Sporophores straight or spiral-shaped. 

207. Streptomyces reticuli 

4. Verticils on secondary branches; 
growth yellowish-red to pink. 

a. Spirals produced. 

218. Streptomyces rub r in - 
ticuli 

b. No spirals formed. 

184. Streptomyces pentati 
cus 

5. Verticils on primary and secondary 
branches. 

a. Growth yellow to brown. 

a 1 . Aerial mycelium grayish-white. 
130. Streptomyces kitasa- 
toensis 



I) 1 . Growth brown to olive-dial). 

176. Streptomyces olivore- 
ticuli 

b. Growth pink. 

120. Streptomyces hiroshi- 
mensis 

c. Growth red to reddish-brown. 

214. Streptomyces roseover- 
ticillatus 
6. Sporophores may also form tufts. 

a. Aerial mycelium greenish-yellow, 
turning gray. 

247. Streptomyces viridofla 
vus 

b. Aerial mycelium white. 

229. Streptomyces spirover 
ticillatus 



C li a i) t e i « 



Description of Species of Streptomyces 



Detailed descriptions of the more im- 
portant, recognizable species of the genus 
Streptomyces are given in this chapter. Most 
of these organisms have been isolated from 
soils, composts, peats, and water basins; 
some have come from dust and food ma- 
terials, from plant disease lesions, and from 
diseased animals and hnnians. Those iso- 
lated from plant disease lesions may or may 
not be the causative agents of such diseases; 
they certainly should be considered on a par 
with the soil-inhabiting forms. In the great 
majority of cases, the cultures isolated from 
diseased animals or from human infections 
as well cannot be considered as the causative 
agents of such diseases, since their patho- 
genic nature has not been established ex- 
perimentally. 

These descriptions vary greatly both in 
the details of the observations reported and 
in the uniformity of treatment of siich ob- 
servations. For many of these observations, 
the author had to depend on other com- 
pilers of the literature, notably Brumpl 
(1939), Lehmann and Neumann (1927), 
Dodge (1935), Krassilnikov (1949), Erikson 
(1935), Ettlinger et al. (1958), and others. 
Unfortunately, one cannot avoid criticizing 
the tendency of certain compilers to de- 
scribe new species, and place others, often 
well recognized and previously described 
forms, as subspecies or as "also belonging 
to this species," or the even worse tendency 
of some classifiers to make certain minor 
variations the basis for establishing varie- 
ties of described organisms. In only a few 
cases was an attempt made to compare newly 



isolated cultures with previously known, 
although unfortunately not always avail- 
able, type cultures. 

Although many of the synonyms have 
been examined, no detailed data are pre- 
sented concerning literature references. 
Additional information can be found in the 
latest edition of Bergey's Manual or in the 
original papers in which the descriptions 
have appeared. 

Because of the growing interest in actino- 
mycetes as producers of antibiotics, numer- 
ous studies of these organisms have been 
made during the last 5 or (> years. Many new 
species and numerous new varieties have 
been described. Old species have been better 
delineated. New systems of classification 
have been proposed. Cooperative experi- 
ments have been carried out. All this ma- 
terial has now been critically examined, and 
much additional information has been in- 
eluded. 

The last edition of Bergey's Manual 
(1 ( .).">7> contains descriptions of lot) Strepto- 
myces species. The number lias nearly 
doubled in the last ."> or (i years, as indicated 
by the descriptions presented here. 

Description of Streptomyces Species 

1. Streptomyces abikoensum Umezawa '/ 
al., L951 (Umezawa, II., Tazaki, T., and 
Fukuyama, S. Japan Med. .1. I: 331 346, 
L951; .1. Antibiotic (Japan) 5: 169 176, 
1952; Okami, Y. ibid. 477-480). 

Morphology: Sporophores straight, short, 
unbranched, bearing chain- of spores. No 
spirals. Certain strains produce verticils. 



166 



THE ACTINOMYCETES, Vol. II 



Sucrose nitrate agar: Substrate growth 
yellowish-brown. Aerial mycelium thin, 
yellowish-white. Soluble pigment yellowish- 
brown. 

Nutrient agar: Substrate growth cream- 
colored to yellow. No aerial mycelium. Solu- 
ble pigment brown. Melanin-positive. 

Gelatin: Growth cream-colored to brown- 
ish. Soluble pigment brown. Liquefaction 
crateriform. 

Milk: Growth brownish. Aerial mycelium 
scant, white. Soluble pigment yellowish- 
brown. Peptonization, but no coagulation. 

Potato: Growth wrinkled, cream-colored 
to brownish. Aerial mycelium yellowish- 
white. Soluble pigment reddish-brown. 

Starch agar: Growth cream-colored to 
yellowish. Aerial mycelium white. Hydroly- 
sis good. No soluble pigment. 

Nitrate reduction: Positive. 

Blood agar: Growth dark cream-yellow. 
Hemolysis strong. 

Egg media: Growth greenish-yellow. No 
aerial mycelium. Soluble pigment reddish to 
violet. 

Cellulose: Not decomposed. 

Carbon utilization: Glucose, maltose, and 
glycerol well utilized. Arabinose, xylose, 
rhamnose, fructose, galactose, mannitol, 
sorbitol, lactose, sucrose, raffinose, and inu- 
lin not utilized. 

H 2 S production: Negative (other strains 
positive) . 

Tyrosinase reaction: Negative. 

Antagonistic properties: Produces an 
antiviral agent, abikoviromycin. 

Habitat: Soil in Japan. 

Remarks: Resembles S. fimicarius and 
S. purpureochromogenes. Gause et al. de- 
scribed a variety of this organism under the 
name of A. abikoensum var. spiralis. The 
above description was based upon strain 
2-1-6. 

* These designations represent the various cul- 
ture collections where the type cultures are de- 
posited. This has been elucidated in Chapter 4, 
p. 78-80. 



Type culture: I. MRU* 3654. 

2. Streptomyces aburaviensis Nishimura 
et al., 1957 (Nishimura, H., Kimura, T., 
Tawara, K., Sasaki, K., Nakajima, K., 
Shimaoka, N., Okamoto, S., Shimohira, M., 
and Isono, J. J. Antibiotics (Japan) 10A: 
205-212, 1957). 

Morphology: Sporophores long and 
straight; no spirals produced. Spores oval. 

Sucrose nitrate agar: Growth yellowish- 
brown. Aerial mycelium well developed, 
velvety, white. Soluble pigment dark yellow- 
ish-brown. 

Glucose-asparagine agar: Growth grayish- 
olive, thin, flat; reverse pale olive. Aerial 
mycelium velvety, almost white, slightly 
grayish. Soluble pigment at first dull yellow, 
later becoming yellowish-brown. 

Starch agar: Growth grayish-yellow- 
brown. Aerial mycelium grayish-white. 
Soluble pigment pale yellow-brown. Hy- 
drolysis weak. 

Calcium malate agar: Growth pale yel- 
lowish-brown. Aerial mycelium thin, white 
to grayish-white. Soluble pigment grayish- 
yellow-brown. 

Nutrient agar: Growth thin, light gray. 
No aerial mycelium. No soluble pigment. 

Milk: Growth grayish- white. Aerial my- 
celium white. Coagulation and peptoniza- 
tion. 

Potato: Growth dull yellow to pale olive. 
Aerial mycelium white to light gray. No 
soluble pigment. 

Gelatin: Positive liquefaction. No soluble 
pigment. Melanin-negative. 

Nitrate reduction: Positive. 

Carbon utilization: Glycerol, dextrin, 
starch, glucose, maltose, galactose, inulin, 
and fructose utilized. Mannitol, arabinose, 
raffinose, a-lactose, inositol, xylose, and 
sucrose not utilized. 

3. Streptomyces achromogencs Okami and 
Umezawa, 1953 (Umezawa, H., Takeuchi, 
T., Okami, Y., and Tazaki, T. Japan. J. 
Med. Sci. Biol. 6: 261-268, 1953). 



DKSCKII'TIOX OF Sl'IX'IKS OF STREPTOMYCES 



L67 



Morphology: Sporophores straight, qo 
spirals. Spores cylindrical. 

Glycerol nitrate agar: Growth colorless to 
brownish. Aerial mycelium scant, white to 
dark grayish. Soluble pigmenl brown. 

Glucose-asparagine agar: Growth yellow- 
ish brown. Aerial mycelium scant, yellowish- 
white. Soluble pigmenl none or slightly 
brown. 

Nutrient agar: Growth wrinkled, elevated, 
colorless to brownish. No aerial mycelium 
or soluble pigmenl . 

Potato: Growth yellowish-brown to 
brownish, fine, wrinkled. Aerial mycelium 
white, powdery. Soluble pigment absent at 
first, later reddish-brown. 

Gelatin: Growth yellowish-brown. Soluble 
pigment slightly brown. Liquefaction very 
weak. Melanin-negative. 

Milk: Surface growth poor. No soluble pig- 
ment. Coagulation and slow peptonization. 

Egg media: Growth reddish-brown, 
wrinkled. No aerial mycelium. No soluble 
pigment. 

Nitrate reduction: Positive. 

Antagonistic properties: Produces an anti- 
viral agent, achromoviromycin. 

Remarks: This culture resembles S. 
diastaticus and S. fimicarius. It is character- 
ized by the brown pigmentation on syn- 
thetic agar only. A strain of this organism, 
which produces the antibiotic streptozotocin, 
was isolated by Vavra et ol. (1959) from a 
soil in Kansas; they have further cultural 
data concerning the original culture and the 
new strain. 

Type culture: [MRU 3730; ATCC 12,767. 

4. Streptomya s acidom ycet icus < >gata < / al., 
L954 (Ogata, K., Miyake, A., and Morimoto, 
A. Japanese Patent No. 204,403, March 5, 
L954). 

Morphology: Sporophores usually do not 
form spirals. Spores cylindrical or oval, 
0.8 to 1.2 by 1.1 to l.S M . 

Sucrose nitrate agar: Growth at firsl 
light yellow, later dark greenish-brown. 



Aerial mycelium greenish to yellow-white. 

Soluble pigmenl slightly violet -colored : 
sometimes absent. 

Glucose-asparagine agar: Growth brown- 
ish-yellow or brownish-red. and partially 
greenish-blue. 

Calcium malate agar: Aerial mycelium 
greenish-white. Soluble pigmenl violet; 
sometimes absent . 

Glucose nutrient agar: Growth brownish, 
partially dark blue. Aerial hyphae gray- 
white. Soluble pigment brownish-black. 

Gelatin: Growth dark green. Aerial my- 
celium greenish-white. Soluble pigment 
greenish-brown. Liquefaction limited. 

Potato: Growth greenish-brown. Aerial 
mycelium at first white, later pinkish-red. 
Soluble pigment dark green. 

Milk: Growth cream-colored, later turning 
light brown. No coagulation. Soluble pig- 
ment light brown. 

Starch: Slow decomposition. 

Tyrosinase reaction: Negative. 

Nitrate reduction: Positive. 

Production of IPS: Positive. 

Carbon utilization: Arabinose, glucose, 
maltose, lactose, salicin, and salts of or- 
ganic acids utilized. Xylose, fructose, raffi- 
nose, inulin not attacked. 

Antagonistic properties: Produces the 
antibiotic acidomycin. 

Remarks: S. acidomyceticus is closely re- 
lated to S. phaeochromogenes, the latter 
forming spirals in gelatin media, but not the 
former. 

Type culture: ATCC 11,611. 

5. Streptomyces acidophilus (Jensen, L928 
Waksman and Henrici, 1 ( .)4S (Jensen, II. L. 
Soil Sci. 25: 226, L928). 

Morphology: Sporophores either few or 
numerous, with sinistrorse spirals. Spores 
oval and spherical, L.O to 1.2 by 1.2 to L.5 

M- 

Agar media: Growth on acid media (pll 
2.0 to 6.0) colorless. Aerial mycelium whitish. 

Sucrose nitrate agar: No growth. 



168 



THE ACTINOMYCETES, Vol. II 



Glucose-asparagine agar: Growth raised, 
somewhat wrinkled, colorless in young 
cultures. Aerial mycelium thin, white at 
first, later gray or yellowish-brown. 

Nutrient agar: No growth. 

Starch agar: Growth at 25°C good, color- 
less. Aerial mycelium abundant, smooth, 
white. Sonic diastatic action. 

Potato: Growth good, raised, folded. No 
discoloration of plug. Melanin-negative. 

Gelatin: Growth after 10 days very scant, 
thin, semitransparent, colorless. Liquefaction 
slow. 

Milk: No growth. 

Nitrate reduction: Trace. 

Sucrose: No inversion. 

Antagonistic properties: Strongly positive. 

Habitat: Soil. 

Remarks: Grows in acid media only, with 
an optimum at pH 3.5 to 4.5. 

6. Streptomyces afghaniensis Shimo et <il. 
1959 (Shimo, M., Shiga, T., Tomosugi, T., 
and Kamoi, I. J. Antibiotics (Japan) 12A: 1, 
1959). 

Morphology: Sporophores form spirals. 

Sucrose nitrate agar: Growth olive- 
colored, with reddish-brown reverse. Aerial 
mycelium pale green to light greenish-gray. 
Soluble pigment brown to reddish-brown. 

Glucose-asparagine agar: Growth olive- 
colored, with reddish-brown reverse. Aerial 
mycelium pale green to light greenish-gray. 
Soluble pigment brown to reddish-brown. 

Calcium malate agar: Growth olive- 
colored. Aerial mycelium pale yellow-orange 
to pale orange. Soluble pigment yellowish- 
brown to reddish-brown. 

Nutrient agar: Growth colorless to olive 
to buff. Aerial mycelium grayish-white. 
Soluble pigment light brown. 

Gelatin: Growth colorless. Aerial my- 
celium white. Soluble pigment brown. 
Medium liquefaction. 

Milk: Growth yellowish-brown. No aerial 
mycelium. Soluble pigment brown to dark 
brown. 



Potato: Growth wrinkled, colorless. Aerial 
mycelium olive to yellowish-brown. Soluble 
pigment yellowish-brown. 

Cellulose: Positive growth. 

Nitrate reduction: Negative. 

Tyrosinase: Doubtful. 

Carbon source: Utilizes rhamnose, raffi- 
nose, and other carbohydrates; does not 
utilize sodium citrate and sodium acetate; 
doubtful growth on dulcitol and sorbitol. 

Antagonistic properties: Produces an 
antibiotic, taitomycin, active upon gram- 
positive bacteria. 

Habitat: Soil in Afghanistan. 

Remarks: Resembles S. colli mis and S. 
erythrochromogenes. 

7. Streptomyces albidoflavus (Rossi-Doria, 
1891, emend. Gasperini, 1894) Waksman and 
Henrici, 1948 (Rossi-Doria, T. Ann. ist. ig. 
sper. Roma, n. s. 1: 399-438, 1894). 

Synonym : A ctinomyces albido-flavus 
Duche, 1934, emend. Krassilnikov, 1949. 

Morphology: Sporophores short, spiral- 
forming, sinistrorse. Spores spherical. 

Glucose-asparagine agar: Growth brown. 
Aerial mycelium white, later becoming 
whitish-yellow. Soluble pigment yellowish. 

Glucose-peptone agar: Growth cream- 
colored, covered with fine white aerial 
mycelium; yellow soluble pigment. 

Tyrosine agar: Growth tine with orange- 
yellow on reverse side; medium becomes 
yellowish to yellowish-rose. 

Gelatin: Punctiform colonies with white 
aerial mycelium on surface. No soluble 
pigment. Rapid liquefaction. 

Milk: Growth rapid, becoming covered 
with whitish aerial mycelium; never fully 
covering the surface; no coagulation; pep- 
tonization begins slowly and is completed in 
13 days; liquid colored yellowish-orange. 

Starch media: Growth cream-colored, 
covered with yellow aerial mycelium. After 
20 days, growth becomes much folded; 
greenish on reverse side; soluble pigment 
slightly amber. Hydrolysis. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 169 

Cellulose: Some growth. thai it grows abundantly on potato. Gause 

Coagulated serum: Cream-colored growth et al. (1957) described a variety of this 

on surface. Aerial mycelium white. Liquefac- organism under the name .1. albidus var. 

fcion rapid. invertens. Ettlinger et al. (1958) considered 

Production of IPS: Negative. ii as a strain of S. griseus. 

Antagonistic properties: Produces strep- ,. .■, , ,, ■ ,■ r \- i 

. . 9. otreptomyces albvreticuh Nakazawa, 

"" 1,nr1 "- , . l'.i.V. (Nakazawa, K. J. Agr. Chem. Soc. 

Habitat: Soil. Japan 29: 644-647; 647-649, 1955). 

Remarks: According to Flaig and Kutz- A , , , ,, , • , ., 

^ B Morphology: Produces spirals m the 

aer (1960), this culture obtained from CBS , .. ., t ., . , .. 

secondary verticils ot the aerial mycelium. 

■s S. coelicolor Miiller. Etthnger et al. < L958) Th(i sporeg jm , cylindrica] () 6 t() 08 by , 4 

considered that Duchess strain ot this , . ,, 

to I .<S lx. 

organism belongs to S. griseus. Q - 4 t ,. ,, ,, • i 

6 sucrose nitrate agar: Growth ilnu, color- 

8. Streptomyces albidus (I)uche, 1934) less; reverse pale ochraceous salmon. Aerial 

Waksman (Duche", .1. Les actinomyces du mycelium white. 

groupe albus. P. Lechevalier, Paris, L934). Glucose-asparagine agar: Growth colorless; 

Morphology: Sporophores form long, later becoming yellow. Aerial mycelium 

open spirals. Spores spherical to oval. white, cottony, later becoming cream-colored. 

Glucose nitrate agar: Growth colorless; Nutrient agar: Growth thin, mouse-gray. 

some drops of colorless guttation. Aerial No aerial mycelium. Soluble pigmenl och- 

mycelium white. Soluble pigmenl yellowish, raceous tawny. 

Peptone agar: Growth colorless. Aerial Potato plug: Growth gray. Aerial my- 

mycelium white; reverse slightly greenish, celium white. Color of plug brown. 

Soluble pigmenl brownish. Gelatin: Liquefaction slow. Soluble pig- 
Potato: Growth flat, colorless. Aerial ment black. 

mycelium white. No soluble pigment. Milk: Growth cream-colored. Peptoniza- 

Gelatin: Growth cream-colored. Rapid tion slow. Soluble pigmenl brown after 24 

liquefaction. No soluble pigment. Melanin- days. 

negative. Starch: Actively diastatic. 

Milk: Growth cream-colored. Coagulation Nitrate reduction: Positive. 

weak; peptonization rapid. Odor cheesy. Production of IPS: Positive. 
Starch: Hydrolysis good. Cellulose: No growth. 
Cellulose: Growth good. Antagonistic properties: Produces euro- 
Fats and waxes: Growth good, according cidin, an antifungal antibiotic. 

to Krassilnikov 1 1949). 

Nitrate: Slow reduction to nitrate. 

( )dor: Strong, earthy. 

Antagonistic properties: According t 

Krassilnikov (1949), it possesses strong l: " L34, 1916; 8: 90, 1919). 

antagonistic activities. Morphology: Sporophores straight, branch- 
Remarks: Closely related to S. albus '"^ wilh V, ''A little tendency to produce 

(Krassilnikov, 1949) ; differs by more delicate spirals. Spores oval-shaped. 

growth, by a reverse that is often yellowish- Sucrose nitrate agar: Growth glossy, 

brown. Also related to S. microflavus, but spreading, colorless, becoming yellowish. 

differs from the form described by Krainsky Aerial mycelium powdery, white, with 

in that its growth is never rose-yellow and yellowish tinge. No soluble pigment. 



10. Streptomyces alboftavus (Waksman and 
Curtis, L916) Waksman and Ilenrici, 1948 
(Waksman, S. A. and Curtis, P. E. Soil Sci. 



170 



THE ACTIXOMYCKTES, V 



Glycerol malate agar: Growth light 
pinkish-cinnamon. Aerial mycelium late, 
white. 

Glucose-asparagine agar: Growth re- 
stricted, much folded, cream-colored with 
sulfur-yellow surface. No aerial mycelium. 
No soluble pigment. 

Nutrient agar: Growth restricted, cream- 
colored. No aerial mycelium. No soluble 
pigment. 

Potato: Growth wrinkled, moist, cream- 
colored. 

Gelatin: Surface growth abundant, color- 
less. Aerial mycelium white or absent. No 
soluble pigment. Slow liquefaction. 

Milk: Surface ring pinkish. No coagula- 
tion; limited peptonization. 

Starch media: Growth thin, spreading, 
yellowish. No aerial mycelium. Good hy- 
drolysis of starch. 

Cellulose: Scant growth. 

Nitrate reduction: Positive. 

Production of H 2 S: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Positive. 

Remarks: Various cultures related to this 
organism have been described under a 
variety of different names. It is sufficient to 
mention A. cremeus, A. griseoloalbus, A. 
flavidovirens, and a variety of the latter, 
fuscus, described by Gause et al. (1957). 
Krassilnikov (1949) considered it as a 
variety of A. flams. 

Type culture: IMRU 3008. 

11. Streptomyces albogriseolus Benedict et 
al, 1954 (Benedict, R. G., Shotwell, 0. L., 
Pridham, T. G., Lindenfelser, L. A., and 
Haynes, W. C. Antibiotics & Chemotherapy 
4: 653-656, 1954). 

Morphology: Sporophores monopodially 
branched, producing short, compact spirals, 
averaging 4 to 6 turns. Spores spherical or 
oval, covered with numerous long, fine hairs 
(PI. II n). 

Sucrose nil rate agar: Aerial mycelium 



white, becoming ash-gray, often with white 
spots. 

Starch agar: Aerial mycelium white to 
dark gray. Hydrolysis. 

Nutrient agar: Aerial mycelium white to 
ash-gray. Melanin-negative. 

Potato: Growth cretaceous to dirty gray- 
ish-white to faint pink. 

Carrot: Vegetative growth white to dirty 
cream; no aerial mycelium. Slant not dark- 
ened. 

Gelatin: Dirty white sediment. Positive 
liquefaction. Not pigmented. 

Milk: Orange-colored ring; partially pep- 
tonized at 14 days. 

Nitrate: Reduction to nitrite. 

Production of H 2 S: Negative. 

Temperature: Good growth at 25-41°C. 
No growth at 50°C. 

Antagonistic properties: Produces a "neo- 
mycin complex." 

Habitat: Soil. 

Type culture: IMRU 3698. 

12. Streptomyces alboniger Hesseltine et al., 
1954 (Hesseltine, C. W., Porter, J. N., 
Deduck, N., Hawk, M., Bohonos, N., and 
Williams, J. H. Mycologia 46: 16-23, 1954). 

Morphology: Sporophores irregularly 
branched, erect to flexuous; no spirals. Ver- 
ticils produced. Spores catenulate, oval, 
0.8 by 1.25 M . 

Sucrose nitrate agar: Growth poor, white. 
Aerial mycelium white to pale olive-buff. 
No soluble pigment. 

Glucose-asparagine agar: Growth black- 
ish-gray. Aerial mycelium white. Soluble 
pigment blackish-gray. 

Nutrient agar: Growth moist, smooth, 
colorless to yellowish, to dark brown or 
black. No aerial mycelium. No soluble 
pigment. 

Starch agar: Growth good. Aerial my- 
celium white to pale olive-buff. Soluble 
pigment black. Good hydrolysis. 

Potato: Growth moist, yellow. Aerial 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



171 



mycelium white. Soluble pigment dark. 
greenish-black. 

Gelatin: Growth fair. Aerial mycelium 
while. Soluble pigmenl lighl yellow. Lique- 
facl ion medium. 

Milk: Surface white ring, with yellow- 
green to lighl yellow-brown below surface. 
Aerial mycelium white. Peptonization slow. 

Cellulose: No growth. 

Production of IPS: Negative. 

Antagonistic properties: Produces puro- 
mycin, an antibiotic active upon certain 
gram-positive bacteria and protozoa. 

Habitat : Forest soil. 

Remarks: Culture is characterized by the 
formation of an olivaceous black soluble 
pigment in some media, such as asparagine- 
glucose agar, but no such pigment is pro- 
duced on certain organic media. 

Type culture: ATCC 12,461. 

13. Streptomyces albosporeus (Krainsky, 
1014) Waksman and Henrici, 1948 (Krain- 
sky, A. Centr. Bakteriol. Parasitenk. Abt. 
[I., 41: 687, 1014; Waksman, S. A. and 
Curtis, II. ]•:. Soil Sci. I: 00, 1916; 8: 90, 
I 'J I!) i. 

Morphology: Sporophores straight, 

branching', with occasional spirals. Spores 
spherical or oval, 0.8 to 1.2 by 1.0 to 1.8m- 

Sucrose nitrate agar: Growth spreading, 
colorless, with pink center, becoming brown- 
ish, vinaceous. Aerial mycelium white, 
covering the whole surface; often none. No 
soluble pigment. 

Glycerol malate agar: Growth rose to 
orange-red. Aerial mycelium white, later 
changing to yellow. No soluble pigment. 

( rlucose-asparagineagar:( Growth wrinkled, 
spreading, red, with colorless margin. Aerial 
mycelium appears late, white. 

Nutrient agar: Small, cream-colored col- 
onies. No aerial mycelium. No soluble 
pigment. 

Starch agar: Growth thin, -pleading. 
transparent, with red tinge. No aerial 
mycelium. Heady hydrolysis. 



Potato: Growth red to brownish-gray. 
No aerial mycelium, or white. Melanin- 
negative. 

Gelatin: Growth yellow, changing to red, 
with hyaline margin. Usually no aerial my- 
celium; when produced, sometimes gray. 
Medium liquefaction. 

Milk: Scant, pink ring. No coagulation; 
no peptonization. 

Cellulose: No growth or scant. 

Nitrate reduction: I'air. 

Production of IPS: Negative. 

Temperature: Optimum :!7°C. 

Antagonistic properties: Positive. 

Habitat : Soil. 

Remarks: Above description is based 
partly upon the isolates of Waksman and 
Curtis, since Krainsky's culture was not 
available. Krassilnikov (1040) considered it 
as a variety of A. ruber. According to 
Ettlinger et al. (1958) this organism should 
be considered as a strain of S. griseus, a 
hardly justifiable assumption. 

Type culture: [MRU 3003. 

14. Streptomyces alboviridis (I)uche, 1934) 
Waksman ( I )uche, J. Pes actinomyces du 
groupe albus. P. Lechevalier, Paris, p. :!I7, 
1934). 

Morphology: According to Krassilnikov 
( 1949), the sporophores produce spirals with 
3 to 4 turns. Spores spherical. 

Glucose nitrate agar: Growth cream- 
colored becoming olive-green. Aerial myce- 
lium white, becoming yellowish-green. Solu- 
ble pigment brownish. 

Glucose-asparagine agar: Growth at first 
white, becoming olive-colored to almost 
dark. Aerial mycelium white to green. Solu- 
ble pigment yellowish. 

Starch agar: Growth cream-colored: re- 
verse brownish-green. Aerial mycelium 
white, becoming green. 

Gelatin: Growth white, becoming green. 
Soluble pigment greenish-brown. Liquefac- 
tion rapid (slow, according to Krassilnikov, 
L949). 



172 



THE ACTINOMYCETES, Vol. II 



Potato: Growth white, becoming brown- 
ish to rust-colored. Plug colored black. 

Tyrosine agar: Growth white with a 
brownish reverse. Soluble pigment brownish. 

Milk: Coagulation and peptonization. 

Coagulated serum: Growth cream-colored. 
No aerial mycelium. No soluble pigment. 
Rapid liquefaction of serum. 

Remarks: This organism is considered as 
a transitional form between S. albas and S. 
viridis. Krassilnikov (1949) considered it as 
a variety of A. viridochromogenes. 

15. Streptomyces albus (Rossi-Doria, 1891; 
emend. Gasperini, 1892) Waksman and Hen- 
rici, 1948 (Rossi-Doria, T. Ann. ist. ig. sper. 
Roma, n. s. 1: 399-438, 1894). 

Synonym: Numerous synonyms of this 
species are found in the literature. They 
belong mostly to the species-group "S. albus.'" 
Many of them are listed in Chapter 6, under 
the corresponding group. 

Morphology: Sporophores produce long 
spirals. Spores spherical to oval. Some strains 
produce, according to Okami, straight sporo- 
phores, depending on the composition of the 
medium. 

Agar media: Growth colorless; may be- 
come yellowish to brown with age. No 
soluble pigment formed, although some 
strains may excrete a brownish substance in 
certain media and under certain conditions. 
Aerial mycelium abundant, white; the shade 
of color varies with composition of medium 
from snow-white to somewhat yellowish. 

Sucrose nitrate agar: Substrate growth 
smooth, colorless. Aerial mycelium cottony 
to powdery; white to snow-white. 

Glucose-asparagine agar: Aerial mycelium 
gray, becoming brownish. 

Nutrient agar: Generally no aerial my- 
celium; chalky white deposit on old colonies. 

Potato: Growth lichenoid, cream-colored. 
Aerial mycelium white. 

Gelatin: Colonies gray. No soluble pig- 
ment. Strong liquefaction. 



Milk: Surface ring cream-colored. Aerial 
mycelium white. Peptonization rapid. 

Starch agar: Aerial mycelium white. 
Rapid hydrolysis of starch in some cultures; 
others show little or no hydrolysis. 

Nitrate: Reduction to nitrite positive. 

Production of H 2 S: Negative. 

Odor: Characteristic, moldy. 

Antagonistic properties: Certain strains 
are active upon gram-positive bacteria. Some 
produce actinomycetin, others form thiolut in 
or endomycin. 

Habitat: Occurs in dust and soil. 

Remarks: The general occurrence of this 
species, the ease of its superficial identifica- 
tion, and the fact that it has been adopted 
as the type species for the genus Strepto- 
myces, justify a more complete characteri- 
zation, as given in Chapter 6. Numerous 
strains of this species, varying in their 
cultural and other properties have been 
reported. Numerous descriptions of closely 
related organisms also are found in the 
literature (Duche). Krassilnikov lists 18 
strains and substrains (A. albus vulgaris, A. 
albus chlamydosporus, etc.). .4. longisporus 
Krassilnikov (1949) and some of the sub- 
strains, like A. longisporus griseus, belong to 
this group. Solovieva and Rudaya (Anti- 
biotiki, 4(6): 5-10, 1959) list a variety fun- 
gatus capable of producing an antifungal 
agent, albofungin. 

Type culture: IMRU 3005. 

16. Streptomyces althioticus Yamaguchi 
et al., 1957 (Yamaguchi, H., Nakayama, 
Y., Takeda, K., Tawara, K., Maeda, K., 
Takeuchi, T., and Umezawa, H. J. Anti- 
biotics (Japan) 10A: 195-200, 1957). 

Morphology: Curved chains or spirals of 
oval spores on ends of aerial sporophores. 
Frequently, tips of aerial mycelium divided 
into tufts of spore chains. 

Sucrose nitrate agar: Growth colorless i<> 
white, later light brown to purplish. Aerial 
mycelium powdery white, later gray. 



DESCRIPTION OF SPECIES OF STPEPTOMYCKS 



17:; 



Aerial mycelium while to 
c pigment. No hydrolysis in 



Glucose-asparagine agar: Growth color- 
less to white, later lighl brown with or 
without dull lighl reddish tinge. Surface 
"lossy. Aerial mycelium scant, white. Light 
brown to dull light reddish-brown soluble 
pigment. 

Starch agar 
gray. No solul 
7 days. 

Glucose-asparagine agar: Growth color- 
less to while, later lighl brown with or with- 
out dull lighl reddish tinge. Surface "lossy. 
Aerial mycelium scant, white. Lighl brown 
to dull lighl reddish-brown soluble pigment. 

Glucose nutrieni agar: Growth lighl 
yellowish-brown. Surface glossy. Aerial 
mycelium white to gray. Soluble pigmenl 
lighl yellowish-brown. 

Potato: ( trowth abundant , lighl yellowish- 
brown. Aerial mycelium white to gray. 

Gelatin: Scant growth. No liquefaction. 
Brown soluble pigmenl . 

Milk: Lighl yellowish-brown surface ring, 
with scant white aerial mycelium. Yellow- 
ish-orange soluble pigment occasionally. 
Peptonization positive. 

Egg medium (37°C) : Growth yellow with 
gray tinge. Aerial mycelium white, later 
lighl purplish occasionally. 

Cellulose: Scant growth with purplish- 
gray aerial mycelium and lighl purplish 
pigmenl . 

Carbon utilization: Abundant growth with 
rhamnose, fructose, galactose, mannitol, and 
glucose; weak growth with xylose, arabinose, 
maltose, sorbitol, and inositol; none or 
very scant with dulcitol, raffinose, and 
inulin. 

Antagonistic properties: Produces anti- 
biotic althiomycin. 

Remarks: Closely related to S. achromo- 
genes and S. rimosus. Spiral formation of 
culture, no nitrile formation, and purplish 
tone of growth and aerial mycelium differ- 
entiate ii from S. achromogenes. Purplish 
tinge of aerial mycelium and growth, soluble 



pigment, no nitrite formation, and no 
cracked surface of the growth differentiate 
it from S. rimosus. 

17. Streptomyccs ambofaciens Pinnert-Sin- 
dico, 1954 (Pinnert-Sindico, S. Ann. inst. 
Pasteur 87: 70:5 707, 1954). 

.Morphology: Sporophores form spirals. 
Spores oval or spherical. 

Sucrose nitrate agar: Substrate growth 
yellow to gray. Aerial mycelium white to 
gray. Soluble pigment weak brownish-yel- 
low. 

Glucose-asparagine agar: Growth yellow, 
covered with white aerial mycelium. Soluble 
pigment weak yellow-brown. 

Calcium malate agar: Growth similar to 
that on sucrose nitrate agar. No soluble 
pigment. 

Potato: Growth clear brown. Aerial 
mycelium powdery gray. Soluble pigmenl 
weakly blown to brownish-red. 

Gelatin: Surface growth yellow; flakes in 
liquefied portion. Medium liquefaction. 
Weak brown-orange pigment in liquefied 
zone. Melanin-negative. 

Milk: No coagulation, partial peptoniza- 
tion in 1 month. Peptonized zone orange- 
brown to red. 

Nitrate: Weak reduction to nitrite in 
synthetic media; none at all in organic 
media. 

Production of IPS: Negative. 

Carbon utilization: Glycerol, arabinose, 
glucose, galactose, levulose, mannose, lac- 
tose, rhamnose, starch, and mannitol well 
utilized. Raffinose, erythritol, dulcitol, and 
sorbitol not utilized. 

Antagonistic properties: Produces two 
antibiotics, congocidin and spiramycin. 

Remarks: Ettlinger et al. (1958) included 
this organism with S. aureofaeiens. 

18. Streptomyces annulatus (Beijerinck, 
1912; emend. Krassilnikov, 1941) Waksman 
( Krassilnikov, N. A. Actinomycetales. [zvest. 
Akad. Nauk, SSSR, Moskau, PHI). 



174 



THE ACTINOMYCETES, Vol. II 



Not A. annulatus Wollenweber, 1920. 

Morphology: Sporophores produce spirals, 
with 3 to 7 turns (sinistrorse) . Spores spheri- 
cal, 0.7 ii. 

Sucrose nitrate agar: Growth colorless, 
Hat, penetrating deep into agar. Aerial 
mycelium white, velvety, growing in the 
form of concentric rings. 

Nutrient agar: Colorless growth. Aerial 
mycelium white, concentric rings less 
marked. Melanin-negative. 

Gelatin: Slow liquefaction. 

Milk: Positive coagulation and slow 
peptonization. 

Starch: Hydrolysis. 

Cellulose: Growth good. 

Invertase: Positive. 

Production of H 2 S: Negative. 

Odor: Strong, earthy. 

Antagonistic properties: Highly antag- 
onistic to mycobacteria and gram-positive 
bacteria; some strains are active against 
fungi. 

Habitat: Soil. 

Remarks: Krassilnikov (1940) considers 
this organism as a variety of S. albus. 

Type culture: IMRU 3307. 

19. Streptomyces antibioticus (Waksman 
and Woodruff, 1941) Waksman and Henrici, 
1948 (Waksman, S. A. and Woodruff, H. B. 
J. Bacteriol. 42: 232, 240, 1941; see also 
Waksman, S. A. and Gregory, F. J. Anti- 
biotics & Chemotherapy 4: 1050-1056, L954). 

Morphology: Sporophores straight, long, 
arranged in clusters or broom-shaped bodies; 
usually not wavy and no spirals; some 
strains may produce a few spirals. Spores 
nearly spherical to somewhat elliptical, 
smooth (PL 11 1). Capacity to produce aerial 
mycelium may be lost upon continued 
cultivation on artificial media (PI. V Kb). 

Sucrose nitrate agar: Growth cream- 
colored to yellowish, tending to darken in 
reverse. Aerial mycelium light to mouse- 
gray, with white patches. Soluble pigment 
t'.iint yellowish to yellow to dark. 



Glucose-asparagine agar: Growth cream- 
colored, with yellowish to orange to dark 
reverse. Aerial mycelium light to ash-gray. 
Soluble pigment absent or yellow to brown- 
ish. 

Calcium malate agar: Growth colorless to 
yellowish. Aerial mycelium white to white- 
gray. 

Nutrient agar: Growth brownish, thin. 
Aerial mycelium yellowish-gray to yellow- 
ish-green. Soluble pigment brown to dark. 
Melanin-positive. 

Potato: Growth thin to heavy, lichenoid; 
brownish to orange in color, sometimes 
olive-green. Aerial mycelium absent or 
thin to patchy, white or gray. Soluble pig- 
ment brownish to dark; absent in many 
cultures. 

Gelatin: Growth yellowish to brown to 
dark brown. Aerial mycelium as patches of 
white to gray. Soluble pigment black. 
Liquefaction at first very slow, later be- 
coming more rapid. 

Milk: Thick surface ring, brownish. Aerial 
mycelium mouse-gray with greenish tinge. 
No coagulation, but gradual peptonization. 
Soluble pigment brownish to black. 

Production of H>S: Positive. 

Tyrosinase: Negative. 

Antagonistic properties: Marked antag- 
onistic effect on bacteria and fungi. Produces 
actinomycin A, the first crystalline anti- 
biotic ever isolated from an actinomycete 
culture. 

Source: Isolated from soil on Escherichia 
coZi-washed plate, using living cells of E. 
coli as the only source of available nutrients. 
Later also isolated from a variety of dif- 
ferent soils. 

Remarks: Ettlinger ct al. (1958) included 
in this group S. bikiniensis, S. cinereoruber, 
S. eurythermus, and S. ipomoeae. Krassil- 
nikov (1949) included this species with A. 
parvus. 

Type culture: IMRU 3435. 

20. Streptomyces antimycoticus Waksman 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



IT.') 



(Leben, ('., Stessel, G. J., Keitt, G. W. 
Mycologia 11: L59 169, L952). 

Morphology: Sporophores with spirals 
situated typically in dense groups. Spirals 
lend to be open, becoming closed and com- 
pact prior to the formation of spores. Spores 
«»val, 0.6 to L.3 by 0.7 to 2.0 n. 

Sucrose nitrate agar: Substrate growth 
at first white, later gray. Aerial mycelium 
abundant, light neutral gray. No soluble 
pigmenl . 

Glycerol malate agar: Aerial mycelium 
abundant, lighl neutral gray. Soluble pig- 
menl taint green. 

Nutrient peptone agar: Growth shiny, 
cream-colored. Aerial mycelium moderate, 
pebbly, white. No soluble pigment. Melanin- 
uegative. 

Potato-glucose agar: Aerial mycelium 
abundant, neutral gray. Soluble pigment 
faint, brown. 

Yeast extract agar: Aerial mycelium 
abundant, neutral gray. No soluble pigment. 

Starch agar: Aerial mycelium abundant, 
white to neutral gray. No soluble pigment. 
Diastatic action weak to moderate. 

Potato: Growth finely wrinkled, cream- 
colored. Aerial mycelium sparse. Plug dark- 
ened slightly. 

Gelatin: Growth translucent, cream- 
colored. Aerial mycelium sparse, white. 
Liquefaction slight at 15 days, moderate 
at 30 days. No soluble pigment. 

Milk: King cream-colored. Coagulation; 
peptonization in 15 to 30 days. Yellowish- 
orange pigmentation. 

Nitrate reduction: Slight. 

Antagonistic properties: Produces an anti- 
fungal agent, helixin. 

21. Streptomyces <ir<n<n Grundy, L954 
(Grundy, W. E. Brit. Pat. 71<),2:;o, Dec l, 
1<)54*). 

Morphology: Monopodia] branching of 
mycelium. Sporophores terminate in tight 
spirals. Spores spherical to oval. 

■ Supplemented by personal communication. 



Sucrose nitrate agar: Growth wrinkled, 
yellow, turning dark orange-brown with age. 
Aerial mycelium grayish-white. Soluble pig- 
ment light yellow-brown. 

Calcium malate agar: Growth cream- 
colored, turning bright reddish-brown with 
age. Aerial mycelium fluffy, cream-colored 
turning gray with pink tinge. Soluble pig- 
ment light pink. Complete dissolution of the 
calcium malate. 

Glucose-asparagine agar: Growth sparse, 

golden brown; a few tufts of white aerial 
mycelium. Soluble pigment yellow. 

Nutrient agar: Growth moderate, golden 
brown. Aerial mycelium gray-white, -pore- 
turning darker gray. Soluble pigment light 
brown. 

Potato: Growth abundant, golden brown, 
turning dark brown. Aerial mycelium fluffy, 
becoming on sporulation dark gray. Potato 
dark gray, turning black. 

Gelatin: Heavy gray pellicle on surface. 
Liquefaction slow. Soluble pigment deep red- 
brown diffusing through the liquefied zone. 
Medium liquefaction. 

Milk: Heavy pellicle. Milk digested in 2.") 
to 28 days with formation of curd just before 
complete digestion. Soluble pigmenl dark 
brown, turning black in MO days. 

Starch: Hydrolysis slow. 

Nitrate: No reduction. 

Carbon utilization: Good growth with 
xylose, glucose, mannose, galactose, lactose, 
maltose, sucrose, starch, mannitol, glycerol 
sodium acetate, sodium citrate, and potas- 
sium sodium tartrate. Sorbitol and calcium 
lactate not utilized. 

Antagonistic properties: Produce- an anti- 
biotic active upon Mycobacterium tuberculo- 
sis. 

Habitat : Illinois soil. 

22. Streptomyces argenteolus Perlman, 
L957 (Perlman, D. I'. S. Patent 2.7<) , .>.7<>:>. 
October 7, 1958 . 

Morphology: Aerial mycelium hyaline, 
generally branched, not forming loop- or 



176 



THE A.CTINOMYCETES, Vol. II 



spirals; individual filaments arc rarely sep- 
tate. Sporophores straight, flexuous, or 
fascicled (in tufts). Spores are oval to oblong, 
1.0 to 1.2 ix. The spore color is light gull-gray. 

Sucrose nitrate agar: Xo growth. 

Nutrient agar: Growth colorless, abun- 
dant, spreading. Aerial mycelium white. No 
soluble pigment. 

Oatmeal agar: Growth good. Aerial myce- 
lium limited, no sporulation. Soluble pig- 
ment slight maize-yellow. 

Casein digest-meat extract agar: Growth 
abundant, dark olive-buff. Aerial mycelium 
well developed, pale smoke-gray. No soluble 
pigment. 

Gelatin: Rapid liquefaction. Melanin-neg- 
ative. 

Milk: Positive coagulation and peptoniza- 
tion. 

Potato: Growth good, creamy-buff, cere- 
briform. Aerial mycelium white; no sporula- 
tion. No soluble pigment. 

Starch: Hydrolyzed. 

Nitrate reduction: Positive. 

Carbon utilization: Mannitol, d-xylose, 
Z-arabinose, /-rhamnose, d-fructose, treha- 
lose, and lactose utilized. No growth or very 
scant growth with inositol, sorbitol, meli- 
biose, sucrose, and dextrin. 

Habitat: Soil. 

Biochemical activities: Certain strains of 
this organism convert progesterone to 10 
a-hydroxyprogesterone. 

2:!. Streptomyces armillatus Mancy-Cour- 
tillet and Pinnert-Sindico, 1954 (Mancy- 
Courtillet, I), and Pinnert-Sindico, S. Ann. 
inst. Pasteur 87: 580-584, 1954). 

Morphology: Aerial mycelium produces 
spirals. 

Glucose- or glycerol-asparagine agar: 
Growth yellow-gray. Aerial mycelium poorly 
developed, white. 

Sucrose nitrate agar: Growth very poor, 
colorless. No aerial mycelium. 

Glucose nitrate agar: Growth pool'. No 
soluble pigment. Xo reduction of nitrate. 



Glucose-peptone agar: ( rrowth very good, 
yellow-gray. Aerial mycelium poorly devel- 
oped, white. Soluble pigment weak rose, 
becoming brownish. Melanin-negative. 

Potato: Growth good, yellow-gray. Aerial 
mycelium poorly developed. No soluble 
pigment. 

Tyrosine medium: Growth flat, yellow- 
gray, becoming beige. Aerial mycelium 
white. Xo soluble pigment. 

( lelatin: Growth in form of pellicle. Aerial 
mycelium white. Soluble pigment rose- 
brown. Rapid Liquefaction. 

Milk: Growth in form of surface ring, 
gray to yellow. Peptonized portion colored 
yellow. Coagulation and rapid peptoniza- 
tion. 

Starch: Xo hydrolysis. 

Antagonistic properties: Produces oxytet- 
racycline. 

Remarks: This organism can be classified 
with the S. bobiliae-S. erythreus group, al- 
though its growth is yellow rather than red. 
It grows poorly upon synthetic media, upon 
which it forms no aerial mycelium. It does 
not reduce nitrate to nitrite. It does not 
produce a purple pigment upon egg media. 
It docs not change the reaction of milk to 
alkaline. It does not hydrolyze starch. It 
differs from S. rimosus and S. griseoflavus, 
which produce yellow to brown pigments; 
S. armillatus under the same conditions does 
not form any pigment. 

24. Streptomyces aurantiacus (Rossi- 
Doria, 1891 emend. Gasperini, 1892; emend. 
Krassilnikov) Waksman (Krassilnikov, X. 
A. Actinomycetales. Izvest. Akad. Xauk. 
SSSR, Moskau, p. 36, 11)41). 

Morphology: Produces an abundance of 
chlamydospores. Sporophores form spirals 
with 3 to 5 turns. Spores spherical to oval. 
0.7 to ().<) by ()A\ to 0.8 m. 

Agar media: Growth lichenoid, dry, com- 
pact; colored bright orange or golden. Pig- 
ment insoluble in medium, but soluble in 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



177 



organic solvents. Aerial mycelium poorly 
developed. Melanin-negal ive. 

Potato: Soluble pigmenl lirown. 

Gelatin: Growth yellow to orange-yellow 
to deep orange. Liquefaction none or slow. 
No aerial mycelium. 

Milk: Surface growth orange. No coagula- 
tion; unchanged or weak peptonization. 

Starch: Slow hydrolysis. 

( lellulose: No growth. 

Nitrate: No reduction. 

[nvertase: None. 

Fats: Hydrolysis and utilization rapid. 

Paraffin: Growth good, with spiral-form- 
ing sporophores and spherical spores. 

Pigment: Red-orange, extracted with 96 
per cent alcohol. The orange pigmenl was 
dissolved in petroleum ether, the red pig- 
menl being insoluble (Kriss). 

Antagonistic properties: Strongly antago- 
nistic to gram-positive bacteria. 

Habitat: Soil, dust. 

Remarks: Some strains deposit ferric 
oxide on the surface of the hyphae. 

25. Streptomyces aureofaciens Duggar, 
I'll!) (Duggar, B. M. Ann. N. Y. Acad. Sci. 
51: 177. 1948; U.S. Patent 2,482,055, Sept, 
14. 1949 . 

Morphology: Sporophores monopodially 
branched, flexuous, producing open spirals. 
Spores spherical to oval, smooth (PI. II m). 

Sucrose nitrate agar: Substrate growth 
only. Occasionally faint brownish pigmenl 
produced. 

Glucose-asparagine-mea1 extracl agar: 
Growth hyaline, changing to orange-yellow 
or purplish-brown. Aerial mycelium, if 
present, white, changing to ash-gray or dark 
gray with tawny reverse. Fainl yellowish 
soluble pigmenl occasionally discernible. 

Nutrient agar: Growth good, translucenl 
In brownish. No aerial mycelium. No soluble 
pigmenl . Melanin-negal ive. 

Potato: Growth lichenoid, lighl orange- 
yellow to brown-red to purplish. No aerial 
mycelium. Color of plug unchanged. 



Gelatin: Cream-colored surface ring. Liq- 
uefaction none to limited. No soluble pig- 
menl . 

Milk: Growth limited, yellow-brown sur- 
face. Coagulation and peptonization variable 
(often none, occasionally presenl I. 

Production of IPS: Mostly uegative. 

Antagonistic properties: Produces chlor- 
tetracycline, an amphoteric compound 
containing both nitrogen and non-ionic 
chlorine, active againsl various bacteria, 
rickettsiae, and the larger viruses. The 
organism also produces, especially in a 
chlorine-poor medium, tetracycline. The 
presence of phosphorus in the medium in- 
fluences not only growth but also antibiotic 
production ( Prokofieva-Belgovskaya and 
Popova, 1 a")!)). 

Habitat: Soil. 

Remarks: The numerous natural and 
induced variants of S. aureofaciens display 
wide variations in color of substrate growth, 
ranging from pale yellow to reddish-brown, 
and even occasionally greenish, depending 
upon the composition of the nutrient sub- 
strates and environmental conditions (Dug- 
gar et al., 1954). Color of aerial mycelium 
is influenced by sporulation. Ettlillger et nl. 
I 1958) included S. ambofaciens in tin- group. 

Type culture: IMRU 3550; ATCC L0/762. 

26. Streptomyces aureus (Waksman and 
Curtis, 1916) Waksman and Henrici, 1948 
(Waksman, S. A. and Curtis, R. K. Soil Sci. 
1: 24, 1916; 8: «.»7. 1919). 

Morphology: Aerial mycelium forms 
sporophores with numerous closed spirals; 
some strains produce flexible sporophores 
with open spirals. Spores spherical to oval, 
0.6, to 1.0 by 0.8 to 1.1m (Fig. 32). 

Sucrose nitrate agar: ( rrowth thin, spread- 
ing, colorless, becoming dark brown. Aerial 
mycelium thin, powdery, mouse-gray, be- 
coming cinnamon-drab. No soluble pigment. 

Malate-glycerol agar: Growth cream- 
colored, with surface almosl black. Aerial 
mycelium lighl brown. No soluble pigment. 



178 



THE ACTINOMYCETES, Vol. II 



Glucose-asparagine agar: Growth light 
orange; raised center, hyaline margin. Aerial 
mycelium light drab. 

Nutrient agar: Growth restricted, gray. 
No aerial mycelium. Soluble pigment deep 
brown. 

Starch agar: Growth thin, transparent, 



spreading. Aerial mycelium buff-colored. 
Good hydrolysis. 

Potato: Growth abundant, wrinkled, 
brown, becoming black. Aerial mycelium 
white to ash-gray. Soluble pigment black. 

Gelatin: Surface growth fair, cream-col- 
ored, becoming brown. Aerial mycelium 




Figure 32. Sporophores of S. aureus, X 30,000 (Courtesy of E. Baldacci, University of Milan, Italy). 



>ESCRIPTION OF SPECIES OF STREPTOMYCES 



179 



absenl or white. Brown soluble pigment. 
Liquefaction rapid, later slowing down. 

Milk: Black ring. Limited coagulation and 
peptonization. 

Nitrate: Reduction to nitrite with certain 
carbon sources. 

[nvertase: None to positive. 

Temperature: Optimum 25 C. 

Antagonistic properties: Produces poly- 
enes, substances active againsl various 
fungi. Some -trains produce luteomycin. 

Habitat: Soil. 

Remarks: Cultures under this name were 
described by DuBois-Severin in L895, by 
Lachner-Sandoval in 1899, and by Sartory 
in 1923. Yamaguchi and Saburi (1955) re- 
ported that the S. aureus culture obtai 1 

from collections produces straighl aerial 
hyphae and no spirals when grown on var- 
ious synthetic media. Okami and Suzuki 
(1958) isolated two strains that produced 
spirals. Ettlinger et al. (1958) considered 
this organism as a strain of S. griseus. Kras- 
silnikov (1949) considered ii as a variety of 
.1 . Ilnrus. 

Type culture: IMRU 3309. 

27. Streptomyces autotrophicus Takamiya 
and Tubaki, 1956 (Takamiya, A., and 
Tubaki, K. Arcli. Mikrobiol. 25: 58 64, 
L956). 

Morphology: Sporophores alternately or 
irregularly branched, breaking up into 
Spores; no spiral formation. Spores colorless 
with smooth surface; varying in shape from 
ellipsoid to long ovoid or cylindrical; usually 
2.5 to 4.:S by 0.5 to 0.8 n, sometimes smaller, 
1.5 by 0.3 ju- 

Nitrate, carbohydrate-free, agar: Aerial 
mycelium powdery and snow-white in ap- 
pearance, consisting of a tough mycelial felt ; 
thicker at central area than at periphery. 
Reverse side of growth wrinkled and pale 
yellowish. No soluble pigment. 

Calcium malate agar: Growth much 
folded, and raised in central area, cream- 
yellow at earlier stages of development; 



reverse side pale brownish. Aerial mycelium 

white. 

Nutrient agar: ( rrowth much folded and 
raised; reverse side relatively smooth and 
pale brownish. Aerial mycelium snow-white. 
In old culture-, a faint brown tint in agar 
layer immediately beneath growing colony. 

Malt agar: Growth irregularly wrinkled 
and folded; reverse side wrinkled and pale 
yellowish. Production of spores rather poor. 

Starch agar: Growth -canty; no hydroly- 
sis. 

Potato: Growth colorless, much folded, 
with thick central area and thin periphery, 
pale brownish. 

( relatin : No liquefaction. 

Milk: Thin white pellicle formed on sur- 
face. Reverse side yellowish. No coagulation. 
No soluble pigment. 

Cellulose: Not decomposed. 

Nitrate reduction : None. 

Habitat: Originally found on the surface 
of phosphate buffer solution left unused in a 
laboratory in Tokyo. Conceivably, it was 
derived from atmospheric dust. 

Remarks: Hirsch (1960) consider- this or- 
ganism as a Nocardia (X. autotrophica) , ca- 
pable of utilizing petroleum. 

28. Streptomyces beddardii (Erikson, 1935) 
Waksman (Erikson, D. Med. Research 
Council (Brit.) Spec. Rept. Ser. 203: 13- 14, 
L935). 

Morphology: Sporophores long, slender, 
tormina; many wavy or closely coiled spirals, 
particularly on glucose agar; spirals less 
marked or lacking on poorer nutritive media 
like synthetic glycerol agar or water agar. 
Aerial hyphae straighter and more branched 
with shorter sporophores on starch agar. 
Spores oval. 

Glucose-asparagine agar: Growth wrin- 
kled, membranous. Aerial mycelium -cant. 
white. 

Nutrient agar: ( rrowth colorless, coherent , 
wrinkled, membranous. Aerial mycelium 
scant, white. Soluble pigmenl deep brown. 



180 



THE ACTINOMYCETES, Vol. II 



Starch agar: Growth spreading, colorless. 
Aerial mycelium abundant, white. Hydroly- 
sis. 

Egg medium: Growth extensive, wrinkled, 
bright yellow. Considerable liquefaction. 

Blood agar: Growth in uniformly striated 
colorless bands; occasional round colonies at 
margin. Hemolysis positive. 

Potato: Growth moist, colorless. Aerial 
mycelium scant, white at top of plug. 

Gelatin: Dull white flakes sinking to bot- 
tom as medium liquefies. Rapid liquefaction. 

Milk : Coagulation followed by peptoniza- 
tion. 

Source: Human spleen in a case of splenic 
anemia. No record concerning actual patho- 
genicity. 

28a. Streptomyces bellus Margalith and 
Beretta (Margalith, P. and Beretta, G. 
Mycopathol. Mycol. Appl. 12: 189-195, 
1960). 

Morphology: Sporophores long, straight, 
flexuous with short, open hooks. 

Sucrose nitrate agar: Substrate growth 
light cherry-pink. Aerial mycelium pinkish- 
white. Soluble pigment light pink. 

Glucose-asparagine agar: Growth colorless 
to light pink with orange tinge. Aerial myce- 
lium white, with small amount of bluish 
spores. Soluble pigment light pink with 
orange tinge. 

Calcium malate agar: Substrate growth 
pink, with pinkish-violet reverse. Sporula- 
tion abundant, pinkish-blue. Soluble pig- 
ment pinkish-violet. 

Nutrient agar: Substrate growth hyaline 
with brownish reverse. Aerial mycelium 
slight or absent. Soluble pigment brown. 

Starch agar: Growth hyaline with color- 
less to pale pink reverse. Aerial mycelium 
limited, with bluish-green spores. Starch 
hydrolyzed. 

Glucose-casein digest-yeast-beef agar: 
Growth pink. Aerial mycelium pinkish with 
bluish-green spores. Soluble pigment light 
pink. 



Glucose-yeast extract-beef-peptone agar: 
Growth pink. Aerial mycelium pinkish- 
white with bluish-green spores. Soluble pig- 
ment reddish-brown. 

Potato: Growth rough, colorless to brown. 
No aerial mycelium. Soluble pigment brown. 

Gelatin: Slow liquefaction. Soluble pig- 
ment brown. 

Milk: Growth in form of brownish ring. 

Nitrate reduction: None. 

Cellulose: Moderate growth. 

Carbon utilization: Most sugars readily 
utilized. Xylose, inulin, and dulcitol not 
utilized in solid media. Succinate, citrate and 
glycine not utilized. 

Antagonistic properties: Produces anti- 
biotic matamycin, active upon gram-posit ive 
bacteria. 

Habitat : Soil in Italy. 

29. Streptomyces bikiniensis Johnstone 
and Waksman, 1948 (Johnstone, D. B. and 
Waksman, S. A. J. Bacteriol. 55: 317-326, 

1948). 

Morphology: Sporophores straight. Spores 
oval (Fig. 33). 

Sucrose nitrate agar: Growth white, be- 
coming pallid neutral gray with white tinge. 
Aerial mycelium abundant, white to gray. 
Soluble pigment light brown. Superficial 
droplets amber-colored. 

Glucose-asparagine agar: Growth abund- 
ant. Aerial mycelium white to mouse-gray. 
Soluble pigment light amber. 

Nutrient agar: Growth luxuriant. Aerial 
mycelium moderate, white. Soluble pigment 
deep brown. 

Starch agar: Growth abundant. Aerial 
mycelium white, becoming gray. Slight hy- 
drolysis. 

Potato: Growth wrinkled and raised, pale 
ochraceous buff. Soluble pigment brown to 
black. 

Gelatin: Slight liquefaction. 

Milk: Surface growth patchy, white. 
Aerial mycelium gray. Gradual peptoniza- 
tion. 



DESCRIPTION OF SPECIES OF STREPTOMYi ES 



181 




Figure 33. S. bikiniensis, grow 
fcesy of K. L. Jones I . 



isem digest I f ex 



r 12 davs. X 3,000 1,000 Cow 



Production of 1 1 _>S : Positive. 
Antagonistic properties: Str< 
si ic. Produce- streptomycin. 
Source: Soil from Bikini Alo 
Type culture: [MRU 351 1. 



Morphology: Sporophores straight. Spores 
;ly antago- oval to spherical, 1 by L.5 /*. 

Sucrose nitrate agar: Growth weak, color- 
less or white. Aerial mycelium poor, white. 
No soluble pigmenl . 

30. Streptomyces blastmyceticus Watanabe Glucose-asparagine agar: Growth good, 
it al., L957 (Watanabe, K.. Tanaka, T., colorless, later cream-colored. No aerial my- 
Fukuhara, K., Miyairi, X., Yonehara, II.. celium. No soluble pigment. 
and LTmezawa, II. J. Antibiotics (Japan) Calcium citrate-glycerol agar: Growth 

10A: 39 1:5,1957). colorless or white, later deep olive-buff. 



182 



THE ACTINOMYCETES, Vol. II 



Aerial mycelium thin, powdery, white or 
pale yellow to pale olive-buff. 

Nutrient agar: Growth white, later cream- 
colored to light brown. Aerial mycelium 
poor, white to gray. Soluble pigment brown. 

Milk: Growth in the form of ring on sur- 
face, cream-colored to brown. Aerial myce- 
lium white. Soluble pigment brown. Rapid 
peptonization. 

Potato plug: Growth gray to olive-gray. 
Aerial mycelium white. Usually no soluble 
pigment. 

Nitrate reduction: Negative. 

Starch: Hydrolyzed. 

Carbon utilization: Utilizes glucose, fruc- 
tose, galactose, or starch. Grows poorly on 
sucrose, lactose, maltose, or inositol. Does 
not utilize xylose, arabinose, raffinose, rham- 
nose, mannitol, sorbitol, dulcitol, or salicin. 

Antagonistic properties: Produces an 
antifungal agent designated as blastmycin. 

Remarks: Related to S. flavochromogenes. 

31. Streptomyces bobiliae (Waksman and 
Curtis, 1916) Waksman and Henrici, 1948 
(Waksman, S. A. and Curtis, R. E. Soil Sci. 
1: 121, 1916; 8: 100, 1919). 

Morphology: Elongated sporophores form 
a few close spirals of a dextrorse type. No 
spirals according to Jensen (1930). Spores 
oval and spherical. 

Sucrose nitrate agar: Growth abundant, 
wrinkled, coral-red becoming deep red. 
Aerial mycelium scant, white; later absent. 
\'o soluble pigment. 

Glycerol malate agar: Growth cinnamon- 
buff. No aerial mycelium. 

Glucose-asparagine agar: Growth coral- 
red. No aerial mycelium. 

Nutrient agar: Growth gray, becoming 
brownish to coral-red. No aerial mycelium. 
Soluble brown pigment in presence of glyc- 
erol (Jensen). 

Potato: Growth thin, dry, and wrinkled, 
yellowish, becoming coral-red. No aerial my- 
celium. Soluble pigment grayish to black. 

Gelatin: Growth cream-colored to orange. 



Aerial mycelium in the form of occasional 
patches of white. Rapid liquefaction. Soluble 
pigment brown. Melanin-positive. 

Milk: Dark brown ring. Peptonization 
without coagulation. 

Starch media: Growth wrinkled, coral-red 
with hyaline margin. Aerial mycelium white. 
Hydrolysis medium. 

Nitrate: Good reduction to nitrite. 

Cellulose: No growth in solution. Good 
growth on plate. 

Invertase: Posit ive. 

Production of H 2 S: Positive. 

Temperature: Optimum 37°C. 

Antagonistic properties: Produces pig- 
mented antibiotic cinerubin. 

Habitat: Common in soil. 

Remarks: S. purpurascens is considered by 
Corbaz et al. (1957) as a synonym of S. 
bobiliae, except that the latter no longer 
produces any aerial mycelium or spores. 
Krassilnikov (1949) considered this species 
as a variety of A. ruber. 

Type culture: IMRU 3310. 

32. Streptomyces bottropensis Konink. 
Nederl. Gist et Spirit. (Konink. Nederland. 
Gist et Spirit. Brit. Pat. 762,73(5, Dec. 5, 
1956). 

Morphology: Aerial mycelium ramified, 
with short, open spirals. Spores cylindrical, 
elliptical to almost spherical, 1 to 4 by 0.6 
to 1.2 m- 

Sucrose nitrate agar: Growth abundant, 
reddish. Aerial mycelium limited. Soluble 
pigment brown. 

Glucose-asparagine agar: Growth good, 
yellow. Aerial mycelium limited, white to 
pale gray. No soluble pigment. 

Calcium malate agar: Growth good, yel- 
lowish-brown. Aerial mycelium white-gray. 
No soluble pigment. 

Starch agar: Growth at first pink, later 
darker (pH sensitive; acid-pink, alkaline- 
blue). Aerial mycelium limited, while to 
gray. Starch hydrolyzed. 

Glucose nutrient agar: Growth folded, 



HI SCREPTION OF SPECIES OF STREPTOMYCES 



183 



yellowish. Aerial mycelium abundant, white. 
No soluble pigment. 

Glucose-yeasi extract-peptone agar: 
Growth yellowish bo buff. Aerial mycelium 
white to gray. No soluble pigment. 

Potato agar: Growth smooth, yellowish- 
brown. Aerial mycelium while to bluish- 
gray. Soluble pigmenl at first absent, later 
dark. 

Gelatin: Growth on surface good. Ae.ial 
mycelium white. Rapid liquefaction. Soluble 
pigmenl dark brown. 

Potato: Growth folded, brown to black. 
No aerial mycelium. 

Milk: Growth moderate. No coagulation; 
no peptonization. 

Antagonistic properties: Produces anti- 
biotic B-mycin, active againsl cocci, gram- 
positive bacteria, and mycobacteria. 

Habitat : Soil. 

Remarks: This is one of the organisms 
thai can be either melanin-negative (nutri- 
ent agar, yeast extract agar) or melanin- 
positive (gelatin, potato agar). 

33. Streptomyces brasiliensis (Spencer, 
1921) Waksnian (Spencer, 1-:. P. Botan. 
(iaz. 72:285-287, 1921). 

Morphology: Aerial mycelium forms 
straight, branched sporophores. Spores borne 
in chains on free ends of hyphae, oblong, 
l.ti. by 0.8 m- 

Sucrose nitrate agar: Growth at first 
white; after 10 days pale pinkish-buff. Aerial 
mycelium white and dense. No soluble pig- 
ment . 

Glucose-asparagine agar: Growth luxuri- 
ant, color same as on sucrose nitrate agar, 
thallus conspicuously zonated. Aerial myce- 
lium powdery, white to pale pinkish-buff. No 
soluble pigment. 

Glycerol malate agar: Growth spreading 
and not zonated, bordered by submerged 
mycelial bands of varying width, pearl- 
white. Aerial mycelium short, loose, and 
pearl-white. 

Potato: Growth vigorous, crumpled, pale 



pinkish-buff. Aerial mycelium abundant, at 
first white, later pale pinkish-buff. No solu- 
ble pigment. Melanin-negative. 

\'ut plugs: Growth vigorous, pale pinkish- 
buff. Aerial mycelium powdery, white. 
Medium not completely destroyed, but 
much shrunken and blackened. 

Gelatin: Rapid liquefaction. No soluble 
pigment. 

Milk: Papid coagulation and peptoniza- 
tion. 

Habitat: Parasitic on kernels of Brazil 
nuts. 

Remarks: This Streptomyces species is to 
be distinguished from Xocurdia brasiliensis, 
a pathogenic organism. 

34. Streptomyces cacaoi (Waksman, 1932) 
Waksman and Benrici, 1948 (Waksman, S. 
A. In Bunting, P. H. Ann. Appl. Biol. 19: 
515-517, 1932). 

Morphology: Sporophores long; spirals 
long and open, not compact. 

Sucrose nitrate agar: Growth thin, yel- 
lowish, later turning reddish-brown. Aerial 
mycelium light gray to mouse-gray, with 
white edge. Xo soluble pigment. 

Nutrient agar: Growth brown, covered 
with tiny patches of ivory-colored aerial 
mycelium. 

Potato: Growth abundant, brownish. 
Aerial mycelium white to mouse-gray. Mel- 
anin-negative. 

Gelatin: Growth flocculent. Xo aerial 
mycelium. Liquefaction rapid. Xo soluble 
pigment. 

Nitrate reduction: Limited. 

Production of IBS: Negative. 

Antagonistic properties: Certain strains 
produce an antibiotic designated as cacao- 
mycetin. 

Source: Three strains were isolated from 
cacao beans in Nigeria. They showed -light 
differences, the foregoing description being 
based on one strain. 

Remarks: Strong proteolytic enzymes, 
strong diastatic action, no sugar or dextrin 



184 



THE ACTINOMYCETES, Vol. II 



being left in 1 per cent starch solution after 
a few days. 

Type cult tire: I MRU 3082. 

35. Streptomyces caelestis DeBoer et al., 
1959 (DeBoer, C, Dietz, A., and Hoeksema, 
H. Canad. Pat. 572,779, March 24, 1959). 

Morphology: Sporophores loosely coiled. 
Spores spherical to oval. 

Sucrose nitrate agar: Growth good. Aerial 
mycelium gray-white. Soluble pigment yel- 
low. 

Nutrient agar: Growth fair to good. Aerial 
mycelium slight pink-white. Soluble pigment 
brown-tan. 

Casein digest-beef agar: Growth good. 
Aerial mycelium pale glaucous blue. Soluble 
pigment brown-tan. 

Starch agar: No growth. 

Tyrosine agar: No growth. No aerial my- 
celium. No soluble pigment. 

Potato: Growth good. Aerial mycelium 
grayish to blue-white. Soluble pigment 
brown. 

Gelatin: Growth good. Aerial mycelium 
blue-gray. Soluble pigment brown. Medium 
liquefaction. 

Milk: Growth fair. No soluble pigment. 
No peptonization. 

Nitrate reduction: Negative. 

Production of IPS: Positive. 

Carbon utilization: Utilizes a variety of 
sugars, ^//-inositol, acetate 1 ; limited utiliza- 
tion of starch, glycerol, citrate, and succi- 
nate; does not utilize dulcitol, mannitol, 
inulin, sorbitol, and various other organic 
acids. 

Antagonistic properties: Produces antibi- 
otic celesticetin. 

Habitat: Soil in Utah. 

Remarks: Similar to S. glaucus and S. 
chartreusis. 

36. Streptomyces caeruleus (Baldacci) 
Waksman (Baldacci, E. Atti ist. botan. 
univ. Pavia 3: ISO L84, 1944). 

Morphology: Sporophores long, straight, 



branched, not forming any spirals. Spores 
cylindrical 1.0 to 1.4 by 2.0 to 2.1 M . 

Agar media: Substrate growth colorless. 
Aerial mycelium pigmented, at first white, 
later becoming blue, and finally dark. Solu- 
ble pigment grayish-green. 

Glycerol agar: Grows slowly; light blue in 
color. 

Carrot agar: Growth at first white; later 
becoming blue. Aerial mycelium blue, be- 
coming gradually deep blue, and finally 
dark blue. 

Oatmeal agar: Color of growth at first 
white and aerial mycelium blue, gradually 
becoming darker in color. The agar is pig- 
mented grayish-green. 

Gelatin: Growth slow, grayish-blue. 
Either no liquefaction or only slow liquefac- 
tion. 

Milk: Weak growth. 

Starch media: Weak greenish growth. 
Bluish-green pigmentation. 

Temperature: Range between 18 and 
30°C. Optimum 24 °C. 

Reaction: Optimum pH 8 to 10. 

Cellulose: Not utilized. 

Antagonistic properties: Produces anti- 
biotic caerulomycin . 

Habitat: Corn straw and decomposing 
rice straw. 

Remarks: Related to S. violaceoruber, S. 
violaceus, and S. viridis. According to Taber 
(1959) the distinctive characteristics of a 
culture that he isolated and identified as 
S. caeruleus are: production of a blue to red 
indicator pigment; requirement of a neutral 
or alkaline reaction for growth and produc- 
tion of oblong to cylindrical spores in straight 
and flexnous chains. It was not chromogenic 
on peptone media but produced H 2 S on 
iron-peptone agar. It did not grow on un- 
buffered potato or carrot plugs, litmus 
milk, and certain synthetic agar media. The 
cult are readily utilized glucose, fructose, 
galactose, mannitol, sucrose, xylose, starch, 
and maltose, but did not utilize, or utilized 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



I s;> 



to :i limited extent, mannose, ^-inositol, 
adonitol, lactose, ribose, raffinose, and cel- 
lulose. 
Type culture: [MRU 3798. 

:!7. Strt ptomyces caespitosus Sugawara and 
Ilata, L956 (Sugawara, R. and Ilata, T. 
J. Antibiotics (Japan) 9A: 1 17 L51, L956). 

Morphology: Primary verticils produced. 
Spores oval, 1.3 to ().."> by ().:! to 0.5 u. 

Sucrose nitrate agar: Growth hyaline, 
colorless to faint yellowish-brown. Aerial 
mycelium white to yellowish-gray to green- 
ish-yellow. Soluble pigmenl taint yellow. 

Calcium tnalate agar: Growth colorless 
with yellow-brownish center, changing to 
dark greenish-yellow to dull yellow. Aerial 
mycelium white, with yellowish tinge, 
greenish-yellow a1 the margin. Soluble pig- 
menl taint yellow, pinkish shade in some 
cultures. 

Glucose-peptone agar: Growth humid, 
wrinkled, cracked in the center; colorless. 
becoming greenish-yellow-gray, reddish- 
gray, to dark gray. Aerial mycelium thin, 
gray. Soluble pigment reddish-brown. 

Starch agar: Growth scanty, colorless to 
taint yellowish-brown, or yellow to orange- 
yellow. Aerial mycelium cottony, white, 
cream with lavender patch in the center, 
becoming yellowish-gray. Soluble pigmenl 
taint yellowish-brown. 

Nutrient agar: Growth yellow-brown to 
gray to dark gray. Aerial mycelium gray. 

Potato: Growth cream to brownish, center 
Ligh.1 greenish-yellow. Aerial mycelium white 
to grayish or gray with pale olive tinge. 
Soluble pigmenl absent, or dark brown, or 
grayish-brown. 

Milk: Surface ring yellow to pale yel- 
lowish-brown. Xo aerial inyceliuni. Soluble 
pigmenl pale brown. 

Gelatin: Growth cream-colored turning 
greenish-yellow to reddish-yellow. Aerial 
mycelium white to yellow. Soluble pigment 
yellowish-brown. Rapid liquefaction. 

Tyrosinase reaction : None. 



Nitrate redud ion : Posil ive. 

Starch: Hydrolysis. 

Carbon utilization: Utilizes various car- 
bohydrates; does not utilize xylose, rham- 
nose, raffinose, arabinose, mannitol, salicin, 
dulcitol, inulin, acetate, and succinate. 

Antagonistic properties: Produce- anti- 
biotic mitomycin, active upon certain 
neoplasms. 

Remarks: Closely related to S. kitasatoen- 
sis and S. hachijoensis. 

38. Streptomyces caiusiae Dhala '/ <il.. 
1957 (Dhala, S. A., Poonawalla, P. M., and 
Bhatnagar, S. S. .1. Sci. & Ind. Res. L6C: 
70 so, L957). 

Morphology: Aerial hyphae short and 
straight; frequently clusters are produced, 
subdividing a1 the distal portions into chains 
of spores. Xo spirals formed either in syn- 
thetic or nonsynthetic media; often tips of 
the aerial hyphae slightly curved. Spore- 
round to oval, 0.0 to 1.4 by 0.1 to 0.8 n- 

Sucrose nitrate agar: Colonies round. 
convex, tough, with smooth surface when 
unsporulated; citron-yellow, later turning 
brown. Aerial mycelium white, turning yel- 
lowish, then gray. Soluble pigmenl at first 
yellow but later darkened to a brown tinge. 

Glucose-asparagine agar: Growth citron- 
yellow. Aerial mycelium white. Soluble pig- 
menl yellowish-brown. 

Xutrient agar: Growth wrinkled, with 
irregular margins and radial ridges in old 
cultures. White aerial mycelium. 

Starch agar: Colonies smooth, colorless 
after 'J days; on further incubation, they 
become large and wrinkled with radiating 
ridges. Aerial mycelium white, turning gray. 
Soluble pigmenl brown. Starch weakly hy- 
drolyzed. 

Potato: Growth luxuriant, citron-yellow. 
Aerial mycelium white, turning gray. Plug 
turns black. 

Milk: Pellicle produced. Coloration of 
milk brownish to black. Peptonization posi- 
tive. 



186 



THE ACTINOMYCETES, Vol. II 



Gelatin: Sediment buff-colored. Liquefac- 
tion medium. 

Tyrosinase reaction: Positive. 

Nitrate reduction: None. 

Carbon sources: Sugars readily utilized, 
with the exception of acetate, benzoate, 
cellulose, dulcitol, /-inositol, and salicylate. 

Antagonistic properties: Active primarily 
upon gram-negative bacteria and fungi. 

Remarks: Closely related to S. antibi- 
oticus. 

39. Streptomyces calif amicus (Waksman 
and Curtis, 1916) Waksman and Henrici 
(Waksman, S. A. and Curtis, R. E. Soil Sci. 
1: 22, 1916; Waksman, S. A. ibid. 8: 104, 
1919). 
Synonyms: 

Streptomyces puniceus Finlay and Sobin, 
1950. 

Streptomyces vinaceus Mayer et al., 
1951. 

Streptomyces floridae Bartz et ed., 1951. 
Streptomyces griseus var. purpureus 
Burkholder et al, 1955. 
Streptomyces purpureus (Burkholder, 
1955) Waksman, 1959. 
Morphology: The original culture was re- 
ported to form sporophores with long, nar- 
row, open sinistrorse spirals. According to 
Okami, however, the sporophores are 
straight. Recent examinations of the original 
culture of Waksman and Curtis (Burkholder 
et al., 1955) did not reveal any spirals either. 
Sucrose nitrate agar: Growth spreading, 
vinaceous-colored. Aerial mycelium 

powdery, light neutral gray to ash-gray. No 
soluble pigment. 

Glucose-asparagine agar: Growth re- 
stricted, much folded, cream-colored, with 
sulfur-yellow tinge. 

Nutrient agar: Growth thin, restricted, 
yellowish to cream-colored, Melanin-nega- 
tive. 

Starch agar: Growth spreading, pink 
center with colorless to gray margin. Hy- 
drolysis rapid. 



Potato: Growth glossy, yellow to red, 
turning red-brown. 

Gelatin: Growth gray, moist, abundant. 
No soluble pigment. Liquefaction medium. 

Milk: Surface growth faint, brownish. 
Coagulation and slow peptonization. 

Nitrate reduction: Positive. 

Production of H 2 S: Negative. 

Cellulose: Growth scant but definite. 

Carbon utilization: According to Burk- 
holder, the various strains utilize D-xylose, 
n-glucose, D-galactose, D-fructose, cellobiose, 
n-maltose, D-mannitol, and starch. Growth 
poor with L-arabinose, L-rhamnose, D-lac- 
tose, sucrose, D-raffinose, dulcitol, /-inositol, 
and salicin. 

Temperature: Optimum 37°C. 

Invertase: Positive. 

Antagonistic properties: Routien and 
Hofmann (1951) first demonstrated that 
cultures of S. californicus are capable of 
producing viomycin. The same antibiotic 
was found to be produced by other strains 
of this organism. 

Habitat: Soil. 

Remarks: Waksman and Curtis reported 
the production of spirals in their original 
description of *S. californicus (see also Waks- 
man, 1919). Several authors who studied 
the S. californicus culture more recently de- 
scribed the aerial mycelium as straight to 
wavy to strongly flexuous (Burkholder et a I.. 
1955; Kutzner, 1956; Ktt linger et al., 1958). 
Since the other properties of the now-avail- 
able S. californicus lit with the original de- 
scription, it might be assumed that the 
strongly flexuous aerial hyphae were con- 
sidered as spirals originally. In 1955 Burk- 
holder et al. made a comparative study of 
several viomycin-producing organisms which 
were originally described under the names 
S. floridae, S. puniceus, S. vinaceus, S. cali- 
fornicus, and several others. All these cul- 
tures behaved in a similar manner, with 
only minor differences between this group 
and N. californicus ATCC 3312; namely. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 187 

"with the exception of ATCC 3312, all Muk: Growth colorless to yellow. Coagu- 

isolates liquefy gelatin rapidly and produce lation and moderate peptonization. 

viomycin or similar antibiotic compounds." Cellulose: Growth yellow. No decomposi- 

Xii studies seem to have been made, how- i ion of cellulose. 

ever, of antibiotics produced by S. californi- Production of H2S: Negative. 

cms A.TCC 3312. Only because the original Carbon utilization: d-fructose, i-inositol, 

description of S. californicus gave spiral for- lactose, d-mannitol, rf-raffinose, ^-rhamnose, 

mation and these organisms did not, the sucrose, d-trehalose, and d(+)-xylose readily 

several viomycin-producing organisms and utilized; Z-arabinose, d-melibiose, and sali- 

the S. californicus A.TCC 3312 were de- cin utilized poorly; dextrin, esculin, deme- 

scribed as a variety of S. tins, us, namely lezitose, and adonitol no1 utilized at all. 

S. griseus var. purpureus. This was due to Antagonistic properties: Produces 

the fact thai S. griseus had the same mor- nucleocidin, an antibiotic possessing anti- 

phology and color of the aerial mycelium, trypanosomal properties. 

and because several streptomycin-producing Habitat: Soil in India. 

strains are known to form also a red-gray Remarks: This organism is closely related 

color in the substrate growth. to S. annulatus. 

Type culture: [MRU (ATCC) 3312. 41 streptomyces candidus (Krassilnikov, 

-10. Streptomyces minis Backus et al., MM 1) Waksman (Not Streptothrix Candida 

L957 (Backus, I':. .1., Tresner, II. I)., and Petruschky) . (Krassilnikov, X. A. Actino- 

Campbell, T. II. Antibiotics & Chemother- mycetales. Ezvest. Akad. Xauk. SSSR, 

apy 7: 532 541, 1957). Moskau, p. 4<), 1941). 

Morphology: Sporophores form short Morphology: Sporophores long, straigb.1 

loose spirals. Spores globose to elongated, or wavy, but never forming spirals; occa- 

0.6 to 1.0 by 1.0 to 1.8 n (Fig. 34). sionally arranged in broom-shaped bodies 

Sucrose nitrate agar: Growth cream-col- or fascicles. Spores oblong to cylindrical 

ored to yellowish. Aerial mycelium scanty, ("fragmentation spores"), '•() to 2.0 by 

white to gray. ().(> to 0.8 m- 

Glucose-asparagine agar: Growth ivory- Sucrose nitrate agar: Growth colorless. 

yellow. Aerial mycelium scanty, white. Aerial mycelium velvety, while. No soluble 

Calcium malate agar: Growth colorless to pigment. 
yellow. Aerial mycelium scanty white to Nutrient agar: Growth good, lichenoid 
gray. Crystalline pellets formed in growth or smooth. Aerial mycelium whitish. Mela- 
zones, nin-negative. 

Starch agar: Growth colorless to yellowish. . Gelatin : Slow liquefaction. Melanin-nega- 
Aerial mycelium white to mouse-gray. 



live. 

Potato: ( rrowth colorless, lichenoid. Aerial 
mycelium poorly developed. X<> soluble pig- 
ment or brownish, 
soluble pigment . A elanin-neuat lve. Ain N ^ , .• , 

1 & Milk: No coagulation; good peptomza- 



Xutrient agar: Substrate growth lighl yel- 
\\ . Aerial mycelium scanty, white. Nc 



tion. 

Starch: Rapid hydrolysis. 



Potato plug: Growth gray. Aerial myce 
lium -canty, white io lighl gray. Plug dis- 
colored. Cellulose: Good growth. 

Gelatin: Growth colorless to yellow. No Nitrate reduction : Positive. 

aerial mycelium. Partial liquefaction. No Sucrose: Inversion. 

soluble pigment. Production of IIS: Negative 



4, 



188 



THE ACTINOMYCOTIC, Vol. II 






<*^v* 



hlmCX&' 



^ 







$ 



t t 



* 









j 



pi 




Figure 34. Sporophores of S. ralvus (Reproduced from: Backus, E. J. et al. Antibiotics & Chemo- 
therapy 7: 530, 1957). 



Antagonistic properties: Weak. 

Habitat : Soil. 

Remarks: Certain varieties of this species 
have also been described. This is true, for 
example, of A. fasciculus, which Krassilni- 
kov himself considered as a variety of S. 
candid 'us; it is also true of .4. farinosus 
Krassilnikov and of .4. candidus var. albo- 
roseus described by Gause et al. (1957). 
S. nitrosporeus Okami (1952) appears to be 
closely related, if not identical to it. Ett- 
lrnger et al. (1958) consider this organism 
as related to S. griseus. 

Type culture: [MRU 3416. 

42. Streptomyces rum sens Hickey et al., 
L952 (Hickey, U. J., Coram, C. J., Hidy, 



P. H., Cohen, I. R., Nager, U. F. B., and 
Kropp, E. Antibiotics & Chemotherapy 
2:472-483, 1952). 

Morphology: Sporophores straight or 
curved, not forming any spirals. Spores 
globose, 1.0 to 1.3 by 1.3 to 2.6 n (Fig. 35). 

Calcium malate agar: Growth gray to 
rose-gray; reverse yellow to tan. No soluble 
pigment. 

Yeast extract-casein digest agar: Growth 
effuse to convex, edge filamentous; reverse 
brown. Aerial mycelium powdery, varying 
from gray-white to gray. No soluble pig- 
ment. 

Acid-glucose-peptone agar: Growth at 
first white, then tan. Aerial mycelium faintly 



DESCRIPTION OF SPECIES OF STREPTOMYCES 189 

greenish, produced after II days. Amber Milk: Soft, rennel curd formed al 36°C 

pigmenl diffused throughoul medium. after is hours; completely peptonized in 12 

Egg medium: Growth tan, wrinkled. No days, 

sporulation after 10 days; limited while Starch: Hydrolysis strong. 

sporulatioD observed in II days. Soluble Nitrate reduction : Negative, 

pigmenl brown. Very slow liquefaction after Production of IPS: Negative. 

28 days. Temperature: < Optimum 36 ( '. 

Potato: Growth lighl gray, wrinkled. Carbon utilization : Utilizes glucose, arabi- 

Soluble pigment deep brown. nose, trehalose, xylose, sucrose, maltose, 

Gelatin: Liquefaction rapid. Soluble pig- galactose, dextrin, soluble starch, mannitol, 

ment deep brown. glycerol, and salicin. No growth with sor- 




Figure 35. S. canescus, grown on casein digesl beefextracl agar for 12 days, X 3,000 I. Courtesy 

of K. L. Jones). 



190 



THE ACTTXOMYCKTES, Vol. II 



hose, melezitose, dulcitol, rhamnose, sorbi- 
tol, melibiose, phenol, raffinose, and lactose. 

Antagonistic properties: Produces anti- 
fungal antibiotic ascosin. 

Source: Contaminated fungus plate. 

Remarks: This species is now included 
with S. coelicolor (Kutzner and Waksman, 
1959). 

Type culture: IMRTJ 3782; NRRL 2419. 

43. Streptomyces canus Heinemann et al., 
1953 (Heinemann, B., Kaplan, M. A., Muir, 
R. D., and Hooper, I. R. Antibiotics & 
Chemotherapy 3: 1239-1242, 1953). 

Morphology: Aerial mycelium forms nu- 
merous loosely wound spirals. Spores spher- 
oidal, 1.0 to 1.2 by 1.6 to 1.8 m- 

Sucrose nitrate agar: Growth moderate, 
wrinkled, yellow-brown. Aerial mycelium 
scant. 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, innlin, raffinose, soluble 
starch, glycerol, inositol, mannitol, and 
sodium salicylate. No growth observed with 
dulcitol, sorbitol, sodium acetate, sodium 



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 
0.75 fj,. 

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 my- 
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. 

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 wrinkled with 
smooth edge. Sporophores in form of short 
clusters; a few tight spirals. Spores oval to 
cylindrical, 1.0 by 1.3 m- 

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 



mi 



Calcium malate agar: ( rrowth pour. Aerial 
mycelium mouse-gray, with some white. Cal- 
cium malate digested. 

Nutrient agar: Growth pale yellow. Aerial 
mycelium white, turning pale gray. Soluble 
pigmenl pale yellow. Nonchromogenic. 

( rlucose-yeasl ex1 ract-beef-peptone agar: 
Growth dark brown. Aerial mycelium olive- 
gray with white. Soluble pigmenl medium 
brown. 

Starch agar: Growth brownish-orange. 
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 anti- 
biotic catenulin. 

Habital : Soil. 

Type culture: ATCC 12,47(5. 

4(k Streptomyces cavourensis Giolitti, 1958 
(Giolitti, G. Belgian Pat. 560,930, March 18, 
L958*). 

Morphology: Aerial mycelium produces 
spirals on certain media. Spores spherical to 
elliptical. 

Sucrose nitrate agar: Growth yellowish. 
Aerial mycelium chalky white to yellowish. 
No soluble pigment . 

Glycerol-asparagine agar: Growth hazel- 
colored. Aerial mycelium whitish. Soluble 
pigment taint brown. 

Calcium malate agar: Growth scanty, 
yellow-brownish. Aerial mycelium scanty, 
white. Soluble pigment scarce, yellow-brown- 
ish. 

Xutrient agar: (Irowth orange-brown. 
Aerial mycelium scanty, chalky white to yel- 
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. 

Oatmeal agar: Growth light brown, 
wrinkled. Aerial mycelium gray with brown 

patches. Soluble pigmenl light brown. 

Starch agar: Growth scanty, yellow- 
brownish. Aerial mycelium scanty, white- 
grayish. Soluble pigment brownish. Strong 
hydrolysis. 

Gelatin: Growth scanty, brown, wrinkled. 
Aerial mycelium scanty, gray. Soluble pig- 
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: Produce- 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 
it <il., 1953 ( Nishimura, II., Kimura, T., and 
Kuroya, M. .1. Antibiotics (Japan) 6A: .~>7 
65, L953). 

Morphology: Sporophores straight with a 
few flexible, hooked spirals. Spores nearly 
spherical, 1.0 by ().«) M . 

Sucrose nitrate agar: Growth glossy, de- 
veloping deej) into medium, later becoming 
yellow. Soluble pigmenl faint sulfur-yellow. 

Glycerol malate agar: Growth yellow, 
later turning white to pale olive-buff with 
blackish center. Aerial mycelium cottony, 
while, with grayish patches, later turning 
olive-buff. Soluble pigment yellow. 

Glucose-asparagine agar: Growth cream 
to yellow. Aerial mycelium scant, cottony, 



192 



THE ACTIXOMYCETES, Vol. II 



white to gray. Soluble pigment sulfur-yel- 
low. 

Nutrient agar: Growth olive-buff, turn- 
ing colorless. Aerial mycelium 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 H 2 S: Negative. 

Antagonistic properties: Produces thio- 
lutin, aureothricin. 

Habitat: Soil. 

48. Streptomyces cellulosae (Krainsky, 
1914) Waksman and Henrici, 1948 (Krain- 
sky, A. Centr. Bakteriol. Parasitenk. Abt. 
II., 41: 683-088, 1914). 

Description after Jensen, H. L. Soil Sci. 
30: 65, 1930. 

Morphology: Sporophores straight; no 
spiral formation. Spores almost spherical, 1.3 
fj. in diameter. 

Sucrose nitrate agar: Growth at first trans- 
parent, becoming lemon-yellow. Aerial my- 
celium light gray, later deep slate-gray. Sol- 
uble pigment may be lemon-yellow. 

Calcium malate agar: Colonies yellowish; 
aerial mycelium gray to white-gray. Soluble 
pigment yellow. 

Glucose-asparagine agar: Growth abun- 
dant; aerial mycelium gray. Soluble yellow 
pigment, especially with high nitrogen con- 
;entration. 



Glucose nutrient agar: Good substrate 
growth, at first cream-colored, later sulfur- 
yellow. Aerial mycelium white, later gray. 

Potato: Growth light cream-colored, later 
often yellow. Aerial mycelium white, later 
slate-gray. 

Gelatin: Growth yellowish-gray to gray- 
ish-black. Rapid liquefaction. 

Milk: Rapid coagulation and peptoniza- 
tion. 

Cellulose: Growth good. 

Esculin: Hydrolysis. 

Starch: Diastatic action strong. 

Nitrate: Reduction weak. 

Invertase: Negative. 

Production of H 2 S: Negative. 

Temperature: Optimum 30-35°C. 

Pigment: Soluble in alcohol and other or- 
ganic solvents. 

Antagonistic properties: Produces anti- 
biotics fungichromin and actinomycin. 

Habitat: Very common in soil. 

Remarks: The culture described by Krain- 
sky (1914) produced an aerial mycelium of a 
gray color ("like diastaticus" >. hater, how- 
ever, Krainsky 's culture was found to pro- 
duce a grayish-yellow aerial mycelium like 
the streptomycin-producing S. griseus (Ett- 
linger et al., 1958); these authors also con- 
sider the S. cellulosae strains obtained from 
ATCC and NRRL as closely related to S. 
griseus. Most probably, one of these cultures 
was used in the studies of 4 "resiier and Danga 
(1958), who mention a yellow-cream-buff 
color of the aerial mycelium. In contrast to 
these cellulosae strains now available for 
comparison, Jensen (1930a) described five 
soil isolates as .1. cellulosae with a distinc- 
tive slate-gray aerial mycelium, in agree- 
ment with Krainsky's description. Krassilni- 
kov (1949) considers this organism as a 
variety of .1 . flavus. 

Type culture: I. MRU 3313, 37S0. 

49. Streptomyces chartreusis Calhoun and 
Johnson, 1956 (Calhoun, K. M. and John- 



DESCRIPTION OF SPECIES OF STREPTOM1 CES L93 

son, L. E. Antibiotics iV Chemotherapy 6 : and Tomiyama, Y. J. Antibiotics (Japan) 

•Jill 298, L956). 1 1 A: si 83, L958). 

Morphology: Aerial hyphae branch pro- Morphology: Sporophores wavy, produc- 
fusely producing closed or open spirals, ing numerous small spirals, 
sinistrorse and dextrorse, depending on com- Sucrose nitrate agar: Growth slow, pene- 
position of medium. Spiral chain- consist of trating into medium, yellow reverse. At firsl 
3 to 7 turns. Spores powdery, blue-gray to no aerial mycelium; later mycelium pro- 
blue-green, depending on the medium. Mos1 duced, white becoming gray, then reddish- 
spirals occur singly; a few are found in gray or black-buff. Soluble pigmenl slighl 
groups of two or three measuring 5 to 20 m yellow. 

in length and -\ to 5 m in width. Spores Nutrient agar: Growth good. Reverse 

spherical to oval, 1.0 to L .5 \i in diameter. cream-colored or yellow. Aerial mycelium 

Sucrose nitrate agar: Growth profuse, powdery, white. Soluble pigmenl absent or 

raised and somewhat wrinkled, honey-col- slightly yellow. 

ored. Aerial mycelium of young colonies Glucose-asparagine agar: Growth good, 

white to pale gray; older colonies have blue yellow, later becoming brown. Aerial myce- 

center. hum powdery, white, later becoming dark 

Glucose-asparagine agar: Growth profuse, brown. Soluble pigmenl yellow, 
blue-gray. Soluble pigment yellow. Starchagar: Growth good, white, later lie- 
Starch agar: Growth profuse. Center of coming olive-yellow. Strong hydrolysis of 
colonies blue-green, edges white. No soluble starch. 
pigment. Good hydrolysis. Potato: Growth good, raised. Aerial mv- 

Xutrient agar: Growth profuse. Center of celium white or cream-colored, powdery. No 

colonies blue-gray, edges powdery white. No change in color of plug, 

soluble pigmenl . ( lelatin: ( irowth on surface and in medium 

Potato: Growth light, raised with some poor. No aerial mycelium. Liquefaction ab- 

wrinkling. Center of colonies blue-gray with sent or limited. Melanin-negative. 

white edges. No soluble pigment. Milk: Growth good; white to cream-col- 

( lelatin: Growth blue-green in center with ored pellicle. No aerial mycelium. No soluble 

white edges. Soluble pigment yellow -green pigment. Peptonization slow. 

to black. Slow liquefaction. Melanin-posi- Carbon sources: Utilizes readily a variety 

tive. of carbon sources, but not intllin. 

Milk: Growth moderate, blue-gray and Antagonistic properties: Produces anti- 
white. Slow peptonization. bid ic cellocidin, active against gram-posit ive 

Nitrate broth: Blue-green ring and white and gram-negative bacteria; this antibiotic 

pellicle. Soluble pigment yellow-tan. Nitrate also possesses ant icancer properties, 

strongly reduced. Habitat : Soil in Japan. 

Production of IPS: Positive. Remarks: The organism is closely related 

Antagonistic properties: Produces ami- to S. flavus. 

biotic chartreusin. - L strepiomyces chrysomallus Lindenbein, 

Remarks: EttlingereUZ. (1958) considered ,,,-._, , Un ,| ( , lh( . iM< w . Arch. Mikrobiol. 17: 

this species to be related to S. viridochromo- ogi qqq J.,--) 

'■'""*■ Morphology: Sub-irate growth soft, con- 

50. Streptomyces chibaensis Suzuki et al., sisting of long, branching hyphae, with mi- 

1958 (Suzuki, S., Nakamura, G., Okuma, K., merous staining granules. Sporophores long. 



194 



THE ACTIXOMYCETES, Vol. II 



straight; no spirals. Spores oval to elliptical; 
surface smooth. 

Glycerol nitrate agar: Growth light yellow. 
Aerial mycelium powdery, white. Soluble 
pigment golden yellow. 

Glucose-asparagine agar: Growth smooth, 
colorless to yellowish. Aerial mycelium 
powdery, white. Soluble pigment faint yel- 
low. 

Glycerol malate agar: Growth thin, 
smooth, colorless to light yellow. Aerial my- 
celium powdery, grayish-white. 

Nutrient agar: Growth poor, shiny, golden 
yellow. Aerial mycelium white, powdery. 
Soluble pigment golden yellow. Melanin- 
negative. 

Glucose-peptone agar: Growth yellowish 
with tinge of orange. Aerial mycelium gray- 
ish-white. Soluble pigment light yellow to 
golden yellow. 

Starch-casein agar: Growth colorless, with 
yellowish reverse. Aerial mycelium powdery, 
chalk-white. Strong hydrolysis of starch. No 
soluble pigment . 

Potato: Growth heavy, yellow, becoming 
brownish-yellow or orange. Aerial mycelium 
cottony white to yellowish-white. 

Gelatin: Surface growth heavy, light to 
dark yellow. Aerial mycelium white. Soluble 
pigment yellow-brown to deep brown, only 
in Liquefied portion. Strong liquefaction. 
Melanin-negative. 

Milk: Growth colorless, with lighi yellow 
reverse. Aerial mycelium cottony, snow- 
white, becoming yellowish. Coagulation 
slight. Strong peptonization. 

Cellulose: Growth very weak. 

Production of H 2 S: Negative. 

Antagonistic properties: Produces actino- 
mycin C; some strains also produce the 
antifungal cycloheximide. 

Habitat: Soil. 

Remarks: Ettlingerr/ al. (1958) considered 
it as a member of the S. griseus group. A 
complete description of this organism was 
also given by Frommer (1058). Frommer 



(1959) described a variety of this organism 
under the name fumigatus. It differed from 
the type species by producing a mouse-gray 
aerial mycelium on synthetic media, no 
aerial mycelium on potato and gelatin, and 
by displaying more limited proteolytic prop- 
erties. 

Type culture: IMRU 3657. 

52. Streptomyces cinereoruber Corbaz et a/., 
1957 (Corbaz, R., Ettlinger, L., Keller- 
Schierlein, W., and Zahner, H. Arch. Mikro- 
biol. 25:325-332, 1957). 

Morphology: Sporophores straight; no 
spirals. Spores slightly elongated, 0.9 to 2 by 
0.7 to lju; surface of spores smooth. 

Glycerol nitrate agar: Growth thin, light 
carmine-red, in 7 days dark red. Aerial my- 
celium ash-gray. Soluble pigment light car- 
mine. 

Glucose-asparagine agar: Substrate 
growth thin, greenish-gray to bluish-gray. 
Aerial mycelium ash-gray. No soluble pig- 
ment. 

Gelatin: Surface growth light carmine to 
light brown. Aerial mycelium light gray. Sol- 
uble pigment red-brown. Medium liquefac- 
tion. Melanin-positive. 

Starch agar: Vegetative growth coral-red. 
Aerial mycelium ash-gray. Soluble pigment 
carmine. Hydrolysis limited. 

Potato: Growth lichenoid, brownish-yel- 
low. Aerial mycelium ash-gray. Soluble pig- 
ment bluish-gray. 

Milk: Pellicle light brown with sparse 
aerial mycelium, powdery, white-gray. Coag- 
ulation and peptonization. Reaction turns 
acid. 

Carbon utilization: Utilizes xylose, arabi- 
nose, and other sugars. Does not utilize l- 
rhamnose, n-fructose, raffinose, inulin, <l- 
sorbitol. 

Antagonistic properties: Produces anti- 
biotic rhodomycin. 

Remarks: This organism is closely related 
to S. bobiliae and S. purpurascens. S. cine- 
reoruber var. fructofermentans is a variety, 



DKscRirnox or spkoies of streptomyces 



195 



based on differences in sugar utilization, and 
produces the antibiotic cinerubin. 

53. Streptomyces cinnamomeus Benedict 
et al, 1954 (Benedict, R. G., Dvonch, \\\, 
Shot well, (). L., Pridham, T. G., and Lin- 
denfelser, L. A. Antibiotics & Chemotherapy 
2: 591, L952; 1: 1 1 10, L954). 

The corred name of this organism is S. 
cinnamomeus l'. cinnamorru us. 

Morphology: Sporophores straight; later 
descriptions indicate verticil formation. 
Spores globose, 0.(3 n (Fig. 36). 

Sucrose nitrate agar: Growth colorless to 
white to cream-colored. Aerial mycelium 
white to ligh.1 cinnamon. 

Glucose-asparagine agar: Growth color- 
less; light greenish-yellow to dull yellowish- 
orange in reverse. Aerial mycelium white to 
cinnamon. 

Nutrient agar: Growth cream-colored to 
light lemon-yellow. No aerial mycelium. No 
soluble pigment. 

Oatmeal agar: Growth tough, leathery, 
yellowish-green to cream-yellow. Aerial my- 
celium floccose, pale violet to faint cinna- 
mon. Exudate tan to white. 

Starch agar: ( rrowth colorless to brownish. 
Aerial mycelium white. Hydrolysis. 

Potato: Growth grayish-white to yellow- 
green to light brown. Aerial mycelium light 
gray to gray. No soluble pigment. 

( lelatin : ( irowth flocculent , dirty yellow to 
white. Aerial mycelium cretaceous. No solu- 
ble pigment. Rapid liquefaction. 

Milk: King light brown. Aerial mycelium 
limited, white. Rapid peptonization. 

('arbon utilization: Utilizes xylose, fruc- 
tose, inositol, starch, dextrin, galactose, and 
maltose. Does not utilize arabinose, rham- 
aose, < lubdt ol, and salicin. 

Nitrate reduction : Negative. 

Production of IbS: Negative. 

Temperature: Good growth al 25 :!7 C. 

Antagonistic properties: Produces cinna- 
mycin, a polypeptide antibiotic. 

Source: Japanese soil. 



* 







Figtjbe 36. Byphae of S. cinnamorrn us showing 
character of verticils of sporogenous branches 
(Reproduced from: Duggar, B. M. et al. Ann. 
X. V. Acad. Sci. 60: 85, Im.M 

Remarks: Pridham et al. (1956) described 
a second form under the name of S. cinna- 
momeus f. azacoluta; it produced a shell-pink 
aerial mycelium on starch agar and an anti- 
biotic, duramycin. 

Type culture: IMRU 3664. 

54. Streptomyces cinnamonensis Okami, 
L953 (Okami, Y., Maeda, K., Kosaka, lb, 
Taya, <)., and Umezawa, 11. Japan. J. Med. 
Sci. Biol. 6: 87-90, 1953). 

Morphology: Sporophores long, flexible, 
hooked, but no true spirals. Spores elliptical 

to oval. 

Nutrient agar: (Irowth colorless to dark. 
No aerial mycelium. Soluble pigment absent 
oi- slightly brown. 

Glycerol agar: (irowth colorless. Scant 
white aerial mycelium or white with pale 
cinnamon-pinkish to lighl brownish-vina- 
ceous tinge. No soluble pigment. 

( rlucose-asparagine agar: ( rrowth colorless 
to light cream-colored. Aerial mycelium 
white to white-pinkish-cinnamon. No soluble 
pigmenl . 



196 



THE ACTINOMYCETES, Vol. II 



Potato: Growth dark to ligh.1 cream-col- 
ored. No aerial mycelium. No soluble pig- 
ment; later, black pigment produced around 
growth. 

Gelatin: Growth colorless to dark brown- 
ish. Aerial mycelium in form of white 
patches. Soluble pigment brown. Xo or very 
slow liquefaction. 

Milk: Growth cream-colored to brownish 
surface ring. Aerial mycelium absent or 
scant white. Soluble pigment absent or 
slightly brown. Coagulation and peptoniza- 
tion absent or very slow. 

Starch agar: Growth colorless. Aerial my- 
celium white with pinkish tinge. No soluble 
pigment. Hydrolysis good. 

Cellulose: No growth. 

Nitrate reduction: None. 

Production of H 2 S: Positive. 

Carbon utilization: Sucrose, mannose, dex- 
trin, galactose, glycerol, fructose, glucose, 
maltose, mannitol, xylose, and sodium suc- 
cinate utilized. Arabinose, esculin, rhamnose, 
dulcitol, sodium acetate, inulin, lactose, sali- 
cin, and raffinose not utilized. 

Antagonistic properties: Produces an anti- 
biotic active against mycobacteria and iden- 
tical with actithiazic acid or thiozolidone. 

Remarks: The culture resembles S. roseo- 
chromogenes in color of growth and in no or 
slow liquefaction of gelatin. It differs in the 
lack of spiral formation and of nitrate re- 
duction. Gause et al. (1957) described a 
variety of this organism under the name of 
.4. cinnamonensis var. proteolyticus. A. dag- 
hestanicus and A. fumanus described by 
these authors apparently also belong to this 
group, although they differ from it in some 1 
respects. According to Benedict and Prid- 
ham (1959) a group of cooperators consid- 
ered this organism as S. cinnamonensis, S. 
virginiae, S. acidomyceticus, S. roseochromo- 
genes, and S. lavendulae; an opinion was ex- 
pressed that all of these are probably re- 
lated to S. lavendulae. 

Type culture: ATCC 12,308. 



55. Streptomyces circulatus ( Krassilnikov, 
1941) Waksman (Krassilnikov, N. A. Aetino- 
mycetales. Izvest. Akad. Nauk. SSSR, Mos- 
kau, p. 60, 1941). 

Morphology: Sporophores produce verti- 
cils with spiral-shaped short branches. Spores 
cylindrical or oblong, 1.5 by 0.7 n, some 
rounding up with age of culture. 

Synthetic agar: Growth good, colorless. 
Aerial mycelium abundant, white. 

Nutrient agar: Growth weak. No aerial 
mycelium. 

Gelatin: Liquefaction weak. 

Milk: No coagulation; slow peptonization. 

Starch: Hydrolysis weak. 

Cellulose: No growth. 

Paraffin: Growth good. Aerial mycelium 
white. 

Nitrate reduction: Weak. 

Sucrose: No inversion. 

Antagonistic properties: Limited. 

Habitat: Soil. 

56. Streptomyces citreus (Krainsky, 1914) 
Waksman and Henrici, 1948 (Krainsky, A. 
Centr. Bakteriol. Parasitenk. Abt. II., 41: 
684, 1914; Waksman, S. A. and Curtis, R. 
E. Soil Sci. 1: 116, 1916; 8: 121, 1919). Not 
Actinomyces citreus Gasperini, 1894. 

Morphology: Sporophores form long nar- 
row, open spirals, dextrorse. Spores spheri- 
cal to oval, 1.2 to 1.5 by 1.2 to 1.8 m- 

Sucrose nitrate agar: Growth abundant, 
raised, wrinkled, citron-yellow. Aerial myce- 
lium white to citron-yellow. No soluble pig- 
ment. 

Malate-glycerol agar: Growth creamy to 
yellow. Aerial mycelium white with mouse- 
gray tinge. No soluble pigment. 

Glucose-asparagine agar: Growth glossv, 
olive-yellow; center elevated. Aerial myce- 
lium white to pinkish. No soluble pigment. 

Nutrient agar: Growth restricted, green. 
No aei'ial mycelium. No soluble pigment. 

Potato: Growth yellowish to gray. Aerial 
mycelium white. No soluble pigment. 

Gelatin: Surface growth restricted, yellow- 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



L97 



ish. Aerial tnycelium white. Liquefaction me- 
dium. Melanin-negative. 

Milk: Surface growth cream-colored. Co- 
agulation followed by rapid peptonization. 

Starch media: Growth abundant, citron- 
yellow to yellowish-green. Aerial mycelium 
pinkish. Rapid hydrolysis of starch. 

Cellulose: No growth. 

lnvertase: Positive. 

Nitrate: Slighl reduction to nitrite. 

Production of H 2 S: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Negative. 

Habitat : ( iarden soil. 

Remarks: Since Krainsky's culture was 
not available for comparison, the above de- 
scription is based upon that of Waksman and 
Curtis (1916) and Waksman (1919) ; some dif- 
ferences exisl between this description and 
thai of Krainsky. Ettlinger et al. ( L958) con- 
sidered this culture as a .-train of S. griseus. 
Krassilnikov (1949) considers this organism 
as similar to Gasperini's culture, both being 
looked upon as varieties of A.flavus. 

Type culture: [MRU :>.">74. 

.")7. Streptomyces clavifer (Millard and 
Burr, L926) Waksman (Millard, W. A. and 
Burr, S. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores Long, straight, 
some terminating in club-shaped structures. 
Spores cylindrical, 1.5 by 1.0 m- 

Sucrose nitrate agar: Growth gray to 
brick-red. Aerial mycelium white, sprinkled 
with cinnamon-drab. Soluble pigmenl yel- 
lowish to brown. 

Potato: Growth wrinkled, gray to orange 
to brown. Aerial mycelium gray t<> olive- 
buff. Color of pluji gray to brown. 

Gelatin: Growth gray to buff. Aerial my- 
celium white. Medium liquefaction. Soluble 
pigmenl yellow to reddish-yellow. 

Starch: Bydrolysis. 

Tyrosinase reaction: Positive. 

Nitrate: No reduction. 



Temp 



('. 



Habitat: Limed soil and common .-cab of 
potatoes. 

Remarks: Kutzner (1956) described the 
original culture obtained from CBS as pro- 
ducing an ash-gray aerial mycelium without 
spirals, and as melanin-negative; this culture 
was indistinguishable from S. craterifer also 
obtained from CBS. Krassilnikov (1949) 
considered it as a variety of .1. scabies. 

58. Streptomyces coelicolor (Muller, 1908 
Waksman and Henrici emend. Kutzner and 
Waksman (Muller, lb Centr. Bakteriol. 
Parasitenk. Abt. I, ( >rig. 46: L95, 1908; 
Kutzner, II. J. and Waksman, S. A. 
J. Bacterid. 78: 528 538, 1959). 
Synonyms: 

Streptothrix coelicolor Muller (Muller, 

1908). 

Actinomyces albidoflavus (strain Hohle, 

CBS). 

Actinomyces alni (strain v. Plotho, CBS). 

Streptomyces cant sens Hickey et al. 

(Hickey et al., L952, X KIM. 2419). 
Possible synonyms: .1. cyaneofuscatus 
( Jause et al. (( rause et al., L957). 

A. levoris Krassilnikov (Krassilnikov, 

1958). 
\oi S. violaceoruber. 

Morphology: Sporophores of mos1 strains 
short, arranged in small tufts, wavy; no 
spirals. Spores spherical to ellipsoidal; sur- 
face smooth. 

Agar media: Substrate growth on most 
media colorless or atypical yellowish-brown- 
ish; sometimes pinkish-red, especially in the 
lower part of the slants. Aerial mycelium 
colored grayish-yellow, often with a green- 
ish or pinkish shade. Soluble pigmenl on 
mosl media either absent or yellowish- 
brown. Blue pigment is produced by some 
-trains on glucose-calcium malate-NH^NOa 
agar, mannitol-calcium malate-peptone agar, 
or glucose-peptone agar. 

Potato: Growth abundant, lichenoid. Aer- 
ial mycelium powdery, white to yellow. 
Characteristic formation of greenish-blue to 



198 



THE ACTIXOMYCKTES, Vol. II 



sky-blue soluble pigment by several strains; 
it may later become deep blue or blue-violet. 
Addition of glycerol delays pigment forma- 
tion. 

Gelatin: Good growth. Rapid liquefaction. 
No soluble pigment. 

Milk: No coagulation, rapid peptoniza- 
tion, complete within 15 days at 22-27°C; 
coagulation within 3 to 5 days, followed by 
peptonization at 36°C. 

Starch hydrolysis: Strong. 

Nitrate reduction : Positive ; none reported 
for S. canescus by Hickey et al. (1952). 

Carbon sources: Utilizes L-xylose, L-arabi- 
nose, D-fructose, D-galactose, D-mannitol, 
salicin; does not utilize L-rhamnose or raffi- 
nose; most strains do not utilize sucrose. 

Hemolysis of blood: Rapid at 37°C. 

Production of H 2 S: Negative. 

Antagonistic properties: Active upon sev- 
eral fungi and yeasts; all strains as far as 
tested produce polyene antibiotics. S. griseus 
(Krainsky) Waksman and Curtis (1916) 
probably belongs to this species, since it is 
now known to produce an antifungal agent 
of the polyene type. 

Ecology: S. coelicolor is widely distributed 
in nature. In a search for polyene-producing 
organisms, Pledger and Lechevalier (1955- 
1956) found 26 strains among 93 isolates 
which produced polyenes and which can be 
regarded as belonging to this species. Among 
the 382 subgroups of Kutzner (1956), the 
S. coelicolor subgroup was the one which 
comprised most strains. Heymer (1957) 
found this organism strikingly often on the 
skin and in the tonsils of men. The first 
culture of this species isolated by Muller 
(1908) and the ascosin-producing organism 
(*S. canescus) were found as chance con- 
taminants; this indicates the wide distribu- 
tion of the organism in air. The relationship 
of blue pigment-forming bacteria, designated 
as Actinobacillus and Actinococcus, to this 
organism was discussed by Beijerinck 
(1913a). 



Numerous cultures isolated by different 
investigators and described as S. coelicolor 
belong to S. violaceoruber. Others, however, 
such as A. tricolor Wollenweber, are related 
to S. coelicolor. 

Type culture: A strain of this organism 
was deposited by R. Muller in the CBS. 

59. Streptomyces collinus Lindenbein, 1952 
Lindenbein, W. Arch. Mikrobiol. 17: 361- 
383, 1952). 

Morphology: Sporophores form spirals. 
Spores oval. 

Glycerol nitrate agar: Growth yellow- 
brown to red-brown. Aerial mycelium chalk- 
white. Soluble pigment yellow-brown, later 
becoming reddish-brown . 

Glucose-asparagine agar: Growth yellow- 
brown to purple-red. Aerial mycelium chalk- 
white, later ash-gray. Soluble pigment car- 
mine-red, later brown-red. 

Glycerol malate agar: Growth yellow- 
brown to red-brown. Aerial mycelium vel- 
vety, chalk-white. Soluble pigment yellow- 
brown. 

Nutrient agar: Growth dark brown. Aerial 
mycelium powdery, gray-white. Soluble pig- 
ment dark brown. Melanin-positive. 

Glucose-peptone agar: Growth yellow- 
brown and red. Aerial mycelium velvety, 
white. Soluble pigment chestnut-brown. 

Starch media: Growth reddish to orange. 
Aerial mycelium white. Hydrolysis medium. 

Potato: Growth good. Aerial mycelium 
white. Xo soluble pigment. 

Gelatin: Growth dark brown. Xo aerial 
mycelium. Soluble pigment dark brown. 
Liquefaction rapid. 

Milk: Growth good; dark brown reverse. 
Aerial mycelium white, later ash-gray. Solu- 
ble pigment dark brown. No peptonization. 

Cellulose: Growth good, colorless. 

Antagonistic properties: Produces an anti- 
biotically active pigment. 

Habitat: Soil. 

Remarks: Closely related to S. erythro- 
chromogenes. ( lause et al. (1957) described a 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



199 



simili 

CI IIS. 



der ill 



1 . albovina- 



60. Stn ptomya s coroniformis I Millard and 
Burr, L926) Waksman (Millard, \Y. A. and 
Hun-, S. Ann. Appl. Biol. L3:580, L926). 

Morphology: Sporophores straight, some 
long and some short. Spores oval, 0.8 by 

().(') /x- 

Sucrose nitrate agar: Growth in form of 
discrete colonics partially coalescing, gray to 
greenish. Aerial mycelium white, covering 
edges of growth. 

Nutrient potato agar: Growth wrinkled, 
grayish. No aerial mycelium. 

Potato: Growth raised, grayish. Aerial 
mycelium white. Plug pigmented brownish 
around and under growth. 

Gelatin: Growth fair. Liquefaction slow if 
any. 

Milk: A few colonies on surface. No coagu- 
1 : 1 1 ion; peptonization limited. 

Starch: No hydrolysis or trace. 

Nitrate: Limited reduction to nitrite. 

Tyrosinase read ion: Negative. 

Temperature: Growth fair at 37.5°C. 

Habitat: Potato scab. 

61. Streptomyces craterifer (.Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, S. Ann. Appl. Biol. 13: 580, L926). 

Morphology: Straight sporophores form 
terminal branches, dichotoniously forked. 
Spores rectangular, L.3 to 0.9 by 1 .0 to 0.8 /x. 

Sucrose nitrate agar: Growth lichenoid, 
abundant, colorless; aerial mycelium mouse- 
gray. Numerous guttation drops, which 
leave blackish craters on surface of growth. 

Nutrient agar: Growth colorless; aerial 
mycelium scant, white. No soluble pigment. 

Starch agar: Growth spreading, thin, col- 
orless; no aerial mycelium. Starch hydro- 
lyzed. 

Potato: Growth cream-colored; aerial my- 
celium white to mouse-gray. Color of plug 
unchanged. 

Gelatin: Surface growth wrinkled; aerial 



■lion. No 
lored. No 

at 



mycelium white. Rapid liquei 
soluble pigmenl . 

Milk: Surface growth cream- 
coagulation, rapid peptonization. 

Tyrosinase reaction: Negative. 

Nitrate reduction : Positive. 

Temperature: Only slighl growtl 

37.5°C. 

Habitat : Raised, smooth scab. 
Type culture: [MRU 3373. 

62. Streptomyces cyaneus (Krassilnikov, 
1941) Waksman (Krassilnikov, N. A. Actin- 
omycetales. Ezvest. Akad. Nauk.SSSP, Mos- 
kau, p. 14, 1941). 

Morphology: Sporophores produce open 
spirals (sinistrorse) , with 2 to 3 turns in 
each. Spores oval, seldom spherical, ().('» to 

0.8 by O.C) n. 

Agar media: Growth pigmented blue at 
both acid and alkaline reactions. The pig- 
ment does not dissolve into medium. Aerial 
mycelium well developed, downy, bluish- 
gray to blue-green in color. 

Sucrose nitrate agar: Colonic- at first 
smooth, becoming lumpy, leathery, com- 
pact, and covered with well developed blue- 
gray aerial mycelium. 

Gelatin: Strong liquefaction. Melanin- 
positive. 

Milk: Peptonization without prior coagu- 
lation. 

Starch: Hydrolysis weak. 

( 'ellulose: No growth. 

Nitrate reduction : Negative. 

Sucrose inversion: Negative. 

Antagonistic properties: Weak. 

Type culture: [MRU 3761. 

63. Streptomyces cyanoflavus Funaki and 
Tsuchiya, 1958 (Funaki, M., Tsuchiya, P., 
Maeda, K., and Kainiya, T. .1. Antibiotics 
(Japan) LI A: 143 I 19, 1958). 

Morphology: Sporophores straight, form- 
ing many branches, but no spirals. 

Sucrose nitrate agar: Growth colorless to 
pale yellowish-brown. Aerial mycelium white 
to light greenish-gray. No soluble pigment. 



200 



THE ACTIXOMYCETES, Vol. II 



Glucose-asparagine agar: Growth pale 
green to yellowish-brown. Aerial mycelium 
brownish-white to brownish-gray. Soluble 
pigment light blue to yellowish-brown. 

Calcium malate agar: Growth light brown- 
ish to brown. Aerial mycelium white to gray. 
Soluble pigment greenish-blue to light 
brown. 

Nutrient agar: Growth yellowish-brown to 
brown. Aerial mycelium grayish-white. No 
soluble pigment. 

Yeast extract agar: Growth pale yellow to 
brown. Aerial mycelium light olive-gray. 
Soluble pigment brown. 

Potato: Growth yellow to brown. Aerial 
mycelium brownish-gray. Soluble pigment 
dark brown. 

Gelatin: Growth yellow. Soluble pigment 
yellow to brownish-yellow. Gelatin lique- 
fied. 

Milk: Produces a sedimented growth with- 
onl any soluble pigment. Milk coagulated 
but not peptonized. 

Carbon utilization: Utilizes various sugars 
and salts of organic acids, but not xylose, 
acetate, or citrate. 

Antagonistic properties: Produces blue 
antibiotic cyanomycin, active against gram- 
positive and gram-negative bacteria; also 
produces aureothricin-like substances. 

64. Streptomyces cylindrosporus (Krassil- 
nikov, L941) Waksman (Krassilnikov, N. A. 

Actinomycetales. Izvest. Akad. Nauk. SSSR, 
Moskau, p. 57, 1941). 

Morphology: Sporophores straight, 
branched. Spores cylindrical or oblong, 1.0 
to 1.7 by 0.7 ij.. 

Sucrose nitrate agar: Colonies velvety, 
dark brown or chocolate. Aerial mycelium 
white-gray to brown-gray. Soluble pigment 
brown. 

\ ut lient agar: ( rrowth dark brown. Aerial 
mycelium white. Soluble pigment brown. 

Glucose-asparagine agar: Growth brown. 
Aerial mycelium white-gray. No soluble pig- 
ment. 



Gelatin: Weak Liquefaction. Melanin-posi- 
tive. 

Milk: Coagulation limited, peptonization 
weak; color of milk brown to almost black. 

Potato: Substrate growth brown. Aerial 
mycelium light gray. Soluble pigment brown. 

Starch: Weak hydrolysis. 

Cellulose: Limited, colorless growth. Aer- 
ial mycelium white. 

Nitrate reduction: Positive. 

Sucrose inversion: Negative. 

Antagonistic properties: None. 

Habitat : Soil. 

Remarks: The description of the organism 
has been supplemented by Hoffmann (1958). 
It appears to be related to S. vinaceus, S. 
purpureochromogenes, and S. purpeoftt.se us. 
( rause et al. (1957) described a related form 
as A. umbrinus. 

Type culture: IMRU 3764. 

(So. Streptomyces diastaticus (Krainsky) 
Waksman and Henrici (Krainsky, A. Centr. 
Bakteriol. Parasitenk. Abt. II, 41: 682, 
1914). 

Morphology: Sporophores form tight spi- 
rals. Spores oval or spherical, 1.0 to 1.2 by 
1.1 to 1.5 ii (Figs. 37, 38). 

Sucrose nitrate agar: Growth thin, gray. 
Aerial mycelium white, becoming drab gray. 

Calcium malate agar: Colonies 2 to 4 mm, 
yellowish when old. Aerial mycelium gray, 
with white outer zone; white specks fre- 
quently produced in gray mycelium. 

Glucose-asparagine agar: Growth yellow- 
ish, spreading. No aerial mycelium. 

Nutrient agar: Growth cream-colored. 
Aerial mycelium white, then gray. Soluble 
brown pigment. 

Potato: Growth white-gray. Aerial myce- 
lium gray and white. 

Gelatin: Liquefaction, with small, cream- 
colored flakes in liquefied part. 

Milk: Brownish ring. Coagulation and 
slow peptonization. 

Starch agar: Growth thin, colorless. 



hi SCRIPTION OF SPECIES OF STREPTOMYCES 



201 




Figure 37. Sporophores of S. tliastalicus, X 4,500, showing 
tesy of E. Baldacci. University of Milan, Italy). 



whole surface i < low 



spreading. Aerial mycelium gray. Ready hy- 
drolysis. 

Cellulose: Good growth. 

[nvertase: Negative. 

Nitrate reduction: Weak. 

Production of 1 1 jS : Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Limited. 

Habitat : Soil. 

Remarks: This species was redescrihed by 
Duche ( L934) under the name .1. roseodiasta- 
tlCUS. Baldacci et al. (1955) raised this spe- 
cies to the status of a "series." Several new 
species or varieties were created: A. virido- 
diastaticus, A. diastaticus var. ardesicicus, A. 
diastaticus var. venezuelae, A. rubrocyano- 
diastaticus var. impiger and var. piger. 

Type culture: [MRU 3315. 

66. Streptomyces diastatochromogi nes 

(Krainsky, 1!I14; Waksman and Curtis, 
L916) Waksman and Henrici, L948 (Krain- 
sky, A. Centr. Bakteriol. Parasitenk. Abt. 
II., 41: 683, L914). 



Morphology: According to Waksman and 
Curtis (1916), sporophores are straight. Ac- 
cording to .Jensen (1930), sinist rorse spirals 
are produced. Spores spherical or oval, L.2 m- 

Sucrose nitrate agar: Growth colorless. 
later yellowish-brown. Aerial mycelium 
abundant, white, later ash-gray. Soluble 
pigment yellowish to light brown. 

Sucrose malate agar: According to Krain- 
sky, growth colorless, with gray aerial myce- 
lium. When glucose is added, center of aerial 
mycelium is colored yellowish, with gray 
margin. 



GllK 



•ose-asparagiue agai 



dor- 



less, with gray aerial mycelium. 

Nutrient agar: Aerial mycelium white to 
gray. Soluble pigment brownish to coffee- 
brown. Melanin-positive. 

Potato: Growth light gray, later grayish- 
black. Aerial mycelium white to gray. Solu- 
ble pigment black. 

Gelatin: Growth cream-colored to yellow- 
ish-brown. Aerial mycelium -cant white. Sol- 



202 



THE ACTINOMYCETES, Vol. II 




Figure 38. Sporophores of S. diastaticus, X 15,000 (Courtesy of E. Baldacci, University of Milan. 
Italy). 



uble pigment brown. Liquefaction fairly Habitat: Very common in soil. 



rapid. 

Starch: Hydrolysis weak. 
Cellulose: No growth. 
Nitrate: Reduction to nitrite strong. 
Tyrosinase reaction: Positive. 
Temperature: Optimum 35°C. 
Antagonistic properties: Very strong. 



Remarks: Krassilnikov (1949) considered 
this species as a variety of A. chromogenes. 
Type culture: ATCC 12,309. 

()7. Streptomyces echinatus Corbaz el al., 
1957 (Corbaz, R., Ettlinger, L., Gasmann, 
Iv, Keller-Schierlein, W., Kradolfer, F., 
Xeipp, L., Prelog, \ T ., Reusser, P., and 



DESCRIPTION OF SPECIES OF STREPTOMYCES 203 

Zahner, II. Belv. Chim. Ada 10: L99 204, Remarks: Closely related to S. griseoflavus 

1957). and S.flaveolus. 

Morphology: Sporophores produce verti- , v <■, , > ,■ tcs~\. ■ 

1 °- , , , . n!v vtreptomyces elasticus (Sohngen and 

cils on sterile aerial hvphae, wit h open, ir- .• i i Q1/ i\ u i ,^.i KT . . 

. • . bol, 101 I i Waksnian (Sohngen, N. L. and 

regular spirals. Spores elliptical to oval; sur- .- . ( < < ,. , u , , • , ,, , A1 

. . ' , . , .. rol, J. G. Centr. Bakteriol. Parasitenk. Abt. 

lace oi spores covered with long, thin spines jj to. 07 no pui, 

(I>1 - I(M - . Morphology: Mycelium typical, branched. 
(dvcer(»l niirate agar: (jrrowth greenis 



yellow to citron-yellow to lighl green. Aerial 



Short, motile rods observed in young cul- 
tures. Spores white, round, diameter aboul 



mycelium white, changing to yellow, to ash- ■ 1 1 . ,1 . ,• .1 

6 & J . 1 /x, double that oi the mycelium, 

gray. Soluble pigmenl greenish-yellow to \ r / ■ ,. ' n • ■ , • 

& • ps & • Agar media: Growth yellowish- white. 

g s-green. Aerial mycelium snow-white. 

Glycerol malate agar: Growth pale yellow, Gelatin: Growth yellow-brown, 

turning greenish-yellow. Aenal mycelium Carbon utilization: Glucose, glycerol, ethyl 

white to pale yellow. No soluble pigment. al( , ()M< mannitolj organic acidSj ( . ;l|( 

Glucose-asparagine agar: Growth golden sa lts readily assimilated. 

yellow to greenish-yeUow to greenish-gray. Stl(T()S(i inversitfn: Positive 

Aenal mycelium ash-ray to reddish-violet. Urea; p roduces urease 

No soluble pigment. Paraffin: Utilized. 

Nutrient agar: Growth lighl yellow. No Rubber; niliz(i( , 1 . ( , :((Hlv 

rial mycelium. No soluble pigment. Temperature: Optimum 28°C, maximun 



•nun 



33 C destroyed at 65°C in 5 minutes. 



Glucose-peptone agar: Growth yellow 

Aerial mycelium white in center, brownish Habitat- S '1 

on periphery, changing to ash-gray. Soluble 

pigmenl golden yellow. (1!) - Streptomyces endus Gottlieb and Car- 
Starch agar: Growth yellow. Aerial myce- ,(>r - l956 (Gottlieb, D. and Carter. II. E. 

limn ash-gray. No hydrolysis, or at most, l ■ s - Paten1 2 > 74,i < 902 > Ma y --• L9 56). 

t races Morphology: Sporophores formed along 

Gelatin: Substrate growth dark brown. entire len S th of mycelium, at righl angles 

Aerial mycelium greenish-gray. Soluble pig- 1() iL ( ' P ac1 s P irals Produced, often with 

mem dark brown. No liquefaction after 31 1() loo P s - V,m,,ti llv P ll; '<' () < to 1.0 M in 

days. Melanin-positive. diameter; old hyphae 1.25 to 1.50 and even 

Potato: Growth greenish to raven-black. -•'' m- 

Aerial mycelium limited, white-gray to Sucrose nitrate agar: Substrate growth 

bluish-gray. Soluble pigmenl brownish to has color of medium, later turning dark, 

pitch-black. Aerial mycelium white, changing to lighl 

Milk: Good coagulation and peptoniza- gray, then to dark gray. No soluble pigment, 

tion. Gelatin: Slow and only slighl liquefaction. 

Tyrosinase reaction: Positive. No soluble pigment. Melanin-negative. 

Carbon utilization: Xylose, lactose, rafli- Starch: I Ivdrolysis rapid. 

nose, acetate, and succinate positive. Su- Potato: Growth good. Aerial mycelium 

crose, inulin, dulcitol, salicin, and sodium light gray. No soluble pigment, 

citrate negative. Milk: Coagulation; no visible peptoniza- 

Temperature: Develops well ai IS 40 C. tion. 

Antagonistic properties: Produce- echino- Carbon utilization: Utilizes starch, man- 

mycin. nose, dextrin, glucose, arabinose, maltose, 



204 



THE ACTINOMYCETES, V 



II 



and levulose. Poor growth with galactose, 
lactose, citric acid, malic acid, succinic acid, 
and cellulose. Does not utilize sucrose, 
sorbitol, dulcitol, inositol, or paraffin. 

Antagonistic properties: Produces an 
antibiotic, endomycin, active largely upon 
fungi. 

Remarks: Tresner and Backus (1956) 
consider this organism as a variant of the 
S. hygroscopicus group. 

70. Streptomyces erythraeus (Waksman 
and Curtis, L916) Waksman and Henrici, 
1948 (Waksman, S. A. and Curtis, P. E. 
Soil Sci. 1: 99, 1910; Waksman, 8. A. Soil 
Sci. 8: 112, 1919). 

Morphology: line, monopodially 

branched aerial mycelium; numerous sporo- 
phores with open and closed spirals. Spores 
spherical to oval, 0.7 to 0.8 M , smooth (PI. 
II m). 

Sucrose nitrate agar: Growth yellowish, 
later becoming red. Pigment insoluble in 
medium. Aerial mycelium thick, white to 
pale rose. 

Glucose-asparagine agar: Growth abun- 
dant, spreading, cream-colored, later turn- 
ing brown chiefly on surface; center raised, 
lobate margin. 

Nutrient agar: Substrate growth cream- 
colored. No soluble pigment. 

Potato: Growth wrinkled, cream-colored, 
becoming yellowish to red to purplish. 
Melanin-negalive. 

Gelatin: Growth abundant, dense, gray 
with pinkish tinge, chiefly on surface of 
slowly liquefied portion. No soluble pigment. 

Milk: Surface zone yellowish. Limited 
coagulation and positive peptonization. 

Starch media: Growth cream-colored with 
faint greenish tinge. Hydrolysis. 

Cellulose: Growth brick-red. 

Invertase: None. 

Nitrate: Reduction to nitrite only with 
starch. 

Production of IPS: Negative. 

Temperature: Optimum 25 ('. 



Antagonistic properties: Marked. Pro- 
duces erythromycin A and B. 

Habitat: Soil. 

Remarks: According to Ettlinger et al. 
(1958) S. rimosus and S. roseochromogenes 
belong to this group. Krassilnikov (1949) 
considers this organism as a variety of .1. 
ruber. A closely related, melanin-positive 
culture has been described as a new species, 
S. bottropensis (Brit. Pat. 762, 736, Nov. 19, 
1953). 

Type culture: IMRU 37M7; ATCC 11,635. 

71 . Streptomya s erythrochromogenes 

(Krainsky, 1914) Waksman and Henrici, 
1948 (Krainsky, A. Centr. Bakteriol. Para- 

sitenk. Abt. II, 41: 079-082, 1914). 

Different strains of this organism have 
been studied by Krainsky (1914), Waksman 
and Curtis ( 1910), Jensen ( 1930), and < )kami 
and Suzuki (1958). 

Morphology: Sporophores flexible, curved; 
spiral formation abundant according to 
Jensen and Okami and Suzuki. Waksman 
and Curtis reported no spirals. Spores oval. 

Sucrose nitrate agar: Growth at first 
cream-colored, later turning red to violet to 
purple. Aerial mycelium white to light gray. 
Soluble pigment red to red-violet according 
to Jensen. 

Calcium malate agar: Growth red to 
violet. Aerial mycelium grayish, with white 
margin. 

Glucose-asparagine agar: Aerial mycelium 
gray to white. Soluble pigment red. 

Nutrient agar: Growth yellowish-gray to 
light brown. Aerial mycelium white to light 
gray. Soluble pigment brown to deep brown. 

Starch agar: Aerial mycelium gray. Solu- 
ble pigment rose-colored. Diastatic action 
weak. 

Potato: Growth yellowish-gray, later al- 
most black. Aerial mycelium gray. Soluble 
pigment black. Melanin-posit ive. 

Gelatin: Growth yellowish to light purple. 
Liquefaction very slow. Soluble pigment 
brown. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 205 

Cellulose: Growth slow or none. Morphology: Substrate growth consists 

[nvertase: Negative. of long hyphae. Aerial mycelium abundant, 

Nitrate: Reduction slight. gray. Sporophores broom-shaped. Spores 

Pigment : Soluble in water, no1 in organic egg-shaped to spherical, smooth, 0.8 to 1.0 

solvents. by 0.6 to 0.7 y.. 

Temperature: Optimum 30 °C. Glycerol nitrate agar: Growth postulate 

Antagonistic properties: Produces sarko- lighl brown. Aerial mycelium sparse, white- 

mycin (Okami and Suzuki). gray, changing to ash-gray. Soluble pig- 
Habitat: No1 very common in soil. menl brown. 
72. Streptomyces eurocidicus Okami ei al, x ""-i<'"' agar: Growth brownish-yellow. 

l'.t:»l (Okami, Y., CTtahara, R., Nakamura, A, ' ml 1,1 . v, ' , ' li " 1 " ash-gray. Soluble pigmenl 



dish 



[■own. 



S.,and Umezawa, II. .1. Antibiotics (Japan) 
7A: 101 L02 1954). Glucose-asparagine agar: Growth thin, 

Morphology: Aerial mycelium short, whitish • V(,|lmv - Arml mycelium white-gray 
branched. Sporophores straight, without to a sh-gray. Soluble pigmenl chestnut- 
brown. 

Starch agar: ( rrOwth golden yellow. Aerial 



spirals; sometimes atypical verticils are 
produced. 

Glycerol nitrate agar: Growth colorless mycelium velvety, a1 first snow-white, later 
to yellowish-brown. Aerial mycelium scant, - r;, - v - Soluble pigmenl lighl brown. Rapid 



hin, w hite. No soluble pigmenl . 



hydrolysis. 



Glucose-asparagine agar: Growth colorless Gelatin: Growth sparse. Soluble pigmenl 

to yellowish-brown. Aerial mycelium white dark browiL Ra P id liquefaction, 

with yellowish tinge. Soluble pigmenl absenl Potato: Growth lichenoid, brownish- 

or slightly brown yellow. Aerial mycelium milky-white, be- 

Nutrienl agar! Growth yellowish-brown coming ash-gray. Soluble pigmenl brownish 

to black. Soluble pigment brown. Melanin- to pitch-black, 

positive i ?) Milk: Brown surface ring. Aerial mycelium 

Starch: Good hydrolysis. ash-gray. Soluble pigmenl dark brown. 

Potato: Growth wrinkled, brownish-yel- Coagulation and peptonization positive, 

low. Aerial mycelium absenl or thin white. Carbon utilization: Xylose, arabinose, 

No soluble pigment. fructose, galactose, saccharose, maltose, 

Gelatin: Growth yellowish-brown. Soluble lactose ' manmtol > salicio well utilized. 



pigmenl brown. No liquefacti 



Rhamnose, inulin, sorbitol, dulcitol, meso- 



Muk: Surface ring yellowish-brown, inositol not utilized. Some strains use acetate, 

NT .. N1 , .. citrate, and succinate. 

Nitrate: No reduction. 

r r ~ . , - 1 emperature: Poor growth al 18 C: very 

1 vrosinasc: Doubtful. , , , ,' 

good growth at 30 C; good growth hut do 



Antagonistic properties: Produce 
antifungal substance, eurocidin, and 
bacterial substances terl iomycin and a 



aerial mycelium at 58 C. 

Antagonistic properties: Produces a basic 
antibiotic, angolamycin, related to erythro 
mycin. 
73. Streptomyces eurythermusCorb&zetal., Habitat: Soil. 
L955 (Corbaz, P., Ettlinger, P., Gaumann, Remarks: Closely related to S. anti- 

!•:., Keller-Schierlein, W., Neipp, P., Prelog, / "'"/"'"*- 

V., Reusser, P., and Zahner, IP Helv. Chim. 74. Streptomyces exfoliatus (Waksman and 
Acta38: 1202 L209, 1955). Curtis, L916) Waksman and Henrici, L948 



206 



THE ACTINOMYCOTIC, Vol. II 



(Waksman, S. A. and Curtis, R. E. Soil Sci. 
1: 116, 1910; 8: 121, 1919). 

Morphology: Colony has tendency to 
crack and surface growth to exfoliate and 
peel off. Sporophores usually straight or 
slightly wavy; on some media there is a 
tendency to produce spirals. Spores oval, 1.0 
to 1.5 by 1.2 to 1.8 m- 

Sucrose nitrate agar: Growth smooth, 
colorless, becoming brown to blue. Aerial 
mycelium white. 

Malate-glycerol agar: Growth cream- 
colored. Aerial mycelium white. No soluble 
pigment. 

Glucose-asparagine agar: Growth cream- 
colored, turning brown. Aerial mycelium 
white, appearing late. 

Nutrient agar: Growth colorless. No 
aerial mycelium. Soluble pigment absent or 
brownish. 

Potato: Growth wrinkled, gray, becoming 
brown. No aerial mycelium. No soluble pig- 
menl . 

Gelatin: Growth cream-colored. Aerial 
mycelium white or absent. Liquefaction 
faint to fair. Melanin-negative. 

Milk: Cream-colored ring. Soft coagula- 
tion and slow peptonization. 

Starch media: Growth restricted, gray 
becoming brown. Aerial mycelium light 
buff -gray. Hydrolysis of starch medium, 
incomplete. 

[nvertase: Positive. 

Cellulose: Growth good. 

Nitrate reduction: Positive. 

Production of H-..S: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Positive. 

Habitat: Soil. 

Remarks: Krassilnikov (1949) considered 
this organism as a variety of one of the 
chromogenic groups . 

Type culture: IMRU 3310. 

75. Streptomyces felleus Lindenbein, 1952 
(Lindenbein, W. Arch. Mikrobiol. 17: 361- 

383, 1952). 



Morphology: Sporophores long, straight, 
branching. Spores spherical, smooth. 

Glycerol nitrate agar: Growth smooth, 
yellow-brown. Aerial mycelium velvety 
gray-white. Soluble pigment yellowish- 
brown. 

Glucose-asparagine agar: Growth colorless 
to brownish-yellow. Aerial mycelium gray- 
white. Soluble pigment brownish. 

Glycerol malate agar: Growth colorless 
to yellowish. Aerial mycelium powdery, 
gray- white. Soluble pigment yellowish- 
brown. 

Nutrient agar: Growth colorless, brown- 
ish-yellow reverse. No aerial mycelium. 
Soluble pigment light brownish-yellow. 
Melanin-negative. 

Glucose-peptone agar: Growth yellowish- 
brown. Aerial mycelium gray-white. Soluble 
pigment light brown. 

Starch media: Growth lichenoid, colorless. 
Aerial mycelium white. No soluble pigment. 
Hydrolysis strong. 

Potato: Growth brownish-yellow. No 
aerial mycelium. Soluble pigment absent or 
pinkish. 

Gelatin: Growth colorless. No aerial myce- 
lium. No soluble pigment. No liquefaction 
by one strain, positive by another. 

Milk: Growth brownish to orange. Aerial 
mycelium gray-white. Peptonization me- 
dium. 

Cellulose: No or weak growth. 

Production of H L .S: Negative. 

Odor: Typical earthy. 

Taste: Gall-bitter. 

Antagonistic properties: Produces anti- 
biotic picromycin. 

Remarks: Related to S. fimicarius. Ett- 
linger et el. (1958) considered this organism 
as belonging to S. olivaceus. 

Type culture: IMRU 3(159. 

70. Streptomyces fervens DeBoer et al., 
1959 (DeBoer, C, Dietz, A., Evans, J. S., 
and Michaels, R. M. Antibiotics Ann. 1959- 
19(10, pp. 220-220). 



DESCRIPTION OF SPECIES OF STREPTOMYCES 207 

Morphology: Sporophores monoverticil- Starch agar: ( rrowth same as on synthetic 

late or biverticillate. Pigment granules agar. Aerial mycelium white or while with 

presenl in mycelium, lighl brownish-salmon-pink tinge. Hydrol- 

Sucrose nitrate agar: Growth faint pink. ysis. 

Aerial mycelium pink. No soluble pigment. Gelatin: Growth yellowish. Aerial myce- 

Calcium malate agar: Growth pink, linm in form of white patches. No soluble 

Aerial mycelium trace, pink. No soluble pig- pigment. Medium liquefaction, 

ment. Potato plug: Growth cream-colored, wrin- 

Glucose-asparagine agar: Growth pink. kled. No aerial mycelium. No soluble pig- 
Aerial mycehum pink. Soluble pigmenl pale ment. 
yellow. Milk: Growth yellowish, surface ring. 

Starch nutrient agar: Growth red-pink. Aerial mycelium white, scant. No soluble 

Aerial mycelium pink. Soluble pigmenl tan. pigment. Coagulation and peptonization. 

Casein digest-beef extract agar: Growth Blood agar: Growth brownish, wrinkled. 

red. Aerial mycelium pink. No aerial mycelium. Hemolysis none or 

Gelatin: Liquefaction medium. Soluble weak. 

pigmenl brown. Antagonistic properties: Produces caryo- 

Milk: Growth brown to cream-pink, my cin, an antitumor substance. 

Aerial mycelium trace pink. No coagulation. 78 _ S treptomyces filipinensis Ammann et 

N,) Peptonization. „ ; ,,,-- (AmmaM1) Ai> Gottlieb, 1)., Brock, 

Production ol E 2 S: Positive. T D>) r . lrter> H . E>> and Whitfield, G. B. 

Starch: llydrolyzed. Phytopathology 15: 559 563, L955). 

Carbon utilization: I blues various carbo- Morphology: Sporophores form spirals 

hydrates, glycerol, inositol, starch, certain that vary from open to tightly closed. Spores 

organic acids (acetate, citrate, succinate); round to oval. 

does not utilize rf-xylose, rhamnose, lactose, Sum)>( . m1i , ;(t(> agaJ . GrQwth ( . X( . (l | 1( . n , 

Z-arabmose, formic, oxalic, and tartaric acids. light V(1 „ mv _ Ama , mV( ,,| mm ( . nl , ((||V- 

Antagonistic properties: Produces an..- wh ite, turning gray. Soluble pigmenl slightly 

biobc fervenulin, active agamsl various ye Uow. Colorless drops of exudate on myce- 

microbes and tumors. [[utd 

Habitat: SoU in California. Starch-nitrate agar: Growth excellent. 

77. Streptomyces filamentosus Okami et al., Aerial mycelium velvety, white, turning 

1953 (Okami, Y., Okuda, T.. Takeuchi, T., gray. No soluble pigment. Hydrolysis weak. 

Nitta, K., and Umezawa, II. J. Antibiotics Glycerol-asparagine agar: Growth excel- 

( Japan) 6A: 153 157, 1!)."):!). lent. Aerial mycehum white, turning gray. 

.Morphology: Sporophores straight, long, Soluble pigmenl slightly yellow. 

without spirals. Spores oval to elliptical. Nutrient agar: Growth very poor, lighl 

Sucrose nitrate agar: Growth colorless. buff. No aerial mycelium. Soluble pigmenl 

Aerial mycelium abundant, cottony, white, brown. 

Xo soluble pigment . Gelatin: Slow but definite liquefaction, 

Glucose-asparagine agar: Growth color- stratiform type. Soluble pigmenl brown. 

less. Aerial mycelium abundant, white with Potato: Growth good. No aerial mycelium, 

pinkish-orange tinge or brownish to almosl Soluble pigmenl purple to black. 

salmon-pink tinge. No soluble pigment. Nitrate reduction: Little, if any. 

Nutrient agar: Growth colorless. Aerial Production of H 2 S: Positive, 

mycelium thin, white. No soluble pigment. Carbon utilization: Utilizes xylose, arabi- 



208 



THE ACTINOMYCETES, Vol. I] 



nose, fructose, galactose, sucrose, maltose, 
lactose, raffinose, inulin, mannitol, inositol, 
sodium acetate, sodium citrate, sodium 
succinate, dextrose, mannose, starch, dex- 
trin, and glycerol. Does not utilize rhamnose, 
sorbitol, dulcitol, salicin, phenol, w-cresol, 
sodium formate, sodium oxalate, sodium 
tartrate, or sodium salicylate. 

Antagonistic properties: Produces anti- 
fungal agent hlipin, of the polyene type. 

Habitat : Philippine soil. 

Type culture: LMRU 3781. 

79. Streptomyces fimbriatus (Millard and 
Burr, 1926) Waksman and Henrici, P.)48 
(Millard, W. A. and Burr, S. Ann. Appl. 
Biol. 13: 580, 1926). 

Morphology: Sporophores form spirals 
with three or more turns. Spores cylindrical 
to oval, 1.2 to 0.9 by 0.9 m- 

Sucrose nitrate agar: Growth gray. Aerial 
mycelium abundant, white to gray. Soluble 
pigment cream-colored. 

Glucose-asparagine agar: Growth very 
good. Aerial mycelium white to mouse-gray. 

Nutrient potato agar: Colonies gray to 
blackish, flat, raised in center. Aerial myce- 
lium a lew specks of white. Soluble pigment 
golden brown. 

Potato: Growth mouse-gray. Aerial myce- 
lium on dried portions of growth scant, white 
to mouse-gray. Pigment around growth 
black. 

Gelatin: Growth good. Aerial mycelium 
white. Liquefaction slow. Soluble pigment 
reddish. 

Milk: Growth good. No coagulation and 
no hydrolysis. 

Starch: Positive hydrolysis. 

Tyrosinase reaction: Strongly positive. 

Nitrate reduction : Positive. 

Habitat: Common scab of potatoes. 

80. Streptomyces fimicarius (Duche, L934) 
Waksman and Henrici, 1948 ( Duche, .1. Pes 
actinomyces du groupe albus. P. Lechevalier, 
Paris, L934). 



Morphology: Sporophores long, tuft- 
forming; no spirals. Spores cylindrical (Hoff- 
mann, 1958). 

Sucrose nitrate agar: ( irowth at first color- 
less, later yellowish to red-brown; reverse 
orange-colored. Aerial mycelium light gray 
with yellowish tone. Soluble pigment faint 
yellowish. 

Glucose-asparagine agar: Growth cream- 
colored with whitish aerial mycelium; re- 
verse cream-colored to slight ocher. 

Nutrient agar: Growth limited, cream- 
colored with white aerial mycelium; reverse 
yellowish. 

Potato: Growth cream-colored to yellow- 
ish to dark brown. Aerial mycelium gray. 
Soluble pigment reddish-brown. 

Gelatin: Punctiform colonies with whitish 
aerial mycelium. Soluble pigment reddish. 
Liquefaction medium. Melanin-negative. 

Milk: (irowth colorless, becoming covered 
with whitish aerial mycelium. Slow pep- 
tonization. Pigment rose, changing to brown- 
ish-red. 

Starch: Hydrolyzed. 

Coagulated serum: Growth cream-colored. 
Aerial mycelium whitish. Liquefaction rapid. 

Cellulose: No growth. 

Tyrosine medium: (irowth white, with 
yellowish reverse. Soluble pigment yellowish. 

Production of H 2 S: Negative. 

Antagonistic properties.- Positive. 

Remarks: Krassilnikov (1949) considers 
this organism as a variety of .1. chromogenes. 

81. Streptomyces flaveolus (Waksman) 
Waksman and Henrici, 1948 (Waksman, 
S. A. No. 168. Soil Sci. 8: 134, PIP)). 

Morphology : Sporophores monopodia lly 
branched. Short, closed and open spirals 
produced on all media. Spores oval to 
elliptical, 0.S by 1.2 n, covered with long, 
fine haii - . 

Sucrose nitrate agar: (irowth light sulfur- 
yellow, turning cadmium-yellow. Aerial 
mycelium white to ash-gray. Soluble pig- 
ment yellow. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 209 

Malate-glycerol agar: Growth colorless to late, at first white, later gray. Soluble pig- 
cream-colored. Aerial mycelium mouse-gray, menl brown. 

Glucose-asparagine agar: Aerial mycelium Starch agar: Growth yellow. Aerial myce- 

pale gray. Soluble pigmenl yellowish-green, lium white. Weakly diastatic. 

Nutrient agar: Growth colorless, ".listen- Potato: Growth yellow. Aerial mycelium 

ing, wrinkled. Aerial mycelium white. Solu- white. Soluble pigmenl black, 

ble pigmenl absenl or yellow. Gelatin: Colonies yellowish. Slight lique- 

Potato: Growth abundant, wrinkled, faction. Soluble pigment brown, 

cream-colored to yellow. Aerial mycelium Cellulose : Growth slow. 

white to pinkish. Soluble pigmenl absent or Nitrate reduction: Strong. 

taint brown. Tyrosinase: Positive. 

Gelatin: Growth abundant, yellowish, Temperature: Optimum 35°C. 
spreading. Aerial mycelium white. Liquefac- Habitat: Garden soil, 
tion rapid. Soluble pigment yellowish-brown, Remarks: Krainsky considered this cul- 
not melanoid. ture as identical to .1. chromogenes (las- 
Milk: Ring sulfur-yellow. Rapid coagula- perini. 
tion and strong peptonization. Type culture: [MRU 3671. 

Starch media: Growth colorless. Aerial my- s; , Strevtomyces ilw , iqns< „, ( Duch< * 

celium hghl gray. Hydrolysis. , u;U) Waksman ( Duch 4 J. Les actinomyces 



Cellulose: Growth scant. du groupe albus. P. Lechevalier, Paris, 1934 

Invertase: Negative. 
Nitrate reduction: Positive. 



Morphology: Sporophores long, straight, 

with a few curling tips. Spores spherical. 

Production ot IP.S: Negative. o„ ,. tl . t n ,i i- ■ i 

6 Sucrose nitrate agar: Growth hunted. 

Antagonistic properties: Some strains yellowish) reverse fcuming black Aeria] 

P roduc e actinomycm. mycelium thin, gray to mouse-gray. 

Habitat: Soil. Xutri(lllt Qrowth thi 



Remarks: Several varieties of this org aJ .. M , <im ,_ A( , na| lium thi white _ X( 



ream 

colored. Aerial mycelium thin, white. 

ism have been described. It is sumcienl to s()lul)](> pigment Melanin-negative. 

mention a culture described by Krassilmkov Glucose-peptone agar: Growth yellow; 

as .1. rectus, which appears to he a variety ,. „ ,. ♦ , u , .,,, i i \ ■ i 

' ' • reverse tending to turn dark. Aerial myce- 

of S. flaveolus. Krassilnikov also believed i- „ , ,, i '. ,, . , , A1 

•' hum abundant, mouse-gray to drab. No 

thai .1. krainskii Duche" belongs to this so i u bl e nigmenl 

lil '" ,l,) ' Starch agar: Growth very limited, similar 

Type culture: IMRU 3319. tn lh;M on sucrose nitrate agar. Hydrolysis. 

82. Streptomyces flavochromogenes (Krain- Potato: Growth abundant, lichenoid. 

sky, L914) Waksman and Henrici, 1948 Aerial mycelium abundant, mouse-gray to 

(Krainsky, A. Centr. Bakteriol. Parasitenk. drab with white edge. No soluble pigment. 

Abt. II, 11: us:,. L914 . Gelatin: Growth flocculent, through me- 

Morphology: Spores oval, 1.7 p. dium. Liquefaction slow. No soluble pig- 

Glucose-asparagine agar: Growth yellow, ment. 

Aerial mycelium gray. Soluble pigmenl Milk: Cream-colored ring. No aerial myce- 

brown. lium. Peptonization very rapid. 

Calcium malate agar: Growth yellow. Remarks: According to Ettlinger et al. 

Aerial mycelium produced late, white to (1958) this organism belongs to the S. 

gray. fradiae group. 

Nutrient agar: Aerial mycelium formed Type culture: IMRU 3322. 



210 



THP] ACTINOMYCETES, Vol. II 



84. Streptomyces flavoreticuli Funaki et al., 
1958 (Funaki, M., Tsuchiya, F., Maeda, 
K., and Kamiya, T. J. Antibiotics (Japan) 
11A: 138-142, 1958). 

Morphology: Aerial mycelium is long with 
many short branches; numerous small 
verticils are produced depending on the 
nature of the medium, especially on starch- 
ammonium agar. 

Sucrose nitrate agar: Growth colorless to 
pale yellow. Aerial mycelium white, cottony. 
Soluble pigment faint yellow. 

Glucose-asparagine agar: Growth pale 
yellow to yellowish-brown. Aerial mycelium 
yellowish-white to olive-gray. Soluble pig- 
ment pale yellow. 

Calcium malate agar: Growth pale yellow. 
Aerial mycelium pale yellow. Xo soluble 
pigment. 

Nutrient agar: Growth yellow to brown. 
Aerial mycelium pale yellow to yellowish- 
gray. Soluble pigment brown. 

Potato: Growth yellow, folded. Aerial 
mycelium yellowish white to olive-gray or 
olive-yellow. Soluble pigment dark brown. 

Milk: Colorless pellicle. Aerial mycelium 
white. Soluble pigment brown. Milk coagu- 
lated, then peptonized. 

Gelatin: Growth yellowish-brown. Aerial 
mycelium white to light gray. Soluble pig- 
ment brown. Gelatin liquefied. 

Starch: Not hydrolyzed. 

Antagonistic properties: Produces anti- 
biotic virocidin, which possesses antiviral and 
antibacterial properties. 

Remarks: Similar to S. reticuli and to S. 
Jim- us, differing from the first by the forma- 
tion of yellow growth and yellow soluble 
pigment and from the second by the forma- 
tion of verticils. 

85. Streptomyces flavovirens (Waksman, 
191'.)) Waksman and Henrici, 1948 (Waks- 
man, S. A. Soil Sei. 8: 117, 1919). 

Morphology: Sporophores coarse, straight, 
and short, relatively unbranched; large 
masses of minute tufts; open spirals may be 



produced in certain substrates. Spores 
spherical, oval to rod-shaped, 0.75 to 1.0 by 
1 .0 to 1 .5 M . 

Sucrose nitrate agar: Growth yellowish 
with greenish tinge. Aerial mycelium gray. 
Soluble pigment greenish-yellow. 

Glucose-asparagine agar: Growth re- 
stricted, developing only to a very small 
extent into the medium, yellow, turning 
black. Soluble pigment golden yellow to 
greenish-yellow. 

Nutrient agar: Growth yellowish; reverse 
dark in center with yellowish zone and 
outer white zone. 

Potato: Growth sulfur-yellow, wrinkled. 

Gelatin: Surface pellicle yellowish-green. 
Good liquet action. Melanin-negative. 

Milk: Cream-colored to brownish ring; 
coagulation and peptonization. 

Starch agar: Growth greenish-yellow, 
spreading, developing deep into the medium. 
Good hydrolysis. 

Invertase: Negative. 

Nitrate reduction: Limited. 

Production of H- 2 S: Negative. 

Cellulose: No growth. 

Antagonistic properties: Produces actino- 
mycin. 

Habitat : Soil. 

Remarks: Certain forms belonging to this 
species, such as A. griseostramineus and A. 
olivaceoviridis, have been described by Gause 
et al. (1957). Ettlinger et al. (1958) considers 
this species as belonging to the S. fradiae 
group. Hirsch (1960) considers this organism 
as an oligonitrophilic form. 

Type culture: IMRU 3320. 

8(>. Streptomyces flavus (Krainsky, 1914) 
Waksman and Henrici, 1948 (Krainsky, A. 
Centr. Bakteriol. Parasitenk. Abt. II, 41: 
(585, 1914; Waksman, S. A. and Curtis, 
R. E. Soil Sci. L: 99, 1916; 8: 71, 1919). 

Not .1. flavus Krainsky emend. Krassil- 
nikov (1941). 

Morphology: Sporophores are long, usn- 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



211 



ally no spirals; some open spirals may be 
produced. Spores oval, 1 .2 m- 

Sucrose nitrate agar: Growth yellow or 
sulfur-yellow. Aerial mycelium straw-yellow. 

Glucose-asparagine agar: Growth sulfur- 
yellow, center shading to brown. Aerial 
mycelium white to gray. 

Nutrient agar: Growth gray, spreading, 
folded. Aerial mycelium white, appears late. 

Starch agar: Growth cream-colored with 
pink tinge. Hydrolysis marked. 

Potato: Growth yellow. Aerial mycelium 
gray. Melanin-negative. 

Gelatin: Growth in form of small, yellow- 
ish masses on surface. Rapid liquefaction. 
Melanin-negative. 

Milk: Rapid coagulation and peptoniza- 
tion. 

Sucrose inversion: Negative. 

Nitrate: No reduction. 

Cellulose: ( Irowth poor. 

Temperature: Optimum 25°C. 

Antagonistic properties: Some strains 
produce actinomycin and certain other 
antibiotics. 

Habitat : Soil. 

Remarks: Represents a large group of 
species, as shown previously (Chapter 3). 
Above description is Rased largely upon that 
given by Krainsky. According to Ettlinger 
ei al. (1958), this organism does not form 
any spirals (as found also by Waksman and 
Curtis) and is related to S. olivaceus. 

Type culture: [MRU 3321. 

87. Streptomyces flocculus (Duche, L934) 
Waksman and Henrici ( Duche, J. Res 
actinomyces du groupe albus. R. Lechevalier, 
Paris, L934). 

Sucrose nitrate agar: Growth cream- 
colored, later covered with white aerial 
mycelium. No soluble pigment. 

Glucose-asparagine agar: Growth limited. 
cream-colored, only slightly raised above the 
surface of the medium; occasionally abun- 
dant growth produced with white aerial 
mycelium, colorless on reverse side. 



Nutrient agar: Growth cream-colored, 

later covered with white aerial mycelium. 
No soluble pigment . 

Rotato: Growth punctiform. Aerial myce- 
lium white. Soluble pigment faint, yellowish. 

Gelatin: Growth limited. Liquefaction 
slow-. Melanin-negative. 

Milk: Growth rose-colored. Peptonization 
slow. 

Coagulated serum: Growth cream-colored. 
Aerial mycelium line, white. Liquefaction 
slow. 

Tyrosine medium: Growth whitish. No 
soluble pigment. 

Production of HjS: Negative. 

Remarks: Belongs to the S. albus series. 

88. Streptomyces fradiae (Waksman and 
Curtis, R)l(i) Waksman and Henrici, L948 
(Waksman, S. A. and Curtis, R. E. Soil Sci. 
1: 99-134, 1916; 8: 1)0, 1919). 

Morphology: Sporophores branched 
monopodially, straight or flexible, but no 
true spirals. On certain media, such as 
glycerol agar, spirals are formed. Ettlinger 
et al. (1958) found open spirals. Spores oval 
to rod-shaped, 0.5 by 0.7 to L.25 m. smooth 
(Fig. 39). 

Sucrose nitrate agar: Growth smooth, 
spreading, colorless, or pale yellow-orange. 
Aerial mycelium thick, cottony, seashell- 
pink. No soluble pigment . 

Malate-glycerol agar: Growth orange. 
Aerial mycelium seashell-pink. 

Glucose-asparagine agar: Growth re- 
stricted, glossy, buff-colored, lichenoid mar- 
gin. Aerial mycelium appears late, seashell- 
pink. 

Nutrient agar: Growth restricted, yellow- 
ish, becoming orange-yellow to buff. No 
aerial mycelium. No soluble pigment. 

Potato: Growth restricted, orange-colored. 
Aerial mycelium white to rose or pink. Solu- 
ble pigment absent or faint brown. 

Gelatin: Growth dense, cream-colored to 
brownish. Aerial mycelium white. Gelatin 
liquefied. No soluble pigment. 



212 



THE ACTINOMYCETES, Vol. II 




Figure 39. Sporophores of S. fradiae I Prepared 
by H. Lechevalier of t he Institute of Microbiol- 
ogy ) . 

Starch media: Growth spreading, colorless. 
Aerial mycelium seashell-pink. Good dia- 
static action. 

Milk: Cream-colored ring; coagulation 
and rapid peptonization. 

Nitrate: Varied reduction. 

Cellulose: No growth in solution, fair 
growth on plates. 

Production of H 2 S: Negative. 

Invertase: None. 

Antagonistic properties: Highly antag- 
onistic. Produces an antibacterial agent, 
neomycin, and an antifungal agent, fradicin. 

Habitat: Soil. 

Remarks: A number of strains of this 
organism have been isolated from various 
soils (see, for example, S. decaris described as 
No. 3719, in Waksman et al., L958). Some 
vary in their pigmentation, rate of gelatin 
liquefaction, and antibiotic production; A. 
longissimus Krassilnikov is one such typical 
strain. Some strains are able to produce 
antiviral substances, as in the case of luridin, 
produced by a strain of S. fradiae designated 
as .1. luridus by Krassilnikov et al. Gause 
et al. (1957) described a spiral-producing 
variety of S. fradiae under the name spiralis. 
Several other such strains have been isolated 
by Waksman and Lechevalier, (Jmezawa, 
and many others. Kit linger et al. (1958) 
claimed that S. rochei, S. filipinensis, S. 



coelicolor, S. flavogriseus, S. tyrosinaticus, S. 
violaceus, and S. violaceoruber belong to this 
group; this claim cannot be accepted on the 
basis of evidence submitted in the descrip- 
tions of these organisms. The characteristics 
of the species are that it is nonchromogenic, 
strongly proteolytic, and produces the 
characteristic seashell-pink aerial mycelium 
on various synthetic media; on organic 
media, orange-colored growth is produced 
without any aerial mycelium. 
Type culture: IMRU 3535. 

89. Streptomyces fragilis Anderson et a/., 
1956 (Anderson, L. H, Ehrlich, J., Sun, 
S. H., and Burkholder, P. K. Antibiotics & 
Chemotherapy 6: 100, L956). 

Morphology: Aerial hyphae simple or 
branched, usually in small clusters; short, 
straight, or slightly curved, with bent or 
curved tips and occasional short spirals. 
Spores spherical to ovoid, 0.8 to 1.5 by 1.0 
to 2.0 m- 

Calcium malate agar: Growth sparse. 
Aerial mycelium light brown. 

Glycerol-asparagine agar: Growth sparse, 
colorless to light yellow. Aerial mycelium 
lighl yellow-pink. 

Starch-ammonium sulfate agar: Growth 
yellow to yellow-orange to orange-brown. 
Aerial mycelium light yellow-pink to light 
brown. 

Nutrient agar: Growth yellow to yellow- 
orange to brown. Aerial mycelium white to 
light yellow-pink. Melanin-negative. 

Glucose-tryptone agar: Growth yellow to 
yellow-orange to brown. Aerial mycelium 
light yellow-pink, occasionally pink to gray- 
pink.' 

Gelatin: Liquefaction slow. No soluble 
pigment. 

Litmus milk: Slow peptonization. 

Starch: Hydrolysis. 

Nitrate reduction : Positive. 

Carbon utilization: btilizes L-arabinose, 
D-cellobiose, dextrin, D-galactose, glucose. 
D-maltose, starch, trehalose, and D-xylose. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 213 

I )<><•> not utilize aesculin, adonitol, cellulose, Habitat: Very common in soil, 

citrate, dulcitol, glycerol, ^'-inositol, inulin, Remarks: The characteristic golden pig- 

D-lactose, D-levulose, D-mannitol, D-man- menl is formed in nearly all media, bul 

nose, melezitose, melibiose, D-ramnose, l- becomes most typical and attains its greatesl 

rhamnose, salicin, D-sorbitol, succinate, or brightness in synthetic agar media. It has 

sucrose. indicator properties, turning red in strongly 

Antagonistic properties: Produces a sub- acid solutions. The species is easily recog- 

stance, azaserine, thai possesses certain uized on agar plates by its bronze-colored 

anticancer properties. colonies, surrounded by halo- of brighl yel- 

Source: Argentine soil. low pigment. 

Remarks: Closely resembles S. fradiae. This species was believed to be identical 

A detailed comparison between tins and with the culture described by Millard and 

closely related organisms has been made by Burr (1926) as .1. flavus. The last name is 

Anderson < / til. (1956). invalid, however, since the culture could be 

Type culture: [MRU : > >7:;'_\ NRRL 2424. readily distinguished from the S. flavus of 

90. Streptomyces fulvissimus (Jensen, Krainsky (1914, emend. Waksman and 

L930) Waksman and Henrici, 1948 (Jensen, Curtis > l916 ) Waksman and Henrici. 

II. L. Soil Sci. 30: 66, L930). T - V l ),> culture: [MRU 3665. 

Morphology: Sporophores short, straight, 91. Streptomyces fumosus (Krassilnikov, 

often trifurcated. Slightly wavy, hut no true 1941) Waksman (Krassilnikov, N . A. Actino- 

spirals. Spore- oblong, smooth, 1.0 to 1.2 mycetales. Izvest. Akad. Nauk. SSSR, 

by 1.2 to l.:> n (PI. I d). Moskau, p. 58, 1941). 

Sucrose nitrate agar: ( rrowth lighl golden, Morphology: Sporophores straighl . Spores 

later deep orange to red-brown. Aerial myce- cylindrical, later round, 1..") to 2.0 by 0.7 m- 

lium scant, white, later grayish-brown. Sucrose nitrate agar : Growth dark brown, 

Soluble pigmenl brighl golden to orange. pigment insoluble. Aerial mycelium well 

Glycerol-asparagine agar: Growth golden developed, cottony, dust-colored, occa- 

to dark brown. Aerial mycelium white to sionallv gray-white. 

lighl cinnamon-brown. Soluble pigment Nutrient agar : Growth dark brown. Aerial 

golden to orange. mycelium white. Soluble pigmenl brown. 

Nutrient agar: Growth wrinkled, deep Potato: Aerial mycelium absenl <>r only 

red-brown. No aerial mycelium. Soluble pig- taint, dark gray. Melanin-negative, 

menl brownish-yellow. Melanin-negative. Gelatin: Liquefaction medium. 

Potato: Growth wrinkled, rust-brown. Milk: No coagulation; slow liquefaction. 

Aerial mycelium absent or white. Soluble Soluble pigment dark brown to almost black. 

pigment gray to faint lemon-yellow. Starch: Good hydrolysis. 

Gelatin: Growth yellowish-brown to red- Cellulose: No growth. 

brown. No aerial mycelium. No soluble pig- Sucrose: Inversion weak. 

ment. Rapid liquefaction. Antagonistic properties: None 

Starch-casein agar: Growth yellowish- TI , .. . Q ., 

i v • , i- i i , Habitat : Soil. 
brown. Aerial mycelium hydrolyzed, smooth, 

lead-gray. Soluble pigment dull yellow to '•'-■ Streptomyces fungicidicus Okaxnietal, 

orange starch. 1954 (Okami, Y., Qtahara, R., Nakamura, 

Production of H 2 S: Positive. S., and Qmezawa, II. J. Antibiotic.- (Japan) 

Antagonistic properties: Produces valino- 7A: 100 101, 1954). 

myein. Morphology: Aerial myceUum produces 



214 



THE A.CTINOMYCETES, Vol. II 



numerous spirals on most synthetic media. 
Spores spherical to oval. 

Glycerol nitrate agar: Growth colorless. 
Aerial mycelium white to grayish. 

Glucose-asparagine agar: Growth color- 
less. Aerial mycelium white to grayish. Solu- 
ble pigment sometimes yellowish. 

Calcium malate agar: Growth colorless to 
yellowish. Aerial mycelium white to grayish. 
Soluble pigment of some strains pink; later 
disappears. 

Nutrient agar: Soluble pigment absent or 
slightly yellowish-brown. Melanin-negative. 

Starch agar: Growth colorless to yellowish. 
Aerial mycelium white to grayish. Strong 
hydrolysis. 

Potato: Growth yellowish to grayish. 
Aerial mycelium absent or white to grayish. 
A deep brown soluble pigment around the 
growth may be produced. 

Milk: Growth colorless to cream-colored. 
Soluble pigment absent or slightly brown. 
Coagulation and peptonization weak. 

Gelatin: Growth colorless to cream- 
colored. Soluble pigment absent or faint 
brown. Positive liquefaction. 

Nitrate: No, or doubtful reduction. 

Tyrosinase: Some strains positive. 

Antagonistic properties: Produces a poly- 
ene-type antifungal substance, fungicidin. 

Remarks: Two groups of this species were 
recognized; Group A produces nonspiral- 
forming sporophores; aerial mycelium white; 
violet pigment on potato. It is thus differ- 
entiated from Group G, described above. 

93. Streptomyces fuscus (Sohngen and Fol, 
llilli Waksman (Sohngen, N. L. and Fol, 
J. G. Centr. Bakteriol. Parasitenk. Abt. II, 
40: 89-98, 1914). 

Morphology: According to Krassilnikov, 
the organism forms short straight sporo- 
phores arranged in fascicles or clusters in the 
form of brushes. 

Agar media: Growth irregular, dry, color- 
less to stone-red. Aerial mycelium initially 
white, later becoming dark brown. Spores 



rose-colored. Some strains excrete a brown 
substance in protein media. 

Carbohydrates: Slight decomposition; 
even glucose is assimilated with difficulty. 

Carbon utilization: Best sources are 
calcium salts of various organic acids, rang- 
ing from malic and citric to stearate and 
palmitate. Formate not utilized. 

Nitrogen utilization: Ammonium chloride 
and asparagine, nitrate, and peptone assimi- 
lated with difficulty. 

Paraffins: Assimilated. 

Rubber: Brown-red growth. Rubber de- 
composed. 

Temperature: Optimum 33°C; maximum 
37°C. Destroyed in 5 minutes at 65°C. 

Habitat: Soil. 

94. Streptomyces galbus Frommer, 1959 
(Frommer, W. Arch. Mikrobiol. 32: 195, 

1959). 

Morphology: Sporophores monopodially 
branched, ending in spirals with 3 to 8 turns. 
On some media, certain strains produce long, 
straight, slightly branched aerial hyphae, 
with short side branches. 

Glycerol nitrate agar: Growth abundant; 
reverse yellow to yellow-green. Aerial 
mycelium cream-colored, mouse-gray, or 
green-gray. Soluble pigment golden yellow, 
later turning green-yellow. 

Glucose-asparagine agar: Growth weak, 
crusty, reverse light yellow or green-yellow, 
later turning brown. Aerial mycelium thin, 
powdery, mouse-gray with white spots. 
Soluble pigment yellowish to yellow-green. 

Calcium malate agar: Growth yellow to 
greenish-yellow. Aerial mycelium white to 
brownish-gray. Soluble pigment yellow to 
yellow-green. 

Nutrient agar: Growth thin, brown. Xo 
aerial mycelium. Soluble pignieni brown. 

Starch-KN< > : , agar: Growth yellow. Aerial 
mycelium white to white-gray. Soluble pig- 
nieni yellow. Slow hydrolysis of starch. 

Potato: Growth heavy, yellow to reddish- 
brown. Aerial mycelium powdery, white. 



Dl S< RIPTION OF SPECIES OF STREPTOMYCES 215 



mouse 



ray to green-gray. Soluble pigmenl 96. Streptomyces galtieri (Cord and Jou- 

black. bert, L951 i Waksman (Goret, P. and Jou- 

Gelatin: Growth abundant. Aerial myce- bert, L. Ann. parasitol. humaine ei comparee 

liuni yellow. Soluble pigmenl dark In-own. 2U: lis L27, 1951). 

Slow liquefaction. Morphology: Two types of colonies are 

Milk: No coagulation; slow peptonization, produced on agar: one small, flat, regular, 

Cellulose: Growth good. white; the oilier large, thick, irregular, 

Antagonistic properties: Produces actino- yellowish. Sporophores form spirals. Spores 

mycin. oval, 0.8 to L.5 by 0.8 n. 

Remarks: Related to S. viridochromogenes, Sucrose nitrate agar: Growth limited. 

S. flavochromogenes, and S. viridoflavus. Ii Aerial mycelium powdery, white. No soluble 

was also said to be related to S. parvullus. A pigment. 

variety of this species designated as achro- Nutrient agar: Growth poor, thin, yellow- 

mogenes, not producing any melanin pig- ish. Aerial mycelium powdery, white. Solu- 

ment, was also described. hie pigmenl brown. 

95. Streptomyces galilaeus Ettlinger et al. t Peptone agar: Growth limited, cream- 

L958 (Ettlinger, L., Corbaz, R., and Hiitter, colored. Aerial mycelium powdery, white. 

P. Arch. Mikrobiol. 31: :;.">ti. 1958). Soluble pigmeni very slight, brown-reddish. 

Morphology: Sporophores tnonopodially Starch agar: Growth thin. Aerial myce- 

branched, with open, regular spirals. Spores limn powdery, while. No soluble pigment. 

smooth (PI. II k). Potato: Punctiform colonies growing to- 

Glycerol nitrate agar: Substrate growth gether as thick crust, orange-reddish in 

at first lighl carmine, later carmine-red. color - Aerial mycelium limited, white, ap- 

Aerial mycelium white-gray. No soluble pig- pearing very slowly. No soluble pigment. 

ment. Gelatin: Growth poor, flaky, white. 

Glucose-asparagine agar: Growth thin. Liquefaction limited. 

white-yellow, later red. Aerial mycelium ^ Inl<: Growth slow. Aerial mycelium 

ash-gray. No soluble pigment. white. At 25°C no coagulation; at 37°C 

Calcium malate agar: Growth thin, white- coagulation after 20 days; no peptonization, 

yellow, later red. Aerial mycelium ash-gray. Nitrate reduction: Positive. 

No soluble pigment. Production of IPS: Negative. 

Starch agar: Growth carmine-red. Aerial Source: Dog septicemia (thoracic, ab- 

mycelium white-gray. Limited hydrolysis. dominal, and brain lesions). 

Potato: Growth brownish-yellow. Aerial Remarks: Said to be pathogenic for 

mycelium ash-gray. Soluble pigment limited, guinea pig and rabbit. Culture grown in the 

chestnut-brown. Melanin-positive. laboratory not pathogenic for dogs. 

Gelatin: Surface growth light red to lighl 

brown. Aerial mycelium sparse, grayish- 97 ' Stre Vtomyces gardneri (Waksman and 

whit... Soluble pigment reddish-brown to Hennci ) Q0V " comb " (Gardner, A. D. and 

dark brown. Liquefaction trace. Melanin- Chain > E - Brit - J - Ex P tL PathoL 2:i: 12:! - 

p 0S itive. L942 S Waksman, S. A., Horning, E. S. 

.Milk: Pellicle thick, light brown. Aerial Welsch, M., and Woodruff, II. B. Soil Sci. 

mycelium ash-gray. Limited coagulation, no 54: 289, L942). 

peptonization. Morphology: When grown on oatmeal 

Antagonistic properties: Positive. agar, aerial mycelium thin, largely at edge 

Habitat : Soil, of growth, consisting <>!' short, straighl to 



216 



THE ACTINOMYCETES, Vol. II 



wavy sporophores, often produced in clus- 
ters; no spirals. 

Glucose-asparagine agar: Growth brown- 
ish, lichenoid, with wide cream-colored 
edge; reverse yellowish. Aerial mycelium 
white to grayish, gradually covering surface. 
Xo soluble pigment . 

Nutrient agar: Growth cream-colored, 
elevated, lichenoid, doughy consistency. No 
aerial mycelium. Soluble pigment faint 
brownish. 

Potato: Growth barnacle-like, reddish- 
brown. No aerial mycelium. Pigment around 
growth grayish-brown. 

Gelatin: Surface ring cream-colored. Liq- 
uefaction medium. Soluble pigment deep 
brown, gradually diffusing through liquefied 
portion. 

Tryptone broth: Growth occurs in form 
of small pellets at the base of the flask; later, 
a thin surface pellicle is produced. Soluble 
pigment produced slowly, black. 

Temperature: Good growth at 25°C; slow 
growth at 37°C. 

Antagonistic properties: Produces on 
synthetic and organic media an antibiotic, 
proactinomycin, active against bacteria. 

Source: Isolated as an air contaminant. 

Remarks: This species was first classified 
as a Nocardia (Bergey's Manual, 7th eel.). 
Recent evidence, comprising both cultural 
and chemical properties (Cummins and 
Harris, 1958), suggests its transfer to the 
genus Streptomyces. It is closely related to 
S. aureofaciens. 

Type culture: IMRU 3834. 

98. Streptomyces garyphalus Harris et ah, 
L955 (Harris, I). A., Etuger, M., Reagan, 
M. A., Wolf, F. J., Peck, P. L., Wallick, H., 
and Woodruff, H. B. Antibiotics & Chemo- 
therapy 5: 183-190, 1955). 

Morphology: Sporophores straight, with- 
out spirals. Spores rod-shaped, 0.8 to 1.1 by 
1.7 to 1.'.) ix. 

Sucrose nitrate agar: Growth colorless. 



Aerial mycelium grayish- white. Xo soluble 
pigment. 

Glucose-asparagine agar: Growth color- 
less. Aerial mycelium white. Xo soluble pig- 
ment. 

Modified glucose-asparagine agar: Growth 
powdery, pinkish- white, reverse buff. Aerial 
mycelium seashell-pink. Xo soluble pigment. 

Calcium malate agar: Growth colorless 
Xo aerial mycelium. 

Nutrient agar: Growth colorless. Aerial 
mycelium grayish-white. Soluble pigment- 
faint brown. 

Yeasl extract -glucose agar: Growth excel- 
lent. Aerial mycelium grayish-white, be- 
coming pinkish-gray and finally seashell- 
pink. Soluble pigment faint brown. 

Starch-tryptone agar: Growth good. Aerial 
mycelium gray. Soluble pigment dark brown. 

Peptone-glucose agar: Growth cream- 
colored. Aerial mycelium grayish-white, be- 
coming pink. Soluble pigment faint brown. 

Starch agar: Growth excellent. Aerial 
mycelium white to gray. Hydrolysis. Soluble 
pigment faint brown. 

Gelatin: Grayish-white ring and sub- 
merged pellicle. Pigmented layer dark 
brown; becomes greenish-brown when 
shaken. Medium liquefaction. 

Milk: Faint grayish-white tinge. Slow 
peptonization turning dark purple at first 
and later brownish-purple. Reaction acid, 
pH 6.4. 

Nitrate reduction: Strong. 

Potato: Growth heavy, wrinkled. Aerial 
mycelium grayish-black. Potato darkened. 

Cellulose: Xo decomposition. 

Antagonistic properties: Produces an anti- 
biotic, D-4-amino-3-isoxazolidone (novobio- 
cin). 

Habitat: Soil. 

99. Streptomyces gedanensis I Lohlein, 
1909) Muller, 1950 (Lohlein, M. Z. Hyg. 
Enfektionskrankh. 63: 1-10, 1909; Muller, R. 
Medizinsche Mikrobiologie, 4th ed. 1950, 
Urban & Schwarzenberg, Munich, p. 294). 



DESCRIPTION OF SPECIES OF STREPTOM1 CES 2\7 

Morphology: Aerial mycelium consists of Milk: Pinkish ring. Coagulation and 

short, gnarled hyphae. Spores short, oval to peptonization. 

spherical. Starch: drouth thin, spreading, cream- 
Synthetic agar: Growth dark to almost colored. Hydrolysis. 
black, with dark reverse. Aerial mycelium Nitrate reduction: Positive. 
abundant, mouse-gray. No soluble pigment. Producti >l' IPS: Negative. 

Nutrient agar: Growth thin, colorless. No Temperature: Optimum 25 C. 

aerial mycelium. No soluble pigment. Antagonistic properties: Produces elaio- 

Glucose agar: Growth cream-colored, be- mycin. 

coming black with lighl margin. Aerial myce- 1 labitat : Soil, 

lium abundant, mouse-gray. Remarks: Various related forms have 

Potato: Growth lichenoid, cream-colored been described by Gause et al. (1957);these 

to brownish. No aerial mycelium. No soluble include .1. griseorubens, .1. rubiginomis, and 

pigment. .1. atroolivaceus. Krassilnikov (1959) con- 

Gelatin: Growth thin, flaky. No soluble siders this organism as belonging to the S. 

pigment. Rapid liquefaction. albus group. 

.Milk: Surface ring cream-colored. No Type culture: IMRU 3323. 

peptonization. , m p, . , , , . 

1 \ . .. -, . .. 101. btreptomyces glaucus (Lehmann and 

otarcn media : Growth yellowish to cream- q i , , ,- •. •, ..... ,,. , 

. . , ,. ■ ,. , bchutze emend. Krassilnikov, 1941) Waks- 

colored. Aenal mycelium lighl gray. Hy- man (Kra ssilnikov, N. A. Actinomycetales. 

dr °* ysis stro ^ g - . X7 Izvest. Akad. Nauk. SSSR, Moskau, p. 16, 

Nitrate reduction: Negative. 1041) 

Production of IIS: Negative. \, , , a , 

1 , °. . , , . Morphology: bporophores form compact 

Source: Sputum oi patienl with chronic gpirals w[th ., t() g turng ^^ oya] fcQ 

l ^ng disease. spherical, 1.0 by 0.8 M - 

1 vpe culture: IMlvl 3417. ^,, „ , , ,. , , , 

•' bucrose nitrate agar: Growth colorless; 

100. Streptomyces gelaticus (Waksman, soluble pigmenl brown. Aerial mycelium at 

1919) Waksman and llenrici, 1948 (Waks- first white, then becoming brighl green, 

man, S. A. Soil Sci. 8: 10."), 1010). Nutrient agar: Growth heavy. Aerial 

Synonym: Streptomyces hepaticus. mycelium green. 

Morphology: Sporophores produce open Potato: Growth heavy. Aerial mycelium 

spirals. According to Anderson et al. (1956), velvety, green. 

the organism does not produce spirals. Gelatin: Liquefaction slow. Melanin-nega- 

Sucrose nitrate agar: Growth colorless, ,1V( '- 

spreading, chiefly deep into the medium. Milk: Peptonization slow, with prior 

Aerial mycelium thin, white, turning grayish. , - ,,; ^ ,ll;l,i " 11 by some strains. 

v,,t,-; ,. . ,. i ■,. .i i .■ Starch: Hydrolysis rapid. 

Nutrient agar: Growth only on surface, ., " " ' 

• , , , , , C ellulose: ( Growth good. 

wrinkled, cream-colored. ^ 

. ,, . , Nitrate reduction : Positive. 

Glucose agar: Growth abundant, spread- 

. . ' Sucrose: Poor inversion, 

in"-, white. dm/- i i 

I aramn : ( rrowth good. 

Potato: (irowth abundant, much wrin- \ , • ,• m. 

Antagonistic properties: All strains 

kled, greenish, becoming black with yellow- stronedv antagonistic 

ishmargin. Habitat: Soil. 

Gelatin: Produces flaky, cream-colored Remarks: Numerous cultures belonging 

sediment. Good liquefaction. to this organism or closely related to it have 



'is 



THE ACTINOMYCETES, V« 



been described under a variety of differenl 
names. It is sufficient to mention S. caelestis, 
which produces an antibiotic, celesticetin, 
described by DeBoer et al. (1954) and A. 
glaucescens, together with a variety badius, 
described by Gause et al. (11)57). 

102. Streptomyces globisporus (Krassilni- 
kov, 1941) Waksman (Krassilnikov, X. A. 
Actinomycetales. Izvest. Akad. Nauk. SSSR, 
Moskau, p. 48, 1941). 

Morphology: Sporophores straight or 
wavy, often gathered in clusters or tufts; no 
spirals. Spores oval ( 1 .2 to 1 .4 by 1 .8 to 
2.0 m) or spherical (0.9 to 1.4 M ). 

Starch-KN0 3 agar: Growth abundant, 
colorless. Aerial mycelium light yellow to 
greenish-yellow with pinkish tinge. No 
soluble pigment. 

Glucose-peptone agar: Growth colorless 
or greenish. Aerial mycelium creamy, seldom 
greenish-yellow. Soluble pigment absent or 
faint yellowish. 

Gelatin: Rapid liquefaction. Soluble pig- 
ment absent or light brownish. 

Potato: Growth colorless or brownish. 
Aerial mycelium greenish-yellow. Ring 
brownish or colorless. 

Milk: No coagulation; rapid peptoniza- 
tion. 

Starch: Weak hydrolysis. 

Invertase: None. 

Nitrate: Reduced to nitrite. 

Cellulose: No or poor growth. 

Antagonistic properties: Some strains 
suppress gram-positive bacteria. 

Habitat: Soil. 

Remarks: Krassilnikov recognized several 
substrains of the species on the basis of 
milk coagulation, proteolysis, and pigmenta- 
tion of aerial mycelium. It is sufficient to 
mention A. globisporus vulgaris, A. globis- 
porus griseus, A. globisporus loch's, A. globis- 
porus diastaticus, A. globisporus flaveolus, A. 
globisporus circulatus, A. globisporus scabies, 
and A. globisporus albus. This heterogeneous 



collection is mosl unfortunate, since these 
"species" show differences in color of aerial 
mycelium, in formation of soluble pigments, 
etc. 

Krassilnikov (1 ( .)4'.)) considered the strep- 
tomycin-producing organism as a variety 
of this species, designating it at first as ^4. 
globisporus streptomycini, and later as A. 
streptomycini; A. griseus Krainsky was 
distinguished from this species on the basis 
of the fact that the sporophores of the latter 
exhibited spiral formation. This again was 
the cause of much confusion in nomenclature 
of the streptomycin-producing organism in 
the literature of the Soviet Union. 

Later, Krassilnikov (1958) divided the 4. 
globisporus group, on the basis of antago- 
nistic effects, into a number of subgroups, 
including A. vulgaris, A. toxicus, A. levoris, 
A. bacillaris, A. fluoresceins, A. raffinosus, A. 
longisporus, and .1 . grisinus. 

Gause et al. (1957) described .4. globis- 
porus in the series "helvolus," comprising 
the S. griseus group; they also listed several 
additional forms belonging to S. globisporus 
under the names A. caucasicus and A. 
cyanofuscatus. The above description is 
based upon the comparison made by Gause 
et al. of six cultures and Krassilnikov 's 
authentic strain. 

103. Streptomyces globosus ( Krassilnikov, 
1941) Waksman (Krassilnikov, N. A. Actino- 
mycetales. Izvest. Akad. Nauk. SSSR, 
Moskau, p. 58, 1941). 

Morphology: Sporophores straight, short, 
slightly branched, wavy. Spores spherical to 
oval. 

Agar media: Substrate growth brown to 
dark brown. Aerial mycelium dark gray, 
velvety. Soluble pigment dark brown. 

Gelatin: Weak liquefaction. No soluble 
pigment. Melanin formation questioned by 
Hoffmann (1958). 

Potato: Soluble pigment red-brown (Hoff- 
mann, 1958). 



Dl SCRIPTION OF SPECIES OF STREPTOMYCES 



219 



Milk: Questionable coagulation; good 
peptonizal ion. 
Starch: Hydrolysis. 
Cellulose: ( rood growth. 
Sucrose: No inversion. 
Production of IPS: Positive. 
Antagonistic properties: No activity. 
Habitat : Soil, food product-, potatoes. 

Type culture: [MRU 3736. 

104. Streptomyces gougeroti (Duche, 1934) 
Waksman and Henrici, L948 (Duche, J. Les 
actinomyces du groupe albus. 1'. Lechevalier, 
Paris, L934). 

Morphology: Aerial hyphae short , gnarled. 
Spores oval. 

Glucose nitrate agar: Growth slow as 
punctiform colonies; cream-colored with 
smooth edge. Xo aerial mycelium. No soluble 
pigment. 

Glucose-asparagine agar: Growth colorless 
to yellowish. Aerial mycelium thin, white. 
Xo soluble pigment. 

Nutrienl agar: Growth cream-colored 
with brownish reverse. Aerial mycelium 
thin, white. Soluble pigment faint yellowish. 

Potato: Growth slow, greenish tinged. 
Aerial mycelium thin, white. Xo soluble pig- 
ment . 

Gelatin: Surface growth heavy, cream- 
colored. Aerial mycelium thin, white. Lique- 
faction strong. Melanin-negative. 

Milk: Growth cream-colored. Aerial my- 
celium thin, white. Peptonization rapid. 

( Joagulated serum: ( rrowth cream-colored. 
Aerial mycelium white. Liquefaction rapid. 

Starch: Hydrolysis rapid. 

Nitrate reduction: Negative. 

Production of H 2 S: Negative. 

Antagonistic properties: Active against 
fungi. 

Remarks: This culture is believed to be 
intermediate between N. albus, with it- 
abundant aerial mycelium, and S. almquisti, 
with its very scant aerial mycelium. 

Type cull lire: [MRU 3590. 



105. Streptomyces gracilis (Millard and 
Burr, 1926) Waksman (Millard, \Y. A. and 
Burr, S. Ann Appl Biol. L3: 580, 1926). 

Morphology: Sporophores form spirals. 
Spores oval or spherical, 0.8 to 0.9 by 0.8 n. 

Sucrose nitrate agar: Growth fern-like, 
pale gray. Aerial mycelium scant, gray to 
buff. Soluble pigment cream-colored. 

Nutrienl potato agar: Growth vinaceous- 
bul'f to dark brown or almost black. Aerial 
mycelium gray. Soluble pigment light golden 
brown. 

Potato: Aerial mycelium abundant, olive- 
gray to buff. Plug pigmented light brown. 

Gelatin: Growth gray. Aerial mycelium 
white. Liquefaction rapid. Soluble pigment 
pink to dark golden blown. 

Milk: Surface growth good. Aerial myce- 
lium white in the form of a ring and specks 
on surface. Coagulation slow, followed by 
rapid peptonization. 

Starch: Positive hydrolysis. 

Nitrate reduction: Positive. 

Tyrosinase reaction: Negative. 

Temperature: Grows well at 37.5°C. 

Habitat : Potato scab. 

106. Streptomyces griseinus Waksman 
(Reynolds, I). M. and Waksman, S. A. 
J. Bacterid., 55: 7:5<> 751, 1948; Okami, Y. 

J. Antibiotics (Japan) 5: '.15 «.»7, 1950). 

Morphology: Straight sporophores pro- 
duced in clusters or tufts, without spirals. 
Spores rod-shaped, 1.0 to 1.8 by 0.8 to 1.0 m- 

Sucrose nitrate agar: Substrate growth 
wrinkled, reverse cream-colored to brownish. 
Aerial mycelium white to cream-colored 
with light greenish tinge (lesser tendency to 
grass-green coloration, more of a cream- 
colon. Xo soluble pigment. 

Starch agar: Colorless to cream-colored 
growth. Aerial mycelium grayish-olive. Hy- 
drolysis rapid. 

Potato: ( Growth wrinkled, yellowish-white. 
Aerial mycelium grayish-white with olive 
tinge. 



220 



THE ACTINOMYCETES, Vol. II 



Gelatin: Growth cream-colored with 
brownish tinge. Aerial mycelium absent, or 
scant, white. Liquefaction rapid. 

Milk: Growth cream-colored. Coagulation 
and peptonization. 

Tyrosine agar: No pigment produced. 

Nitrate reduction: Positive. 

Production of H 2 S: Negative. 

Carbon utilization: Okami (1950) re- 
ported that the grisein-producing organism 
(S. griseinus) grows more readily in synthetic 
media containing glucose, glycerol, and 
sucrose than the streptomycin-producing S. 
griseus. According to Benedict et al. (1955), 
the former utilizes xylose, L-arabinose, and 
rhamnose, but S. griseus utilizes only xylose. 

Phage sensitivity: Not sensitive to phages 
effective against S. griseus strains. 

Pigments: No soluble pigments on calcium 
malate or succinate media, whereas S. gri- 
seus forms green and yellow pigments on 
these media, according to Benedict and Lind- 
enfelser (1951). 

Antagonistic properties: Produces the an- 
tibiotic grisein. Albomycin, produced by A. 
subtropicus (Gause, L955), is an identical or 
closely related compound (Waksman, 1957; 
Thrum, 1957). 

Remarks: S. griseus and N. griseinus show 
other striking differences. There are some 
close resemblances between these and the 
viomycin-producing cultures. Thus, on tyro- 
sine-starch agar, certain S. griseus strains 
form a dark pigment in the agar, whereas S. 
griseinus strains resemble the viomycin-pro- 
ducing cultures by not forming this pigment . 
S. griseinus and the viomycin group grow 
well on NaN0 3 , but S. griseus utilizes this 
compound poorly. .1. subtropicus, described 
by Kudrina and Kochetkova (1958), is 
closely related to, if not identical with S. 
(/risci nus. 

Type culture: [MRU 3478. 

107. Streptomyces griseobrunneus Waks- 
man, L919 (Waksman, S. A. Soil Sci. 8: 125 
127, 10 19). 



Morphology: Sporophores usually straight 
on most media; often a few short, open 
spirals are formed; 1 ufts are produced on 
certain media. Spores oval-shaped. 

Sucrose nitrate agar: Growth cream-col- 
ored to yellowish-brown. Aerial mycelium 
appears early; powdery, olive-buff to water- 
green. No soluble pigment. 

Glycerol-calcium malate agar: Growth 
cream-colored. Aerial mycelium water-green 
in color. 

Glucose-asparagine agar: Growth yellow- 
ish-brown. Aerial mycelium pale olive-buff. 
No soluble pigment. 

Nutrient agar: Growth cream-colored, be- 
coming brown. Aerial mycelium abundant, 
white. Soluble pigment brown. 

Starch agar: Growth cream-colored to yel- 
lowish. Aerial mycelium white. Good hydrol- 
ysis. 

Egg media: Growth cream-colored with 
brownish tinge. Aerial mycelium olive-buff. 
Soluble pigment purple. 

Potato: Growth brownish. Aerial myce- 
lium white, turning olive-buff. Soluble pig- 
ment faintly brown. 

Gelatin: Growth cream-colored, turning 
brown. Aerial mycelium white. Soluble pig- 
ment deep brown. Medium liquefaction. 

Milk: No coagulation; rapid peptoniza- 
tion. 

Nitrate reduction: Positive. 

Sucrose: Xo inversion. 

Cellulose: Good growth. 

Habitat: Sewage. 

Remarks: This organism had been de- 
scribed by Waksman (1919) as Actinomyces 
218, but never named before. It was said to 
be closely related to S. griseus, differing from 
it by lesser proteolysis and production of a 
brown pigment on protein media. 

Type culture: [MRU 3068. 

108. Streptomyces griseocarneus Benedict 
et al, 1951 (Benedict, R. G., Lindenfelser, 
P. A., Stodola, F. IP, and Traulier, I). PI. 
J. Bacteriol. 62: 487-497, 1951; see also 



DESCRIPTION OF SPECIES OF STREPTOMYCES 22] 

Grundy, W. E. Antibiotics & Chemother- Antagonistic properties: Produces hy- 

apy 1:309 317, L951). droxystreptomycin. 

Morphology: Sporulation occurs besl on a Type culture: [MRU 3557; ATCC 12,628. 

carbon-free sail agar, to which 0.5 per ecu , ()() streptomyces griseochromogenes Fuku- 

soluble starch has been added. Sporophores naga tf ^ ,,,-- , Fukunagaj K>> Misat0] T< 

straight, forming no spirals. Spores coccoid Mui _ , ;m(1 Asakawa> M . B ull. Agr. Chem 

fcooval > Ll fc0 1-6 by O. 1 : to 1.1 m. Soc. Japan L9: L81 L88, L955). 

Agar media: Aerial mycelium on some Morphology: Sporophores form closed spi- 

media powdery, becoming gray, bul no spor- ralg ())1 gtarch agar . th( , n . are 1|(( s))iraU ny 

illation. When sporulation ..ecus the myce- only curling tipSj t(imi( , ( , ()M sum)S(i liilr;it( . 

Il,1,n becomes lighl pmk. ;m( j g i UC0S e-asparagine agars. Spores spheri- 

Suen.se nitrate agar: Growth limited, ca i or oval, about 1.0 to 1.5 M - 

white. Aerial mycelium white, no sporula- Surms(1 m11 . al( . agar . ( ;,. mvth spreadingj 

" <)IK orange-cinnamon. Aerial mycelium white or 

Glucose-asparagine agar: Growth moder- light neut ral gray. No soluble pigment. 

ate. Aerial mycelium powdery, white, no Glucose-asparagine agar: Growth .<- 

sporulation. stricted, ivory-yellow, penetrating into the 

Calcium malate agar: Growth moderate, medium . No aeria] myce i mmj i ater white. 

white. Aerial mycelium white, no sporula- No soluble pigment. 

"' ,n ' Nutrient agar: Growth restricted, opales- 

Nutrient agar: Growth moderate, cream- cent Nq ;i( ,,. |;(| mycelium- Soluble pigment 

colored. No aerial mycelium. Soluble pig- brown 

met lighl yellow-brown. ,, ()tat(): Qrowth abundantj wri „kl,,l. 

Oatmeal agar: Growth luxuriant, brown. snuff „ brown . Ama) myce lium white to 

Aerial mycelium abundant, fluffy, white; no mouse _ gray . Color of plug dark brown to 

sporulation. No soluble pigment. 1)1;(rk ;(1 . (mn(1 growth 

Potato: Growth cream-colored. Aerial my- Gelatin: Growth wrinkled, yellowish in 

cehum gray. Soluble pigment light brown, liquefied portion . Aeria] myC elium whit,-. 

turning dark brown. S( . :mt Lique f actioi] medium. Soluble pig- 
Gelatin: Growth cream-colored to brown. m( , nt ( lark brown to black. 

Rapid liquefaction. Soluble pigmenl dark Milk: Growth as surface ring, brown. No 

,rown - coagulation; peptonization begins in 8 days 

Milk: Growth .lark brown to black. No ;i , :i7 - r:11()t completed in 21 days. 

coagulation; rapid peptonization. Soluble Nitrate reduction: Positive. 

I ,1 -" 1< ' 1 " brown - Cellulose: No growth. 

Starch: Hydrolysis. Tyrosine agar: Growth orange-colored. No 

Carl.,,,, utilization: Glucose, dextrin, so i ub i e pigment . 

-larch, glycerol, calcium malate, and sodium rnvprtas •' 1* «itivp 

succinate rapidly utilized. Mannose, mal- Carbon utilization: (duc.se, D-fructose, 

tose, mositol, and sodium acetate utilized D _ galactosej ma i t0 se, lactose, raffinose, d- 

slowly. Xylose, galactose, sorbose, sucrose, ma nnitol, DL-inositol utilized. Rhamnose, 

cellobiose, melibiose, lactose, mannitol, sor- iliu | uii D . sor bitol, dulcitol, salicin, sodium 

bitol, sodium citrate, and potassium sodium acetate, sodium citrate, sodium succinate not 

tartrate not utilized. ut ilized. 

Nitrate reduction : Negative. Antagonistic properties: Produce- blasti- 

Production of IIS: Positive. cidinsA, B, and C, active againsl fungi. 



222 



THE ACTINOMYCETES, Vol. II 



Remarks: S. griseochromogenes belongs to 
the group of chromogenic actinomycetes. S. 
resistomycificus differs from S. griseochromo- 
genes in the color of its aerial mycelium ob- 
served on various media, and also in that it 
produces an aerial mycelium on nutrienl 
agar and a soluble pigment in glucose-aspara- 
gine agar. S. mirabilis has a different form 
of aerial mycelium and a different optimum 
temperature. S. flavochromogenes produces 
an aerial mycelium on nutrient agar and a 
grayish soluble pigment. S. olivochromogenes 
assumes a dark brown or black color of 
growth and shows an alkaline reaction in 
milk medium. S. diastatochromogenes is quite 
similar to S. griseochromogenes in the ap- 
pearance of its growth on several media, but 
differs from it by producing a white or gray 
aerial mycelium on nutrient agar, and also 
by producing tyrosinase. 

110. Streptomyces griseoflavus (Krainsky, 
L914) Waksman and Henrici, L948 (Krain- 
sky, A. Centr. Bakteriol. Parasitenk. AM. 
II, 41: (584, L914). 

Morphology: Sporophores straight, mono- 
podially branched; no curvatures and uo 
spirals produced. Spores oblong, 1.0 to 1.2 n, 
covered with short spines. (According to 
Ettlinger et al. (\ { .)r>S), open, regular spirals 
are formed.) 

Sucrose nitrate agai : Growth reddish- 
brown to orange. Aerial mycelium gray to 
yellowish-gray. Faint greenish-yellow soluble 
pigment . 

Glucose-asparagine agar: Growth citron- 
yellow. Aerial mycelium powdery, greenish- 
yellow changing to gray (Hoffmann, L958). 

Calcium malate agar: Growth yellowish- 
green-gray. 

Nutrienl agar: Growth cream-colored, 
covered with white to gray aerial mycelium. 
Soluble pigment absent or, according to 
Hoffmann (1958), greenish-gray. 

Starch agar: Growth cream-colored with 
brownish center. Aerial mycelium absent or 
powdery, gray. Hydrolysis limited. 



Potato: Growth lichenoid, yellowish. Ae- 
rial mycelium powdery, white to gray. Mela- 
nin-negative. 

Gelatin: Growth cream-colored to brown- 
ish, covered with white to yellowish -gray 
aerial mycelium. Positive Liquefaction. Mela- 
nin-negative. 

Milk: Growth cream-colored to yellowish; 
aerial mycelium thin, white. Xo coagulation; 
rapid peptonization. 

Cellulose: Growth good. Greenish-yellow 
soluble pigment, according to Hoffmann 
(1958). 

Nitrate: Strong reduction to nitrite. 

Production of H 2 S: Negative. 

Invertase: Negative. 

Antagonistic properties: According to 
Waga (1953), a member of this group pro- 
duced an antibiotic, griseoflavin; this anti- 
biotic was later (Kuroya et al., 1958) found 
to be identical with novobiocin. Another 
antibiotic, grisamine, has also been reported. 

Remarks: According to Jensen (1930), the 
species is characterized by the grayish-yellow 
color of its aerial mycelium, which never 
assumes the distinct green shade of S. griseus. 
A detailed study of the life cycle of this 
organism has been made by Saito and Ikeda 
(1958). They found that between the pri- 
mary (vegetative phase) and the secondary 
(sporulation phase) mycelium, there may be 
a transitional stage which comprises "nests," 
"swollen bodies," and "clubs, " correspond- 
ing to the "initial cells" reported in the litera- 
ture. 

111. Streptomyces griseolus (Waksman, 
1923) Waksman and Henrici, 1948 (Waks- 
man, S. A. Actinomyces '.Mi. Soil Sci. 8: 121, 
L919). 

Morphology: Sporophores short, straight, 
without spirals, some curling found on side 
branches. Spores spherical to oval-shaped in 
cylindrical. 

Sucrose nitrate agar: Growth colorless, 
thin, spreading, chiefly in the medium. 
Aerial mycelium at first gray, later becoming 



DESCRIPTION OF SPECIES OF STREPTOM1 CBS 223 

pallid neutral gray, with yellowish tone. Maeda, K., Ogata, Y.. and Okami, V. J. 

Faint brownish soluble pigment. Antibiotics (Japan) t: 34 10, L951; Okami, 

Mfalate-glycerol agar: Growth brownish. Y. ibid. 5: 477 180 L952). 

Aerial mycelium lighl mouse-gray. Soluble Morphology: Sporophores with mono- 

pigmenl taint brownish. podial and irregular branching, flexible and 

Glucose-asparagine agar: Growth cream- hooked. Spores oval to cylindrical, L.O to 1.2 

colored, turning dark. Aerial mycelium deep by i .s to 2.2 M . 

dull gray. No soluble pigment. Sucrose nitrate agar: Growth thin, color- 

Xuirient agar: Brownish growth, with less to cream-colored. Margin plumose, pene- 

smooth surface. Aerial mycelium white with trating into medium. Aerial mycelium pow- 

gray tinge. Soluble pigmenl brown. Mela- dery, grayish-white to lighl drab. Soluble 

nin-negative. pigmenl absent or yellowish-brown. 

Potato: Growth cream-colored, becoming Glucose-asparagine agar: Growth wrin- 

black. Aerial mycelium white with greenish kled, cream-colored. Aerial mycelium thin, 

tinge. Soluble pigmenl brown to black. white. Pigmenl reddish-brown. 

Gelatin: Yellowish flaky pellicle and sedi- Nutrient agar: Growth wrinkled, trans- 
ment. Aerial mycelium white. Gradual lique- parent. Aerial mycelium thin, white, pow- 
taction. Paint browning of medium. dery. Soluble pigmenl absent or yellowish- 
Milk: Growth abundant, pink pellicle, brown. 
Slow coagulation and good peptonization. Potato: Growth abundant, wrinkled, 

Starch media: ( Irowth grayish-brown with cream-colored. Aerial mycelium dusty white, 

dark liiiu;. Aerial mycelium gray. Slight hy- thin. Plug becoming slightly brownish. 

drolysis of starch. Gelatin: No growth. 

Cellulose: Scant growth. Milk: Surface ring cream-colored. Aerial 

Invertase: Negative. mycelium in form of white patches. 

Nitrate reduction: Positive. Starch: Hydrolysis. 

Production of IPS: Negative. Nitrate reduction: Positive. 

Temperature: Optimum 25 C. Production of IPS: Negative. 

Antagonistic properties: Some strains Antagonistic properties: Produces griseo- 

show considerable activity againsl various lutein 

bacteria. Several antibiotics (phagomycin, Type culture: [MRU 3674; 3729. 
fermicidin, anisomycin, oxytetracycline, gris- 

eomycin) were isolated from culture, de- l13 " Streptomyces griseoplanus Backus ei 

scribed as strains of S. griseolus. " l - L957 (Backus, E. J, Tresner, IP I)., and 

Habitat: Soil. Campbell, T. IP Antibiotics cV- Chemother- 

Remarks: Ettlinger et al. I L958) considered ;l P>' ": 532 541, 1957 . 

this organism a- related to S. olivaceus. Morphology: Sporophores arise as tangled 

Krassilnikov (1949) considered it a- a -train and curved and often loosely spiraled chains 

of A. candidus. Hoffmann (1958) isolated a of spores. Spore- globose to elliptical, 0.6 to 

strain of this species from potato scab. Al- 1 .1' by 1.2 to 1.5 n (Fig. 10). 

though this organism is usually described as Sucrose nitrate agar: (irowth colorless, 

melanin-negative, Krassilnikov (1941) and Aerial mycelium scant, white to gray. 

Hoffmann consider it as melanin-positive. Glucose-asparagine agar: (irowth -ray to 

Type culture: [MRU 3325. [ighi pinkish. No aerial mycelium. 

112. Streptomyces griseoluteus Dmezawa Nutrient agar: (irowth ivory-yellow. No 

et <il., L951 (Umezawa, IP, Hayano, S., aerial mycelium. Melanin-negative. 



224 



THE ACTINOMYCETES, Vol. II 





e ■ ^Kjtr.. 



Figure 40. Sporophores of S. griseoplanus (Reproduced from: Backus, E. J. et al. Antibiotics & 
Chemotherapy 7: 537, 1057). 



Starch agar: ( Irow 

ish. Aerial mycelium 

Potato plug: Grow 

rial mycelium scant, 

plug browned. 

Milk: No growth. 

Cellulose: No grow 

Production of H 2 S: 

Carbon utilizatioi 

with ammonium suit' 

gen. With aspartic ; 

L-arabinose, D-xylos< 

moderate growth on 

trehalose, and salicii 

D-mannitol, /-inositol. 



th colorless to yellow- 
scant, white. 
th light brownish. Ae- 
white to gray. Apex of 



th. 

Negative. 

: Extremely limited. 

ite as source of nitro- 
icid, organism utilizes 
, and glucose. Fair to 

lactose, D-raffinose, n- 
l. Sucrose, D-fructose, 

idonitol, D-melezitose, 



L-rhamnose, esculin,n-melibiose, and dextrin 
utilized poorly or not at all. 

Antagonistic properties: Produces anti- 
biotic alazopeptin. 

Habitat : Grassland soil. 

Remarks: This organism is closely related 
to ,s. flavogriseus. 

114. Streptomyces griseoruber Yamaguchi 
and Saburi, 1955 (Yamaguchi, T. and Sa- 
buri, V. J. Gen. Appl. Microbiol. 1: 201-235, 
1955). 

Morphology: Aerial hyphae produce open 
and closed spirals on sucrose nitrate and on 
starch agars. Spores short, cylindrical, 0.5 to 
().<) by ().*) to 1.2 fx. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 225 

Sucrose nitrate agar: Growth reddish- et al, 1956 (Anderson, L. E., Ehrlich, J., 

orange when freshly isolated, bul changes to Sun, S. II.. and Burkholder, P. 1{. Antibiot- 

colorless or whitish on repeated transfer, ics & Chemotherapy 6: 100 115,1956). 

Aerial mycelium powdery, drab-gray. No Not S. griseoviridus. 

soluble pigment. Morphology: Sporophores straighl or 

Calcium malate agar: Growth at first curved, with open and closed spirals on 

glossy, pinkish-gray, later becoming dull lateral branches. Spores spherical to ovoid, 

purplish. Aerial mycelium powdery, olive- 0.6 to 1.5 by 0.8 to 2.1 /*■ 

gray. Soluble pigmenl absenl or taint yel- Starch-ammonium sulfate agar: Growth 

lowish-brown. tan-gray to black. Aerial mycelium tan to 

Nutrient agar: Growth a1 firsl lighl olive- lighl brown, 

gray, later becoming brown. Aerial myce- Glycerol-asparagine agar: Growth lighl 

hum absent or scanty, white. Soluble pig- yellow to gray. Aerial mycelium pink-tan to 

ineni brown. gray-green. 

Starch agar: Growth lighl reddish-orange Calcium malate agar: Growth yellow-tan 

to reddish-purple. Aerial mycelium powdery, to gray. Aerial mycelium lighl brown. Slight 

olive-gray. Soluble pigmenl taint yellow to hydrolysis of starch, 

faint yellowish-pink. Strong hydrolysis. Nutrient agar: Growth yellow-tan to 

Potato: Growth wrinkled, at firsl olive- green-gray to brown. Aerial mycelium lighl 

gray to dark yellowish-brown, later becom- gray-pink to light gray-green. Soluble pig- 

ing dark reddish or black. Aerial mycelium menf light brown. 

absent or scanty, while. Soluble pigment Glucose-tryptone agar: Growth lighl 

deep purple to black. brown to red-brown to dark brown or black. 

Gelatin: Growth light yellowish-brown on Aerial mycelium pink-gray to lighl brown 

surface. No aerial mycelium. Soluble pig- to green-brown. Soluble pigment brown; red- 

ment brown to light yellowish-green. Lique- brown near growth, 

faction weak to medium. Gelatin: Fairly rapid liquefaction. Soluble 

Milk: Growth deep brown al 37°C, but pigment lighl brown to dark brown, 

cream-colored to lighl yellowish-brown, and Milk: Peptonization. 

occasionally with pinkish tone at 25°C. Sol- Starch: Hydrolysis, 

uble pigment grayish-yellow-brown al 37°C, Carbon utilization: Utilizes arabinose, 

but sometimes faint yellowish-brown with cellobiose, dextrin, galactose, glucose, glyc- 

pinkish shade at 25°C. Coagulation begins in erol, lactose, levulose, maltose, mannitol, 

3 days, followed by peptonization. mannose, rhamnose, starch, trehalose, and 

Cellulose: No growth. xylose. Doe.- not utilize esculin, adonitol, 

Carbon utilization: Utilizes D-xylose, l- dulcitol, /-inositol, inulin. melezitose, meli- 

arabinose, L-rhamnose, lactose, inositol, sali- biose, raffinose, salicin, sorbitol, and sucrose. 

• •in. -odium acetate; does not utilize sucrose, Antagonistic properties: A source of <j;n>- 

raffinose, inulin, mannitol, sorbitol, citrate, eoviridin and viridogrisein (etamycin). 

and succinate. Habitat : Texas soil. 

Antagonistic properties: Active againsl Type culture: IMRU 3735. 
gram-positive and acid-fasl bacteria; pos- ,„. Streptomyces griseus Waksman and 
antitnchomonal activity. Eeari^ 1948 (Waksman and Benrici, Ber- 

aemarks: Related to the S. ruber -roup gey)g M:mll:lIj ,. |h ( , ( , ,, lls _ p 94g; Waks _ 

and to S. erythrochromogenes. m;m _ s A ;m(1 rmU ^ ,, ,.- S(i]] S( ., ,. 

Mo. Streptomyces griseoviridis Anderson 11 ( .» 120, 1916; Waksman, S. A., Eteilly, 



226 



THK ACTINOMYCETES, Vol. II 



II. C, and Harris, I). A. J. Bacterid. 56: 
259, 1948; Waksman, S. A. Proc. Natl. 
Acad. Sci. U. S. 45: 1043-1047, 1959). 

Synonyms: Actinomyces globisporus Kras- 
silnikov, 1941. Actinomyces globisporus sub- 
sp. streptomycini (Waksman) Krassilnikov, 
1949. Actinomyces streptomycini Krassilni- 
kov, 1957. 

Morphology: Sporophores straight, pro- 
duced in tufts (Fig. 41). Spores spherical to 
oval, 0.8 by 0.8 to 1.7 m; surface smooth (PI. 

iii). 

Sucrose nitrate agar: Growth thin, spread- 
ing, colorless, becoming olive-buff. Aerial 
mycelium thick, powdery, water-green. Pig- 
ment insoluble. 

Nutrient agar: Growth abundant, almost 
transparent, cream-colored. Aerial mycelium 
powdery, white to light gray. No soluble 
pigment. 

Glucose agar: Growth elevated in center, 
radiate, cream-colored to orange, erose mar- 
gin. 




Figure 41. Substrate and aerial mycelium of 
S. grist us. 



Starch media: Growth thin, spreading, 
transparent. Hydrolysis strong. 

Tyrosine agar: Dark pigment often pro- 
duced. 

Potato: Growth wrinkled, yellowish to 
brownish, covered with white, powdery aer- 
ial mycelium. 

Gelatin: Greenish-yellow or cream-colored 
surface growth with brownish tinge. Rapid 
liquefaction. 

Milk: Cream-colored ring; coagulation 
with rapid peptonization, becoming alkaline. 

Cellulose: Scant to fair growth. 

Nitrate reduction: Positive. 

Pigments: Produces green or yellow solu- 
ble pigment on calcium malate and succinate 
media. 

Production of H 2 S: Negative. 

Carbon sources: See S. griseinus. 

Antagonistic properties: Strongly antago- 
nistic. Produces antibiotic streptomycin, ac- 
tive against a large number of bacteria and 
actinomycetes, but not against most fungi or 
viruses; also produces cycloheximide, active 
upon fungi. Resistant to streptomycin-pro- 
ducing organisms and to streptomycin. 

Remarks: An extensive literature has ac- 
cumulated on the nature of this organism 
(Koreniako and Nikitina, 1959), on its phage 
sensitivity (Koerber et al., 1950), antibiotic 
production (Waksman, 1949), etc. 

Habitat: Soils, river muds, throat of 
chicken. 

Type culture: [MRU 3463. 

117. Streptomyces hachijoensis Vamaguchi, 
1954 (Yamaguchi, T. J. Antibiotics (Japan) 
7 A: 10-14, 1954). 

Morphology: Aerial hyphae short , straight, 
0.6 to 1.2 m- Secondary verticils produced. 
Spores cylindrical, 0.8 to 1.0 by 1.5 to 1.8 m- 

Sucrose nitrate agar: Growth restricted, 
colorless; reverse yellowish. Aerial mycelium 
white, changing to pinkish-buff. No soluble 
pigment. 

Calcium malate agar: Growth colorless to 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



227 



yellow. Aerial mycelium white to pale pink- 
ish-buff. Soluble pigmenl absent. 

Nutrienl agar: Growth cream-colored, 
wrinkled. Aerial mycelium powdery, shade of 
pale ochraceous-buff. No soluble pigment. 

Potato: Growth cream-colored to yellow- 
ish, wrinkled, raised. Aerial mycelium white. 
Soluble pigment around growth faint pur- 
plish. 

Blood agar: Growth yellow to brownish- 
yellow. Aerial mycelium flocculent, white. 
Soluble pigment dark. Positive hemolysis. 

Gelatin: Growth yellow to brown. No 
aerial mycelium. Rapid liquefaction. 

Milk: Surface ring yellow to brown. Aerial 
mycelium in form of white patches. Soluble 
pigment pinkish to orange. Coagulation fol- 
lowed by peptonization. 

Nitrate reduction: Negative. 

( 'ellulose: No growth. 

Antagonistic properties: Produces an anti- 
fungal agent, trichomycin, a member of the 

candicidin group of antibiotics. 

Remarks: Resembles S. rubrireticuli. Bli- 
nov (1958) described a variety (fuscatus) of 
this species, as a producer of candicidin- 
type antibiotics. 

118. Streptomyces halstedii (Waksman and 
Curtis, L916) Waksman and Henrici, 1948 

(Waksman, S. A. and Curtis, R. K. Soil Sci. 
1: 124, 1916;8: 121, 1919). 

Morphology: Sporophores form closed 
spirals. Spores oval or rod-shaped, 1.0 to 
1.2 by 1.2 to 1.8 /x- 

Sucrose nitrate agar: Substrate growth 
abundant, spreading, raised, at first light 
colored, becoming dark to almost black. 
Aerial mycelium white, turning dull gray. 
No soluble pigment. 

( rlycerol malate agar: ( trowth dark. Aerial 
mycelium deep mouse-gray. 

Nutrient agar: Growth restricted, wrin- 
kled, cream-colored. No aerial mycelium. 
Melanin-negat ive. 

Glucose-asparagine agar: Growth wrin- 



kled, center elevated, edge lichenoid, color- 
less, becoming brown. No aerial mycelium. 

Potato: Growth abundant, moist, wrin- 
kled, cream-colored with green tinge. 

Gelatin: Small, cream-colored masses of 
growth in bottom of tube. Rapid liquefac- 
tion. No soluble pigment . 

Milk: Cream-colored ring. Coagulation 
and slow peptonization. 

Starch media: Growth abundant, glossy, 
brownish. No aerial mycelium. Rapid hy- 
drolysis. 

( ellulose: No growth. 

Nitrate: Reduction to nitrite. 

Production of IBS: Negative. 

Temperature: Optimum :-)7°C. 

Antagonistic properties: Strongly antag- 
onistic; some strains show only antifungal 
activity; some strains produce carbomycin. 

Habitat: Soil. 

Remarks: Several closely related forms 
have been described. According to Ettlinger 
et al. (1958), the strains examined produce 
no spirals and belong to S. olivaa us. Ac- 
cording to Okami and Suzuki (1958), the 
sporophores are wavy, seldom forming hooks 
or primitive spirals. Gause et al. (1957) de- 
scribed a closely related form as .1. griseoirtr 
car not us. 

Type culture: [MRU 3328. 

IP.). Streptomyces hawaiiensis Cron et el., 
1956 (Cron, M. J., Whitehead, D. F., Hooper, 
B R., Heinemann, B., and Lein, .1. Anti- 
biotics & Chemotherapy 6: (i:; 67, 1956). 

Morphology: Sporophores produce spirals 
on some media. Spores oval, 0.6 to 0.8 by 
0.7 to 1.3/*. 

Sucrose nitrate agar: ( rrowth faint yellow. 
Aerial mycelium sparse, while to flesh-col- 
ored. Soluble pigmenl faint tan or absent. 

Glucose-asparagine agar: Growth light 
brownish. Aerial mycelium moderate, white 
to gray. Soluble pigment faint tan or absent . 

Nutrient agar: Growth gray with light 
brown reverse. Aerial mycelium limited. 



228 



THE ACTINOMYCETES, Vol. II 



wood-ash to steel-gray. Soluble pigment 
brown. 

Potato: Growth gray. Aerial mycelium 
limited, gray. Soluble pigment dark brown, 
almost black. 

Milk: No coagulation or peptonization. 
Slight acid reaction. Soluble pigment green- 
ish-brown. 

Gelatin: Slight liquefaction at 26°C in 19 
days. Soluble pigment brown. 

Si arch: Slight hydrolysis in 7 days at 28- 
30°C. 

Blood agar: No hemolysis. Soluble pig- 
ment black. 

Production of H 2 S: Positive. 

Carbon utilization: Utilizes arabinose, 
rhamnose, glucose, galactose, fructose, su- 
crose, maltose, lactose, xylose, raffinose, cell- 
obiose, dextrin, inulin, soluble starch, glyc- 
erol, inositol, mannitol, sodium acetate, 
sodium citrate, and calcium malate. Does 
not utilize sorbitol, dulcitol, sodium oxalate, 
sodium salicylate, sodium tartrate, and so- 
dium succinate. 

Antagonistic properties: Produces a poly- 
peptide antibiotic, bryamyein. 

Remarks: S. hawaiiensis is a chromogenic 
form which produces a soluble, dark brown 
pigment on protein media and a white to 
gray aerial mycelium. The organism is fur- 
ther characterized by spiral formation in the 
aerial mycelium and weak proteolytic ac- 
tivity in gelatin and milk. 

Streptomyces phaeofaciens possesses cul- 
tural and morphological characteristics simi- 
lar to those of S. hawaiiensis, fait differs in 
its rapid peptonization of milk and produc- 
tion of an antifungal substance inactive on 
bacteria. 

S. hawaiiensis resembles S. aureus in that 
spiral formation occurs with both cultures 
and both form soluble brown pigments in 
organic media. They differ in that S. aureus 
liquefies gelatin to ;i greater extent. S. ha- 
waiiensis is also similar to S. bikiniensis in 
some of its cultural properties; both produce 



a white aerial mycelium which becomes 
gray-colored; the sporulation of S. hawaiien- 
sis takes place in the form of spirals in its 
aerial mycelium, whereas S. bikiniensis is 
completely devoid of spirals and produces an 
alkaline reaction accompanied by hydroly- 
sis in milk. 

Type culture: ATCC 12,236. 

120. Streptomyces hiroshimensis Shinobu, 
1955 (Shinobu, R. Seibutsugakkaishi 6: 43- 
46, 1955). 

Morphology: Sporophores produce verti- 
cils of the Nitella type, both primary and 
secondary. No spirals. Spores elliptical to 
oval, 0.8 to 1.2 M (PI. V, Gb). 

Sucrose nitrate agar: Growth poor, re- 
stricted, pink. Aerial mycelium scant, pale 
pink to pinkish-white. 

Calcium malate agar: Growth slow. Aerial 
mycelium pale cinnamon-pink. Soluble pig- 
ment brownish. 

Glucose-asparagine agar: Growth good, 
reddish-pink. Aerial mycelium pink to pur- 
plish-pink. Soluble pigment usually absent, 
sometimes pale brown. 

Nutrient agar: Growth reddish-brown. 
Aerial mycelium absent, or scant, pale pink 
to pinkish-white. Soluble pigment brownish- 
orange. 

Starch agar: Growth red to purplish-red. 
Aerial mycelium pink to pale pink. 

Potato plug: Growth deep pinkish-red to 
brownish-black. Aerial mycelium scant, 
pinkish- white. Soluble pigment brownish- 
black. 

Gelatin: Growth pale reddish-brown. Aer- 
ial mycelium absent, or scant, pinkish- 
white. Soluble pigment pale reddish-brown. 
Rapid liquefaction. 

Milk: Growth dee]) pinkish-red. Aerial 
mycelium pinkish-white to pink. Soluble pig- 
ment brown with reddish tinge. No coagu- 
lation; rapid peptonization. 

Nitrate reduction: Strong. 

Starch: Rapid hydrolysis. 

Tyrosinase reaction: Positive. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



229 



Cellulose: No1 attacked. 

Carbon utilization: Fructose and inositol 

well utilized; xylose, rhanim >se, sucrose, lac- 
lose, raflinose, and mannitol not utilized; 
galactose and trehalose slightly utilized. 

Antagonistic properties: Inhibits growth 
of gram-positive bacteria and fungi. 

Source: Isolated from soil in Hiroshima, 
Japan. 

Remarks: Resembles S. rubrireticuli. 

\'2\. Streptomyces hirsutus Ettlinger et al., 
L958 (Ettlinger, L., Corbaz, R., and Hiitter, 
R. Arch. Mikrobiol. 31: 344, L958). 

Morphology: Sporophores long, mono- 
podially branched; shorl open spirals with 
about three coils are produced. Spores cov- 
ered with narrow, long spines (PI. II, k). 

Glycerol nitrate agar: Growth colorless. 
Aerial mycelium at first milky white, later 
leek-green. 

Glucose-asparagine agar: Growth color- 
less. No aerial mycelium. 

Glycerol malate agar: Growth at first 
milky white; later covered with aerial my- 
celium gradually colored leek-green. 

Starch-K\( );; agar: Growth milky white. 
Aerial mycelium leek-green. Starch hydro- 
lyzed. 

Gelatin: Growth whitish-yellow, covered 
with 1 i t»; 1 1 1 green aerial mycelium. Slow lique- 
faction. No soluble pigment. Melanin-nega- 
tive. 

Potato: ( rrowth colorless. Aerial mycelium 
at first white, later leek-green. 

.Milk: Pellicle heavy, lighl yellow. Aerial 
mycelium white-gray. Rapid coagulation; no 
peptonizat ion. 

Antagonistic properties: None. 

I labitat : Soil in Switzerland. 

Remarks: Some of the cultures described 
by < rause et al. 1 1957), such as .1. acrimycini 
and .1. acrimycini var. globosus, are closely 
related to this organism. 

122. Streptomyces hominis (Bostroem 
L890; emend. Waksman, 1919) Waksman 



and Ilenrici, L948. (Bostroem, E. Beitr. 
pathol. Anat. allgem. Pathol. 9: 1-240, 
1890; Waksman, S. A. Soil Sci. 8: 12!) 130, 
I'M!). 

Synonyms: Streptothriz hominis Fouler- 
ton, 1899. Oospora hominis Ridet, L911. 

Morphology: Sporophores straight. A few 
dextrorse spirals on glycerol synthetic me- 
dia. 

Sucrose nitrate agar: Growth white with 
shade of yellow, turning brownish with age. 
Aerial mycelium white with olive tinge. No 

soluble pigmenl . 

Glycerol malate agar: Growth yellowish. 
Aerial mycelium with olive-green tinge. 

Nutrient agar: Growth yellowish. Aerial 
mycelium white. No soluble pigment. 

Starch: Hydrolysis good. 

Potato: Growth yellowish to orange, be- 
coming brown. Aerial mycelium white. Color 
of plu»; unchanged, later becoming brown. 

Gelatin: Growth cream-colored. No aerial 
mycelium. No soluble pigment. 

Milk: Rapid coagulation and peptoniza- 
tion. 

Nitrate reduction: Positive. 

Sucrose: Xot inverted. 

Production of IPS: Negative. 

Habitat: Supposed to have been isolated 
from abscess of palm of hand; probably an 
air contamination. Appears to be related to 
the S. griseus series. 

123. Streptomyces humidus Nakazawa and 
Shibata, 1956 (Belgian Patent 533,386. Ta- 
keda Pharmaceutical Industries bid., .la- 
pan. March 21, L956; Proc. Japan Acad. 
32: (lis 653, 1956). 

Morphology: Sporophores form spiral-. 
Spores oval, 1 to 1 .."> by 1 .2 to 2 m- 

Sucrose nitrate agar: Growth colorless. 
Aerial mycelium white. No soluble pigment. 

Nutrient agar: Growth colorless. No aer- 
ial mycelium. No soluble pigment. 

Glucose-asparagine agar: Growth color- 
less. Aerial mycelium white to smoke-gray 
or vinaceous-chamois. No soluble pigment. 



230 



THE ACTINOMYCETES, Vol. II 



Calcium malate agar: Growth colorless, 
becoming yellowish. Aerial mycelium white. 
No soluble pigment. 

Starch agar: Growth colorless to cream- 
colored. Aerial mycelium white to pale 
smoke-gray. No soluble pigment. 

Potato: Growth colorless. Aerial mycelium 
white to smoke-gray; black, moist speckles. 
No soluble pigment. 

Gelatin: Growth colorless. No aerial my- 
celium. Xo soluble pigment. Moderate lique- 
faction. 

Milk: Growth colorless. Aerial mycelium 
white. No soluble pigment. Slow peptoniza- 
tion. 

Nitrate reduction: Positive. 

Carbon utilization: n-xvlose, L-arabinose, 



w 



imy 



ttt% 



o 



B 



Figure \'l. S. hygrosco' t 


oicus (B 


eproduc 


ed fro 


Tresner, H. 1). and Bac 


kus, E. 


.). A,,„l 


. Mici 


biol. 4: 246, 1956). 









L-rhamnose, D-fructose, galactose, maltose, 
lactose, D-mannitol, salicin utilized. Sucrose, 
n-raffinose, inulin, D-sorbitol, dulcitol, i-ino- 
sitol, sodium acetate, sodium citrate not uti- 
lized. 

Antagonistic properties: Produces an anti- 
biotic, acidomycin, said to be dihydrostrep- 
tomycin (see also Imamura el al., 1956). 

Remarks: S. humidus is closely related to 
S. hygroscopicus; growth of the latter on 
agar media and on potato is cream-colored 
to yellow to brown. 

124. Streptomyces hygroscopicus (Jensen, 
1931) Waksman and Henrici, 1948 (Jensen, 
H. L. Proc. Linnean Soc. N. S. Wales 56: 
357-358, 1931). 

Morphology: Sporophores monopodially 
branched, with narrow compact, sinistrorse 
spirals, situated as dense clusters on the 
main stems of the sporophores (Fig. 42). 
Spores oval, 0.8 to 1.0 by 1.0 to 1.2 M , 
smooth (PI. II nf, PI. IV Gb). 

Sucrose nitrate agar: Growth folded, 
white to cream-colored, later sulfur-yellow 
to yellowish-gray, with golden to light orange 
reverse. Aerial mycelium scant, white to ash- 
gray. Soluble pigment golden to light orange. 

Glucose-asparagine agar: Growth cream- 
colored to straw-yellow, later dull chrome- 
yellow to brownish-orange. Aerial mycelium 
dusty white to pale yellowish-gray; later 
small, moist, dark violet-gray to brownish 
patches produced, gradually spreading over 
the whole surface. Soluble pigment light 
yellow. 

Nutrient agar: Growth wrinkled, cream- 
colored, later yellowish-gray with yellowish- 
brown reverse. Aerial mycelium scant, white. 

Potato: Growth raised, wrinkled, cream- 
colored, later yellowish-gray to dull brown- 
ish. Aerial mycelium absent or trace of 
white. Melanin-negative. 

Gelatin: Liquefaction slow. No soluble 
pigment. 

Milk: Xo coagulation; positive peptoniza- 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



l'.;i 



lion. The reaction becomes faintly acid (pH been indicated by Nomi I 1960b). Vavra et al 

6.0orless). (1959) described a variety decoyicus that 

Starch: Hydrolysis. differed in certain minor properties. A num- 

Cellulose: Ready decomposition by some ber of other varieties of this organism have 

strains. been described, such as odoratus (Yiintsen 

Nitrate reduction: None with sucrose as et al., L956) and angustmyceticus (Takahashi 

source of energy. and Amano, L954). 
Sucrose inversion: Positive. ,._>- Streptomyces intermedins (Kruger- 

Production -.1 H 2 S: Negative , 7// , w W ollenweber, L922) Waksman (Wol, 

Antagonists propert.es: Produces hygro- lenweberj H . Ber . (1( , lt ,„„.,„. Ges 39: ._, (1 

L922). 



mycin, an antibiotic active agamsl mycobac- 
teria and roundworms (see also Pagano et <//., 
L953). 

Ilabital : Soil. 

Remarks: Tresner and Backus (1956) 
made a comprehensive study of IS cultures 
representing S. hygroscopicus and closely re- 
lated forms. They came to the conclusion 
that the following three properties are the 
fundamental characteristics of the organ- 
ism: (1) sporophores terminate in tight spi- 



Morphology: Sporophores straight, wavy, 
frequently arranged in fascicles or clumps 
\o spirals. Spores round to oval; ().'.) to 1.0 
by 0.7 m- 

Glycerol nitrate agar: Substrate growth 
cream-colored to brown; sometimes dark 
green to greenish-brown. Aerial mycelium 
thin, gray to dark gray. 

Glucose-asparagine agar: Growth brown- 
ish with greenish shade. Aerial mycelium 



rals oi a tew to many turns, plus a clustering dark gray X() S(( , llM( , pigmei) 

ot such sporophores along hyphae; (2) Nutrien1 agar: Growth much foldedj 

brownish-gray (mouse-gray to benzo-brown) cream _ colored . Aeria] myce li U m white. Sol- 



spore color on favorable media; (3) distinc- 
tive hygroscopic character on some agar 
media. The characteristic feature, not 
equally distind in all strains, however, is the 
fact that the aerial mycelium on synthetic 
media becomes moistened and exhibits dark, 
glistening patches; when touched with a 
needle, these patches prove to be moist, 
smeary masses of .-pores. Shape and size of 
spores, color of substrate growth, formation 
of soluble pigments, growth on potato, 
growth on milk, cellulose decomposition, and 
carbon and nitrogen utilization were con- 
sidered by Tresner and Backus as variable 
properties. They considered S. platensis and 

S. <nd us as closely related. 

Other related form-, such as .1. kurssano- 
mand .1. nigrescens, have been described by 

Cause et al. (1957). Ettlinger et al. (1958) [ a ] mycelium dark gray, 
also included S. platensis and S. rutgersensis Sucrose: [nversion -low 
Var. castelarense in tin- group. The relation 
ship of this species to S. violaceoniger ha: 



uble pigment faintly golden, occasionally 
green to olive-green; on continued cultiva- 
tion, green color tends to become cream- 
colored to brownish. 

Glucose-peptone agar: Growth good, 
brownish. Aerial mycelium heavy, cream- 
colored to dark gray. No soluble pigment. 

Potato: ( rrowth folded, brown to green i.-h- 
brown. Aerial mycelium dark gray. Soluble 
pigment olive-green. Melanin-negative. 

( relatin: ( rrowth thin, colorless to faintly 
brown, dropping to bottom. Slow liquefac- 
tion. ( rreenish-brown pigment . 

.Milk: Surface growth heavy, cream-col- 
ored. No aerial mycelium. No coagulation, 
good peptonization. 

Starch: Good hydrolysis. 

( 'ellulose: ( irowth good, olive-green. Aer- 



Nitrate reducl ion : Limited. 

Habitat : Potato -cab. 



2:!2 



Till; ACTINOMYCOTIC, Vol. II 



Remarks: Above description was supple- 
mented by Hoffmann (1958). Krassilnikov 
( L949) considers this organism as a variety of 
.1 . cretaceus. 

Type culture: IMRU 3329. 

L26. Streptomyces ipomoeae (Person and 
Martin, 1940) Waksman and Henrici, 1948 
(Person, L. H. and Martin, W. J. Phyto- 
pathology 30: 913, 1940). 

Morphology: Spores oval to elliptical, 0.9 
to L.3 by L.3 to 1.8 y.. 

Sucrose nitrate agar: Growth abundant, 
wrinkled, olive-yellow. Xo aerial mycelium. 

Xiitrieni agar: Growth moderate, in form 
of small, shiny colonies, both on the surface 
and imbedded in the medium, silver-colored. 

Starch agar: Growth moderate, smooth, 
ivory-colored. Aerial mycelium white with 
patches of bluish-green. Xo soluble pigment. 
Complete hydrolysis after 12 days. 

Potato: Growth moderate, shiny, wrin- 
kled, light brown. No aerial mycelium. No 
soluble pigment. 

Gelatin: Growth scanty, after 25 days at 
20°C. No aerial mycelium. No soluble pig- 
ment. Some liquefaction. 

Milk: Ring on surface. No visible coagu- 
lation; positive peptonization. 

C ellulose: Xo growth. 

Nitrate reduction: Positive. 

Antagonistic properties: Positive. 

Habitat: Lesions caused by sweet-potato 
disease. 

Type culture: [MRU 3476. 

127. Streptomyces kanamyceticus Okami 
and Umezawa, 1957 (Umezawa, H., Ueda, 
M., Maeda, K., Yagishita, Iv., Kondo, S., 
Okami, Y., Utahara, R., Osato, Y., Nitta, 
K., and Takeuchi, T. J. Antibiotics (Japan) 
I0A: 181-188, 1957). 

Morphology: Sporophores flexible and 
hooked, no true spirals. 

Glycerol nitrate agar: Growth at first 
colorless, later lemon-yellow. Aerial myce- 
lium white to yellow, occasionally with a 



greenish or faint pinkish tinge. Soluble pig- 
ment occasionally produced, faint brown. 

Glucose-asparagine agar: Growth color- 
less to yellow with faint pinkish-white, and 
yellow or hay-colored reverse. Aerial myce- 
lium scant; develops from center of colony, 
white to faint pinkish-white to greenish- 
yellow or yellow. Soluble pigment occasion- 
ally produced, faint brown. 

Calcium malate agar: Growth yellow. 
Aerial mycelium white-yellow. 

Nutrient agar: Growth cream-colored. 
Aerial mycelium absent or white. Xo soluble 
pigment. 

Potato: Growth wrinkled, faint yellowish- 
brown to yellow. Aerial mycelium scant, 
white. Xo soluble pigment. Substrate be- 
neath growth occasionally changes to brown. 

Gelatin: Liquefaction positive. Xo soluble 
pigment. Melanin-negative. 

Milk: Growth colorless. Aerial mycelium 
absent or white. Coagulation and peptoni- 
zation doubtful. 

Blood agar: Growth wrinkled, grayish- 
reddish-brown. Xo aerial mycelium. Xo sol- 
uble pigment. 

Starch: Hydrolyzed. 

Carbon utilization: Utilizes arabinose, 
dextrin, fructose, galactose, glycerol, mal- 
tose, mannitol, mannose, raffinose, starch, 
sucrose, and succinate. Does not utilize ino- 
sitol, inulin, lactose, rhamnose, sorbose, xy- 
lose, and acetate. Some strains grow slightly 
on esculin, salicin, sorbitol, and citrate. 

Antagonistic properties: Produces kana- 
mycin, an antibiotic related to the neomycin 
group. 

Remarks: Closely related to S. albido- 
flavus, S. lieskei, and S. alboflavus. Okami 
et al. (1959a) made a detailed study of the 
mutants produced by this organism. 

128. Streptomyces kentuckensis Parr and 
Carman, 1956 (Han-, V. S., and Carman, 
P. E. Antibiotics & Chemotherapy 6: 286 
289, 1950). 

Morphology: Aerial mycelium thick- 



DESCRIPTION OF SPECIES OF STREPTOMYCES 233 

walled, generally no1 branched; sporophores Milk: Coagulation, slow peptonization. 

straight; do nol form spirals. Spores oblong Surface ring pinkish-brown; medium later 

to oval, 0.5 by 0.5 to 1.5 n. Spores produced becomes dark brown. 

by fragmentation of the hyphae in substrate Starch: No hydrolysis. 

mycelium arc generally smallei than those Source: Blood culture of a woman with 

formed from aerial hyphae. acholuric jaundice. No record concerning 

Nutrient agar: Growth gray to yellow. No actual pathogenicity. 

soluble pigment. Melanin-negative. ,.»,, ^, , i -, ■ ,, , , 

f & . \ . 130. StreptomycestotasatoensisHa,ta,etal., 

Gelatm: Slow liquefaction. No soluble pig- ,,,-■> ,ii * -r i- i- i i- 

1 ' & L953 (llata, I.. Koga, I'., and Kaiiainon. 

''"■ II. .1. Antibiotics (Japan) 6A: L09 1 12, 

Potato: Mycelium gray, Plug darkened. pr -> ) 

Milk: Peptonization positive. m i i o i i 

1 . ... Morphology: Sporophores produce pn- 

Nitrate reduction: Positive. i i - - , . .. . , 

mary and secondary verticils. A few spirals 
El 2 b: Negative. ,..,. f , Q ■ k.. ..• , 



inent . 



Product ion i 

Carbon utilization: Readily utilizes var 

•us pentoses, hexoses, disaccharides, aci 



were also found. Spores oval or cylindrical. 
1.9 to L.3 by 0.9 At. 
Sucrose nit late agar: Growth yellow to 

,o 



i^mium- 1 1 1 i i ; u i • ; i ji,; 1 1 . \ 1 1 ( i w l n \ e 

late and citrate; slight utilization of rham- r i . ■■ • i ■ . 

& . light yellowish-brown. Some strains lwx. 

Dose, inulin, glycerol, inositol, mannitol ; does ■ i <■ ■•« ■ ■ , • 

aerial mycelium even alter prolonged culti- 

not utiuze dextran or salts of oxalic, succinic, ,• ,i ,- .i- i • i " i •* 

..,.., vation; others form thick grayish-white ae- 

11,1,1 sallr - vl,( ' :l, ' 1(ls - rial mycelium. Soluble pigment light yellow- 
Antagonistic properties: Effective against ■ i 

gram-positive and some gram-negative bac- /<i,, • ,. ,, , 

1 & & Glucose-asparagine agar: Growth brown 

teria. Produces antibiotic raisnomycin. + i ,i i , • , i -,i 

to dark brown, restricted, with raised center. 

Remarks: Pridham et al. (1958) consider » ■ , , „ ,-, (1 • • , 

Aerial mycelium thin, grayish or mouse- 

this organism as a membei <>t the biverticilla- • i o i ii . i 

grayish. Soluble pigment In-own. 

= " "' Nutrient agar: Growth brown, restricted, 

Type culture: ATCC 12,691. •., , , v ,. 

• ' with raised center. No aerial mycelium. 

129. Streptomyces kimberi (Erikson, 1935) Soluble pigment brown. 

Waksman (Erikson, 1). Med. Research Starch agar: Growth colorless; reverse 

Council (Brit.) Spec. Kept. Ser. No. 203: yellow to yellowish-brown. Aerial mycelium 

II 15, 1935). yellowish- white, cottony or flocculent. 

Morphology: Growth made up of long, Tyrosine agar: Growth brown to dark 

straight, profusely branching filaments. Ae- lirown. Aerial mycelium grayish-white, thin 

rial mycelium produce- short and straight later becoming cottony. Tyrosinase reaction 

sporophores. Spores small, round. positive. 

Sucrose nitrate agar: Growth moist. Potato: Growth yellowish-brown and 

cream-colored. Aerial mycelium powdery, wrinkled. No aerial mycelium. Color of plug 

white. light brown. 

Glucose-asparagine agar: Growth cream- Gelatin: Growth dark brown. Soluble pig- 
colored. Aerial mycelium white. meni dark brown. Liquefaction slow at be- 

Nutrienl agar: Growth moist, cream-col- ginning, bul complete liquefaction I weeks 

orcd. Aerial mycelium powdery, white. later. 

Gelatin: Colonies smooth, shining, float- Milk: No coagulation; slow peptonization, 

ing on liquefied medium. Aerial mycelium No clearing of milk, but heavy brown pre- 

powdery, white. Good liquefaction. No sol- cipitate on bottom; color of liquid in upper 

uble pigment. portion brownish. 



234 



THE ACTINOMYCETES, Vol. II 



Starch: Hydrolysis positive. 

Cellulose: No decomposition. 

Nitrate reduction: Positive. 

Production of H 2 S: Positive. 

Carbon utilization: Utilizes glucose, 
starch, dextrin, glycerol, galactose, maltose, 
sucrose, trehalose, inositol, sorbitol, sodium 
succinate, sodium citrate, and sodium ace- 
tate. Does not utilize xylose, rafnnose, rham- 
nose, lactose, arabinose, mannose, mannitol, 
inulin, dulcitol, fructose, salicin, or esculin. 

Antagonistic properties: Produces an anti- 
biotic, leucomycin. 

Remarks: S. kitasatoensis is similar to S. 
reticuli in morphology of the mycelium, cul- 
tural characteristics, and utilization of car- 
bon sources, but different in several other 
respects. 

131. Streptomyces kitasawacnsis Harada 
and Tanaka, 1956 (Harada, Y. and Tanaka, 
S. J. Antibiotics (Japan) 9A: 113-117, 1956). 

Morphology: Sporophores straight; no spi- 
rals. 

Sucrose nitrate agar: Growth cream to 
yellow. Aerial mycelium white with pale 
pinkish tinge. 

Calcium malate agar: Growth cream-col- 
ored. Aerial mycelium white. No soluble 
pigment . 

Glucose-asparagine agar: Growth has 
brownish tinge. Aerial mycelium white with 
grayish tinge. Soluble pigment pale yellow- 
ish-brown to pale greenish-yellow. 

Nutrient agar: Growth brownish. Aerial 
mycelium absent or scarce. Soluble pigment 
dark brown. 

Starch agar: Growth pale grayish-brown 
to pale blackish-brown. Aerial mycelium 
white. Soluble pigment pale greenish-yellow 
to pale yellowish-brown. 

Gelatin: Growth white to gray. Aerial my- 
celium absent or scarce. Soluble pigment 
dark brown. No or weak liquefaction. 

Potato: Growth brown. Aerial mycelium 
white. Soluble pigment dark brown. 



Milk: Growth in form of dark brownish 
ring. Coagulation and peptonization. 

Carbon utilization: Utilizes i)(+)xylose, 
D-mannitol, L-arabinose, salicin. Does not 
utilize L(+)rhamnose, D-maltose. 

Antagonistic properties: Produces an anti- 
tumor substance, carzinocidin. 

Habitat :Soil. 

132. Streptomyces lanatus Frommer, 1959 
(Frommer, W. Arch. Mikrobiol. 32: 203, 
1959). 

Morphology: Sporophores long, straight 
or wavy, with short side branches; the ends 
of these are more entangled than spiral- 
shaped. 

Glycerol-sucrose agar: Growth abundant, 
cottony, with red-brown reverse. Soluble 
pigment brown to dark red-brown. 

Glucose-asparagine agar: Growth rose- 
brown. Aerial mycelium velvety to cottony, 
rose to gray-green. Soluble pigment brown- 
ish. 

Calcium malate agar: Growth colorless to 
yellowish. Aerial mycelium powdery, gray to 
gray-green. No soluble pigment. 

Nutrient agar: Growth yellow-brown. Ae- 
rial mycelium cream-colored or lacking. Sol- 
uble pigment yellow-brown to dark brown. 

Starch: Weak hydrolysis. 

Potato: Growth yellow-brown. Aerial my- 
celium powdery, white. Soluble pigment 
black. 

Gelatin: Growth yellow. Aerial mycelium 
yellow. Soluble pigment brown to red-brown. 
Liquefaction medium. 

Milk: Growth abundant, dark brown. 
Aerial mycelium powdery, cream-colored. 
Coagulation, slow peptonization. 

Cellulose: No growth. 

Antagonistic properties: Produces actino- 
mycin. 

Remarks: Closely related to S. purpureo- 
chromogenes and S. phaeochromogenes. 

133. Streptomyces lavendulae (Waksman 
and Curtis, L916) Waksman and Henrici, 



DESCRIPTION OF SPECIES OF STREPTOMYCBS 



235 



L948 (Waksman, S. A. and Curtis, R. E. 
Soil Sci. I: 126, L916; 8: L30, L919). 

Morphology: Sporophores long, m - 

podially branched; short, compacl spirals of 
the dextrorse type, 5 to 8 m in diameter; 
spirals sometimes open. Sonic strains form 
no spirals, according to Okami (1956). 
Spores oval, 1.0 to 1.2 by l.li to 2.0 m, 
smooth ( PI. V, Ea). 

Sucrose nitrate agar: ( Growth thin, spread- 
ing, colorless to cream-colored. Aerial my- 
celium cottony, white, becoming vinaceous- 
lavender. No soluble pigment. 

Glycerol malate agar: Growth cream-col- 
ored. Aerial mycelium lavender. Xo soluble 
pigment. 

Glucose-asparagine agar: Growth yellow- 
ish. Aerial mycelium white with lavender 
tinge. Xo soluble pigment. 

Nutrient agar: Growth wrinkled, gray. Xo 
aerial mycelium. Soluble pigment brown. 

Starch agar: Growth restricted, glistening, 
transparent, rose-colored. Aerial mycelium 
lavender. Good hydrolysis of starch. 

Potato: Growth thin, wrinkled, cream to 
yellowish. Xo aerial mycelium. Soluble pig- 
ment black. 

Gelatin: Surface growth creamy to brown- 
ish. Aerial mycelium absenl or white. Lique- 
faction slow. Soluble pigment brown. 

Milk: Cream-colored ring. Xo coagulation; 
good peptonizat ion. 

Cellulose: ( Irowth scant. 

Nitrate reduction: Positive. 

Production of H 2 S: Positive. 

Temperature: < Optimum 37°C. 

Antagonistic properties: Various strains 
of this organism produce antibiotics. One 
such antibiotic, streptothricin, is active 
both in vitro and in vivo againsl various 
gram-positive and gram-negative bacteria, 
fungi, and actinomycetes. Certain other 
strains produce an antiviral agent, ehrlichin. 

Habitat : Soil. 

Remarks: Numerous strains and varieties 
of this organism have been isolated. It is 



sufficient to mention S. lavendulcn var. 
japonicus, and several of the cultures listed 
by Cause et al. (1957), notably .1. flavotri- 
cini, .1. toxytricini, and .1. violascens. Ett- 
linger et <il. I 1958) considered S. aeidomyceti- 
cus and S. virginiae as members of this 
group. Krassilnikov (1<)4!)) considered this 
species as a variety of .1. chromogi'ne.s. 
Okami (1956) and Rangaswami (1958) made 
a detailed study of numerous representatives 
of this species or species-group. 

Morais et al. (1958) described a variety of 
S. lavendulae as brasilicus, the aerial my- 
celium being red-pink or red-brown but not 
lavender and not utilizing salicin. 

Type species: IMRU 3440. 

134. Streptomyces lieskei (Duche, 1934) 
Waksman and Benrici, 1948 ( Duche, .1. Les 
actinomyces du groupe albus. I\ Lechevalier, 
1'aiis, 1934). 

.Morphology: Sporophores form oval 
spores. 

Glucose-asparagine agar: Growth cream- 
colored, later yellowish to green. Aerial my- 
celium white, later yellowish, growing from 
the edge toward the center. Soluble pigment 
dirty yellow to yellow-green. 

Nutrient agar: Growth cream-colored. Ae- 
rial mycelium white. Soluble pigment yellow- 
ish. 

Gelatin: Growth cream-colored. Aerial 
mycelium white. Xo soluble pigment. Liq- 
uefaction rapid. 

Milk: (Irowth cream-colored. Xo aerial 
mycelium. Peptonization without coagula- 
tion. After 20 days the whole milk becomes 
a clear yellowish liquid. 

Tyrosine medium: (irowth rapid. Aerial 
mycelium whitish-yellow. Soluble pigment 
yellowish to orange-yellow. 

Coagulated serum: (irowth colorless. Liq- 
uefaction rapid. 

Remarks: Related t<> S. alboflavus and S. 
albidoflavus. 

135. Streptomyces limosus Lindenbein, 



236 



THE ACTIXOMVCETES, Vol. II 



L952 (Lindenbein, \V. Arch. Mikrobiol. 17: 
361-383, L952). 

Morphology: Substrate mycelium divides 
completely into coccoidal pieces. Some simi- 
larity to Nocardia. Aerial mycelium gray, 
produced in certain media. 

Glycerol nitrate agar: Growth colorless, 
later becoming deep yellow. No aerial my- 
celium. Soluble pigment citron-yellow. 

Glucose-asparagine agar: Growth lemon- 
yellow, later becoming black with yellow 
reverse. Aerial mycelium white, later ash- 
gray. Soluble pigment lemon-yellow. 

Glycerol malate agar: Growth dark yel- 
low. Aerial mycelium white, later ash-gray. 
Soluble pigment golden yellow. 

Nutrient agar: Growth light brown. No 
aerial mycelium. Soluble pigment light 
brown. Melanin-positive. 

Glucose-peptone agar: Growth yellow- 
brown. Aerial mycelium ash-gray. Soluble 
pigment yellow-brown. 

Starch-nitrate agar: Growth brownish- 
yellow. Aerial mycelium gray- white. Soluble 
pigment lighl yellow. Hydrolysis strong. 

Potato: Growth brownish-yellow. Aerial 
mycelium gray-white. Soluble pigment 
lemon-yellow to sulfur-yellow. 

Gelatin: Growth yellow-brown. No aerial 
mycelium. Soluble pigment dark brown. 
Liquefaction complete. 

Milk: Growth lichenoid, light yellow. 
Aerial mycelium gray-white. Soluble pig- 
ment light brown. Strong peptonization. 

( 'ellulose: No growth. 

Antagonistic properties: None. 

Source: Isolated from the slime of a river 
bank. 

Remarks: Related to S. flavovirens. 

136. Streptomyces lipmanii (Waksman and 
Curtis, 1916) Waksman and Ilenrici, 1948 
(Waksman, S. A. and Curtis, II. E. Soil 
Sci. I: 123, 1916; 8: 121, 1919). 

Morphology: Sporophores si raight , no spi- 
rals. Spores oval, 0.8 to 1.1 by 1.0 to 1.5 p. 



Sucrose nitrate agar: Growth abundant, 
raised, colorless, becoming light brown and 
wrinkled. Aerial mycelium white, turning 
gray to dark gray. No soluble pigment. 

Glycerol malate agar: Growth colorless, 
becoming dark brown. Aerial mycelium 
mouse-gray. No soluble pigment. 

Glucose-asparagine agar: Growth spread- 
ing, light yellow. No aerial mycelium. No 
soluble pigment. 

Nutrient agar: Growth wrinkled, glossy, 
yellow. No aerial mycelium. No soluble pig- 
ment. 

Potato: Growth abundant, wrinkled, 
cream-colored. Aerial mycelium white to 
gray. Soluble pigment purplish. 

Gelatin: Cream-colored, flaky sediment. 
Aerial mycelium while-gray. Liquefaction 
medium to rapid. Melanin-negative. 

Milk: Cream-colored ring. Coagulation 
and peptonization. 

Starch media: Growth transparent, be- 
coming dark with age. No aerial mycelium. 
Hydrolysis medium. 

Cellulose: No or very scant growth. 

[nvertase: Positive. 

Nitrate reduction: Positive. 

Production of II..S: Negative. 

Temperature: Optimum 25°C. 

Antagonistic properties: Good, though 
some strains show no activity. 

Habitat : Soil. 

Remarks: Ettlinger et al. (1958) consider 
this organism as a strain of S. griseus. 
Tresner and Danga reported that their 
strain produced a grayish-yellow-buff aerial 
mycelium. Kra-silnikov (194'.)) considered it 
as a variety of . 1 . viridis. 

Type cult me: [MRU 3331. 

lo7. Streptomyces loidensis (Millard and 
Burr, L926) Waksman (Millard, W. A., and 
Burr, S. Ann. Appl. Biol. 13:580, 1926). 

Morphology: Sporophores straight and 
spiral-forming. Spores cylindrical to spheri- 
cal, 0.9 to 1.0 by 0.9 to 0.95 m- 



DESCRIPTION OF SPECIES OF STREPTOMYCES 237 

Sucrose nitrate agar: Growth thin, flat, L39. Streptomyces lucensis Arcamone et al., 
gray to yellowish-olive. Aerial mycelium L957* (Arcamone, F., Bertazzoli, ('., Cane- 
scant, olive-colored. Soluble pigment yellow, vazzi, (>., DiMarco, A., Ghione, M., and 

Nutrient potato agar: Growth good, gray. Grein, A. Giorn. Microbiol. 1: 119 128, 

Aerial mycelium olive-buff. Soluble pigmenl 1957). 
golden brown. Morphology: Aerial hyphae long, 

Gelatin: Growth gray. Aerial mycelium branched, and hooked at the tip. Spirals 

scant, white. Liquefaction rapid. Soluble produced abundantly, 
pigment yellow. Glycerol-glycine agar: Growth abun- 

Milk: Surface growth excellent. Aerial dant, lemon-yellow. Aerial mycelium gray- 
mycelium white. Coagulation and rapid pep- brown. Some soluble pigmenl produced. 
tonization. Glucose-asparagine agar: Growth abun- 

Starch: Hydrolysis. dant. Aerial mycelium hazel-brown; scanty 

Nitrate reduction : None. clusters of white, short, sterile hyphae. No 

Temperature: Grows well at :i7."> ( '. soluble pigment. 

Habitat: Potato scab. Potato-Glucose agar: Growth abundant, 

,, /T , smooth, yellowish. Aerial mycelium abun- 

138. Streptomyces longisporoflavus (Kras- . a 

.. .. „ ,, „, , .... ., .. dant, bun-gray to hazel-brown. Soluble pig- 

silnikov, 11)4 1 ) Waksman (Krassilmkov, N. . , > 

, . . . . . , N . , ment ash-gray, later turning gray- brown. 

A. Actmomycetales. Izvest. Akad. Nauk. _. ° ■ ,.,,',, 

Yeast-glucose agar: drowth dark brown. 



SSSH, Moskau, p. 30, 1941). 

Morphology: Sporophores produce long 
open spirals. Spores cylindrical or elongated, 

1.0 to 1..") by 0.7 /u. some rounded at ends 
and swollen in center. 

Agar media: Growth yellow to lemon-yel- 
low or dirty yellow, seldom golden yellow. 
Pigment insoluble. Aerial mycelium well de- 



veloped, velvety, whitish-yellow to brow.. 

. . .. soluble pigm 

ish-vellow. ,, , 



Aei'ial mycelium whitish. Soluble pigmenl 
dark brown. 

Starch agar: Growth abundant, colorless 
to yellowish-brown. Aei'ial mycelium pow- 
dery, buff-gray to light brown. No soluble 
pigment. Moderate starch hydrolysis. 

Oatmeal agar: Growth yellowish and 
smooth. Aerial mycelium hazel-brown. No 



Potato plug: Growth abundant, wrinkled. 

Aerial mycelium lighl gray to hazel-brown. 
Milk: Coagulation and slow peptonization. _. J , ' , ,' , , 

. ' . , , PI1112; surface turns dark la-own around 



Gelatin: Liquefaction mediui 



Starch: Hydrolysis weak. 

Cellulose: No growth. 

Nitrate reduction : Positive. 

Sucrose: No inversion. 

Antagonistic properties: Weakly antago- 
nistic. 

Remarks: Some strains, such as S. flavo- 
viridis, sometimes have a greenish or green- 
ish-yellow color instead of a yellow color. 
.... . . . . . I ype culture: I AIKI 3783. 

1 his organism and related forms belong to 

the same group as S. griseoflavus and S. 140. Streptomyces luridus (Krassilniko\ 

microflavus. The form described by Gause l( " L - l!,: > 7 ' Waksman (Krassilnikov, N. A. 



culture. 

Gelatin: Growth abundant, blown. Aerial 
mycelium white, turning grayish-brown. 
Substrate is strongly darkened within 3 
days. No liquefaction. 

Antagonistic properties: Produces an 
antifungal antibiotic, etruscomycin, of the 
let raene i ype. 



<t al. (1 ( .)")7) as A. aurini also belongs to this 



Koreniako, A. [., Meksina, M. M., Vale- 



group. * Supplemented by personal communicatio 



238 



THE ACTINOMYCETES, Vol. II 



dinskaia, L. K., and Vesselov, N. M. Mikro- 
biologiya 26: 558-564, 1957). 

Morphology: Substrate mycelium mono- 
podially branched, 0.7 to 0.8 n in diameter. 
Sporophores produce spirals with 1 to 3 
turns. Spores spherical, oval, seldom elon- 
gated. Sporulation is generally weak, oc- 
curring only on certain media; spiral forma- 
tion occurs seldom, largely on synthetic 
media with a limited amount of sucrose, and 
on starch media. 

Sucrose nitrate agar: Growth yellow- 
orange. Aerial mycelium white with rose 
tinge. 

Nutrient agar: Substrate growth colorless, 
free of aerial mycelium. No soluble pigment. 
In old cultures, clumps of aerial hyphae may 
be formed. 

Potato agar: Growth yellow-orange. Aerial 
mycelium white with rose tinge. Crystals of 
salts deposited along the mycelium in the 
substrate. 

Milk: Coagulation weak; rapid peptoniza- 
tion. 

Gelatin: Not liquefied in 10 days. 

Starch: Moderate hydrolysis. 

Nitrate: Reduced. 

Sucrose: Not inverted. 

Cellulose: No growth. 

Carbon utilization with acid formation: 
arabinose, inositol, sorbitol; no acid from 
glucose, lactose, rhamnose, xylose, inulin, 
inositol, mannitol, or dulcitol. 

Antagonistic properties: Produces anti- 
bacterial and antiviral (luridin) substances. 

Remarks: Cannot be distinguished from 
S. fradiae in its morphological and cultural 
properties, but is different in its biochemical 
and its antibiotic activities. 

141. Streptomyces luteoverticillatus Shin- 
obu, 1956 (Shinobu, R. Mem. Osaka Univ. 
B (N.S.) 5: 84 93, L956). 

Morphology: Primary and secondary 
verticils produced on various synthetic 
media. Spores coccoid to elliptical, about 0.8 
M long. 



Sucrose nitrate agar: Growth pale brown. 
Aerial mycelium cottony, brownish-white 
to brown. 

Glucose-asparagine agar: Growth thin, 
brown. Aerial mycelium cottony, yellow- 
white. Soluble pigment pale brown. 

Nutrient agar: Growth excellent, deep 
brown. Aerial mycelium white to yellow to 
green. Soluble pigment deep brown. 

Potato plug: Growth heavy, brown. Aerial 
mycelium yellow to greenish. Soluble pig- 
ment brown. 

Milk: Aerial mycelium heavy, brown. 
Coagulation uncertain; peptonization strong. 
Soluble pigment brown. 

Gelatin: Strong liquefaction. 

Diastase reaction: Strong. 

Tyrosinase reaction: Strong. 

Carlton utilization: fructose, mannitol, 
and inositol utilized. Xylose, rhamnose, 
sucrose, lactose, and raffinose not utilized. 

Habitat: Soil. 

Remarks: Resembles S. verticillatus. 

142. Streptomyces lydicus DeBoer et al., 
L955 (DeBoer, G, Dietz, A., Silver, W. S., 
and Savage, G. M. Antibiotics Ann. 1955- 
L956, p. 886-892). 

Morphology: Sporophores long, slightly 
coiled at tip. Spores spherical to oval. 

Sucrose nitrate agar: Substrate growth 
buff-colored. Aerial mycelium white. 

Nutrient agar: Some substrate growth. 
No aerial mycelium. Soluble pigment yellow- 
ish. 

Casein-yeast extract -beef agar: Growth 
olive-tan. Aerial mycelium gray-white with 
flecks of black. Soluble pigment olive-tan. 

Glucose-peptone agar: Aerial mycelium 
gray-white. Soluble pigment yellow. 

Starch agar: Growth good. Aerial myce- 
lium pink-gray-white. Hydrolysis good to 
excellent. 

Gelatin: Some growth. No aerial myce- 
lium. Liquefaction positive. Soluble pigment 
olive-colored. 



DESCRIPTION OF SPECIES <>K STREPTOMYCES 



■2:\\) 



Milk: Ring around surface. Peptonization 
positive. 

Nitrate reduction: Positive. 

Carbon utilization: Most sugars and 
organic acids utilized, hut not rhamnose, 
inulin, dulcitol, inositol, or the sodium salts 
of formic, oxalic, tartaric, and salicylic acids. 

Production of H 2 S: Negative. 

Antagonistic properties: Produces an anti- 
biotic, streptolydigin, active against various 
bacteria. 

Remarks: Related to S. diastaticus. 

143. Streptomyces macrosporeus Ettlinger 
et al, 1958 (Ettlinger, L., Corbaz, R., and 
Hiitter, R. Arch. Mikrobiol. 31: 346, 1958). 

Morphology: Sporophores monopodially 
branched along the whole axis with open, 
irregular spirals. Spores large, 1.7 to 2 by 
1 .5 to 2 /x; short spines (Plate II m). 

Glycerol nitrate agar: Substrate growth 
yellow. Aerial mycelium white-yellow to ash- 
gray. Soluble pigment golden yellow. 

Glucose-asparagine agar: Growth milk- 
white. No aerial mycelium. Soluble pigment 
whitish-yellow. 

Calcium malate agar: Growth yellow. 
Aerial mycelium white-yellow to ash-gray. 
Soluble pigment white-yellow. 

Starch agar: Growth light yellow. Aerial 
mycelium white-gray. Good hydrolysis of 
starch. 

Potato: Growth abundant, light yellow to 
golden yellow. Aerial mycelium ash-gray. 

Gelatin: Growth sparse. Liquefaction 
slow. Xo soluble pigment. 

Milk: Pellicle light yellow to yellow- 
brown. Aerial mycelium white to white-gray. 
Coagulation strong; no peptonization. 

Antagonistic properties: Produces an 
antibiotic, carbomycin. 

Habitat : Soil in Madras, India. 

144. Streptomyces maculatus (Millard and 
Burr, 1926) Waksman and Henrici, I'.MN 
(MiUard, W. A. and Burr, S. Ann. Appl. Biol. 
13: 580, 1926). 



Morphology: Growth tough, shiny, carti- 
laginous. Aerial mycelium rarely produced, 
though in certain strains it may frequently 
occur, especially when grown on organic 
media. Sporophores straight, short. Spores 
spherical, 0.5 to 0.6 /x- 

Sucrose nitrate agar: Growth orange- 
yellow to orange-red; as the culture ages it 
may change to dark green or black. Xo 
aerial mycelium. 

Nutrient potato agar: Growth vinaceous- 
tawny. Soluble pigment vinaceous-tawny. 

Potato: Growth restricted, raised, pinkish. 
Aerial mycelium scant, white. Soluble pig- 
ment gray to brown. 

Gelatin: Growth limited. Liquefaction 
slow. 

Milk: Growth slight. Xo coagulation; no 
peptonization. 

Starch : Hydrolyzed. 

Nitrate reduction: Negative. 

Tyrosinase reaction: Negative. 

Oxygen requirement: Said to grow well 
under anaerobic conditions. 

Paraffin: Not utilized. 

Temperature: Grows well at 37.5°C. 

Habitat: Potato scab and soil. 

Type culture: IMRU 3376. 

145. Streptomyces madurae (Vincent, 
1894) now comb. (Vincent, H. Ann. inst. 
Pasteur 8: 129, 1894). 

Synonyms: N. babiensis, N. brumpti, X. 
madurae, and A', salmonicolor (A. salmoni- 
color Millard and Burr, 1926). Baldacci (1944) 
listed 17 synonyms. 

Strains of this organism were reported by 
various investigators, most recently by 
Gonzalez Ochoa and Sandoval (1951), to 
form, under certain conditions of culture 
and on certain media, such as grain, an 
aerial mycelium, with slraight or spiral- 
shaped sporophores; the spores were cylindri- 
cal or oval. This led them to consider this 
organism as a Streptomyces. Mariat (1957) 
was also of the same opinion. MacKinnon 
and Artagaveytia-Allende (1956) consider 



240 



THE ACTIXOMYCETES. Vol. II 



the generic position of this species as far 
from settled. 

Morphology: Growth red to red-brown or 
pink. In tissues it forms grannies consisting 
of radiating actinomycosis. Initial branched 
mycelium is said to be nonsegmented. Not 
acid-fast. Aerial mycelium white and pink 
in color. 

Glucose-asparagine agar: Growth cream- 
colored. Some cultures give reddish pig- 
mentation. 

Protein media: Growth good, pinkish. 
Soluble pigment brown. 

Gelatin: Growth glistening, at first white, 
then buff to rose or crimson. Soluble pigment 
irregular and unpredictable, occasionally red. 
Gelatin slowly liquefied. 

Milk: No change, or slight; coagulation 
slow, if any; peptonization slow. 

Carbon utilization: Utilizes starch, glu- 
cose, mannitol, and xylose, but not lactose 
or paraffin. 

Nitrate reduction: Positive. 

Pathogenicity: This property was vari- 
ously reported. Topley and Wilson (1946) 
stated that this organism causes a local 
tissue disease when inoculated under the skin 
in guinea pigs. Often reported as not patho- 
genic for the usual laboratory animals; 
pathogenic for monkeys. 

Source: Wide geographical distribution. 
Madura foot and other substrates. 

146. Streptomyces marginatus (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, S. Ann. Appl. Biol. 13: 580, 11)20). 

Morphology: Sporophores straight. Spores 
oval to spherical, 0.0 by 0.8 fi. 

Sucrose nitrate agar: Growth thin, echi- 
nate. Aerial mycelium olive-buff. Soluble 
pigment cream-colored. 

Nutrient potato agar: Growth heavy, 
gray. Aerial mycelium white to whitish- 
yellow. Soluble pigment light golden brown 
to deep golden brown. 

Potato: Growth good, raised. Aerial myce- 



lium abundant, buff to olive-buff. Plug at 
first gray, later becoming black. 

Gelatin: Growth thin, pale olive-gray. 
Aerial mycelium abundant, pale gray to 
olive-buff. Soluble pigmenl buff. Liquefac- 
tion rapid. 

Milk: Growth good. Aerial mycelium 
white. Coagulation and peptonization. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Temperature: Grows well at 37.5°C. 

Habitat: Potato scab. 

147. Streptomyces marinolimosus (Zobell 
and Upham, 1044) Waksman (ZoBell, C. E. 
and Upham, H. G. Bull. Scripps Inst. 
Oceanogr. Univ. Calif. 5: 230-202, 1044). 

Morphology: Aerial mycelium consists of 
branching filaments with chains of spores. 
Spores 0.0 m in diameter. 

Agar media: Growth dull. Aerial myce- 
lium white to pinkish, powdery, rough, in 
concentric circles. Odor of freshly turned 
soil. 

Potato: Growth yellow, becoming white, 
powdery, raised, rugose. Potato darkened. 

Gelatin: Growth in form of flat, yellowish, 
circular colonies, with tendency to grow 
together. Liquefaction crateriform. Melanin- 
negative. 

Sea water broth: Growth in form of light 
yellow clumps; pellicle produced on surface. 
Earthy odor. 

Milk: Growth in form of pellicle. Com- 
plete peptonization in 20 days. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Production of H 2 S: Positive. 

Source: Marine mud. 

Remarks: All differential media were pre- 
pared with sea water. 

148. Streptomyces marinus (Humra and 
Shepard, 1946) nov. com)). (Humm, H. J. 
and Shepard, K. S. Duke Univ. Marine Sta. 
Bull. 3: 77, 1046). 

Morphology: Sporophores sometimes form 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



241 



loose spirals. Spores spherical to oval, 0.8 
to 1.2 fi, produced as a dark gray area in 
center of colonics. 

Agar media: Growth sparingly branched, 
dense, entangled, frequently forming con- 
centric rings in response to alternate periods 
of lighl and darkness. Aerial mycelium 
white. Spores gray to dark gray. No soluble 
pigment. 

Gelatin : ( Growth arborescenl . Liquefaction 
stratiform, slow. Melanin-negative. 

Milk: Peptonization complete, usually 
within 1 month. 

Starch: Vigorous hydrolysis. 

Cellulose: Xot attacked. 

Chitin and alginic acid: Attacked. 

Agar: Slowly digested, softened, not lique- 
fied. Growth on agar in culture dish sur- 
rounded by rather wide, gently sloping 
depression. Gelase held relatively wid(\ with 
distinct margin. Irish moss and Hypnea gels 
also slowly digested. 

Nitrate reduction: Usually negative. In 
some media, slight nitrite is produced after 

10 days' incubation, especially if glucose is 
present. 

Production of H 2 S: Positive. 

Indole: Xot formed. 

Carbon utilization: Acid produced from 
galactose, glucose, fructose, mannose, cel- 
lobiose, lactose, maltose, sucrose, and 
glycerol. Arabinose, xylose, rhamnose, and 
sorbitol utilized without acid production. 
Xo growth with raffinose, salicin, inulin, 
dulcitol, inositol, ethyl alcohol, or ethylene 
glycol. Utilizes acetic, citric, lactic, propi- 
onic, succinic, and iso-valeric acids. Does no1 
utilize butyric, gluconic, maleic, malonic, 
and oxalic acids. 

Habitat: Marine sediments. 

149. Streptomyces mashuensis Sawazaki 
et al, 1955 (Sawazaki, T., Susuki, S., Naka- 
mura, G., Kawasaki, M., Yamashita, S., 
[sono, K., Anzai, K., Serizawa, Y., and 
Sekiyama, Y. J. Antibiotics (Japan) 8A: 

11 17, 1955). 



Morphology: Sporophores straight, no 
spirals; numerous primary and secondary 

verticils. 

Sucrose nitrate agar: Growth yellow; 
reverse yellow-green. Aerial mycelium abun- 
dant , powdery. 

Glucose-asparagine agar: Growth pow- 
dery, grayish- white, reverse yellow-brown. 
Xo aerial mycelium. 

Nutrient agar: Growth cream-colored; 
reverse brown. Xo aerial mycelium. Xo 
soluble pigment . 

Starch agar: Growth marguerite-colored; 
margin cottony, primrose-pink; reverse yel- 
low-brown, margin white. Aerial mycelium 
white. Strongly diastatic. 

Potato: Growth spreading, dark cream- 
colored. Aerial mycelium limited. Limited 
discoloration of plug. 

Gelatin: Growth white. Xo aerial myce- 
lium. Soluble pigment pinkish. Liquefaction 
medium. 

Nitrate reduction: Negative. 

Carbon utilization: Xylose, glucose, su- 
crose, trehalose utilized. Rhamnose, raffi- 
nose, salicin, mannitol, lactose, arabinose 
not utilized. 

Antagonistic properties: Produces two 
antibiotics, streptomycin and a labile sub- 
stance active againsl mycobacteria, fungi, 
and B. .subtilis. 

Remarks: Okami et al. (1959b) made a 
detailed study of this organism. They re- 
ported, instead of the yellow growth on 
synthetic media, poor colorless growth. 

150. Streptomyces matensis Margalith et 
al., 1959 (Margalith, P., Beretta, G., and 
Timbal, M. T. Antibiotics & Chemotherapy 
9: 71-75, 1959). 

Morphology: Sporophores produce verti- 
cils, the branches forming spirals. Spore- 
spherical. 

Sucrose nitrate agar: ( irowth colorless, the 
reverse being hyaline to lighl violet-gray. 
Aerial mycelium powdery, gray. Faint 
bluish pigmenl . 



242 



THE ACTIXOMYCETES, Vol. II 



Glucose-asparagine agar: Growth hyaline 
with pinkish reverse. Aerial mycelium pres- 
ent. No soluble pigment. 

Calcium malate agar: Growth poor. No 
aerial mycelium. 

Nutrient agar: Growth abundant, color- 
less. Aerial mycelium whitish. Soluble pig- 
ment amber. 

Starch: Strong hydrolysis. 

Potato: Growth abundant. Aerial myce- 
lium light gray. No soluble pigment. 

Gelatin: Partial liquefaction. No soluble 
pigment. 

Milk: No coagulation; some peptoniza- 
tion. 

Nitrate reduction: Negative. 

Cellulose: Good growth. 

Carbon utilization: Utilizes glucose, su- 
crose, lactose, galactose, rhamnose, xylose, 
inositol, sodium succinate, and others. Does 
not utilize sucrose, rafrinose, glycine, or 
sodium citrate. 

Antagonistic properties: Produces an anti- 
bacterial agent, matamycin. 

Habitat: Soil. 

Remarks: Related to S. noboritoensis and 
S. spiralis. 

Characteristic properties: Culture said to 
be melanin-positive; it produces gray to 
violet spore masses. 

151. Streptomyces mediocidicus Okami et 
al, 1954 (Okami, Y., Utahara, R., Naka- 
mura, S., and Pmezawa, H. J. Antibiotics 
(Japan) 7 A: 98-103, 1954). 

Morphology: Aerial mycelium sometimes 
produces verticils, depending on composition 
of medium; no spirals. 

Glycerol nitrate agar: Growth colorless to 
yellowish. Aerial mycelium absent, or white 
patches. Soluble pigment absent, or slightly 
yellowish brown. 

Glucose-asparagine agar: Same as above. 

Starch agar: Same as above. Diastatic 
action weak or medium. 

Nutrient agar: Growth colorless or slightly 



yellowish. No aerial mycelium. Soluble pig- 
ment brownish. Melanin-positive. 

Potato: Growth yellowish or light yellow- 
ish-brown. Aerial mycelium absent or white. 
No soluble pigment. 

Gelatin: Growth yellowish-brown. No 
aerial mycelium. Soluble pigment brown. 
Strong liquefaction. 

Blood agar: Growth yellowish-brown to 
reddish-brown. Aerial mycelium absent or 
white. Hemolytic action strong. 

Milk: Surface ring colorless to yellowish. 
No aerial mycelium. Soluble pigment 
slightly brownish. Slow coagulation and 
peptonization. 

Nitrate: No reduction. 

Antagonistic properties: Produces an anti- 
fungal substance, mediocidin, a polyene of 
the hexaene type. 

Type culture: IMRU 3777. 

152. Streptomyces melanocyclus (Merker, 
1911, emend. Krainsky) Waksman and 
Henrici, 1948 (Merker, E. Centr. Bakteriol. 
Parasitenk. Abt, II, 31: 589, 1912; Krainsky, 
A. ibid. 41:649-688, 1914). 

Morphology: Spores spherical, 0.9 /x. 

Agar media: Growth much folded, red. 
Aerial mycelium dark brown. Soluble pig- 
ment dark brown, turns whole culture red- 
brown to almost black with a shade of red. 

Calcium malate agar: Colonies small, flat, 
orange-red. Aerial mycelium black, occur- 
ring along the edges. 

Gelatin: Growth poor. Liquefaction rapid. 

Milk: Coagulation and rapid peptoniza- 
tion. 

Starch: Hydrolysis. 

Cellulose: Good decomposition; black cir- 
cles produced on paper. 

Nitrate reduction: Positive. 

Sucrose: Inverted. 

Pigment: Insoluble in water and in or- 
ganic solvents. Considered by Kriss to be 
related to the melanins. 

Antagonistic properties: Strong effect 



DESCRIPTION OF SPIX'II'.S OF STREPTOMYCES 



•_'i:i 



upon various bacteria; some strains show- 
no activity. 

Habitat : Soil. 

Remarks: .4. melanosporeus (Krainsky, 
1914) and A. melanogenes (Rubentschik 
1928) arc related to above species. 

153. Streptomyces melanogenes Sugawara 
and Onuma, 1957 (Sugawara, R. and 
Onuma, M. J. Antibiotics (Japan) 10A: 138- 
1 r_\ L957). 

Morphology: Sporophores monopodially 
branched; no spirals, sometimes slighl cur- 
vature. Spores cylindrical, 1.7 to 0.8 by 
li.'J in 0.5 v. 

Sucrose nitrate agar: Growth moist, fold- 
ing, colorless to grayish-red-brown; reverse 
yellow-orange. Aerial mycelium thin, brown- 
ish-white. Soluble pigment brownish-yellow. 

Glucose-asparagine agar: Growth colorless 
to cream-colored with dark reddish center; 
reverse dark yellow-orange. Aerial my- 
celium pale grayish-white. Soluble pigment 
yellowish-brown. 

Calcium malate agar: Growth colorless 
to brownish-yellow to grayish-blue-black. 
Aerial mycelium yellow-white. Soluble pig- 
ment greenish-yellow to brown. 

Nutrient agar: Growth cream-colored to 
brown. No aerial mycelium. Soluble pigment 
reddish-brown. 

Potato: Growth folded, colorless to yel- 
lowish-brown. Aerial mycelium brownish 
or grayish-white. Soluble pigment dark 
yellowish-brown. 

Gelatin: Growth colorless to dark brown. 
Aerial mycelium white to gray. Soluble pig- 
ment pale yellowish-brown. Liquefaction 
weak. 

Milk: Cream-colored to dark brown ring. 
Soluble pigment pinkish-brown. 

Blood agar: Growth glistening, yellowish- 
gray to dark olive-gray. No aerial mycelium. 
Soluble pigment dark brown. Hemolysis 
positive. 

Antagonistic properties: Produces a mela- 
nin-like tumor-inhibiting substance. 



Remarks: Resembles S. phaeochromo- 
genes, S. griseocarneus, and S. cinnamonen- 

sis. 

L54. Streptomyces michiganensis Corbaz 
et al., L957 (Corbaz, R., Ettlinger, L., 

Keller-Schierlein, \Y., and Zahner, II. Arch. 
Mikrobiol. 26: L92 208, L957). 

Morphology: Sporophores straight, ar- 
ranged in sympodially branched clusters; 
no spirals. Spores smooth (PL II l). 

Glycerol nitrate agar: Growth whitish- 
yellow. Aerial mycelium velvety, white to 
yellowish to greenish-gray. 

Calcium malate agar: Growth thin, golden 
yellow. Aerial mycelium chalk-white, be- 
coming light yellow. 

Glucose-asparagine agar: Growth thin, 
white to yellow, changing to light yellow- 
red. Aerial mycelium velvety, white-yellow. 

Glucose-peptone agar: Growth wrinkled, 
at first light brown, then copper-red, finally 
reddish-brown. Aerial mycelium velvety, 
greenish-gray. Soluble pigment reddish- 
brown. 

Gelatin: Pellicle light brown. Aerial my- 
celium powdery, chalk-white. Liquefaction 
slow. Soluble pigment brown. 

Starch: No hydrolysis. 

Potato: Growth light yellow. Aerial my- 
celium velvety, white-gray to white-yellow. 
Soluble pigment gray-black. 

Milk: Pellicle light brown. Aerial myce- 
lium sparse. Coagulation and peptonization. 

Tyrosinase reaction: Positive. 

Antagonistic properties: Produces actino- 
mycin X. 

Carbon utilization: Xylose, arabinose, 
fructose, galactose, maltose, mannitol, salicin 
utilized. Rhamnose, sucrose, lactose, ralli- 
nose, inulin not utilized. 

Habitat: Soil. 

155. Streptomyces micrqflavus (Krainsky, 
1914) Waksman and Henrici, 1948 (Krain- 
sky, A. Centr. Bakteriol. Parasitenk. Abt. 
II., U:086, 1914). 



244 



THE ACTINOMYCKTES, Vol. II 



Morphology: Spores spherical to rod- 
shaped, often produced in pairs or in chains, 
2.0 by 2 to 5 /x- 

Calcium malate agar: Colonics minute, 
yellow. No aerial mycelium. 

Glucose-asparagine agar: Aerial myce- 
lium produced late (12 days), rose-yellow. 

Nutrient agar: Colonies yellow. Aerial 
mycelium produced late, yellowish-rose. 

Potato: Growth yellowish, slimy mass. 
No aerial mycelium. Melanin-negative. 

Gelatin: Colonies small, yellowish. Lique- 
faction rapid. 

Milk: Rapid coagulation and peptoniza- 
tion. 

Invertase: Negative. 

Starch: Diastatic action strong. 

Cellulose: Growth scant, white. 

Nitrate reduction: Positive. 

Production of H 2 S: Negative. 

Antagonistic properties: Said to produce 
a form of strep tothricin. 

Habitat: Soil. 

Remarks: According to Ettlinger et al. 
(1958), this organism belongs to the S. 
griseus series. 

Type culture: IMRU 3332; A TCC 13,231. 

156. Streptomyces mirabilis Ruschmann, 
1952 (Ruschmann, G. Pharmazie 7: 542 
550, 639-648, 823 -831, 1052). 

Morphology: Sporophores straight, with- 
out spirals or curvature. 

Agar media: Aerial mycelium white, 
cottony. 

Nutrient agar: Growth poor, forming 
slimy surface. No aerial mycelium. 

Glucose agar: Growth grayish-brown. No 
aerial mycelium. Soluble pigment brown. 

Potato: Growth good, lichenoid. Soluble 
pigment dark brown to black. 

Gelatin: Good flaky growth. Rapid lique- 
faction. Soluble pigment dark brown to 
black. 

Milk: Surface growth covered with white, 
fluffy aerial mycelium. Coagulation and 



peptonization positive. Liquefied portion 
colored black. 

Fats: Ready utilization. 

Temperature: Optimum 2 ( .)°C. No growth 
at 37°C. 

Antagonistic properties: Antagonistic ef- 
fect strongest in freshly isolated cultures. 
Property lost on cultivation; activity lost 
first against gram-negative, rod-shaped bac- 
teria, cocci remaining most sensitive. Pro- 
duces antibiotic miramycin. 

Remarks: Highly proteolytic and lipoly- 
tic. Grows best on complex organic media, 
at slightly acid reaction, pH 6.0 to 6.6. 

157. Streptomyces mitakaensis Ami 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- 
cal, 1.2 to 1.5 fx. 

Sucrose nitrate agar: Growth good, color- 
less or white to dark yellowish-brown. Aerial 
mycelium powdery, abundant, light gull- 
gray. No soluble pigment. 

Glucose-asparagine agar: Growth good, 
white to brown; reverse becomes dark 
brown. Aerial mycelium powdery, abundant, 
gray. No soluble pigment. 

Glycerol citrate agar: Growth good, 
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 
pale yellowish-brown. Aerial mycelium pow- 
dery, abundant, white, with or without 
gray 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 good, wrinkled, 
cream-buff. Aerial mycelium poorly devel- 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



2 15 



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, \Y. Arch. Mikrobiol. 32: 198, 
L959). 

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 agar: Growth yellow 
to brown-yellow. Aerial mycelium white- 
gray to gray-brown. Soluble pigment golden 
yellow. 

Glucose-asparagine agar: Growth yellow, 
occasionally brown-violet. Aerial mycelium 
powdery white to gray-white. Soluble pig- 
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 pigmenl 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 cream-colored to gray. 
Soluble pigment yellow to golden yellow. 
Liquefaction limited. Melanin-negative. 

Milk: Growth abundant, golden yellow to 



yellow-brown. Aerial mycelium powdery, 

cream-colored. Questionable coagulation and 
liquefaction. 

Cellulose: Growth weak. Aerial mycelium 
gray-brown. Soluble pigmenl yellowish. 

Antagonistic properties: Produces actino- 
mycin. 

l.")«). Streptomyces naganishi Yamaguchi 
and Saburi, 1955 (Yamaguchi, '1'. and 
Saburi, Y. J. Gen. Appl. Microbiol. I: 201 
235, 1955). 

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 m- 

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 light brown, sometimes 
reddish-brown. Coagulation and peptoniza- 
tion. 



246 



THE ACTINOMYCETES, Vol. II 



Carbon utilization: Utilizes D-xylose, 
L-arabinose, L-rhamnose, D-galactose, lactose, 
ramnose, mannitol, inositol, salicin, acetate, 
citrate, and succinate; docs not utilize su- 
crose, inulin, sorbitol, or cellulose. 

Antagonistic properties: Active againsl 
gram-positive and acid-fast bacteria, fungi, 
;ind trichomonads. 

Remarks: Related to S. antimycoticus. 

1(10. Streptomyces narbonensis Corbaz et 
a/., 1955 (Corbaz, R., Ettlinger, L., Gau- 
mann, E., Keller, W., Kradolfer, F., Ky- 
burz, E., Xeipp, L., Prelog, V., Reusser, R., 
and Zahner, H. Helv. Chim. Acta 38: 935- 
942, L955); 

Morphology: Spirophores straight; no 
spirals. Spores smooth, cylindrical, 0.8 to 
1.1 by 0.7 to 0.9 M - 

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. Xo soluble 
pigment. 

Calcium malate agar: Growth colorless. 




Figure 43. Hyphae of isolate AA 877, re- 
sembling S.netropsis, showing character of verticils 
of sporogenous branches (Reproduced from: Dug 
gar, IV M. etal. Ann. X. V. Acad. Sci. 60:85, 1954). 



Aerial mycelium white-gray. Xo soluble 
pigment. 

Glucose-peptone agar: Growth light yel- 
lowish, punctiform. Aerial mycelium pro- 
duced late, powdery, gray-white. Xo soluble 
pigment. 

Nutrient agar: Growth punctiform, yel- 
lowish. Xo aerial mycelium. Xo soluble 
pigment. Melanin-negal ive. 

Starch agar: Growth thin, colorless to 
yellowish. Aerial mycelium powdery, white. 
Xo 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. Xo aerial mycelium. Soluble 
pigment dark brown. 

Milk: Surface ring whitish-yellow. Pep- 
tonization without coagulation. 

Production of PES: Positive. 

Antagonistic properties: Produces basic 
antibiotic, narbomycin, related to picromy- 
cin. 

Carbon utilization: Etilizes xylose, arabi- 
nose, rhanmose, fructose, galactose, sac- 
charose, maltose, raffinose, inulin, salicin, 
sodium acetate. Does not utilize mannitol, 
sorbitol, dulcitol, mesoinositol. 

Habitat: Soil. 

Remarks: Ettlinger et al. (1958) consider 
this organism as belonging to S. <>lirac< us. 

161. Streptomyces netropsis Finlay and 
Sobin, 1952 (Finlay, A. C. and Sobin, B. A. 
E. 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 1.3 m, smooth (PI. I a). 

Sucrose nitrate agar: Growth thin, pale 
olive-buff. Aerial mycelium pale vinaceous- 
fawn. Xo soluble pigment. 

Glucose-asparagine agar: Growth moder- 
ate, wrinkled. Aerial mycelium white. Solu- 
ble pigment brown. 

Calcium malate agar: Growth moderate, 



DESCRIPTION OF SPIX'IKS OF STREPTOMYCES 



247 



cream to huff. Aerial mycelium white. No 
soluble pigment. 

Nutrient agar: Growth moderate i<> 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. Xo aerial mycelium. Soluble pig- 
ment dark brown. 

Gelatin: Moderate surface growth. Aerial 
mycelium white. Soluble pigment dark 
brown. Xo liquefaction. 

Milk: Growth poor. Xo peptonization. 

Nitrate reduction: Negative. 

Production of H.S: Variable. 

Antagonistic properties: Produces a basic 
antibiotic, netropsin. 

Remarks: Ettlinger et al. (1958) report 
this organism to be melanin-negative. They 
also consider S. cinnamomeus as closely re- 
lated. 

102. Streptomyces niger (Rossi-Doria, 
1891; emend. Krassilnikov, 1949) Waksman 
(Rossi-Doria, E. Ann. igiene, 1: 399-438, 
L891; Krassilnikov, X. A. Antinomy cetales. 
[zvest. 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. Xo soluble pigment. 

Nutrient agar: Growth black. Soluble 
pigment brown. 

Gelatin: Slow liquefaction, in 30 days. 
Melanin-negative (?). 

Milk: Xo change. 

Starch: No growth. 

Cellulose: No growth. 

Nitrate reduction: Negative. 

Sucrose: Xo inversion. 

Temperature: Optimum 25-30°C. 



Antagonistic properties: None. 

Remarks: This is a very unstable species 
which dies out rapidly. It easily mutate.-, 
giving rise to colorless cultures, producing 
no aerial mycelium. It appears to be a 
transition form, if not a true Nocardia. .1. 
nigi r aromaticus Beresl new and .1 . nigrificans 
(Kriiger) Wollenweber are listed by Krassil- 
nikov as varieties of .1. niger. In view of the 
formation of a soluble brown substance on 
certain protein media, this organism may 
belong to one of the chromogenic groups. 

163. Streptomyces nigrifaciens Waksman, 
H)l 9 (Waksman, S. A. Soil Sci. 8: 107-168, 
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: Growth cream-colored to 
yellow. Aerial mycelium light buff-gray. 
Hydrolysis imperfect. 

Egg media: Growth abundant, dark 
brown. Xo aerial mycelium. Purplish zone 
around 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. 

( Jellulose: Xo growth. 

Habitat: Pineapple soil in Hawaii. 



248 



THE ACTIXOMYCETES, Vol. II 



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, rc-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, supplied alone or with glucose in 
the basal medium, supported growth. 

The organism grew as well on carbamate 
when first isolated from a carbamate-enrich- 
meni 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- 
ity 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. Ilirsch 
I L960) considers this organism as a Nocardia 



(N. nitrificans) capable of utilizing petro- 
leum. 

165. Streptomyces nitrosporeus Okami, 
1952 (Okami, V. 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- 
gray. 

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 H 2 S: None. 

Loeffler's serum media: Growth thin. 
Aerial mycelium gray. Soluble pigment 
limited. Rapid liquefaction of serum. 

Carbon utilization: Utilizes arabinose, 
galactose, glucose, maltose, rhamnose, xy- 
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 
<il., 1958 (Ettlinger, L., Corbaz, R., and 
IIiittei\ 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 light yellow 
or carmine-red. Aerial mycelium sparse, 
chalk-white. 

Glucose-peptone agar: Growth white-yel- 



DESCRIPTION (>!■• SN.CIKS <>F STREPTOMYCES 



24! > 



low )o carmine-red. Aerial mycelium abun- 
dant, white. Soluble pigment somewhat 
carmine-red. 

Calcium malateagar: Growth lighl yellow 
to light carmine. No aerial mycelium. 

Starch agar: Growth light yellow-red to 
carmine-red. Aerial mycelium abundant, 
white. Limited hydrolysis. 

Potato: Growth lighl brown. Aerial myce- 
lium powdery, chalk-white. Melanin-nega- 
tive. 

Gelatin: Growth carmine-red. Aerial my- 
celium sparse. Trace <>i' liquefaction. Xo 
soluble pigment. 

Milk: Pellicle light yellow. Aerial myce- 
lium sparse. Coagulation limited; no pep- 
tonization. 

Antagonistic properties: Produces anti- 
biotic cinerubin. 

Habitat: Soils in England and Germany. 

Hi". Streptomyces niveus Smith et al, 1956 
(Smith, C. G., Dietz, A., Sokolski, W. T., 
and Savage, G. M. Antibiotics A: Chemo- 
therapy 6: 135-142, L956). 

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. Xo soluble pigment. 

Nutrient starch agar: Growth yellow. 
Aerial mycelium cream-pink. Hydrolysis 
good. 

Tyrosine agar: Soluble yellow pigment. 

Milk: Ring on surface; flocculenl growth 
at bottom. Positive peptonization. 

Production of IPS: Negative. 

Carbon utilization: D-xylose, D-arabinose, 



rhamnose, D-fructose, D-galactose, D-glucose, 
D-mannose, maltose, sucrose, lactose, cello- 
biose, rail inose, dextrin, in ul in, soluble si arch, 
glycerol, dulcitol, n-mannitol, D-sorbitol, in- 
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. griseoflaviis. 

168. Streptomyces noboritoensis Isono et 
al., 1957 (Isono, K., Yamashita, S., Tomi- 
yama, V., Suzuki, S., and Sakai, H. J. Anti- 
biotics (Japan) 10A: 21-30, 1957). 

Morphology: Aerial mycelium long and 
wavy; no regular spirals. 

Sucrose nitrate agar: Growth colorless. 
Aerial mycelium slight. Soluble pigment 
absent or pale yellow. 

Glucose-asparagine agar: Growth pale 
brown to dark brown. Aerial mycelium 
pale violet-gray. Xo soluble pigment or 
slightly brownish. 

Nutrient agar: Growth fiat, pale gray, 
smooth and restricted. No aerial mycelium. 
Soluble pigment dark red-brown. 

Starch agar: Growth dry, wrinkled, pale 
grayish-brown. Aerial mycelium pale-gray, 
coi tony. 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 ramnose well utilized. 
Utilization of arabinose, inositol, salicin, 
and xylose limited. Rhamnose and sucrose 
no1 utilized. 



250 



THE ACTINOMYCETES, Vol. II 



Antagonistic properties: Produces anti- 
biotic homom y cin-hygromy cin . 

Remarks: S. noboritoensis belongs to the 
group of chromogenic actinomycetes closely 
related to S. cinnamonensis, S. fiavochromo- 
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. now 
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 /i. 

Sucrose nitrate agar: Growth white-green- 
ish. Aerial mycelium pearl-gray to dawn- 
gray. 

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. Docs 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). 



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 
(PL Ie). 

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 
gray. Aerial mycelium white, then reddish- 
gray, 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- 
granate-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 liquet' action. Melanin- 
negative. 

Milk: Coagulation, followed by peptoni- 
zation. 

Cellulose: Growth poor. 

Nitrate: Traces of nitrite produced. 

Production of H..S: Negative. 

Blood agar: Growth consists of convex, 
lobate colonies, with central perforation. 
Aerial mycelium heavy, chalky white. No 
hemolysis, but darkening of blood. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



251 



Antagonistic properties: Produces an 
antifungal agent, nystatin. 
Type culture: [MRU .".771. 

171. Streptomyces novaecaesarecu (Waks- 
nwiii and Curtis, L916) Waksman and Hen- 
rici, 1948 (Waksman, S. A. and Curtis, 1!. 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, he- 
coming bluish, glossy, much wrinkled. Aerial 
mycelium white, appears late. Soluble pur- 
ple pigment formed. 

Glucose-asparagine agar: Growth re- 
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. violaceorubt r . 

172. Streptomyces odorifer (Rullman 
emend. Lachner-Sandoval, 1898) Waksman 
(Lachner-Sandoval, A'. 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- 



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: Growth cream-colored to 
brown. Aerial mycelium abundant, cream- 
colored to straw-colored. No soluble pig- 
ment. Hydrolysis good. 

Maltose-peptone agar: Foulerton and 
Price-Jones (1902) described growth as 
"raised, drab-colored, semi-translucent, the 
sin face 1 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 H 2 S: 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, 
frequenl ly forming broom-shaped structures. 
Spores 1.0 to 1.8 by 0.5 to 1.0 n, 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. 

Antagonistic properties: None. Jolly 
(1956) obtained positive effects for his 
strain. 

Habitat: Rarely found in soil. 

Remarks: The organism resembles S. 
ruber and S. longispororuber. Some strains 
were obtained as variants of Nocardia 
rubra. Jolly (1956) reported the isolation of 
a strain of *S. oidiosporus from an Italian 
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 n\ surface 
smooth (PI. 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. Alelanin-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 H 2 S: 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 sources: According to Shinobu 
(1958), S. olivaceus rapidly utilizes xylose, 
rhamnose, fructose, galactose, sucrose, lac- 
tose, and mannitol; slow utilization: treha- 
lose, rafhnose, and inositol. 

Antagonistic properties: Various strain- 
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. olivaceus: 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 



253 



Sucrose nitrate agar: Growth while, 
spreading. Aerial mycelium ash-gray with 
brownish tinge. No soluble pigment. 

Glycerol malate agar: Growth colorless. 
Aerial mycelium light grayish-olive to dark 
gray. 

Glucose-asparagine agar: Growth abun- 
dant, natal-brown to almost black. Aerial 
mycelium white with gray tinge. Soluble 
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. 
Xo 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: [nvertase 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. 
Krassilnikov (1949) considered it as a vari- 
ety of .1 . chromogenes. 

176. Streptomyces olivoreticuli Arai et "/., 
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- 



coming cottony, white with yellowish tinge. 
Soluble pigment faint brown or absent . 

Glucose-asparagine agar: Growth thin, 
light brown to olive-drab. Aerial mycelium 
cottony, white with faint yellow to grayish- 
pink t inge. 

Nutrient agar: Growth limited, brownish. 
Aerial mycelium grayish- white. Soluble pig- 
menl light blown. 

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: King on surface brown. Coagulation 
with limited peptonization. Soluble pigment 
brown. 

Nitrate reduction: Negative. 

Si arch: Hydrolysis. 

Cellulose: No decomposition. 

Antagonistic properties: Produces anti- 
biotic viomycin. 

177. Streptomyces olivoverticillatus Shi- 
nobu, 1956 (Shinobu, R. Mem. Osaka Univ. 
B (N. S.) 5: 84-93, 1956). 

Morphology: Sucrose-ammonium agar 
mosl suitable for microscopic study. Pri- 
mary and secondary verticils produced, 
branches issuing sometimes closely, near the 
top of the sporulating hyphae, forming 
cluster-like or tuft-like branches. Spores 
spherical to elliptical, 0.6 to 0.8 n. 

Sucrose nil rate agar: Trace of growth. 

Glucose-asparagine agar: Growth thin, 
modeiate, pale olive to pale dark yellow. 
Aerial mycelium thin, partially yellowish- 
gray. 

Nutrient agar: Growth heavy, deep 
brown. Aerial mycelium olive-gray to yellow 
to green. Soluble pigment brown. 

Potato: Growth heavy, brown. Aerial 
mycelium yellow-white to yellow-gray. Solu- 
ble pigment brown. 



254 



THE ACTINOMYCETES, Vol. II 



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 Umezawa 
et al,, 1949 (Umezawa, H., Tazaki, T., 
Okami, Y., and Fukuyama, S. J. Antibiotics 
(Japan) 3:294-296, 1949). 

Morphology: Aerial mycelium shows 
scant branching. Sporophores straight, no 
spirals. Spores 1.0 to 1.2 by 2 to 3 m- 

Sucrose nitrate agar: Growth thin, trans- 
parent, cream-colored to dark. Aerial my- 
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. cacaoi. 

179. Strcptomyns 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- 



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 /x. 

Sucrose nitrate agar: Growth scant to 
moderate, pale cream color. Aerial mycelium 
trace of off-white. No pigment or pale yel- 
lowish-brown to light biown 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 Hocculent, 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. 

Remarks: S. orientalis is most closely re- 
lated to species intermediate between S. 
albus and S. fiavus, such as S. alboflavus, S. 
fllobisporus, and N. longisporofiavus. S. albo- 
gavus cannot utilize cellulose, hydrolvzes 
gelatin far less effectively than S. orientalis, 
but attacks starch readily. Milk is feebly 



DESCRIPTION OF SPFCIFS OF STREPTOMYCES 



255 



digested by S. alboflavus but rapidly hy- 
drolyzed by S. orientalis. Production of ae- 
rial mycelium by the two cultures (lifters on 
several media. 

ISO. Streptomyces paraguayensis (Al- 
meida, L940) now comb. (Almeida, F. 
Mycopathologia 2: 201-203, 1940). 

Morphology: Thin, ramified mycelial fila- 
ments; aerial mycelium consists of thicker 
and darker filaments, 1 m 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, white; 
growth dry and friable. 

Gelatin: Growth on surface. No liquefac- 
tion. 

Milk: Surface membrane, the milk colored 
pink; no peptonization. 

Habitat : Thoracic mycetoma; dark heavy 
grains. 

181. Streptomyces parvullus Waksman and 
Gregory, 1954 (Waksman, S. A. and Greg- 
ory, F. J. Antibiotics & Chemotherapy 4: 
1 o;,() L056, L954). 

Morphology: Sporophores long, mono- 
podially branched, twisting into long closed 
spirals. Spores spherical, smooth (PI. I g). 

Sucrose nitrate agar: Growth abundant 
with yellow reverse. Aerial mycelium ash- 
gray. 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: Surface pellicle covered with 



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 H 2 S: Negative. 

Antagonistic properties: Produces actino- 
mycin D. 

Habitat: Soil. 

Type culture: IMRU 8077. 

182. Streptomyces parvus (Krainsky, 1914) 
Waksman and Henrici, 1948 (Krainsky, A. 
Centr. Bakteriol. Parasitenk. Abt. II., 41: 
685-686, 1914). 

Morphology: Sporophores straight, 
branched, or wavy; no true spirals; some 
strains, however, produce spirals. Spores 
oval, 0.9 to 1.3 by 1.2 to 1.8 /x- 

Sucrose nitrate agar: Growth yellow, rose 
or red. Aerial mycelium light yellow to 
white-rose. Soluble pigment rose-colored to 
bright yellow. 

Calcium malate agar: Small yellow colo- 
nies. Light yellow aerial mycelium. 

Nutrient agar: Growth yellow. Aerial my- 
celium light yellow. Soluble pigment bright 
yellow. 

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 2 S: Negative. 

Antagonistic properties: Produces actino- 
tnycin. 

Habitat: Soil. 

Remarks: Ettlinger et al. (1958) con- 



156 



THE ACTINOMYCETES, Vol. II 



sidered S. parvus as belonging to the S. 
griseus series. According to Gause et al. 
(1957), S. parvus is a member of the scries 
Fradiae. 

Type culture: IMRU 3686. 

183. Streptomyces pelletieri ( Laveran, 1906) 
nov. comb. (Laveran, S. Compt. rend. soc. 
biol. 61: 340, 1906). 

Morphology: Growth red, smooth, con- 
sisting of small, dense, pink colonics. 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- 
onics. Xo 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 masses. Aerial mycelium 
scant, white. 

Blood agar: Colonies at first a few pin- 
head, cream-colored; no hemolysis. Later, 
colonies are 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, 
mealy, with considerable liquefaction. 

Gelatin: Few pink flakes. At first slow, 
later almost complete liquefaction. 

Milk: Soft curd; gradual peptonization. 

Starch: Xo hydrolysis. 

Production of H 2 S: Negative. 

Source: Mycetoma in Nigeria. 

Remarks: In the original description of 
tlii- culture by Laveran, the organism was 
called Micrococcus pelletieri, because no my- 
celium was seen, only coccoid bodies. A'. 
indica was regarded as identical by Pinoy. 
A', genesii Froes was described as closely 



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. 
pelletieri hydrolyzes gelatin, serum albumin, 
casein, and egg albumin; it utilizes urea but 
not (XH 4 ) 2 S04 and KN0 3 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. pelletieri. 

184. Streptomyces pentaticus Umezawa 
and Tanaka, 1958 (Umezawa, S. 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 color- 
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- 
ular margin. No aerial mycelium. Soluble 
pigment faint brown. 

Nutrient agar: Growth wet, colorless or 
brownish-white. Xo aerial mycelium. Solu- 
ble pigment brown. 

Starch agar: Growth colorless or pale 
yellow, penetrates deeply into medium. Ae- 
rial mycelium white, partially pinkish cot- 
tony colonies. No soluble pigment. Starch 
hydrolyzed. 

Gelatin: Growth consists of reddish colo- 
nies produced on surface. Xo aerial myce- 
lium. Soluble pigment deep brown. Rapid 
liquefaction. 

Potato: Growth wrinkled, wet, grayish- 
brown. Xo aerial mycelium. Soluble pig- 
ment brownish-black. 



INSCRIPTION OF Sl'KCIKS OF STREPTOMYCES 



257 



Milk: Surface 1 ring dull yellow. Coagula- 
tion and peptonization. 

Antagonistic properties: Produces an anti- 
fungal polyenic antibiotic, pentamycin. 

Habitat : Soil in Japan. 

Remarks: Resembles S. rubrireticuli, 
which differs from the strain producing 
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, 1017) Waksman and Ilenrici, 1948 
(Conn. II. J. X. V. Agr. Expt. St a. Tech. 
Bull. 00, 1017). 

This culture has been studied by Conn 
(1017), Waksman (1010), Jensen (1931), 
Krassilnikov (1949), Kutzner (1956), and 
Ettlinger et al. (1958). 

Morphology: Sporophores form narrow, 
open, elongated, sinistrorse spirals (Conn, 
Waksman, Jensen, Krassilnikov). Kutzner 
(1056) examined 25 strains belonging to this 
species; only five of them produced spirals. 
Ettlinger et al. (1058) could not find any 
spirals on any of the strains obtained from 
various culture collections. Spores spherical 
to short rods; surface smooth (PI. I d). 

Sucrose nitrate agar: Growth brown to 
almosl black. Aerial mycelium abundant, 
white with brownish shade. Soluble pigment 
brown to dark brown. 

Calcium nialate agar: Growth buff to 
brown. Aerial mycelium white. Soluble pig- 
ment brown. 

Nutrient agar: Growth gray to brown, 
later turning nearly black. Aerial mycelium 
white to gray, often absent. Soluble pigment 
deep red-brown. 

Starch agar: Growth brown. Hydrolysis 
medium. 

Potato: Growth brown to almosl black. 
Xo aerial mycelium. Soluble pigment dark 
brown to black. 

Gelatin: Surface growth abundant. 



-pleading, cream-colored, becoming brown. 
Liquefaction slow. Soluble pigment brown. 

Milk: Dark, almost black ring: coagula- 
tion with slow peptonization. 

Nitrate: Reduction limited. 

Production of IPS: Positive. 

Temperature: Optimum 25°C. 

Antagonistic properties: Strong. 

Habitat: Soil. 

Type culture: [MRU 3338. 

186. Streptomyces phaeopurpureus Shi- 
nobu, 1057 (Shinobu, R. Mem. Osaka Univ., 
B. Nat. Sci. 6:6.3-67, 1057). 

Morphology: Substrate mycelium mono- 
podial, 0.4 to 0.6 n in diameter; no frag- 
mentation. Aerial mycelium straight, usu- 
ally short. Spores spherical to elliptical, 0.6 
to 0.8 m; rarely 1 /u. 

Sucrose nitrate agar: Growth good, brown 
to dark red. No aerial mycelium. Soluble 
pigment brown. 

Glycerol malate agar: Growth good, 
orange to purple. Aerial mycelium powdery, 
yellowish-gray to pinkish-gray. Soluble pig- 
ment red-purple to brown-purple. 

Glucose-asparagine agar: Growth moder- 
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. 

I Hastase: Weak. 

( 'ellulose: Negative. 

Nitrate reduction: Negative. 

Carbon sources: Utilizes xylose, rhamnose, 



THE ACTINOMYCETES, Vol. II 



fructose, sucrose, lactose, raffinose, 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 n in diameter, no fragmentation. 
Aerial mycelium short; monopodial branch- 
ing; some spirals, sinistrorse, 1 to 3 turns. 
Spores elliptical, 0.6 to 0.8 fj,. 

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. 

188. Streptomyces pilosus Ettlinger et <il., 
L958 (Ettlinger, L., Corbaz, R., and Hiitter, 
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- 
lieb, 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 M . 

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 
growth ochraceous-buff becoming tawny. 
Aerial mycelium white becoming grayish- 
olive to almost black. Soluble pigment ;il>- 
sent or slight, brown. 

* Supplemented by personal communication. 



DESCRIPTION OF SPECIES OF STREPTOM YCKS 



259 



Calcium malate agar: ( rrowth ochraceous- 
salmon, becoming cinnamon-buff. Aerial 
mycelium quaker-drab with areas <>l black 
and while. Soluble pigmenl slight, greenish- 
yellow. 

Nutrient agar: Poor substrate growth, 
cream-yellow becoming buff to drab. Xo 
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 scant, 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 format* 1 , 
sodium tartrate, xylose poorly utilized. 
Asparagine, rhamnose, o-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. A. Actinomycetales. 
Izvest. Akad. Xauk. SSSR, Moskau, p. 17, 
1949). 



Morphology: Sporophores produce nu- 
merous spirals, with .'5 to 5 turns (sinistrorse). 
Spores oval, 0.9 by 0.7 y.. 

Synthetic agar: ( rrowth at first pigmented 
yellow-red, Inter becoming bine to blue- 
green. Aerial mycelium white-gray. The blue 
pigment dissolves into the medium. 

Nutrient agar: Soluble pigmenl greenish, 
fluorescent. 

Potato: Growth and soluble pigment 
sharp blue. 

Gelatin: Liquefaction rapid. 

Milk: Peptonization positive; no coagula- 
tion. 

Starch: Hydrolysis. 

Cellulose: No growth. 

Nutrient broth: Soluble pigment green, 
fluorescent . 

Sucrose: Inversion. 

Antagonistic properties: None. 

Habitat: Soil. 

Remarks: Closely related to S. violaee- 
oruber. A. pluricolor diffundens Berestnew is 
considered by Krassilnikov as a synonym. 

191. Streptomyces phuicolorescens Okami, 
Y. and Umezawa, H.* n. sp. 

Morphology: Aerial hyphae not flexuous, 
few branches; no spirals. 

Glycerol nitrate agar: Growth at first 
colorless and yellowish, then yellowish- 
brown with reddish tone. Aerial mycelium 
white to olive or pinkish. Soluble pigment 
slightly yellowish-brown or light wine-color 
with aging. 

Glucose-asparagine agar: Growth at first 
colorless and yellowish, then yellowish- 
brown with reddish tone. Aerial mycelium 
white to olive or pinkish. Soluble reddish- 
purple pigment occasionally produced. 

Calcium malate agar: Same as on glycerol 
nitrate agar. 

Nutrient agar: :>7°C. Colorless or slight 
yellowish-brown growth with fine wrinkles. 
Aerial mycelium white. Soluble pigmenl ab- 
sent at first, later a brown pigment appears. 

* Personal communication from Okami. 



260 



THE ACTINOMYCETES, Vol. II 



Potato plug: Growth colorless, then 
slightly yellowish or brownish. Aerial myce- 
lium white to olive-colored. Color of plug 
unchanged. Melanin-negative. 

Gelatin: 18-20°C. Growth colorless. No 
aerial mycelium. Soluble pigment slightly 
yellowish-brown. Gelatin liquefied. 

Milk: 37°C. Colorless to slight yellowish 
growth. No aerial mycelium. Coagulation 
with acid reaction, then peptonization. 

Starch: Hydrolysis weak to medium. 

Carbon utilization: Good growth with 
arabinose, dextrin, fructose, galactose, glu- 
cose, glycerol, maltose, mannitol, mannose, 
raffinose, rhamnose, salicin, sorbose, starch, 
sucrose, xylose, and sodium succinate. Scant 
growth with esculin, inositol, lactose, sor- 
bitol, sodium acetate, and citrate. 

Antagonistic properties: Produces anti- 
tumor substances pluramycin A and B 
(Maeda et al, 1956). 

Remarks: This culture is said to be re- 
lated to S. vinaceus, but it does not produce 
blue-red pigment in reverse of growth on 
nutrient agar. 

192. Streptomyces poolensis (Taubenhaus, 
1918) Waksman (Taubenhaus, J. J. J. Agr. 
Research 13: 446, 1918). 

Morphology: Sporophores straight. Spores 
oval to elliptical. 

Sucrose nitrate agar: Growth thin, color- 
less. Aerial mycelium white to gray. 

Glucose-asparagine agar: Growth abun- 
dant, glossy, light brown. 

Nutrient agar: Growth translucent, yel- 
lowish to brown. Soluble pigment brown. 

Potato: Growth thin, reddish-brown. Sol- 
uble pigment purplish. 

Gelatin: Liquefaction, with small, brown- 
ish flakes in fluid. 

Milk: Brownish ring. Coagulation and 
peptonization. 

Starch: Growth restricted, cream-colored. 
No hydrolysis. 

Nitrate reduction: Positive. 

Antagonistic properties: Positive. 



Babitat: Sweet-potato disease known as 
'•pox." 

19:;. Streptomyces praecox (Millard and 
Burr, 1926) Waksman and Henrici, 1948 
(Millard, W. A. and Purr, S. Ann. Appl. 
Biol. 13: 580, 1926). 

.Morphology: Sporophores produce short, 
open spirals. Spores spherical or oval, 0.8 
n in diameter. 

Sucrose nitrate agar: Growth thin, color- 
less. Aerial mycelium gray to olive-buff. ( )n 
continued cultivation, aerial mycelium tends 
to become white. 

Nutrient agar: Growth colorless. Aerial 
mycelium white. 

Starch media: Growth thin, cream-col- 
ored. Aerial mycelium white with greenish 
tinge. Hydrolysis positive. 

Potato: Growth lichenoid, cream-colored 
to light brown. Aerial mycelium white to 
olive-buff. Soluble pigment olive-buff to 
drab. On continued cultivation, no soluble 
pigment produced. 

Gelatin: Growth good. Aerial mycelium 
white. Liquefaction medium. Melanin-nega- 
tive. 

Milk: Surface growth cream-colored, in 
form of ring. Aerial mycelium white. Coagu- 
lation slow; peptonization rapid. 

Nitrate: Reduction variable. 

Cellulose: Good growth, colorless. Aerial 
mycelium dark gray. 

Tyrosinase reaction: Negative. 

Production of H 2 S: Negative. 

Temperature: Grows well at 37.5°C. 

( >dor: Very strong. 

Antagonistic properties: Represses growth 

of S. SCIlllil.S. 

Habitat: Knoblike scab of potatoes. 

Remarks: According to Ettlinger et al. 
(1958), this organism belongs to the S. 
griseus series. Hoffmann (1958) described a 
culture of S. praecox that produced a light 
to dark gray aerial mycelium and many 
spirals; nonchromogenic. 

Type culture: IMRU 3374. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



261 



194. Streptomyces praefecundus (Millard 
and Burr, 1926) Waksman (Millard, \Y. A. 
and Burr, S. Ann. Appl. Biol. L3: 580, L926). 

Morphology: Sporophores straight, fre- 
quently forming brushes. Spores spherical 
to oval, 0.8 by 0.85 p. 

Sucrose aitrate agar: ( Jrowth good, cream- 
colored. Aerial mycelium cottony, olive- 
buff. Soluble pigment cream-colored. 

Nutrient potato agar: Growth lichenoid, 
gray. Aerial mycelium smooth, white to 
yellowish. Soluble pigment golden brown. 

Potato: Growth good, wrinkled. Aerial 
mycelium white to yellowish to olive-buff. 
Soluble pigment gray to brown. 

Gelatin: Surface growth good. Aerial my- 
celium white. Soluble pigment light pink to 
dark golden brown. Liquefaction rapid. 

Milk: Surface growth good. Aerial myce- 
lium scant, white. Coagulation and peptoni- 
zation. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Temperature: Grows well at 37.5°C. 

Habitat : Potato .-cab and soil. 

195. Streptomyces pra&inophilus Ettlinger 
et a/., L958 (Ettlinger, L., Corbaz, R., and 
Hutter, R. Arch. Mikrobiol. 31: 345, L958). 

Morphology: Sporophores monopodia I ly 
branched, long, straight, with open spirals, 
usually I to 3 coils. Spores covered with 
long, fine hair (PI. II, K). 

Glycerol nitrate agar: Growth red or red- 
brown. Aerial mycelium leek-green with 
white spots. Soluble pigment pink. 

Glucose-asparagine agar: Growth brick- 
red. Aerial mycelium white to leek-green. 
Soluble pigment brick-red. 

Glycerol malate agar: Growth brick-red. 
Aerial mycelium leek-green. Soluble pig- 
ment pink. 

( rlucose-peptone agar: Growth brick-red. 

Starch-KN( b agar: Growth pink. Aerial 
mycelium white to leek-green. Soluble pig- 
ment light pink. Good hydrolysis. 

Gelatin: Bottom hakes red to yellowish, 



later brick-red. Slow liquefaction. No soluble 
pigment . .Melanin-negat ive. 

Potato: Growth slow, flesh-red. No solu- 
ble pigment. 

Milk: Strong coagulation and peptoniza- 
tion. 

Antagonistic properties: Weak activity 
against gram-positive bacteria. 

Habitat: Soil in Mallorca, Spain. 

196. Streptomyces prasinus Ettlinger et "/., 
1958 (Ettlinger, L., Corbaz, P., and Hutter, 
R. Arch. Mikrobiol. 31:343, 1958). 

Morphology: Sporophores monopodia I ly 
blanched, long, straight, with open spirals, 
forming 1 to 2 coils. Spores covered with 
short spines (PI. II, K). 

Glycerol nitrate agar: Growth colorless. 
Aerial mycelium grass-green, later dark 
green. 

Glucose-asparagine agar: Growth whitish- 
yellow. 

Glycerol malate agar: Growth copper-red. 
Aerial mycelium velvety, leek-green. 

Gelatin: Growth limited, whitish-yellow. 
No soluble pigment. Liquefaction positive. 

Starch agar: Growth reddish-brown. 
Aerial mycelium leek-green. Strong hy- 
drolysis. 

Potato: Growth limited, light brown. 
Aerial mycelium leek-green. Melanin-nega- 
tive. 

Milk: Heavy pellicle. Aerial mycelium 
whitish-gray to greenish-gray. No coagula- 
tion ; slow peptonization. 

Antagonistic properties: None. 

Habitat: Soils in Mallorca and Belgian 
Congo. 

I!»7. Streptomyces pseudogriseolus Okami 
et al., 1955 (Okami, Y., [Jtahara, P.. Oyagi, 
II., Nakamura, S., and Umezawa, II. .1. 
Antibiotics (.Japan) 8A: 126-131, L955). 

Morphology: Sporophores produce nu- 
merous closed spirals. Spores oval to cylin- 
drical, 0.8 to 1.2 by 1.0 t«, 1.5 fj.. 

Glycerol nitrate agar: Growth colorless to 



262 



THE ACTIXOMYCETES, Vol. II 



grayish-buff. Aerial mycelium grayish-huff, 
powdery. No soluble pigment. 

Nutrient agar: Growth colorless. Aerial 
mycelium white, thin. Soluble pigment ab- 
sent or slight, brown. 

Potato: Growth colorless to slightly yel- 
lowish, elevated, wrinkled. Aerial mycelium 
white, cottony to velvety. No soluble pig- 
ment. 

Milk: Surface growth orange. Aerial my- 
celium velvety, white. Coagulation and pep- 
tonization completed in 25 to 30 days. 

Gelatin: Growth yellowish-brown. Aerial 
mycelium white to grayish. Soluble pigment 
brownish. Liquefaction weak to medium. 
Melanin-negative. 

Starch: Hydrolysis strong. 

Carbon utilization: Utilizes arabinose, 
dextrin, iso-duleitol, fructose, galactose, 
glucose, glycerol, inositol, lactose, maltose, 
mannitol, mannose, rhamnose, salicin, 
starch, sucrose, sodium acetate, sodium 
citrate, and sodium succinate. Does not 
utilize esculin, raffinose, sorbitol, or sorbose 
(inulin). 

Antagonistic properties: Produces xantho- 
mycin-like substance. 

Habitat: Isolated from soil in Japan. 

Remarks: This culture resembles S. gri- 
seolus, but it differs in spiral formation and 
growth on potato and milk media. These 
differences may not be enough to warrant 
establishing a new species, but the produc- 
tion of xanthomycin is a property not found 
in the culture liquid of S. griseolus. 

Type culture: ATCC 12,770. 

198. Streptomyccs purpureofuscus Yama- 
guchi and Saburi, 1955 (Yamaguchi, T. and 
Saburi, Y. J. Gen. Appl. .Microbiol. I: 201- 
2:;5, 1955). 

Morphology: Aerial hyphae long and 
straight; on synthetic and starch agars, they 
show a tuft-forming tendency at the margin. 
Spirals not produced. Spores cylindrical, 
I.I to 2.2 by 0.7 to 1.1 n. 

Sucrose nit rate agar: Growth thin, color- 



less, later becoming purple to dark purple; 
this property may be lost after repeated 
transfer, in which case growth remains white 
to light purple. Aerial mycelium powdery, 
white, later smoke-gray. Soluble pigment 
faint purple. 

Glycerol malate agar: Growth brownish 
to purplish-brown. Aerial mycelium white, 
smoke-gray to light grayish-olive. Soluble 
pigment light brownish-purple. 

Nutrient agar: Growth wrinkled, colorless. 
Aerial mycelium absent, or scant, white. 
Soluble pigment changes from reddish-brown 
to dark vinaceous-brown. 

Starch agar: Growth yellowish-brown to 
dark olive-buff; sometimes with hygroscopic, 
black patches. Aerial mycelium velvety, at 
first white, later olive-gray. Usually no sol- 
uble pigment is produced, but sometimes 
faint pinkish-purple is seen. Strong hy- 
drolysis. 

Potato: Growth vigorous, finely wrinkled, 
at first purplish-brown or yellowish-brown, 
later becoming black. Aerial mycelium abun- 
dant, powdery, grayish. Soluble pigment 
purplish. 

Gelatin: Growth dark brownish-gray. 
Aerial mycelium coarse, powdery, grayish- 
white. Soluble pigments yellowish-brown to 
brown and a more diffusible yellowish-green. 
Strong liquefaction. 

Milk: Growth at first dull reddish-brown, 
later purplish-brown. Soluble pigment faint 
purplish and more diffusible faint yellowish. 
Coagulation and peptonization. 

Cellulose: No growth. 

Carbon utilization: n-xylose, L-arabinose, 
D-galactose, sucrose, lactose, raffinose, so- 
dium succinate readily utilized. L-rhamnose, 
inulin, mannitol, sorbitol, inositol, acetate, 
and citrate not utilized. 

Antagonistic properties: Active against 
gram-positive and acid-fast bacteria; pos- 
sesses antitrichomonal activity. 

Remarks: Related to S. vinaceus, S. 
cylindrosporus, and S. purpureochromogenes. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



263 



1!)!). Streptomyces purpurascens Linden- 
bein, L952 (Lindenbein, \V. Arch. Mikrobiol. 
L7:361 383, L952). 

Morphology: Sporophores long, straight, 
with open and closed spirals, 2 to 5 turns as 
side branches. Spores covered with long 
spines (PI. I b). A detailed electron micro- 
scope study of this organism has been made 
by Petras I 1959). 

Glycerol nitrate agar: Growth carmine- 
red to purple. Aerial mycelium cottony, 
white to purplish. Soluble pigment brown- 
red. 

Glucose-asparagine agar: Growth car- 
mine-red to purple. Aerial mycelium white 
to pinkish. Soluble pigment orange to car- 
mine-red. 

Glycerol malate agar: Growth carmine- 
red. Aerial mycelium white. Soluble pigment 
orange to brick-red. 

Nutrient agar: Growth light brown, with 
dark brown reverse. Aerial mycelium white. 
Soluble pigment dark brown. Melanin- 
positive. 

Glucose-peptone agar: Growth lichenoid, 
red to red-brown. Aerial mycelium white. 
Soluble pigment light brown. 

Starch media: Growth light carmine to 
yellow-red. Aerial mycelium while. No sol- 
uble pigment. Hydrolysis strong. 

Potato: Growth brownish to reddish. 
Aerial mycelium white to gray. Xo soluble 
pigment. (Kutzner (1956) observed on six 
strains a gray to black pigment on potato 
plug.) 

Gelatin: Growth light brown. Aerial my- 
celium white. Soluble pigment red-brown. 
Liquefaction medium. 

Milk: Growth red to dark brown. Aerial 
mycelium white. No proteolysis. 

Cellulose: Growth white to red. 

Production of IPS: Positive. 

Antagonistic properties: Produces rhodo- 
mycin. 

Remarks: On continued growth on syn- 
thetic media, the culture may lose the 



property to produce the typical pigment. It 
can be regained, however, by growth on 
organic media. This organism is considered 
by Corbaz et «l. (1957) as a synonym of S. 
bobiliae, excepl that the latter lost the prop- 
erty of producing aerial mycelium or spores. 
Lindenbein (1952) and Frommer (1959) ob- 
tained colorless mutants from S. purpuras- 

C( lis. 

Type culture: IMRU :;<i(iO. 

200. Streptomyces purpureochromogt nes 
(Waksman and Curtis, 1916) Waksman and 
Henrici, P. MS (Waksman, S. A. and Curtis, 
R. E. Soil Sci. 1: 113, 1916; 8: 132, 1919). 

Morphology: Long sporophores produce 
few imperfect spirals. Spores spherical, 0.75 
to 1.0 m in diameter. 

Sucrose nitrate agar: Growth restricted, 
smooth, gray, becoming brown with purplish 
tinge; center raised, margin yellow. Aerial 
mycelium dark brown to dark gray. 

Glucose-asparagine agar: Growth abun- 
dant, gray, becoming brown to dark brown. 

Nutrient agar: Growth gray to brownish, 
becoming dark brown, almost black. Soluble 
pigment dark brown. Melanin-positive. 

Potato: Growth orange to orange-red. 

Gelatin: Surface growth slow, brownish. 
Liquefaction slow. 

Milk: Dark brown ring. Coagulation and 
slow peptonization. 

Starch media: Colonies small, dark brown. 
Slight hydrolysis. 

( 'ellulose: Moderate growth. 

Sucrose: Inversion. 

Nitrate reduction: Negative. 

Production of IPS: Negative. 

Temperature: Optimum 25°C. 

Antagonistic properties: Active against 
various bacteria. 

Habitat: Soil. 

Type culture: [MRU 3343. 

201. Streptomyces putrificus (Nikolaieva, 

nil.".) Waksman (Nikolaieva, E. Arch. Biol. 
Nauk. 18:220, 1914). 



2(14 



THE ACTINOMYCETES, Vol. II 



A I nephology : Sporophores spiral-shaped. 
Spores spherical. 

Nutrient agar: Growth colorless to gray- 
ish. Aerial mycelium white. No soluble pig- 
ment. 

Potato: Growth folded, sulfur-yellow. 
Aerial mycelium chalk- white. No soluble 
pigment . Melanin-negative. 

Milk: Pellicle heavy. Aerial mycelium 
white. Peptonization gradual without pre- 
vious coagulation. 

Loefrler's serum: Growth yellow. No aerial 
mycelium. Serum liquefied and colored yel- 
lowish-brown. 

Odor: Strong, putrefactive. 

Habitat: Spring water. 

Remarks: Decomposes proteins ener- 
getically, with the formation of bad-smelling 
products (H 2 S, NH 3 ). Morphological prop- 
erties given by Krassilnikov (1949), who 
considers this organism as a member of the 
.4. albus group. 

202. Streptomyces pyridomyceticus Okami 
et a/., 1957 (Okami, Y., Maeda, K., and 
Umezawa, H. J. Antibiotics (Japan) 7A: 
55-50, 1954; 10A: 172, 1957). 

Morphology: Sporophores form flexible, 
open spirals. Spores of irregular size. 

Glycerol nitrate agar: Growth colorless 
to dark. Aerial mycelium thin, white, some- 
times gray to brownish-gray. No soluble 
pigment. 

Nutrient agar: Growth colorless. Aerial 
mycelium absent, or scant, white. No soluble 
pigment. 

Potato: Growth wrinkled, dark yellowish- 
brown. Aerial mycelium absent, or later 
while. No soluble pigment. 

Gelatin: Growth colorless. Aerial myce- 
lium white, sometimes grayish. No soluble 
pigment. No liquefaction. 

Starch: Hydrolysis. 

Milk: Growth yellowish, in the form of 
surface ring. No aerial mycelium. Coagula- 
tion and peptonization absent or very slow. 

Blood: No hemolysis. 



Nitrate reduction: Negative. 

Carbon utilization: Utilizes arabinose, 
dextrin, fructose, galactose, glucose, glycerol, 
maltose, xylose, and sucrose. Does not utilize 
dulcitol, esculin, inulin, lactose, mannose, 
raffinose, rhamnose, salicin, sorbitol, sodium 
citrate, sodium acetate, and sodium suc- 
cinate. 

Habitat: Soil in Japan. 

Antagonistic properties: Produces anti- 
biotic pyridomycin. 

Remarks: Isolated by means of chlortetra- 
cycline-containing agar. Related to S. cacaoi 
and S. flocculus, as well as to S. hygroscopicus. 
Above description was first given under the 
name S. olbidofuscus. It was later found that 
this name was preempted by Neukirch and 
Berestnew, and was, therefore, changed to 
S . pyridom ycet ic us. 

203. Streptomyces rameus Okami et al., 
1959) (Okami, Y., Suzuki, M., Takita, T., 
Ohi, K., and Umezawa, H. J. Antibiotics 
(Japan) 12A: 257-202, 1959). 

Morphology: Aerial mycelium forms in- 
complete spirals or loops or hooks. Spores 
oval to oblong. 

Glycerol nitrate agar: Growth yellow. 
Aerial mycelium white. Soluble pigment 
absent or yellowish. 

Glucose-asparagine agar: Growth yellow- 
ish. Aerial mycelium white. Soluble pig- 
ment absent or yellowish. 

Calcium malate agar: Growth colorless to 
yellowish. Aerial mycelium off-white. No 
soluble pigment. 

Nutrient agar: Growth colorless to brown- 
ish. Aerial mycelium white. Soluble pigment 
absent or slight brownish. 

Starch agar: Growth colorless to yellowish. 
Aerial mycelium white. Soluble pigment 
yellowish. Hydrolysis weak to medium. 

Potato: Growth brownish. Aerial myce- 
lium white. Soluble pigment brown to black. 

Gelatin: Growth brownish. Aerial myce- 
lium white. Soluble pigment brown. Liquefac- 
tion doubtful. 



DESCRIPTION OF SPECIES OF STREPTOMYCBS 



265 



Milk: Coagulation weak; peptonization 
doubtful. 

Nitrate reduction: Negative. 

Carbon sources: Utilizes arabinose, dex- 
trin, fructose, galactose, glucose, glycerol, 
inositol, maltose, niannitol, mannose, raffi- 
nose, starch, and sucrose. Lactose and xylose 
gave less response. Poor growth on inulin, 
rhamnose, salicin, sorbitol, sorbose, sodium 
acetate, and sodium citrate. 

Antagonistic properties: Produces strepto- 
mycin. 

Remarks: belated to S. alboflavus, S. 
xanthophaens, and S. orientalis. 

204. Streptomyces ramnaii Bhuiyan and 
Ahmad, L956 (Bhuiyan, A. M. and Ahmad, 
K. Ann. Biochem. Exptl. Med. India 16: 
101-104, 1956). 

Morphology: Open spirals, with 2 or ."> 
turns. Spores spherical, 0.S /i in diameter. 

Sucrose nitrate agar: Growth whitish. 
Aerial mycelium white to pale rose. No 
soluble pigment. 

Glucose-asparagine agar: Growth color- 
less to pale rose. Aerial mycelium white, 
later pale rose. 

Calcium malate agar: Growth smooth, 
cream-colored. Aerial mycelium white. Me- 
dium becomes clear. 

Nutrient agar: Growth cream-colored, be- 
coming light brown. Aerial mycelium pow- 
dery, white. Soluble pigment slight blown 
coloration. Melanin-negative. 

Starch agar: Growth cream-colored to 
yellowish brown. No aerial mycelium. Xo 
soluble pigment. Hydrolysis rapid. 

Potato: Growth abundant, cream-colored. 
Aerial mycelium white, turning pale rose. 

Potato nutrient agar: Growth rapid, color- 
less to cream-colored. Aerial mycelium deep 
rose. Soluble pigment deep reddish-brown 
to almost red. 

Gelatin: Growth cream-colored. Xo aerial 
mycelium. Soluble pigment absent or light 
brown. Liquefaction medium. 

.Milk: Growth cream-colored. Xo aerial 



mycelium. Coagulation, followed by pep- 
tonization. Read ion acid. 

Nitrate reduction: Positive. 

Cellulose: ( irowth good. 

< )ptimum temperature: 37°C. 

Antagonistic properties: Produces anti- 
biotic ramnacin. 

205. Streptomyces ramulosus Ettlinger <t 
til., 1958 (Ettlinger, L., Gaumann, P., Flut- 
ter, P., Keller-Schierlein, W., Kradolfer, 
P., Xeipp, P., Prelog, V., and Zahner, H. 
Ilelv. Chun. Acta 41: 216-219, 1958). 

Morphology: Sporophores monopodia I ly 
branched, straight with many side branches. 
Spores smooth (PI. II h ). 

Glycerol nitrate agar: Growl h al first 
carmine-red, later turning greenish-brown. 
Aerial mycelium ash-gray with greenish 
tinge. Substrate pigmented carmine-red. 

Glucose-asparagine agar: Growth yellow- 
ish-red. Aerial mycelium gray. Substrate 
carmine-red. 

Calcium malate agar: ( irowth light yellow. 
Aerial mycelium chalky white to gray with 
greenish tinge. 

Glucose-peptone agar: Growth yellowish- 
red, partly greenish to greenish-black. Aerial 
mycelium powdery, ash-gray. Substrate 
greenish to brownish-black. 

Starch agar: Growth light yellow. Sub- 
strate light carmine. Gradual hydrolysis. 

Potato: Growth yellowish-red. Aerial my- 
celium chalk-white to ash-gray. Substrate 
carmine-red. 

Gelatin: Growth light yellow. Soluble pig- 
ment light brown. Xo liquefaction. 

Milk: Light yellow pellicle. Xo aerial 
mycelium. Coagulation limited; peptoniza- 
tion good. 

Carhon utilization: Glucose, L-xylose, d- 
fructose, sucrose, inulin, D-sorbitol well 
utilized. Does not utilize raflinose, L-arabi- 
nose, D-mannitol, mesoinositol. Question- 
able utilization of L-rhamnose, salicin. 

Antagonistic properties: Produces anti- 
biotic acetomycin, active against gram-posi- 



2GG 



THE ACTINOMYCETES, Vol. II 



tive and gram-negative bacteria, as well as 
against triehomonads and amoebae. 

206. Streptomyces resistomycificus Linden- 
bein, 1952 (Lindenbien, W. Arch. Mikro- 
biol. 17: 361-383, 19.52). 

Morphology: Sporophores long, with 
curling tips. Spores short, oval. 

Glycerol nitrate agar: Growth yellow- 
brown to dark brown. Aerial mycelium ash- 
gray. Soluble pigment red-brown. 

Glucose-asparagine agar: Growth yellow- 
brown. Aerial mycelium ash-gray. Soluble 
pigment yellow-brown. 

Glycerol malate agar: Growth dark brown. 
Aerial mycelium ash-gray to red-gray. 
Soluble pigment gray to dark brown. 

Nutrient agar: Growth dark brown. 
Aerial mycelium absent or lead-gray. Soluble 
pigment dark brown. Melanin-positive. 

Glucose-peptone agar: Growth dark 
brown. Aerial mycelium white. Soluble pig- 
ment reddish to dark brown. 

Starch agar: Growth light yellow to red- 
dish-brown. Aerial mycelium gray-white, 
later red-gray. Soluble pigment lacking or 
reddish-brown. Hydrolysis strong. 

Potato: Growth brownish-black. Aerial 
mycelium reddish-white. Soluble pigment 
dark brown. 

Gelatin: Growth dark brown. Aerial my- 
celium white-gray. Soluble pigment chest- 
nut-brown. Good liquefaction. 

Milk: Growth dark brown. Aerial myce- 
lium white, later yellowish-red. Soluble pig- 
ment dark brown. Peptonization none or 
slight. 

Cellulose: No growth. 

Antagonistic properties: Produces resisto- 
niycin, which is active against gram-positive 
bacteria. 

Remarks: Gause et al. (1 ( .)57) have de- 
scribed certain closely related forms, such as 
.1. griseorubiginosus with a variety .spiralis, 
.•iihI .1. variabilis with a variety roseolas. 

Type culture: IMRU 3058. 



207. Streptomyces reticiili (Waksman and 
Curtis, 19 Hi; Waksman, 1919) Waksman and 
Henrici, 1 ( .)48 (Waksman, S. A. and Curtis, 
R. E. Soil Sci. 1: 118, 1916; Waksman, S. A. 
Soil Sci. 8: 143, 1919). 

Morphology: Aerial mycelium gives rise 
to simple verticils. Sporophores straight or 
spiral-shaped (sinistrorse) on different media. 
Spores spherical or oval, smooth, 1.0 to 1.4 
H in diameter (PI. I a). 

Sucrose nitrate agar: Growth colorless, 
with yellowish tinge, becoming brownish. 
Aerial mycelium thin, cottony, white to ash- 
gray. No soluble pigment. 

Glycerol malate agar: Growth colorless. 
Aerial mycelium yellowish. No soluble pig- 
ment. 

Nutrient agar: Growth wrinkled, gray, 
becoming brownish. No aerial mycelium. 
Soluble pigment dark brown. 

Potato: Growth gray, with black center. 
Aerial mycelium ash-gray. Soluble pigment 
black. 

Gelatin: Growth gray to brown. Aerial 
mycelium white. Soluble pigment faint 
brown to dark brown. Good liquefaction. 

Milk: Coagulation rapid; peptonization 
slow. 

Starch: Growth brownish-gray. Hydrol- 
ysis. 

Cellulose: Scant growth. 

Nitrate reduction: Positive. 

Production of H 2 S: Positive. 

Invertase: Positive. 

Temperature: Optimum 25°C. 

Antagonistic properties: Some strains 
product 1 neomycin or a neomycin-like sub- 
stance. Some strains reduce double bonds in 
certain steroids. 

Habitat: Soil. 

Remarks: According to Ettlinger et al. 
(1958), the verticils are both primary and 
secondary; no spirals were observed. They 
also report the species to be melanin-nega- 
tive. One wonders whether they had a typi- 
cal culture. This culture was later found to 



DKSCHII'TIOX OF SPKCIICS ol' STItKI'TOUYCES 



207 



be identical with S. abikoensum. Sakagami 
et al. (1958) described a variety latumcidicus 
thai produced no aerial mycelium on mosl 

media, and formed the antibiotic latumcidin. 
Type culture: IMRU 3344. 

208. Streptomyces rimosus Sobin et al., 
1950 (Sobin, B. A., Finlay, A. ('., and Kane, 
J. H. U. S. 2,510,080, July IS, 1050; see also 
Koehi, M., et al. Proc. Natl. Acad. Sci. U. S. 
38:883-891, 1952). 

Morphology: Sporophores long, usually 
straight, occasionally open or closed spirals 
depending on composition of medium. Spores 
cylindrical, 0.6 to 0.7 by 0.8 to 1.4 M - A 
microscopic study of S. rimosus (strain 3558 i 
grown on yeast extract-glucose agar, after 
14 days incubation, revealed the following: 
Aerial hyphae were long and fairly straight, 
segmenting into chains of even, bead-like 
-poics. Other aerial hyphae were long, 
tangled, branching, twisting into spirals, 
also segmented into chains of bead-like 
spores. 

Sucrose nitrate agar: Growth thin, cream- 
colored, developing slowly at first, later be- 
coming abundant, much folded or lichenoid; 
reddish-brown to orange. Aerial mycelium 
appears first over the drier edge of the 
growth or in the form of thin white patches. 
When the culture becomes older, a faint 
bluish zone appears around the edge of the 
growth. Soluble pigment faint yellowish. 

Glucose-asparagine agar: Growth at first 
cream-colored, becoming brownish to orange- 
brown with age. Aerial mycelium white. 
Soluble pigment yellowish to golden. 

Yeast-glucose agar: Growth much more 
rapid than in synthetic media; lichenoid, 
cream to brownish. Aerial mycelium appears 
at an early stage of growth, white, later 
tending to become mouse-gray. Soluble pig- 
ment yellowish. 

Nutrient agar: Growth poor, cream- 
colored to yellowish-brown to mouse-gray. 
Aerial mycelium white or absent. Soluble 
pigment absent or yellowish. 



Starch agar: Growth limited, cream- 
colored, with deeper brown center. No aerial 
mycelium. Limited hydrolysis. 

Potato: Growth lichenoid, cream-colored 
t<> reddish-brown. Aerial mycelium white to 
gray to dark brown. Soluble pigmenl yel- 
lowish-brown. 

Gelatin: Growth cream-colored to brown- 
ish. Aerial mycelium white. Slow liquefac- 
tion. No soluble pigment, only a faint yel- 
lowish coloration of liquefied portion. 

Milk: Heavy surface pellicle, cream- 
colored to yellowish. Aerial mycelium gray- 
ish-white. Peptonization, without coagula- 
tion. 

Cellulose: Xo growth. 

Nitrate reduction: Positive. 

Production of H 2 S: Negative. 

Antagonistic properties: Produces an anti- 
bacterial antibiotic, oxy tetracycline, and an 
antifungal agent, rimocidin. 

Habitat : Soil. 

Remarks: A variety of S. rimosus (forma 
paromomycinus) was briefly described (Brit. 
Pat. 797,568, July 2, 1958). This variety was 
isolated from a soil in South America. It dif- 
fers from S. rimosus in certain minor cultural 
properties (somewhat lighter color on agar 
media) and in poorer utilization of arabinose. 
Both form dense clusters of spirals on various 
synthetic media and on glucose-tryptone 
agar. The variety produces an antibiotic, 
paromomycin, apparently closely related to 
the neomycin group. 

Type culture: IMRU 3558; ATCC 1(),<>7() 

209. Streptomyces rochei Berger et al., 
L949 (Berger, J., Jampolsky, L. Al., and 
Goldberg, M. W. Arch. Biochem. 22: 476- 
478, 1949; Waksman, S. A. and Lechevalier, 
H. Guide to the classification and identifica- 
tion of the actinomycetes and their anti- 
biotics. The Williams and Wilkins Co., 
Baltimore, 1953, p. 40). 

Morphology: Sporophores straight, 1.5 ^ 
in diameter; often, but no1 always spirally 
twisted; spirals usually shorl and loose with 



208 



THE ACTINOMYCETES, Vol. II 



rarely more than 2 to 3 coils. Spores oval to 
elliptical, sometimes spherical, 1.2 to 2.8 by 
0.8 to 1.5 /i. 

Sucrose nitrate agar: Growth thin, color- 
less, covered with sandy lavender to dark 
gray aerial mycelium. Reverse of growth 
light gray, later becoming grayish-yellow. 
No soluble pigment. 

Nutrient agar: Growth cream-colored. 
Aerial mycelium white. No soluble pigment. 

Calcium malate glycerol: Growth good, 
raised in center. Aerial mycelium gray, buff 
around the edges, having a fuzzy appearance. 

Glucose agar: Growth smooth, yellowish, 
covered with white to gray aerial mycelium. 
Yellowish soluble pigment. 

Potato: Growth abundant, lichenoid, 
cream-colored. Aerial mycelium abundant, 
cottony, white to gray. Color of plug be- 
comes reddish-tan. 

Gelatin: Cream-colored ring, covered with 
white aerial mycelium. Rapid liquefaction. 
Faint yellow soluble pigment. 

Milk: Cream-colored to brownish ring. 
Coagulation and rapid peptonization. 

Starch: Growth brownish. Aerial myce- 
lium mouse-gray. Diastatic action strong. 

Production of H 2 S: Negative. 

Antagonistic properties: On certain com- 
plex nitrogenous media, the organism shows 
a wide range of antimicrobial activity, 
partly because of borrelidin. 

Remarks: Morphologically, the culture 
resembles S. albidoflavus, S. calif ornicus, S. 
lipmanii, and certain others, but it is not 
believed to be identical to any of them. 
Ettlinger et al. (1958) considered this or- 
ganism as a strain of S. fradiae. Okami and 
Suzuki (1958) could not demonstrate any 
spirals on several media tested. 

Type culture: IMRU 3602; ATCC 10,739. 

210. Streptomyces roseochromogenes (Jen- 
sen, 1931) Waksman and Henrici, 1948 
(Jensen, H. L. Proc. Linnean Soc. X. S. 
Wales 56: 359, 1931). 

Waksman and Curtis (1916) and Waks- 



man (1919) described an organism as .1. 
roseus Krainsky. This culture was, in con- 
trast to Krainsky's organism, chromogenic. 
Jensen (1931) compared it with his own 
isolates and changed the name .4. roseus to 
roseochromogenes, because of the fact that 
the name roseus had previously been used by 
Namyslowsky (1912). 

Morphology: Sporophores form numerous 
open and closed sinistrorse spirals; some- 
times 3 to 5 branches issue together from 
end point of main stem, giving impression 
of brooms or verticils. Spores spherical, 1.0 
to 1.2 by 1.3 to 3.0 M (PL V, 2b). 

Sucrose nitrate agar: Growth thin, spread- 
ing, colorless to pale yellow. Aerial mycelium 
pale grayish-rose. 

Glucose-asparagine agar: Growth pale 
yellow. Aerial mycelium white, later be- 
coming rose-cinnamon, with many small 
white tufts. 

Nutrient agar: Growth wrinkled, yellow- 
ish-gray, later brown-red. Aerial mycelium 
white, then rose-gray. Soluble pigment deep 
brown. 

Potato: Growth wrinkled, yellowish-gray 
to grayish-black. Aerial mycelium absent or 
white. Soluble pigment black. Melanin- 
positive. 

Gelatin: Colonies small, cream-colored, in 
bottom of liquefied zone. Soluble pigment 
blown. Liquefaction medium. 

Milk: Coagulation limited; peptonization 

slow. 

Starch media: Growth colorless, spreading. 
Hydrolysis good. 

Nitrate reduction: Positive. 

Production of IPS: Positive. 

Antagonistic properties: Active against 
various bacteria; produces antibiotic roseo- 
mycin. 

Habitat: Soil. 

Remarks: Jensen (1931) obtained, on 
plating the tufts of white aerial mycelium 
arising on agar media, a variant with pure 
white aerial mycelium. 

Type culture: ATCC 13,400. 



DESCRIPTION OF SIMX.'IKS OF STREPTOMYCES 



211. Streptomyces roseocitreus Kato, I'.'");! 
(Kato, II. J. Antibiotics (Japan) 6A: L43; 
6B: 206-208, L953). 

Morphology: Sporophores produce nu- 
merous open and closed spirals of the dex- 
trorse type. Spores oval, 1.2 to l.."> by L.6 to 
LS M . 

Sucrose nitrate agar: Growth pale olive- 
buff, later changing to deep olive-buff, ivory- 
yellow, <>r colonial huff. Aerial mycelium 
scant, white. Soluble pigmenl at first faint 
creamy, later changing to colonial huff. 

Glycerol-calcium malate agar: Growth at 
first transparent with gray to blackish-blue 
patches, later becoming light yellowish- 
olive to reed-yellow. Aerial mycelium thin, 
white, at lirst having tinge of gray. Soluble 
pigment yellowish with tinge of green. 

Nutrient agar: Growth olive-buff, later 
changing to deep olive-buff with bluish 
patches. No aerial mycelium. Soluble pig- 
ment brown. 

Starch agar: Growth hyaline, cottony, 
reverse becoming faint bluish. Aerial myce- 
lium white, later becoming livid pink, and 
finally pale grayish-vinaceous. Enzymatic 
zone fair to good. 

Potato: Growth thick, folded, pale olive- 
buff, later deep olive to dark olive. Aerial 
mycelium at first white, later becoming livid 
pink to vinaceous-buff. Color of plug black- 
ish-brown. 

Gelatin: Whitish colonies on surface of 
tube. Aerial mycelium scant, white. Soluble 
pigment brown. Liquefaction slow. 

Milk: Growth in yellow ring with patches. 
Soluble pigment yellowish. Xo coagulation; 
peptonization slow. 

( 'ellulose: Xo growth. 

Carbon utilization: Utilizes sucrose, L- 
arabinose, D-sorbitol, salicin, and sodium 
acetate; not sodium succinate. 

Antagonistic properties: Produces anti- 
biotics roseociti'in A and B. 

Habitat: Soil. 

212. Streptomyces roseodiastaticus (Duche, 



L934 ) now comb. ( I )uche, J. Les actinomyces 
du groupe albus. P. Lechevalier, Paris, 
p. 329, L934). 

Morphology: Growth consists of fine my- 
celium 0.5 to 0.7 fj. in diameter. Aerial my- 
celium of larger diameter, but usually less 
than 1 ju. 

Glucose nitrate agar: Growth limited, 
cream-colored, becoming white with a 
brownish reverse; on prolonged incubation 
the culture becomes rose-gray. Soluble pig- 
ment brownish. 

Glycerol nitrate agar: Growth cream- 
colored, becoming rose- violet ; reverse red. 

Asparagine agar: Growth cream-colored, 
becoming rose-white; reverse of growth 
reddish-brown. Soluble pigment yellow. 

Tyrosine medium: Pigment brownish, 
later becoming brown. 

Gelatin: Liquefied. Xo soluble pigment. 
Melanin-negative. 

Potato: Growth cream-colored, becoming 
brownish-white. Soluble pigment brown, 
only in presence of glycerol. 

Milk: Growth limited. Peptonization 
slow. 

Starch: Diastatic action weak. 

Remarks: Closely related to N. halstedii 
and considered as a transitional form. Ac- 
cording to Ettlinger el ol. (1958), this or- 
ganism is related to S. griseus. 

2b"). Streptomyces roseoflavus Arai, 1 ( .).">1 
(Arai, T. J. Antibiotics (Japan) 4: 215 221, 
L951 ). 

Morphology: Sporophores form spirals. 
Spores oval to oblong, 0.X to 1.0 by 1.0 to 

1.8 m- 

Sucrose nitrate agar: Growth colorless to 
yellowish. Aerial mycelium powdery, white 
to yellow-rose. 

Glucose-asparagine agar: Growth color- 
less to yellowish-white. Aerial mycelium 
rose-colored. 

Xutrient agar: Growth much folded, 
white-gray to golden yellow. Aerial myce- 



270 



THE ACTINOMYCETES, Vol. II 




Figure 44. Verticil formation by S. roseover- 
ticillatus (Reproduced from: Shinobu, R. Mem. 
< >saka Univ. Ser. B, No. .">, p. 93, 1956). 



Hum limited to center of colonies, white to 
rose. 

Starch agar: Growth golden yellow. Aerial 
mycelium whitish. 

Potato: Growth yellow. No aerial myce- 
lium. No soluble pigment. 

Gelatin: Liquefaction strong. Colonies at 
bottom of liquefied zone orange-brown. No 
soluble pigment. Melanin-negative. 

Milk: Ring cream-colored. Coagulation 
and peptonization rapid, medium becoming 
strongly alkaline. 

Cellulose: Growth on paper fair; cellulose 
decomposed. 

Nitrate reduction: Positive. 

Production of H 2 S: Negative. 

Antagonistic properties: Produces a basic 
antibiotic, flavomycin, similar to neomycin. 

Remarks: Culture similar to S. micro- 
flavus. Gause et al. (1957) described other 
closely related cultures, such as A. roseoful- 
vis. 

Type culture: [MRU 3672 ; ATCC 13,1(17. 

214. Streptomyces roseoverticillatus Shin- 
obu, 1956 (Shinobu, R. Mem. Osaka Univ., 
B (N.S.)5:84 93, L956). 

Morphology: Sporophores produce abun- 



dant primary and secondary verticils (Fig. 
44) . Spores spherical to elliptical, 0.8 to 1.0 /*. 

Sucrose nitrate agar: Growth thin, mod- 
erate, pinkish-red. Aerial mycelium cottony, 
pink to pale pink. 

Glucose-asparagine agar: Growth red to 
purple-red. Aerial mycelium cottony, dull 
red to reddish-brown. 

Nutrient agar: Growth reddish-brown to 
deep brown. Aerial mycelium thin, pinkish 
to red. Soluble pigment reddish-brown to 
deep brown. 

Potato: Growth brownish-red to dull red. 
Aerial mycelium powdery, pink to reddish- 
purple. Soluble pigment brown. 

Milk: Growth red. Aerial mycelium pale 
pink. Coagulation and peptonization strong. 
Soluble pigment pale brown. 

Gelatin: Rapid liquefaction. 

Starch: Rapid hydrolysis. 

Tyrosinase reaction: Weak. 

Cellulose: No growth. 

Nitrate reduction: Positive. 

Carbon utilization: Utilizes fructose; 
inositol uncertain. Does not utilize xylose, 
rhamnose, sucrose, lactose, raffinose, or 
mannitol. 

Habitat: Soil in Japan. 

Remarks: Resembles S. rubrireticuli. 

215. Streptomyces roseus (Namyslowsky, 
1909; emend. Krainsky, 1914; emend. Waks- 
man and Curtis, 1916) Waksman and 
Henrici, 1948 (Namyslowsky, B. Centr. 
Bakteriol. Parasitenk. Abt. I, Orig. 62: 564, 
1909; Krainsky, A. Centr. Bakteriol. Para- 
sitenk. Abt. II, 41: 082-683, 1914; Waks- 
man, S. A. and Curtis, R. E. Soil Sci. 1: 125, 
1910). 

Morphology: Sporophores produce num- 
erous open and closed dextrorse spirals. Ac- 
cording to Okami, sporophores are straight, 
without spirals. Spores oval to elongate, 1.5 
to 2.0 by 1.1 fi. 

Sucrose nitrate agar: Growth colorless. 
Aerial mycelium pale brownish-vinaceous. 
No soluble pigment. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



271 



Glycerol malate agar: Growth colorless. 
Aerial mycelium white to rose. 

Xutrient agar: Growth white, turning 
yellowish. Xo aerial mycelium. No soluble 
pigment. 

Starch agar: ( rrowth colorless. Aerial my- 
celium white with shade of pink. No soluble 
pigment. Hydrolysis medium. 

Gelatin: Growth yellowish-brown. Aerial 
mycelium white. Soluble pigment brown. 
Liquefaction slow; in some cases no liquefac- 
tion. Melanin-negative. 

Potato: Growth brownish. No aerial my- 
celium. Soluble pigment brownish or absent. 

Milk: No coagulation; gradual peptoniza- 
tion. 

Cellulose: No growth. 

Invertase: None. 

Nitrate reduction: Rapid. 

Habitat: Soil. 

Remarks: Various cultures have been 
described under this name. Krassilnikov 
(1949) considered it as a varietal strain of 
S. ruber. 

Type culture: IMRU 3772. 

21(i. Streptomyces ruber (Krainsky, L914) 
Waksman and Henrici, 1948 (Krainsky, A. 
Centr. Bakteriol. Parasitenk. Abt. 11., II: 
649-688, 1914). 

Not Actinomyces ruber Ruiz-Cazabo, 1894; 
not Actinomyces ruber (Kruse, 1896) San- 
felice, 1904. 

Morphology: Sporophores straight, 
blanching; a few spirals may be formed. 
Spores spherical and oval, 0.7 to 0.8 by 0.8 
to 1.0/z. 

Sucrose nitrate agar: Growth abundant, 
orange to coral-red. Aerial mycelium red to 
red-orange to dark red. Pigment insoluble 
unless vegetable oil present in medium. 

Nutrient agar: Growth elevated, wrinkled, 
olive-green. No aerial mycelium. 

( rlucose agar: ( rrowth abundant, coral-red. 

Potato: Xo growth. 

Gelatin: Growth scant, yellow, flaky. 
Liquefaction slow, increasing with growth. 
Melanin-negative. 



Milk: Dark ring with red tinge. Coagula- 
tion ; peptonization gradual. 

Starch: Hydrolysis weak. 

Sucrose: Inversion positive. 

Cellulose: Growth in form of red spots. 

Nitrate reduction: Positive, depending on 
carbon source. 

Production of IPS: Negative. 

Temperature: Optimum 37°C. 

Pigments: Soluble in organic solvents; 
alcohol extracts a red-violet pigment and 
petroleum-ether a red-orange pigment ( Kriss, 
1936). 

Antagonistic properties: Strongly effective 
upon gram-positive bacteria. Various anti- 
biotics are produced by different strains. 

Habitat: Soil. 

Remarks: Above description is based 
largely upon that given by Krainsky. Closely 
related forms include A. longisporus ruber 
Krassilnikov, which is said to form some- 
what longer spores, and to give sometimes a 
brown coloration in protein media. A. auran- 
tiogriseus Gause et al. also appears to be 
closely related. 

217. Streptomyces rubescens (Jarach, 1931) 
Umezawa et al., 1952 (Jarach. Boll. sez. ital. 
soc. intern, microbiol. 3:43, 1931 ; Umezawa, 
H., Tazaki, T., and Fukuyama, S. .1. Anti- 
biotics (Japan) 5: 469, 1952). 

Morphology: Sporophores short, curved, 
well branched; no spirals. Spores spherical or 
oval. 

Sucrose nitrate agar: Growth at first 
white, changing to salmon-pink. Aerial my- 
celium powdery, white. Xo soluble pigment. 

Nutrient agar: Growth same as on sodium 
nitrate agar. 

Blood agar: After 10 days' incubation. 
growth becomes salmon-pink. Aerial myce- 
lium powdery, white. Xo soluble pigment. 
Xo hemolysis. 

Egg media: Growth colorless, changing to 
coral-pink. Aerial mycelium powdery, white. 

Potato: Growth coral-pink. Aerial myce- 
lium powdery, white. Plug changes slightly 



272 



THK ACTINOMYCETES, Vol. II 



to brown. No soluble pigment. Melanin- 
negative. 

Gelatin: Surface growth coral-pink. No 
liquefaction. No soluble pigment. 

Milk: Growth coral-pink. Aerial mycelium 
powdery, white. No coagulation and no 
peptonization. Soluble pigment sometimes 
slightly reddish. 

Starch: No hydrolysis. 

Nitrate reduction: None. 

Production of H 2 S: Negative. 

Carbon utilization: Glycerol and glucose 
utilized, but not other carbohydrates. 

Antagonistic properties: Produces an anti- 
viral agent, abikoviromycin. 

Remarks: .4. griseoruber of Gause et al. 
(1957) appears to be a closely related form. 
This organism is considered by R. Gordon as 
a Nocardia, related to N. asteroides. 

Type culture: IMRU 3655. 

218. Streptomyces rubrireticuli (Waksman, 
L919) Waksman and Henrici, 1948 (Waks- 
man, S. A. Soil Sci. 8: 146, L919). 

Synonyms: Actinomyces, reticulus-ruber 
Waksman. L919; A. reticulus Bergey, 2nd 
ed., 1925. 

Morphology: Sporophores produce both 
primary and secondary verticils; composi- 
tion of medium influences structure of 
sporophores, glucose-asparagine agar favor- 
ing spiral formation. Spores oval-shaped, 
smooth (PI. I a). 

Sucrose nitrate agar: Growth abundant, 
spreading, usually pink. Aerial mycelium 
white, later rose to pink. 

Glucose-asparagine agar: Entire growth 
abundant, spreading, rose-red. 

Nutrient agar: Growth red, with yellowish 
margin, becoming red. Soluble pigment dark 
brown. 

Starch agar: Growth white with red tinge. 
Hydrolysis fair. 

Potato: Growth cream-colored, later pink 
to dark red. Melanin-positive. 

Gelatin: Surface growth yellowish-red to 
pink. Ready liquefaction. Brown pigment. 



Milk: Growth abundant. Coagulation and 
peptonization. 

Invertase: Positive. 

Cellulose: Growth good. 

Nitrate reduction: Rapid. 

Production of H 2 S: Positive. 

Antagonistic properties: Certain strains 
produce an antibiotic designated as streptin; 
others produce trichonin. 

Habitat: Soil. 

Remarks: Numerous cultures that pro- 
duce a rose to pink substrate growth, a 
soluble brown pigment in organic media, and 
both primary and secondary verticils in the 
sporophores have been described. It is suf- 
ficient to mention A. biverticillatus by Gause 
et al. (1957). 

Type culture: IMRU 3631. 

219. Streptomyces rutgersensis (Waksman 
and Curtis, 1916) Waksman and Henrici, 
1948 (Waksman, S. A. and Curtis, R. E. 
Soil Sci. 1: 123, 1916; 8: 152, 1919). 

Morphology: Sporophores produce abun- 
dant close and open spirals. Spores spherical 
and oval, 1.0 to 1.2 /x. with tendency to bi- 
polar staining. 

Sucrose nitrate agar: Growth thin, color- 
less, becoming brownish to almost black. 
Aerial mycelium white, becoming dull gray. 

Glucose-asparagine agar: Growth abun- 
dant, brown, becoming black with cream- 
colored margin. No aerial mycelium appears 
within 15 days. 

Nutrient agar: Growth thin, wrinkled, 
cream-colored. 

Starch agar: Growth gray, spreading. 
Hydrolysis good. 

Potato: Growth abundant, much folded. 
Aerial mycelium white-gray. Melanin-nega- 
tive. 

Gelatin: Growth cream-colored. Liquefac- 
tion medium. No soluble pigment. 

Milk: Cream-colored ring. Coagulation 
and slow peptonization. 

Cellulose: Growth scant. 

Sucrose: Inversion weak. 



)ESCRIPTION OF SPECIES OF STREPTOMYCES 



27:; 



Nitrate reduction : ( rood. 

Production of IPS: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Various strains 
produce xanthomycin-like substances; others 
produce ruticin. 

Remarks: The pigmenl formed is not 
soluble. Krassilnikov (1949) considered the 
organism, quite incorrectly, as a variety of 
.1 . chromogenes. 

Type culture: [MRU 3350. 

220. Streptomyces sahachiroi Hat a et r//., 
1954 (Ilata, T., Koga, F., Sano, Y., Kana- 
mori, K., Matsumae, A., Sugawara, R., 
Hoshi, T., and Shima, T. J. Antibiotics 
(Japan) 7A: 107-112, 1954). 

.Morphology: Sporophores form numerous 
closed spirals with a iv\\ open spirals. Spores 
oval or cylindrical, 0.7 to 1.3 by 0.5 to 0.8 l x. 

Sucrose nitrate agar: Growth folded, dark 
orange. Aerial mycelium velvety, white to 
pale grayish-brown. Soluble pigment yel- 
lowish-brown. 

Calcium malate agar: ( irowth thin, cream- 
colored with orange-yellow reverse. Aerial 
mycelium thin, white, powdery. Soluble pig- 
ment pale orange-yellow. 

Nutrient agar: Growth glistening, white- 
gray. Xo aerial mycelium. Soluble pigment 
light brown to yellow. 

Starch agar: ( Irowth thin, yellowish-white. 
Aerial mycelium thin, powdery, pale red- 
brown. Xo soluble pigment. Slow hydrolysis. 

Potato: Growth wrinkled, pale yellowish- 
brown. Aerial mycelium thin, white. Soluble 
pigment absent or faint brown. 

Gelatin: Growth limited, white. Xo aerial 
mycelium. Xo soluble pigment. 

Milk: Surface growth white to pale yellow. 
Coagulation; no peptonization. Strongly 
alkaline. Soluble pink pigment. 

Nitrate reduction: Positive. 

Carbon utilization: Xylose, arabinose, 
lactose, trehalose, mannitol, sucrose, salicin, 
glucose, maltose, mannose, glycerol, dextrin, 
fructose, starch, galactose, sorbitol utilized. 



Rhamnose, raffinose, inositol, esculin, dulci- 

tol, inulin, sodium acetate, sodium citrate, 
sodium succinate not utilized. 

Antagonistic properties: Produces anti- 
tumor agent carzinophilin. 

221. Streptomyces sampsonii (Millard and 
Burr, 1926) Waksman (Millard, \Y. A. and 
Burr, S. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Aerial mycelium produces 
long, straight sporophores, rarely spiral- 
shaped. Spores cylindrical, 0.8 to 1.0 by 0.5 
fx (spores oval to spherical, Waksman and 
Gordon). 

Sucrose nitrate agar: Growth wrinkled, 
pale gray to white. Aerial mycelium very 
scant, white. Soluble pigment green to buff. 

Potato: Growth wrinkled, grayish. Aerial 
mycelium white. Soluble pigment golden 
brown (none, Waksman and Gordon). 

Gelatin: Surface growth scant, gray. 
Aerial mycelium trace, whitish. Liquefaction 
rapid. Melanin-negative. 

Milk: Surface growth good, whitish. Xo 
aerial mycelium. No coagulation; no pep- 
tonization (rapid peptonization, Waksman 
and Gordon). 

Starch: Xo hydrolysis (rapid hydrolysis, 
Waksman and Gordon). 

X'itrate reduction: Positive. 

Tyrosinase reaction: Negative. 

Temperature: 28°C. 

Habitat: Potato scab. 

Type culture: [MRU 3371. 

222. Streptomyces sayamaensis Arishima 
et al, 1955 (Arishima, M., Sekizawa, A., Sato, 
T.,and Miwa, K. J. Agr. Chem. Soc. Japan 
29:810-817, 1955). 

Morphology: Sporophores straight, spores 
short rods to cylindrical, 1.0 by 1.5 m- 

Sucrose nitrate agar: Growth pale yellow. 
Aerial mycelium gray with brownish tinge. 
Soluble pigment yellow. 

Glucose-asparagine agar: Growth white to 
pale yellow, turning pale brown. Aerial my- 
celium white, becoming pale brown. 

Calcium malate agar: (Irowth limited. 



274 



THE ACTINOMYCETES, V 



Aerial mycelium gray. Soluble pigment 
brownish. 

Starch agar: Growth brownish-yellow. 
Aerial mycelium white, turning gray. Hy- 
drolysis. 

Nutrient agar: Growth pale orange-yellow. 
No aerial mycelium. 

Potato: Growth heavy, pinkish-gray with 
purplish tinge. Soluble pigment reddish- 
brown. 

Gelatin: Surface pellicle pale yellow. No 
soluble pigment. No liquefaction in 15 days 
at 26°C. Melanin-negative. 

Milk: Yellow-gray ring. Coagulation and 
peptonization. 

Nitrate reduction: Negative. 

Cellulose: No growth. 

Optimum temperature: 35-37°C. 

Tyrosinase reaction: Negative. 

Carbon utilization: Utilizes n-galactose, 
sucrose, maltose, sodium citrate and succi- 
nate; does not utilize xylose, arabinose, 
lactose, rhamnose, raffinose, inulin, manni- 
tol, sorbitol, inositol, and salicin. 

Antagonistic properties: Produces chlor- 
tetracycline. 

Habitat: Soil in Japan. 

Remarks: Related to S. aureofaciens. 



223. Streptomyces scabies (Thaxter, 1891) 
Waksman and Henrici, 1048 (Thaxter, R. 
Ann. Kept. Conn. Agr. Expt. Sta. 1891, p. 
153). 

Morphology: Sporophores much branched, 
wavy or slightly curved; occasionally form 
spirals. Spores cylindrical, 0.8 to 1.0 by 1.2 
to 1.5 M (Fig. 45). 

Sucrose nitrate agar: Growth abundant, 
wrinkled, raised, gray to cream-colored. 
Aerial mycelium cottony, white to gray. 

Glucose -asparagine agar: Growth re- 
st ricted, folded, cream-colored. Aerial my- 
celium scant, white to gray. 

Nutrient agar: Growth wrinkled, white to 
straw-colored, opalescent to opaque. No 
aerial mycelium. Soluble pigment deep 
golden brown. 

Potato: Growth gray, opalescent, becom- 
ing wrinkled, black. Aerial mycelium scant, 
grayish-white. Color of plug brown. 

Gelatin: Surface growth cream-colored, 
becoming brown. Liquefaction slow. Soluble 
pigment yellowish. 

Milk: Surface ring brown, with greenish 
tinge. Coagulation and peptonization lim- 
ited. 

Starch: Hydrolysis. 



Figure 45. Sporophort 
of E. Baldacci, University 




of transparent spores, X 15,000 (Courtesy 



DKSCRII'TIOX OF Sl'KCIKS OF STREPTOMYCES 



275 



Sucrose: Inversion. 

Nitrate reduction: Positive. 

Tyrosinase reaction: Strong. 

Antagonistic properties: Certain strains 
show positive antimicrobial action; others 
are negative. 

Habitat: Numerous strains of this organ- 
ism have been isolated from various forms of 
potato seal) and sugar beet seal) throughout 
the world. True causative agent of seal). 

Remarks: According to Hoffmann (1058), 
growth on synthetic agar is reddish with 
dark gray aerial mycelium; on glucose agar, 
growth is colorless with blue-gray aerial my- 
celium; on asparagine agar, growth is dark 
red with no aerial mycelium. 

Closely related forms include S. clavifer, 
S. spiralis, S. carnosus, and S. sampsonii 
described by Millard and Burr; also .1. 
xanthostromus and .1. ochroleucus of Wollen- 
weber. Various strains differ in the amount 
of aerial mycelium produced and in their 
biochemical properties. 

Type culture: [MRU 3018. 

224. Streptomyces setonii (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, S. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores straight, wavy, 
formed in clumps. Spores oval, 0.6 to 0.8 by 
0.85 a. 

Sucrose uitrate agar: Growth abundant, 
smooth, yellow to brown. Aerial mycelium 
gray to olive-buff. Soluble pigment faint 
yellowish to brown. 

Nutrient agar: Growth colorless. Aerial 
mycelium smooth, white. Soluble pigment 
brownish. Melanin-negative. 

Glucose agar: Growth lichenoid, gray to 
brown. Aerial mycelium abundant, white to 
olive-buff. Soluble pigment golden brown. 

Potato: Growth heavy, wrinkled, brown 
to red-violet. Aerial mycelium abundant, 
white to green to olive-buff. 

Gelatin: Surface growth gray. Aerial my- 
celium white. Rapid liquefaction. Soluble 
pigment brownish. 



Milk: Surface growth, covered with ring 
of white aerial mycelium. Questionable co- 
agulation, followed by rapid peptonization. 

Starch agar: Growth cream-colored. 
Aerial mycelium patchy, white. Hydrolysis. 

( lellulose: ( rrowth colorless. 

Nitrate reduction: Positive. 

Temperature: Grows well at 37.5 C. 

Habitat : Scabby potatoes. 

Remarks: Millard and Burr also described 
a similar form under the name .1. setonii 
flavus. Ettlinger et al. (1058) consider this 
organism as a strain of S. griseus. Hoffmann 
(1058) described an organism with lighl gray 
aerial mycelium as a strain of S. setonii. 

Type culture: [MRU 3375. 

225. Streptomyces somaliensis (Brumpt, 
10011; emend. Erikson, 1935) Waksman 
(Brumpt, E. Arch. Parasitol. Paris 10: 480, 
100(1; Precis de Parasitologic. Paris, 2nd ed., 
p. 0(17, 1913; Erikson, D. Med. Research 
Council (Brit.) Spec. Rept. Ser. Xo. 203: 
1035, p. 17-18). 

Morphology: Substrate growth made up of 
simple branching, unicellular mycelium with 
long straight filaments. Aerial mycelium 
forms straight nonsegmented sporophores 
with typical chains of spores, 1.25 yu in 
diameter. 

Glucose-asparagine agar: Growth thin, 
smooth, and soft. 

Glycerol nitrate agar: Growth abundant, 
colorless to dark gray and black. 

Nutrient agar: Growth abundant, granu- 
lar, yellowish, with small discrete colonies al 
margin; later growth colorless, colonies 
umbilicated. 

Potato: Colonies round and oval, partly 
piled up in rosettes. Aerial mycelium whitish- 
gray. Plug discolored. Pater, aerial myce- 
lium becomes transient, growth nearly black. 

Blood agar: ( Irowth in form of small, dark 
brown colonies. Pound and umbilicated, 
piled up in confluent bands. Reverse red- 
black. Blood hemolyzed. 

Dorset's ege medium: (Irowth colorless. 



276 



THE ACTINOMYCETES, Vol. II 



becoming opaque, cream-colored, very wrin- 
kled. Later, rough, yellow; medium lique- 
fied. 

Gelatin: Growth cream-colored. Black 
.sediment at bottom. Rapid liquefaction. 

Milk: Surface pellicle heavy, wrinkled. 
Milk coagulated and completely peptonized. 

Starch: Hydrolysis. 

Habitat: Frequently found in Africa. 

Remarks: Although S. somaliensis has 
long been known, there has been, until re- 
cently, no detailed description of the or- 
ganism beyond the fact that it possesses 
around the grain a distinctly hard sheath 
which is insoluble in potash and eau de 
javelle. The rare occurrence of septa and 
occasional intercalary chlamydospores is re- 
ported by Brumpt, but has not been con- 
firmed by Erikson. Chalmers and Chris- 
topherson merely mentioned the growth on 
potato as yellowish-white and lichenoid, 
without describing any aerial mycelium. 
According to Mariat, S. somaliensis hy- 
drolyzes gelatin, serum albumin, and egg 
albumin; utilizes casein hydrolyzate, but not 
urea, (NH 4 ) 2 S0 4 and KN0 3 as nitrogen 
sources; utilizes glucose, maltose, and fruc- 
tose, but not xylose, starch, mannitol, or 
paraffin as carbon sources. 

22(i. Strcptomyccs spectabilis Dietz, 1957* 
(Brit. Pat, 811,757, April 8, 1959; Am. Rev. 
Tuberc. 75:576, 1957). 

Morphology: Sporophores monopodially 
branched, long, straight. Pigment granules 
produced in both substrate and aerial myce- 
lium. 

Sucrose nitrate agar: Growth mottled 
orange to cream-orange. Aerial mycelium 
mottled orange to orange. 

Starch-nitrate agar: ( rrowth cream-colored, 
flecked with orange. Aerial mycelium pale 
pink to orange. Starch hydrolyzed. 

Starch-peptone-beef extract agar: Growth 
cream-colored, turning orange. Aerial myce- 

* Personal communication. 



Hum deep orange to pale pink. Soluble pig- 
ment yellow. 

Gelatin: Medium liquefaction. Soluble 
pigment slightly yellow to dark brown. 
Melanin-negative. 

Milk: Growth orange. Soluble pigment 
brown. Peptonization varies with strain. 
Acid formation by some strains. 

Carbon utilization: Utilizes various sugars 
and organic acids, depending on strain. Does 
not utilize rhamnose, sucrose, inulin, ducitol, 
D-sorbitol, fumarates, oxalates, or salicylates. 

Nitrate reduction: Negative. 

Production of H 2 S: Positive. Some strains 
negative. 

Antagonistic properties: Produces antibi- 
otic streptovaricin. 

Remarks: Closely related to S.fulvissimus. 

227. Streptomyces spheroides Wallick et al., 
1955 (Wallick, H., Harris, D. A., Reagan, 
M. A., Ruger, M., and Woodruff, H. B. 
Antibiotics Ann. 1955-1950, p. 909-917). 

Morphology: Sporophores form spirals, 
the majority of which are closed and com- 
pact; in some areas the spirals appear ball- 
like. Spores oval, 0.7 to 1.1 by 1.5 to 2.0 id. 

Sucrose nitrate agar: Substrate growth 
white, becoming straw-colored. Aerial my- 
celium abundant, white, tinged with cream 
to olive-gray. No soluble pigment, 

Glucose - asparagine agar: Substrate 
growth pale yellow. Aerial mycelium white, 
becoming gray. No soluble pigment. 

Glucose-peptone agar: Growth moderate, 
yellow. Aerial mycelium grayish-white. No 
soluble pigment. 

Starch agar: Growth heavy, cream- to 
straw-colored. Aerial mycelium white. 

Potato: Growth slow, scant, white, later 
becoming heavy, gray. Aerial mycelium 
gray. Soluble pigment dark brown. 

Gelatin: Cream-colored, flaky sediment. 
Rapid liquefaction. No soluble pigment. 

Milk: Slow coagulation and peptonization. 
Slight acidification. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



277 



> gas from adonito 
dextrin, dextros' 
se, maltose, maim 
imnose, salich 



Prodi 



am 



■t al. 



Cellulose: No growth. 

Carbon utilization: Xi 
arabinose, cellobiose, 
galactose, lactose, levulose, 

sucrose, or xylose. 

Antagonistic properties: 
biotic novobiocin. 

Habitat: Soil. 

Remarks: According to 
(1958), this organism is related if not identi- 
cal to S. griseoflavus. 

228. Streptomyces spiralis (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, S. Ann. Appl. Biol. 13: 580, L926). 

Morphology: Sporophores straight or 
spiral-shaped. Spores cylindrical, 1.0 to 1.7 
by 0.9 m (Fig. 46). 

Sucrose nitrate agar: Growth rough or 
granular, yellowish-golden. Aerial mycelium 
vinaceous-buff to dark grayish-olive. Soluble 
pigment pale vinaceous to fawn-colored. 

Potato: Growth poor, wrinkled, grayish- 
vinaceous. Aerial mycelium white to grayish- 
vinaceous. Plug colored brown around and 
below growth. 

Gelatin: Growth limited, gray. Aerial my- 
celium scant, white. Liquefaction rapid. 
Melanin-negative. 

Milk: Surface growth good. Aerial myce- 
lium abundant, white. Coagulation and 
rapid peptonization. 

Starch: No hydrolysis. 

Nitrate reduction: None. 

Tyrosinase reaction: Negative. 

Habitat : Potato scab. 

Remarks: Krassilnikov (1949) considered 
this organism as belonging to the A. scabies 
group. 

229. Streptomyces spiroverticillatus Shin- 
obu, 1958 (Shinobu, R. Botan. Mag. Tokyo 
71:87-93, L958). 

Morphology: Verticil formation usually 
occurs near base of aerial mycelium, but 
generally not so remarkable as in the other 




Figure 46. Sporophores of S. spiralis, showing 
that not all arc transformed into spun-, x 8,000 
(Courtesy of E. Baldacci, University of Milan. 
Italy). 

verticil-forming species. Occasionally, very 

few tufts on the skirt of the colony. Nitella 
type verticils, generally primary only, seldom 
secondary. About 2 to 4 short radial 
branches. On synthetic media, many spirals 
in form of curled tips with 1 to2 turns, seldom 
.") turns; diameter of spirals about 5 to 8 n; 
sometimes snail-like and hook-like curls, 
ly loose or closed spirals with 2 to 



o, 



iona 
3 verticil t 
somewhat 
(Fig. 47). 



urns, sinistrorse. Spores spheroid, 
ellipsoid; about 0.8 n in length 



nil rate agar: ( 



th colorless t. 



278 



THE ACTINOMYCETES, Vol. II 




Figure 47. S. spiroverticillatus (Reproduced 
from: Shinobu, R. Botan. Mag. (Tokyo) 71: 88, 

1958). 

pale brown to yellowish-orange. Aerial my- 
celium thin, somewhat cottony, white. 

Glucose-asparagine agar: Growth pale 
yellow-orange. Aerial mycelium good, cot- 
tony, white to brownish. 

Calcium malate agar: Growth yellow- 
orange to light brown. Aerial mycelium 
abundant, cottony, white. 

Nutrient agar: Growth golden yellow to 
buff. No aerial mycelium. Soluble pigment 
pale brown. 

Potato: Growth yellowish-brown to brown. 
Aerial mycelium abundant, cottony, white 
to brownish- white. Soluble pigment brown. 

Milk: Growth yellow to yellow-orange. 
Aerial mycelium poor, light brownish-gray. 
Soluble pigment yellowish-orange. No co- 
agulation; rapid peptonization. 

Gelatin: Growth poor; liquefaction strong. 

Tyrosinase reaction: Somewhat unstable, 
generally positive, weak. 

Diastase reaction: fairly strong. 

Nitrate reduction: Positive. 

Carbon utilization: Utilizes lactose, fruc- 



tose, and xylose. Sucrose and inositol un- 
certain. Does not utilize rhamnose, man- 
nitol, and raffinose. 
Habitat: Soil. 

230. Streptomyces sulphureus (Rivolta, 1882 
emend. Gasperini, 1894) Waksman (Rivolta, 
S. Arch. path. Anat. Phys. 88: 389, f882; 
Gasperini, G. Centr. Bakt. Abt. 1, 15: 684, 
1894; Waksman, S. A. Soil Sci. 8: 102-104, 
1919). 

Synonym: Actinomyces bovis (Harz) 
Waksman, 1919. 

Not .4. sulphureus Berestnew, 1897. 

This organism is usually found in culture 
collections under the name of Actinomyces 
boris. Baldacci (1937, 1947) emphasized the 
synonymy of this organism, listing as many 
as 13 different names. The following de- 
scription is based upon the data of Waks- 
man (1919), who also spoke of it as .4. bovis. 

Sucrose nitrate agar: Growth white, turn- 
ing yellow. Aerial mycelium light, powdery, 
sulfur-yellow. No soluble pigment. 

Calcium malate-glycerol agar: Growth 
brownish. No aerial mycelium. 

Nutrient agar: Growth at first cream- 
colored, later becoming fawn-colored, brown, 
then almost black. Aerial mycelium pale 
yellow-green. No soluble pigment. Melanin- 
negative. 

Glucose agar: Growth yellowish, later be- 
coming dark. Aerial mycelium thin, sulfur- 
yellow. 

Starch: Fair hydrolysis. 

Potato: Growth abundant, much wrin- 
kled, gray to canary -yellow. Aerial myce- 
lium yellow, turning sulfur-yellow. Plug at 
first not pigmented, later turning brownish. 

Gelatin: Growth gray to brownish. No 
aerial mycelium. No soluble pigment. 
Liquefaction rapid at 37°C; slow at 18°C. 

Milk: Surface growth thin, yellowish. 
Coagulation and peptonization. 

Carbon utilization: Ready utilization of 
glucose, lactose, sucrose, maltose, glycerol, 
and various organic acids. 



DESCRIPTION OF SPECIES OF STREPTOM1 CES 



279 



Nitrate reduction: Positive. 

Production of H 2 S: Negative. 

[nvertase: None reported. 

Remarks: Ettlinger et al. (1958) considered 
certain strains of this organism as belonging 
to 1 lie S. griseus series. 

231. Streptomyces tanashiensis Hata et <rf., 
1952 (Hata, T., Ohki, X., and Higuchi, T. 
J. Antibiotics (Japan) 5: 529 534, 19.32). 

Morphology: Sporophores almost straight. 
Spores spherical to oval, 1.0 by 1.2 /x- 

Sucrose nitrate agar: Growth grayish- 
yellow. Aerial mycelium white-gray, turning 
brownish-gray. Soluble pigment light yellow. 

Potato: Growth brown. Aerial mycelium 
dark gray to whitish-gray. Soluble pigment 
dark brown. 

Gelatin: Soluble pigment brown. Rapid 
liquefaction. 

Milk: Yellowish surface ring. Coagulation 
and peptonization. 

Starch: Hydrolysis. Most suitable for anti- 
biotic production. 

Nitrate reduction: Negative. 

Tyrosinase reaction: Positive. 

Production of H 2 S: Positive. 

Optimum pH: 5.8 to 6.5. 

Antagonistic properties: Produces luteo- 
mycin. 

Habitat: Soil. 

Remarks: Resembles S. aureus and *S. 
antibioticus. 

232. Streptomyces tendae Ettlinger et ui, 
1958 (Ettlinger, L., Corbaz, R., and H utter, 
R. Arch. Mikrobiol. 31: 351, 1958). 

Morphology: Sporophores form verticils; 
chains of spores as open, regular spirals. 
Spores smooth (PI. I c). 

Glycerol nitrate agar: Growth thin, light 
yellow. No aerial mycelium. No soluble pig- 
ment . 

Glucose-asparagine agar: Growth light 
yellow to light carmine. Aerial mycelium 
cottony, cinnamon-brown. 

Calcium malate agar: Growth light yellow 



to brownish-yellow. Soluble pigment brown- 
ish-yellow. 

Gelatin: Growth Ligh.1 yellow. Aerial my- 
celium sparse. Liquefaction limited. Soluble 
pigment dark brown. 

Starch agar: Growth thin, light yellow. 
Limited hydrolysis of starch. 

Potato: Growth brown to dark. Aerial 
mycelium powdery, chalk-white. 

Milk: Growth brownish-yellow. Aerial my- 
celium sparse. Xo coagulation: weak pep- 
tonization. 

Antagonistic properties: Produces anti- 
biotic carbomycin. 

Habitat: Soils in France. 

Remarks: Organism said to be melanin- 
negative, although dark brown pigment re- 
ported on gelatin. 

233. Streptomyces tenuis (Millard and 
Burr, 1926) Waksman (Millard, W. A. and 
Burr, S. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores straight. Spores 
cylindrical, 0.9 by 0.8/x- 

Sucrose nitrate agar: Growth thin, Hat, 
yellowish-drab. Aerial mycelium dee]) olive- 
buff. Soluble pigment pale orange-yellow. 

Glucose agar: Growth thin, flat. Aerial 
mycelium olive-buff. Soluble pigment green. 

Potato: Growth good. Aerial mycelium 
deep olive-buff. Soluble pigment gray to 
olive to black. 

Nutrient potato agar: Growth wrinkled, 
grayish. Aerial mycelium white to vinaeeous- 
fawn. Soluble pigment golden brown. 

Gelatin: Growth pale gray. Aerial myce- 
lium scant, white. Rapid liquefaction. Solu- 
ble pigment yellow. 

Milk: Growth good. Aerial mycelium 
white. Coagulation; incomplete peptoniza- 
tion. 

Starch: Hydrolysis. 

Nitrate reduction: Negative. 

Tyrosinase reaction: Negative. 

Habitat: Potato scab. 

234. Streptomyces thioluteus Okami, L952 



280 



THE ACTINOMYCKTES, Vol. II 



(Okami, Y. Taxonomic study of antibiotic 
streptomyces. Thesis, Hokkaido University, 
Japan, 1952). 

Morphology: Aerial hyphae with few 
branches. No spirals, bu1 verticils produced 
occasionally, depending on composition of 
medium. 

Glycerol nitrate agar: Growth yellowish- 
brown, penetrates deep into medium. Aerial 
mycelium thin, white, with dark yellowish 
tinge. Soluble pigment yellowish -brown. 

Nutrient agar: Growth wrinkled, yellow- 
ish-brown. Aerial mycelium scant, white. 
Soluble pigment slight, yellowish-brown. 

Starch agar: Growth thin, cream-colored. 
No aerial mycelium. No hydrolysis. 

Gelatin: Growth yellowish-brown at bot- 
tom of liquefied portion. No aerial mycelium. 
Soluble pigment slight, yellowish-brown. 
Slow liquefaction. 

Potato: Growth wrinkled, cream to yel- 
lowish. No aerial mycelium. Soluble pigment 
slight, yellowish-brown. 

Milk: Growth on surface of milk yellowish. 
Aerial mycelium scant. Coagulation occurs 
in 2 to 3 days, followed by slow peptoniza- 
tion. 

Blood agar: Growth dark gray with green- 
ish tinge. Aerial mycelium dark. No hemoly- 
sis. 

Nitrate reduction: Negative. 

Production of H 2 S: Negative. 

Antagonistic properties: Produces anti- 
fungal substance, aureothricin (Maeda, 
1953). 

Type culture: ATCC 12,310. 

235. Streptomyces tumuli (Millard and 
Beeley, 1927) Waksman (Millard, W. A. 
and Beeley, F. Ann. Appl. Biol. 14: 296-311, 
1927). 

Sucrose nitrate agar: Growth gray, later 
becoming opaque dark. Aerial mycelium 
arises at center of growth, at first white, 
later becoming pale gray. Surface of growth 
covered with colorless drops leaving small 
black craters. No soluble pigment. 



Glucose-asparagine agar: Growth wrin- 
kled, pale gray. Aerial mycelium white, 
arising in concentric rings around a dark 
bare center. Soluble pigment olive-colored. 

Nutrient agar: Growth good, lustrous, 
slimy, gray. No aerial mycelium. No soluble 
pigment. Melanin-negative. 

Potato: Growth heavy, slimy, black. No 
aerial mycelium. Soluble pigment grayish- 
brown. 

Gelatin: Growth beaded. No aerial myce- 
lium. Liquefaction rapid. No soluble pig- 
ment. 

Milk: Growth good; no aerial mycelium. 
Coagulation and slight peptonization. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Habitat: Mound scab of mangels. 

236. Streptomyces venezuelae Ehrlich et al., 
1948 (Ehrlich, J., Gottlieb, D., Burkholder, 
P. R., Anderson, L. E., and Pridham, T. G. 
J. Bacterid. 56: 407 -477, 1948; Pridham, 
T. G. and Gottlieb, D. J. Bacteriol. 56: 
107-114, 1948). 

Morphology : Sporophores monopodially 
branched, straight or slightly and irregularly 
curved. Spores oval to oblong, 0.4 to 0.9 by 
0.7 to 1.6 /i, smooth (PI. II p). 

Sucrose nitrate agar: Aerial mycelium 
light lavender. 

Nutrient agar: Substrate growth yellow 
to brown. Aerial mycelium gray. Soluble 
pigment dark brown to black. 

Calcium malate agar: Substrate growth 
yellow to brown; aerial mycelium gray. 

Glucose agar: Soluble pigment dark 
brown. 

Potato: Growth abundant, gray to dark 
brown. Aerial mycelium gray. Soluble pig- 
ment dark brown or black. 

Gelatin: Liquefaction rapid. Soluble pig- 
ment dark brown. 

Milk: Peptonization. Soluble pigment 
dark brown. 

Starch agar: Growth white to lavender. 
Hydrolysis. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



281 



Nitrate reduction: Positive. 

Tyrosinase reaction: Positive. 

Production of IPS: Positive. 

Carbon utilization: Good growth: xylose, 
arabinose, rhamnose, D-glucose, D-mannose, 
D-fructose, D-galactose, cellobiose, starch, 
dextrin, glycerol, acetate, citrate, succinate, 
and salicin. Slighl or no growth: D-ribose, 
sucrose, raffinose, inulin, erythritol, dulcitol, 
niannitol, sorbitol, inositol, and malate. Xo 
growth: formate, oxalate, tartrate, salicylate, 
phenol, o-cresol, w-cresol, p-cresol. 

Antagonistic properties: Produces chlor- 
amphenicol, an antibiotic active against 
various gram-positive and gram-negative 
bacteria, rickettsiae, and psittacosis group. 

Habitat: Different soils. 

Remarks: This organism is variable, re- 
sembling in some respects S. lavendulae, al- 
though Okami (1956) found it to be mark- 
edly different. Krassilnikov described a form 
under the name .1 . rectus, and a related form, 
A. rectus brunneus, which belong to this 
group. Gause et al. (1957) described a form 
as A. venezuelae var. spiralis. S. phaeochromo- 
genes var. chloromyceticus Okami is identical 
with S. venezuelae. 

Morais et al. ( 1958) described a variety of 
S. venezuelae as roseospori with a rose- 
colored rather than lavender aerial myce- 
lium, not producing any antibiotic and not 
chomogenic on organic media. 

Type species: IMRU 3534; ATCC 10,711'. 

237. Streptomyces verne (Waksman and 
Curtis, L916) Waksman and Henrici (Waks- 
man, S. A. and Curtis, Pi. E. Soil Sci. 1: 120, 
1910; 8: 156, L919). 

Morphology: According to Jensen (1931), 
sporophores are long, spiral-shaped. Spores 
spherical and oval. 

Sucrose nitrate agar: Growth abundant, 
spreading, lichenoid, glossy, yellowish, be- 
coming brownish. Capacity to produce aerial 
mycelium lost on cultivation. 

Glucose-asparagine agar: Growth abun- 
dant, lichenoid, center raised, gray with 



purplish tinge. No aerial mycelium. Soluble 
pigment faint brown. 

Nutrient agar: Colonies small, grayish, 
with depressed center, becoming wrinkled. 
Xo aerial mycelium. Xo soluble pigment. 

Potato: Growth wrinkled, cream-colored, 
becoming gray. Aerial mycelium absent or 
scant , white. 

Gelatin: Colonies small, cream-colored. 
Xo aerial mycelium. Soluble pigment green, 
a property lost on continued cultivation. 
Papid liquefaction. 

Milk: Ring pinkish-brown. Coagulation 
and rapid peptonization. 

Starch: Growth scant, restricted, brown- 
ish; hydrolysis rapid. 

Cellulose: Growth good. 

Nitrate reduction: Positive. 

Production of H 2 S: Negative. 

Temperature: Optimum 37°C. 

Antagonistic properties: Limited activity 
against some bacteria. 

Remarks: Soluble green pigment produced 
by freshly isolated cultures; in time, this pig- 
ment becomes brown. According to Ettlin- 
ger et al. (1958), this organism should be 
regarded as a strain of S. olivaceus. 

Type Culture: [MRU 3353. 

238. Streptomyces verticillatus (Kriss, 
1938) Waksman (Kriss, A. Mikrobiologiya 7: 
105-111, 1938). 

Morphology: Substrate mycelium pro- 
duced by monopodial branching. Aerial my- 
celium characterized by primary verticils 
produced on straight sporophores. The 
number of verticils at the proximal ends of 
the primary sterile hyphae is much larger 
than in the younger portions. Secondary 
verticils are also produced at the ends of the 
primary. Spores cylindrical and oblong, 1.0 
to 1.7 by 0.8 n. 

Sucrose nitrate agar: Aerial mycelium well 
developed, velvety, at lirst white, later dark 
gray or gray-green. 

Xutrienl agar: Growth brown. No aerial 
mycelium. Soluble pigment brown. 



282 



THE ACTINOMYCETES, Vol. II 



Potato: Soluble pigment brown. 

Gelatin: Rapid liquefaction. 

Milk: Coagulation and peptonization. 

Starch: Hydrolysis. 

Cellulose: No growth. 

Nitrate reduction: Rapid. 

Sucrose: Inversion. 

Production of H 2 S: Positive. 

Antagonistic properties: Weak. 

Habitat: Rhizosphere of wheat grown in a 
salinized soil. 

Remarks: ,4. verticiUatus viridans was de- 
scribed by Krassilnikov (1941) as a substrain 
of this organism. 

239. Streptomyces violaceoniger (Waksman 
and Curtis, 1916) Waksman and Henrici, 
1948 (Waksman, S. A. and Curtis, R. E. 
SoilSci. 1: 111, 1916). 

Synonym: S. violaceus-niger. 

Morphology: The sporogenous hyphae are 
frequently sterile. Sporophores monopodially 
branched. Waksman and Curtis (1916) re- 
ported no spirals, but Ettlinger et al. (1958) 
found compact spirals. Spores spherical and 
oval, 1.2 to 1.5 by 1.2 to 2.3 n, smooth (PI. 
II n). 

Sucrose nitrate agar: Growth at first dark 
gray, turning almost black. Aerial mycelium 
white to gray after the colony is well de- 
veloped. Soluble pigment at first bluish, later 
turning almost black. 

Potato: Growth at first very slight, but 
after 48 hours develops into continuous, 
thick yellowish-gray smear, which later 
turns brown, with white aerial mycelium 
covering the growth. Melanin-negative. 

Gelatin: Growth gray; no aerial mycelium. 
Liquefaction rapid. No change in color. 

Production of H 2 S: Negative. 

Antagonistic properties: Produces anti- 
biotic nigericin. 

Habitat: Soil. 

Remarks: According to Ettlinger et al. 
(1948) the color of the aerial mycelium is 
carmine-red to cinnamon-brown; with age, 
t he aerial mycelium liquefies and turns black. 



This organism was believed to belong to the 
S. hygroscopicus group. Nomi (1960) came 
to similar conclusions. 

240. Streptomyces violaceoruber (Waks- 
man and Curtis, 1916) Waksman (Waksman, 
S. A. and Curtis, R, E. Soil Sci. 1: 110-111, 
1916; 8: 160-163, 1919). 

This organism has an interesting history. 
In the original description of Waksman and 
Curtis (1916), it was listed in the text (p. 
110) as A. violaceus, the word "ruber" being 
left out due to poor proof-reading; in the 
key, however (p. 130), as well as in the 
following paper by Waksman (1919), in 
which a complete description was given, it was 
correctly listed as A. violaceus-ruber . The 
above error was unfortunately repeated in 
the first and second (p. 374) editions of 
Bergey's Manual. In the third edition of this 
manual (1930), Bergey himself changed the 
name of this organism to Actinomyces Waks- 
manii (p. 489). In the fourth and fifth (p. 
867) editions, it was changed to Actinomyces 
coelicolor (Miiller) Lieske, and finally in the 
sixth and seventh editions to Streptomyces 
coelicolor (Miiller) Waksman and Henrici. 
Only the recent studies in which both or- 
ganisms, S. coelicolor Miiller and S. violaceo- 
ruber Waksman and Curtis, were directly 
compared (Kutzner, 1956; Zahner and 
Ettlinger, 1957; Kutzner and Waksman, 
L959) demonstrated that they are distinctly 
different. 

There are marked physiological and bio- 
chemical differences between S. coelicolor 
and S. violaceoruber. They particularly in- 
clude differences in color and morphology of 
the aerial mycelium, antagonistic properties, 
and pigment production. S. coelicolor is ac- 
tive upon fungi and yeasts, as first shown by 
Miiller; several strains of S. violaceoruber 
produce antibacterial antibiotics, such as 
actinorhodin, coelicolorin, and mycetin. The 
pigment of S. coelicolor changes to green at 
an alkaline reaction, that of S. violaceoruber 
to blue. The nature of the pigment has been 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



28:-! 



studied by Conn (1943) and by Cochrane 
and Conn (1947). 

Various strains closely related to S. vio- 
laceoruber have been isolated all over the 
world; some have been listed as varieties, 
such as achrous and flavus (Cause et al., 
1957). Most of the strains now in the culture 
collections, designated as *S'. codicolor, ac- 
tually belong to S. violaceoruber. 

Type culture: Waksman and Curtis strain 
Xo. 3030, available in the IMRU culture 
collection. 
Synonyms : 

A. violaceus Waksman and Curtis 

(Waksman and Curtis, L916). 
.4. violaceus-ruber Waksman and Curtis 

(Waksman, L918). 
A. waksmanii Bergey (Bergey's Man- 
ual, 3rd ed. 1930). 
A. codicolor (Miiller) Lieske (Bergey's 
Manual, 4th and 5th ed., 1934, 1939). 
S. codicolor (Miiller) Waksman and 
Henrici (Bergey's Manual 6th and 
7th ed., 1948, 1957). 
.1. codicolor (Miiller) Krassilnikov 

(Krassilnikov, 1941). 
A. codicolor Krassilnikov (Cause et al, 
1957). 
Possible synonym: A. pluricolor Berest- 
new emend. Krassilnikov. 

Morphology: Aerial mycelium mono- 
podially branched; abundant formation of 
spirals with 3 to 8 turns, sinistrorse. Ac- 
cording to Naganishi and Xomi (1954), two 
or more sporulating branches may grow from 
the same spot on the main sporophore. 
Secondary branches may also be produced. 
Terminal branches are often arranged in 
clusters or umbellate forms. Terminal hy- 
phae carry many spirals. Spores spherical to 
oval, 0.7 to 1.0 by 0.8 to 1.5 n (PI. IV Ah). 
Surface of spores smooth. Asporogenous, 
nonpigmented strains can be obtained by 
plating out cultures on carbohydrate-free 
synthetic media containing nontoxic surface- 
acting agents (Erikson, 19551)). 



Sucrose nitrate agar: Substrate growth 
abundant, colorless al first, becoming red, 
then blue to dark blue. Aerial mycelium 
thin, powdery, white, becoming ash-gray, 
with a bluish tinge; on some media, light 
pink to cinnamon; sometimes blue drops can 
be observed on the surface of the aerial my- 
celium. Soluble rv{\ pigment on acid media, 
changing to dark blue as medium becomes 
alkaline. 

Glycerol-asparagine agar: Growth good, 
violet to deep blue. Soluble pigment diffuse- 
through medium. 

Glucose-asparagine agar: Growth poor; 
red pigment does not diffuse readily. 

Nutrient agar: Growth white, becoming 
red with white margin. Xo soluble brown 
pigment. Melanin-negative. 

Potato: Small, brownish, lichenoid colonies. 
Aerial mycelium white. Mycelium and plug 
gradually colored red and blue. 

Gelatin: Growth cream-colored, becoming 
pink or blue. Liquefaction slow. 

Milk: Gray surface ring, with red or blue 
tinge. Coagulation limited ; peptonization 
rapid. 

Starch agar: Growth pink. Hydrolysis 
rapid. 

Cellulose: Growth good. 

Nitrate reduction: Excellent. 

Sucrose: Inversion. 

Carbon sources: Utilizes L-xylose, L- 
arabinose, L-rhamnose, D-fructose, rafhnose 
(some strains only faintly), D-mannitol. Xone 
or poor utilization by most strains: sucrose, 
inulin. 

Antagonistic properties: Most strains do 
not show any strong antagonistic effect; 
several cultures, which seem to belong or are 
closely related to S. violaceoruber, produce 
coelicolorin, actinorhodin, streptocyanin, 
and mycetin. 

Habitat: Very common, especially in field 
soils. 

Remarks: Ettlinger et al. I L 958) considered 
S. violaceoruber, quite incorrectly, as a 



284 



THE ACTINOMYCETES, Vol. II 



strain of S. fradiae. Krassilnikov (1949) 
considered it as a synonym of S. coelicolor. 
Type culture: IMRU 3030. 

241. Streptomyces violaceus (Gasperini, 
1X91, emend. Krassilnikov) Waksman (Gas- 
perini, G. Centr. Bakteriol. Parasitenk., 
Abt. I 15: 084, 1894; Krassilnikov, X. A. 
Actinomycetales. Izvest. Akad. Xank. SSSR, 
Moskau, p. 15, 1941). 

Morphology: Aerial hyphae long, straight, 
seldom branching; also short, branched 



hyphae. Sporophores produce open, sinis- 
trorse spirals with 2 to 3 coils. Spores spheri- 
cal and oval (Fig. 48). 

Agar media: Substrate growth lichenoid, 
at tiist red, becoming red-blue, finally 
purple- violet. Some cultures produce fa I 
droplets in the colony, pigmented red or 
purple. Aerial mycelium white to gray, pro- 
duced poorly or not at all; some substrates, 
like cellulose, paraffin, or fats, favor forma- 
tion of aerial mycelium. Different pigments 





«4 
3 



i 



Figure 48. Sporophores and spores of >s\ violaceus, grown for II days on glucose-asparagine agar 
iop, hyphae); for S days on yeast-glucose agar (bottom left, sporophore); for 1 days on potato agar 
(bottom right, spores) x 13,500 (Reproduced from: Lechevalier, II. A. and Tikhonienko, A. S. Mikro- 
biologiya 29: 43-50, I960). 



)ESCRIPTION OF SPECIES OF STREPTOMYCES 



285 



arc formed in different media and under dif- 
ferent conditions of growth. Pigments dis- 
solved in medium do not change with 
reaction. 

Sucrose nit rale agar: ( rrowtb dark brown. 
Aerial mycelium white. Soluble pigmenl lie- 
comes violet to dark violet . 

Potato: Growth red-brown to la-own. 
Aerial mycelium, if present, white. Soluble 
pigmenl grayish-1 >r< >wn . 

Gelatin: Aerial mycelium white. Liquefac- 
tion slow. Soluble pigment gray-brown. 

Milk: Growth-grayish brown. Coagulation 
questionable; peptonization slow. 

Starch: Hydrolysis weak. 

Cellulose: Growth weak, violet. Aerial 
mycelium light gray. 

Nitrate reduction: Positive. 

Sucrose: Rapid inversion. 

Pigment: According to Kriss (1936), the 
pigmenl is soluble in water and in 96 per 
cent alcohol. 

Melanin: According to Hoffmann (1958), 
this species is melanin-positive. 

Antagonistic properties: Exerts strong 
antagonistic effect upon various gram-posi- 
tive bacteria. 

Remarks: According to Krassilnikov, this 
species includes .1. violacea Rossi-Doria, .1. 
violaceus-caesari Waksman and Curtis, Ac- 
tinomyces 103 and 109 Lieske; also A. 
incanescens Wollenweber and A. brasiliensis 
Lindenberg (the last is probably a Nocardia). 
A subspecies, A. violaa us chromogenes is also 
included. Some of the cultures described by 
Gause et al. (1957) may also be included 
here, such as .1. lateritius, A. roseoviolaceus, 
A. violaceorectus, A. viridoviolaceus, and .1. 
violaceus var. mhcscens. 

Type culture: [MRU 3497. 

242. Streptomyces virgatus (Krassilnikov) 
Waksman (Krassilnikov, X. A. Actino- 
mycetales. Izvest. Akad. Xauk, SSSIi. 
Moskau, p. 32, 1941). 

Morphology: Sporophores short, in form 



of tufts. Spirals produced rarely. Spore- 
cylindrical, elongated ; in some st rains round 
to oval. 

Agar media: Substrate growth yellow- 
green to citron-yellow or pure yellow; on 
some media pale green. Pigment insoluble. 
Some strains produce a brown substance in 
protein media. Aerial mycelium weakly de- 
veloped, white or pale yellow . 

( lelatin: Liquefaction rapid. 

Milk: Coagulation and peptonization 
rapid. 

Starch: Hydrolysis rapid. 

Cellulose: Xo growth. 

Sucrose: Inversion. 

Nitrate reduction : Positive. 

Antagonistic properties: None. 

Habitat: Soil. 

243. Streptomyces virginim Grundy et r//., 
1952 (Grundy, W. E., Whitman, A. L., 
Rdzok, E. .1., Hanes, M. Iv, and Sylvester, 
.1. C. Antibiotics A: Chemotherapy 2: 399 

408, 11)52). 

Morphology: Sporophores usually 
straight ; occasionally spirals are observed at 
or near the tips of the hyphae. Spores cylin- 
drical, 1.1 to 1.5 by 0.75 to L.0/i. 

Sucrose nitrate agar: Growth sparse, 
cream-colored. Aerial mycelium light gray- 
ish-lavender. Xo soluble pigment. 

Glucose-asparagine agar: Growth sparse, 
cream-colored to light brown. Xo aerial my- 
celium. 

Calcium malate agar: Growth abundant, 
cream-colored. Aerial mycelium white, be- 
coming tinged with grayish-pink to lavender. 

Nutrient agar: Growth sparse, white, 
turning cream-colored to light brown. Aerial 
mycelium white, turning light grayish-pink 
to lavender. Soluble pigment light brown. 

Oatmeal agar: Growth abundant, cream- 
colored, turning golden brown. Aerial my- 
celium abundant, light rose, turning lavender 
and gray. Soluble pigmenl pale yellow, turn- 
in"; light brown. 



286 



THE ACTINOMYCETES, Vol. II 



Starch agar: Growth thin, colorless. Aerial 
mycelium rose to lavender-colored. Hy- 
drolysis. 

Potato: Growth abundant, spreading, 
brownish. Aerial mycelium grayish-pink to 
lavender. Browning of the potato. 

Gelatin: Surface pellicle gray to brownish. 
Aerial mycelium thin, white. Soluble pig- 
ment brown. Liquefaction slow. 

Milk: Growth brown. Coagulation none; 
peptonization slow. Milk becomes dark gray- 
brown or black. 

Nitrate reduction: Limited or absent. 

Production of H 2 S: Positive. 

Carbon utilization: Utilizes glucose, man- 
nose, galactose, maltose, starch, glycerol, 
sodium acetate, sodium citrate. Does not 
utilize xylose, lactose, sucrose, mannitol, 
sorbitol, potassium sodium tartrate. 

Antagonistic properties: Produces an 
antibiotic, actithiazic acid. 

Remarks: Various related organisms have 
been listed. It is sufficient to mention A. 
gobitricini, A. roseolus, A. syringini, and A. 
roseolilacinus, described by Cause el al. 
(1957). 

Type culture: IMRU 3051. 

244. Streptomyces viridans (Krassilnikov, 
1 ( .)41) (Krassilnikov, X. A. Actinomycetales. 
[zvest. Akad. Xauk. SSSR, Moskau, 1941). 

Morphology: Sporophores branched, spi- 
ral-shaped. Spores cylindrical. 

Glycerol nitrate agar: Growth olive-green 
with soluble green pigment. Aerial mycelium 
dark gray, olive-colored, or gray-green, vel- 
vety, covering the whole growth. 

Nutrient agar: Growth brown-green. Sol- 
uble pigment brownish. Melanin-negative. 

Potato: Growth brown. Aerial mycelium 
light gray. Soluble pigment olive-green 
(Hoffmann, 1958). 

Gelatin: Rapid Liquefaction. 

Milk: Xo coagulation; rapid peptoniza- 
tion; soluble brown pigment. 

Starch: Hydrolysis rapid. 

Cellulose: Growth poor. 



Nitrate: Reduction to nitrite. 

Sucrose: Inversion rapid. 

Antagonistic properties: None; some 
si rains are weakly active. 

Remarks: Related to S. intermedins. 
Drechsler (1919) described two similar 
strains, Xos. X and XIV. Gause et al. 
( 1957) described a related strain as A. roseo- 
viridis. 

245. Streptomyces viridis (Lombardo-Pel- 
legrino, 1903) Waksman (Lombardo-Pelle- 
grino, P. Riforma med. 39: 1005-1067, 1903. 
Summarized by Baldacci, E. Atti ist. botan. 
"Giovanni Briosi" e lab. crittogam. nniv. 
Pavia (Ser. IV) 11: 221-223, 1939). 

Morphology: Sporophores long or short, 
straight, undulated; frequently producing 
broom-shaped clumps. Spores ovoid, 0.7 to 
1 .4 n in diameter. 

Agar media: Substrate growth on all media 
at first hyaline, later turning green to dark 
green. Soluble pigment green. The cultures 
also grow under anaerobic conditions, but 
produce no soluble pigment. Aerial mycelium 
on all media cottony, whitish to grayish. 

Potato: Growth dark violet. Aerial my- 
celium white. Melanin-negative. 

Production of H 2 S: Positive. 

Antagonistic properties: Not reported, or 
negative. 

Habitat: Soil. 

Remarks: Baldacci and Comaschi (1956) 
concluded that the culture described by 
Krainsky (1914) as .1. griseus belongs more 
accurately to the S. viridis series. According 
to Hoffmann (1958), the A. griseus Krainsky 
appears to belong to this group, although he 
refers to it as .1. griseus Krassilnikov. It is 
said to produce broom-shaped sporophores 
with spirals. Growth colorless, turning light 
brown. Aerial mycelium velvety, light gray 
turning dark dray. Xo soluble pigment. 
Melanin-negative. Growth on potato lichen- 
oid. Milk not coagulated, but peptonized. 
Gelatin liquefied. Starch hydrolyzed. It 
grows on cellulose. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



L'ST 



Certain other forms belonging to this 
group have been described, such as .1 . griseus 
variabilis and A. griseus zonatus of Krassil- 
nikov (1949), .1. badius and A. malachiticus 
of Gause et al. (1957). Krassilnikov also 
listed viridis sterilis as a strain that lost the 
capacity to produce aerial mycelium. 

Millard and Burr (1926) described, under 
A. viridis, an organism thai produces dark 
to black growth on sucrose-nitrate agar, 
with a mouse-gray aerial mycelium, gradu- 
ally becoming black. On nutrient agar, 
growth is at first colorless, gradually becom- 
ing gray; aerial mycelium gray to mouse- 
gray. On gelatin, it produces a thin colorless 
growth and a faint brownish pigment; rapid 
liquefaction. 

Duche ( 1934) described an organism under 
the name .4. viridis; he later changed this 
name to .1. baarnensis. This organism was 
isolated as a contaminant of cultures of S. 
albus and S. lavendulae. 

Type culture: [MRU 3372 (strain of 
Millard and Burr). 



246. Streptomyces viridochromogenes (Kra- 
insky, L914) Waksman and Henrici, L948 
(Krainsky, A. Centr. Bakteriol. Parasitenk. 

Abt. II., II: 684 lis;,, Mil l,. 

Morphology: Sporophores monopodially 
branched, with numerous open or compact, 
sinistrorse spirals, :; to 5 M in diameter, oc- 
curring as side branches. Spores short, oval 
or spherical, 1.25 to 1.5 n (Figs. 49-51); sur- 
face covered with long spines (PI. II k). 

Sucrose nitrate agar: Growth cream-col- 
ored with dark center, becoming dark green; 
reverse yellowish to light cadmium. Aerial 
mycelium white, becoming light green to 
light blue. 

Glucose-asparagine agar: Growth abund- 
ant, spreading, wrinkled, gray, becoming- 
black. Aerial mycelium appeal's late; white, 
later becoming green to light blue. 

Nutrient agar: Growth restricted, gray, 
with greenish tinge. Xo aerial mycelium. 
Soluble pigment brown. 

Potato: Growth abundant, gray-brown. 




Figure 49. Chains of spores of S. viridochromogenes, grown for 10 days on glucose asparagine-CaCh 
agar, X 13,500 (Reproduced from: Lechevalier, H. A. and Tikhonienko, A. S. Mikrobiologiya 2<>: 13 :.n 
1960). 



THE ACTIXOMYCKTKS. Vol. II 




Figvre 50. S. viridochromogenes grown for 30 
Lechevalier, H. A. and Tikhonienko, A. S. Mikrol 



days on potato agar, X 13,500 (Reproduced from: 
iologiya 29: 43-50, 1960). 



Aerial mycelium white. Soluble pigment 
black. 

Gelatin: Surface growth cream-colored, 
becoming greenish. Positive liquefaction. 
Soluble pigment brown. 

Milk: Surface growth dark brown; coagu- 
lation and peptonization. 

Starch agar: Colonies circular, spreading, 
yellowish. Hydrolysis. 

Cellulose: No growth. 

Sucrose inversion: Positive. 

Nitrate reduction: Positive. 

Production of H2S: Positive. 

Tyrosinase reaction: Positive. 

Temperature: Optimum 37°C. 

Antagonistic properties: Active upon 
fungi. 

Habitat: Soil. 



Remarks: This group occurs abundantly 
in nature. Gause et al. (1958) described 
various forms under different names, such 
as A. bicolor, A. coeruleofuscus, A. coeruleo- 
rubidus, and A. coerulescens, and a variety, 
longisporus. According to Ettlinger et al. 
(1958), S. chartreusis also belongs to this 
group. 

247. Streptomyces riridoflavus Waksman 
and Taber, \ { .)»:] (Waksman, S. A. and 
Lechevalier, H. A guide to the classification 
and identification of the actinomycetes and 
their antibiotics. The Williams & Wilkins 
Co., Baltimore, 1953, p. 66). 

Not A . viridiflavus Duche. 

Morphology: Sporophores formed in fas- 
cicles; t nfts, with some curling of tips, are 
produced on certain media. Tendency 1<> lose 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



289 




Figure 51. Sporophores of S. viridochromogenes, X 30,000 (Courtesy of E. Baldacci, University of 
Milan. Italy). 



property of producing aerial mycelium. Sub- mycelium usually absent. No soluble pig- 
merged sporulating lateral branches form ment. 

single spores at the tips. Glucose nutrient agar: Growth lichenoid. 

Sucrose nitrate agar: Growth limited, yellowish-brown to olive-brown. Aerial my- 

cream-colored to yellowish green. Aerial celium abundant, later covering the whole 



290 



THE ACTINOMYCETES, Vol. II 



surface of growth with a mat, yellowish to 
gray. Soluble pigment brownish or absent. 

Glucose-asparagine agar: Growth moist, 
yellow to yellow-green. Aerial mycelium 
abundant, grayish-yellow to sulfur-yellow, 
later overgrown by white sporulating hy- 
phae. Soluble pigment absent or faint yellow. 

Nutrient agar: Growth moist, gray to 
light green with green to almost bluish tinge 
at bottom of slant. Nonsporulating aerial 
mycelium appears much later; it is white to 
gray. No soluble pigment. 

Potato: Growth lichenoid, brownish to 
greenish-yellow to dark olive-green. Aerial 
mycelium absent, or formed as thin, yelkrw- 
ish layer on drier portions of growth. Soluble 
pigment absent or dark brown. 

Gelatin: Growth in form of surface ring, 
canary-yellow. Slight liquefaction. Soluble 
pigment brown to dark brown, a property 
that may be lost on cultivation. 

Starch : Hydrolysis. 

Cellulose: Limited growth, no destruction 
of cellulose. 

Production of H 2 S: Negative. 

Carbon utilization: No growth with su- 
crose, lactose, or rhamnose; good growth on 
mannose and glucose. 

Antagonistic properties: Produces an anti- 
fungal substance, candidin. 

Habitat: Soil. 

Type culture: IMRU 3685. 

248. Streptomyces viridogenes (Millard and 
Burr, 1926) Waksman (S. viridis of Millard, 
W. A. and Burr, S. Ann. Appl. Biol. 13: 
580, 1926). 

Morphology: Sporophores long, sympodi- 
ally branched, straight. Spores spherical, 
0.9 M , smooth (PI. II o). 

Sucrose nitrate agar: Growth abundant. 
Aerial mycelium olive-gray. Soluble pigment 
greenish-yellow to blackish-green. 

Glucose-asparagine agar: Growth smooth, 
raised, olive-buff. Aerial mycelium abund- 
ant, light gray to deep mouse-gray. Soluble 
pigment yellowish to greenish-yellow. 



Nutrient agar: Growth lichenoid, cream- 
colored. No aerial mycelium. No soluble 
pigment. 

Nutrient agar with glucose: Growth gray 
to black. Aerial mycelium gray. Soluble pig- 
ment dark brown. 

Starch agar: Growth gray to brown. Aerial 
mycelium thin, white. Hydrolysis positive. 

Potato: Growth gray to olive-gray. Aerial 
mycelium either absent or white, turning 
gray. Soluble pigment brown. 

Gelatin: Growth grayish. Aerial mycelium 
scant, white to gray. Liquet' action rapid. 
Soluble pigment light golden brown. 

Milk: Surface growth good. Aerial my- 
celium scant, white. Coagulation rapid and 
peptonization gradual. 

Nitrate reduction: Positive. 

Tyrosinase reaction: Negative. 

Temperature: Grows well at 37.5°C. 

Habitat: Potato scab. 

Remarks: Ettlinger et al. (1958) reported 
that this species is melanin-negative. 

249. Streptomyces wedmorensis (Millard 
and Burr, 1926) Waksman (Millard, W. A. 
and Burr, S. Ann. Appl. Biol. 13: 580, 1926). 

Morphology: Sporophores straight, 
branched. Spores oblong, 0.8 to 0.9 by 0.6 to 
0.8/x- 

Sucrose nitrate agar: Growth flat, thin, 
grayish. Aerial mycelium white to gray. 

Nutrient potato agar: Growth wrinkled, 
grayish. No aerial mycelium. Melanin- 
negative. 

Potato: Growth wrinkled, grayish. Aerial 
mycelium white. Plug pigmented drab. 

Gelatin: Growth fair. No aerial mycelium. 
Liquefaction medium. 

Milk: Growth greenish. Coagulation and 
slow peptonization. 

Starch: Hydrolysis. 

Nitrate reduction: Positive. 

Tyrosinase reaction: Negative. 

Temperature: Grows well at 37.5°C. 

Habitat : Potato tubers. 



DESCRIPTION OF SPECIES OF STREPTOMYCES 



291 



•_'.')(). Streptomyces willmorei (Erikson 
L935) Waksman and Eenrici, L948. (Erik- 
son, I). Med. Research Council (Brit.) 
Spec. Rept. Ser. No. 203: 19 20, L935). 

Morphology: Submerged growth con- 
sists of unicellular mycelium frequently 
branched at short intervals, presenting pe- 
culiar clubbed and budding forms with 
occasional separate, round, swollen cells. 
The filaments are characteristically long 
homogeneous, and much interwoven. Aerial 
mycelium profuse in most media, with a 
marked tendency to produce 1 loose spirals 
with chains of ellipsoidal spores. Thick aerial 
clusters may also he formed. 

Glucose-asparagine agar: Growth color- 
less, wrinkled, confluent, with smooth entire 
margin; large discrete colonies like flat 
rosettes. Aerial mycelium scant, white. 

Glycerol nitrate agar: Round, smooth, 
cream-colored colonies, heavy texture, mar- 
gin submerged. Stiff, sparse aerial spikes. 

Peptone-beef extract or nutrient agar: 
Growth heavy, colorless, lichenoid, rounded 
elevation covered with white aerial myce- 
lium. Later, submerged margin, round con- 
fluent growth; aerial mycelium marked in 
concentric zones. 

Potato agar: Fair growth, partly sub- 
merged. Aerial mycelium grayish-white. 

Gelatin: Colonies minute, colorless. Posi- 
tive liquefaction. 

.Milk: Coagulation and slow peptonization. 

Dorset's egg medium: Large, round, color- 
less, scale-like colonies, radially wrinkled, 
later growth brownish; medium discolored. 

Serum agar: Smooth colorless discoid col- 
onies; marked umbilication after 2 weeks. 

Production of H 2 S: Negative. 

Antagonistic properties: Positive. 

Source: Streptothricosis of liver. 

Remarks: Ettlinger et al. (1958) place 
this species in the S. griseus series. 

Type culture: [MRU 3332. 

251. Streptomyces xanthophaeus Linden- 



,bein, 1952 (Lindenbein, W. Arch. Mikro- 
l.iol. 17: 361 383, L952). 

Morphology: No description. 

Glycerol nitrate agar: Growth brownish. 
Aerial mycelium white-gray or reddish-gray. 
Soluble pigment yellow-brown. 

Glycerol malate agar: Growth deep 
orange. Aerial mycelium white-gray to red- 
gray. Soluble pigment dee]) orange. 

Glucose-asparagine agar: Growth diffuse, 
light yellow. Aerial mycelium white. Solu- 
ble pigment light yellow. 

Nutrient agar: Growth light brown. Aerial 
mycelium ash-gray to white. Soluble pig- 
ment yellow to yellow-brown. Melanin- 
negative. 

Glucose-peptone agar: Growth light yel- 
low. Aerial mycelium ash-gray. Soluble pig- 
ment yellow. 

Starch agar: Growth lichenoid. Aerial 
mycelium violet -gray. Hydrolysis rapid. 

Potato: Growth lichenoid. Aerial myce- 
lium gray. No soluble pigment. 

Gelatin: Growth In-own. Aerial mycelium 
ash-gray. Soluble pigment yellow-brown. 
Liquefaction strong. 

Milk: Growth lichenoid. Aerial mycelium 
gray to violet. Soluble pigment dark brown. 
Peptonization strong. 

Cellulose: No growth. 

Antagonistic properties: Produces geo- 
mycin, active against gram-negative bac- 
teria. 

Habitat: Limestone deposit in Germany. 

Remarks: Related to S. erythraeus and 
S. erythrochromogenes. Kutzner (1956) stud- 
ied five soil isolates. Four strains did not 
form any spirals; one did. The spores were 
smooth. The soluble pigment on glucose- 
peptone agar was dark brown. He thus con- 
sidered this species as melanin-positive. 

Addt ikI urn 

After the text of this volume was com- 
pleted, the following newly described forms 
appeared in print: 

Streptomyces aerocolonigenes Shinobu and 



292 



THE ACTINOMYCETES, Vol. II 



Kawato (Botan. Mag. Tokyo 73: 212-216, 
1960). 

A ctinomyces aureoverticillatus Krassilnikov 
and Dzi-Shen (Mikrobiologiya 29: 482-489, 
1960). 

Streptomyces herbaricolor Kawato and 
Shinobu (Mem. Osaka Univ. B 8: 114-119, 
1959). 



Streptomyces massasporeus Shinobu and 
Kawato (Botan. Mag. Tokyo 72: 853-854, 
1959). 

Streptomyces ostreogriseus (Antibiotic 
E-129) Brit. Pat. 799,053, July 30, 1958. 

Streptomyces psammoticus Virgilio and 
Hengeller (Farmaco, Ed. Sci. 15: 164-174, 
1960). 



Chap t e r 9 



The Genus Micromonospora 



The genus Micromonospora is character- 
ized by the production in nutrienl media of a 
well developed substrate mycelium, 0.2 to 
0.6 n in diameter, partly penetrating into 
the medium. The substrate or vegetative 
hyphae arc straight or curved, branching, 
without cross walls. Aerial mycelium is not 
formed at all or only in a rudimentary, non- 
sporulating form, when the hyphae arise 
upward directly from the substrate myce- 
lium. 

Multiplication occurs by means of frag- 
ments of mycelium and special spores formed 
singly. A swelling takes place at the end of 
the sporophore; later the swelling is sepa- 
rated by a cross wall, giving rise to spherical, 
oval, or oblong spores, 1.0 to 1.5 by 0.8 to 
1.2 m- The sporophores are often branched, 
each branch forming a spore at the end, 
giving rise to a grape-like bunch of spores. 
These germinate in a manner similar to the 
spores of Streptomyces. The mycelium and 
>poivs are gram-positive, not acid-fast (Fig. 
52). 

The colonies of Micromonospora are simi- 
lar to those of Streptomyces. They are com- 
pact, leathery, smooth or lichenoid, raised or 
flat. They are frequently colored red or 
orange or yellow, occasionally brown or green 
To almost black or bine. The pigments, ex- 
cept the dark brown, are not dissolved into 
the medium. 

In characterizing species of Micromono- 
spora, T'ao Bo and Potter (1960) considered 
morphological properties as primary criteria 
for identification of the organisms. The 



important physiological characteristics in- 
cluded the disintegration of cellulose, in- 
version of sucrose, and the reduction of 
nitrate. The investigators emphasized that 
the color of the growth and the form of the 
colony could not serve as basic characteris- 
tics. Certain strains may show different 
colors for the mass of growth and for the 
spores. Reproducibility of colony color for a 
given organism could not be obtained on the 
same medium. The color itself was not con- 
sistent, varying through every shade of yel- 
low, orange, pink, red, and brown. Many 
species gave more than one colonial form. 
The large spores of M. globosa were very 
helpful in differentiating it from .1/. fusca or 
M. chalcea. 

Micromonospora species are aerobic or 
anaerobic, and mesophilic. They grow- 
readily at 25-40°C. Thermal death point of 
the mycelium is 70°C in 2 to 5 minutes; 
spores resist 80°C for 1 to 5 minutes. They 
utilize various carbon and nitrogen sources, 
both organic and inorganic (Fig. 5:!). 

The type Species is Micromonospora chair, a 

(Foulerton) 0rskov. 

The genus Micromonospora comprises nine 
species, which can be classified as follows: 

Classification of the genus Micromonospora 

A. Aerobic. 

I. Sporophores lnn^- 

1. Sporophores showing Hi lie branching, 
a. No aerial mycelium. 

'_'. Micromonospora chalcea 
1). Rudimentary aerial mycelium. 
(i. Micromonospora gallica 



294 



THE ACTINOMYCETES, Vol. II 



2. Sporophores form extensive branching. 

a. Growth colorless; brown spores 
appear in mass. 

7. Micromonospora globosa 

b. Growth pigmented. 

a 1 . Growth green to dark green. 
a 2 . Spores blue. 

3. Micromonospora coerulea 
b 2 . Spores black or brown. 

1. Micromonospora bicolor 
b 1 . Growth pink to orange-colored. 
a 2 . Pigment not excreted into 
substrate. 

8. Micromonospora parva 

I) 2 . Red-brown pigment excreted 
into substrate. 
5. Micromonospora fusca 
II. Sporophores short. 

1. Growth brown; spores dark brown. 

4. Micromonospora elongata 
B. Anaerobic. 

9. Micromonospora pro- 
pionics 

Various other micromonosporas have been 
observed in natural substrates, but either 
have not been isolated or only insufficiently 



f 




***»••» *> 



% 



to 



Figure 52. Micromonospora (clumps of spores) 
growing in a compost. 




;« 



Figure 53. Micromonospora (double spores) 
growing in a compost. 

studied. This is true, for example, of the 
cellulose-decomposing, facultative anaerobic 
form studied by Meyer, 1934 (Prevot, 1955); 
and of M. cabaelli Maquer and Comby 
(Prevot, 1955). It is also true of some of the 
forms reported by Waksman et al. (1939). 
Some of the micromonosporas (M. mono- 
spora and M. vulgaris) have been placed, 
because of their temperature optima, among 
the thermophilic forms. 

Description of Micromonospora Species 

1. Micromonospora bicolor Krassilnikov, 
1941 (Krassilnikov, N. A. Actinomycetales. 
[zvest. Akad. Nauk. SSSR, 1941, p. 131). 

Morphology: Sporophores long, branch- 
ing, 10 to 25 /x; spores oval, 1.0 to 1.2 by 
0.8 p. 

Synthetic agar: Growth green, smooth; 
covered with a dark brown to black hue of 
spore-bearing hyphae. Pigment insoluble. 

Nutrient agar: No growth. 

Potato: No growth. 

Gelatin: Xo growth. No liquefaction. 

Milk: Unchanged. 

Starch: Not liquefied. 



THE GENUS MICROMONOSPORA 



205 



Sucrose: Inverted. 

Cellulose: Good growth and decomposi- 
tion. 

Carbon sources: ( rlucose, sucrose, levulose, 
acet ic and cit ric acids. 

Nitrogen sources: Ammonium salts and 
nitrates. 

Habitat : Soil. 

2. Micromonospora chalcea (Foulerton, 
1905) Orskov, L923 (Foulerton, A. Lancet 
1: 1200, 1005; 0rskov, J. Investigations into 
the morphology of the ray fungi. Levin and 
Munksgaard, Copenhagen, 1923). 

Morphology: Grows well on all media, 
especially on glucose-asparagine agar. 
Growth heavy, compact, raised, pale pink to 
dee]) orange 1 , not spreading much into the 
medium. Hyphae long, thin, branching, 
nonseptate. Surface of growth smooth or 
folded, dull or shining. Spore layer well de- 
veloped, moist and glistening, brownish- 
black to greenish-black; color sometimes 
spreading through the whole mass of growth. 
Spores oval or spherical, formed individually 
on relai ively nonbranching sporophores (Fig. 
54). 

Gelatin: Liquefaction positive. No soluble 
pigment. 

Milk: Coagulation and peptonization 
positive. 

Starch: Hydrolyzed. 

Cellulose: Rapid decomposition. 

Chitin: Decomposed. 

Nitrate reduction: Positive. 

Sucrose: Inverted. 

Proteolytic action: Strong. 

Temperature: Optimum for growth, 30- 
35°C. Thermal death point of mycelium, 
70°C in 2 to 5 minutes. Spores resist 80°C 
for 1 to 5 minutes. 

Source: Soil, lake mud, and other sub- 
strates. 

.3. Micromonospora coerulea Jensen, 1032 
(Jensen, II. Proc. Linnean Soc. X.S. Wales 
57: 173, 1032). 

Morphology: Growth smooth, lustrous, 



' 



■ \ 

J : 






; 






f f ■ - 






Figure 54. Micromonospora (single spores) 
growing in a compost . 

greenish-blue; pigmentation only on free 
admission of oxygen. Pigment insoluble. 
Surface of colonies hard and glossy; thin, 
white veil on surface resembles aerial my- 
celium. Spherical blue spores produced 
on branching short sporophores. 

Nutrient media: Slow growth. 

Liquid media: Growth at bottom, in the 
form of firm, round, white to pink granules. 

Gelatin liquefaction: Rapid. 

Milk: Positive coagulation, but very slight 
peptonization. 

Starch: Hydrolyzed. 

Cellulose: Not decomposed. 

Nitrate reduction: None. 

Sucrose: Not inverted. 

Source: Occurs rarely in soil. 

4. Micromonospora elongata Krassilnikov, 
1941 (Krassilnikov, X. A. Act inomycet ales. 
Izvest. Acad. Nauk. SSSR, 1041, p. 130). 

Morphology: Sporophores short (2 to 3 
/x), little branched. Spores oval, 1.0 to 1.:! by 
0.8 M (Fig. 55). 

Agar media: Growth poor, adhering to 
substrate in form of minute pale yellow 
smooth colonies. Surface is dark brown. 

Potato, gelatin, and milk : No growth. 



296 



THE ACTINOMYCETES, Vol. II 






t 






Figure 55. Micro, 
growing in a compost. 



ipom (single sp 



Cellulose: Good growth and decomposi- 
tion. 

Sucrose: Inverted. 

Nitrate reduction: Positive. 

Habitat: Soil. 

5. Micromonospora fusca Jensen, L932 
(Jensen, H., Proc. Linnean Soc. N.S. Wales, 
57: 178, 1932). 

Morphology: Growth heavy, compact, 
orange-colored, rapidly changing to deep 
brown and nearly black. Spore layer moist, 
glistening, grayish to brownish-black. Spores 
oval or spherical. 

Gelatin: Liquefaction slow. Soluble pig- 
ment very slight. 

Milk: No coagulation, slow peptonization; 
grayish-brown discoloration of milk. 

Starch: Hydrolyzed. 

Cellulose: Limited decomposition. 

Nitrate reduction: Positive or negative. 

Sucrose: Inverted. 

Antagonistic properties: Produces the 
antibiot ic micromonosporin. 

Source: Soil. 

('). Micromonospora gallica (Erikson, L935) 
Waksman (Erikson, I). Med. Pes. Council, 
London, Pub. No. 4582, L935, p. 24). 

Morphology: Aerial hyphae retarded and 
rudimentary. Typical single spores produced. 

Sucrose nitrate 1 agar: No growth. 

( Uycerol agar: No growth. 



( Hucose agar: No growth. 

Potato agar: Growth pale pink, moist, 
granular. 

Potato: Growth slow in form of pink, 
translucent colonies, tending to become 
umbilicated and heaped up. 

Egg media: Colonies minute, becoming 
confluent , tangerine-colored. 

Blood agar: Colonies minute, discrete, 
pink. No hemolysis. 

Gelatin: Growth scant, irregular, pink. 
Liquefaction slow. 

Broth: Pinkish flakes. Small rounded red 
granules at bottom. 

Milk: Surface ring yellowish-pink. Positive 
coagulation and peptonization. 

Habitat: Isolated from blood culture. 

7. Micromonospora globosa Krassilnikov, 
L939 (Krassilnikov, X. A. Mikrobiologiya 8 : 
179, 1939; Actinomycetes. Izvest. Akad. 
Nauk, SSSP, Moskau, 1941, p. 129). 

Morphology: Spores spherical, 1.0 m, ar- 
ranged in clusters on long branching sporo- 
phores. 

Agar media: Growth at first colorless, 
leathery, lichenoid; covered with a dark 
brownish tarnish of spores. 

Gelatin: Liquefaction slight. 

Milk: Coagulated and peptonized. 

Starch: Hydrolyzed. 

Cellulose: Satisfactory growth. 

Nitrate reduction: Positive. 

Sucrose: Inverted. 

Source: Soil. 

8. Micromonospora parva Jensen, L932 
(Jensen, H. Proc Linnean Soc. X.S. Wales 
57: 177, 1932). 

Morphology: Growth pale pink to orange, 
compact. Substrate mycelium thin, spread- 
ing widely into the agar. Sporulation scant, 
giving rise to thin, grayish, moist crusts on 
the surface. Spores oval; in mass gray- 
colored. 

Gelatin: Liquefied slowly. 

Milk: Unchanged; or coagulated and 
slowly peptonized with faintly acid reaction. 

Starch: Hydrolyzed. 



THE GENUS MICROMONOSPORA 



297 



Cellulose: No decomposition. 

Nitrate reduction: None. 

Sucrose: Not inverted. 

Source: Soil. 

tt. Micromonospora propionica Hungate, 
L946 (Hungate, R. E. J. Bacteriol. 51 : 51 
56, L946). 

Morphology: Grows very slowly. White 
spherical colonies produced on cellulose. 
Colony consists of gradually expanding hol- 
low shell, its outer surface consisting of sub- 
strate hyphae, the adjacent inner portion 
containing numerous spores, and the center 
relatively devoid of protoplasm. 



iplex media required. 
>wth. ( rood decomposi- 



tion 
ui'ce 



: Obligate 

( llucose a 



taerobe. 

! cellulose are 



Media: Eighty 

Cellulose: Cood 

tion. 
( >xyg< 

Carl., 
utilized. 

Fermentation product 
acetic and propionic acids. 

Temperature: 30-40°C. 

Habitat : Cut of termites, rumen of cattle. 

Remarks: Prevot (1957) considers this 
species as an anaerobic Actinomyces, which 
he includes in the genus Actinobacterium. 



Carbo 



Chapter 10 



The Genus Waksmania {Microbispora) 



The genus Waksmania (Microbispora) is 
characterized by the paired spores produced 
on aerial hyphae (Fig. 56). 

The fine mycelium (about 1 n in diameter) 
is differentiated into (a) primary or substrate 
mycelium which grows into, and forms a 
compact layer on top of agar media, and (b) 
secondary or aerial mycelium which arises 
from the primary mycelium but grows into 
the air, away from the agar surface. The 
substrate mycelium does not bear spores of 
any type; the aerial mycelium bears spores 
which are formed in longitudinal pairs. The 
spores are produced either directly on the 
aerial hyphae or on sporophores which 
branch from the aerial hyphae. 

The sporophores may be so short that the 
spores appear to be produced directly on the 
mycelium. The aerial mycelium forms a bud 
at the side, and later the bud, or occasionally 
the tip of the side branch, swells and is 
separated by a cross wall giving rise to two 
spherical or oval conidia, 1.4 to 1.6 m in 
diameter (Fig. 57). 

The germination of the spores and the 
structure of substrate mycelium are similar 
to those of Streptomyces. 

The type species is Waksmania rosea 
Lechevalier and Lechevalier. 

Waksmania rosea Lechevalier and Leche- 
valier, 1957 (Lechevalier, M. P. and Leche- 
valier, II. J. Gen. Microbiol. 17: 104-111, 
L957). 

Synonym: Microbispora rosea Xonomura 
and Ohara, 1957 (Nonomura, H. and Ohara, 
V., .1. Ferm. Technol., 35: 307 -311, 1057). 

Morphology: The dominant form of this 



organism on some media, after 14 days at 
30°C, may consist of chlamydospores. 
Hyphae do not segment, even in old cultures. 
"Fairy rings," or alternating areas of aerial 
mycelium with zones which have none, occur 
on some media. "Coremia-like" aggregations 
of aerial hyphae are formed on a variety of 
substrates. Small branches are produced 
monopodially in respect to the main axis of 
the aerial sporogenous hyphae. Spores are 
borne at the tip of these branches or sporo- 
phores. Spores are spherical, 1.5 to 2.0 m, 
usually about 1.7 to 1.8 m- Spores are borne 
terminally on sporophores, as well as at the 
tips of the main sporogenous hyphae and 
branches. They are also borne directly on 
the sporogenous hyphae. Spores are formed 
in pairs and, when mature, are very easily 
detached from the sporophore and from each 
other. 

Agar media: Growth slow on nutrient 
agar. Color of substrate growth pale pink to 
coral-orange, changing to chest nut -brown. 
Diffusible pigment very slight. Aerial myce- 
lium white, powdery, with tendency to form 
fairy rings on some media. 

Sucrose nitrate agar: Growth thin, yel- 
lowish-white. Aerial mycelium scant. Some 
malformed spores appear. 

(ilucose-asparagine agar: Growth meager, 
colorless. No aerial mycelium. 

Yeast-glucose agar: Growth white-tan, 
glistening, becoming dark brown and con- 
voluted. Xo aerial mycelium. 

Oatmeal agar: Orange-pink vegetative 
growth. Aerial mycelium white, with light 
pink spores. Earthy odor. 



2! IS 



THE GENUS WAKSMANIA {MICROBISPORA 



2! Hi 



Potato: Growth dark reddish-brown. 
Aerial mycelium has a slight trace of white. 

Gelatin: Growth on the bottom of tube 
white, fluffy. Liquefaction slight. 

Milk: Growth on the bottom of the tube 
white. Surface colonies orange-pink, at- 
tached to sides of test tube. Coagulation 
none ; peptonization complete after 1 month. 
NO change in pH. 

Starch: Not hydrolyzed. 

Sucrose: No1 inverted. 

Cellulose: Attacked to a very limited de- 
gree. 

Nitrate reduction: Negative. 

Temperature: The organism is a meso- 
phile, growing well at 25-35°C. It produces 
only sparse growth at 40°C, and does not 
grow at all at 55°C. 

Antagonistic properties: None. 

Habitat: Soil. A culture of this organism 
causing pericarditis and pleuritis has been 



-^W- 




W)r^ 



sfnr^ 



CO 



ocW 




h*J$** 



Figure 56. Waksmania rosea, schematic repre- 
sentation of the formation of aerial mycelium and 
spores (Reproduced from: Lechevalier, M. P. and 
Lechevalier, H. J. Gen. Microbiol. 17: 108, 1957). 



4^1 




10/lt 

Figure 57. Waksmania (Microbispora) rosea: A. 
sporulation; B. chlamydospores; C. germination 
of conidia (Reproduced from: Xonomura, H. and 
Ohara, Y. J. Fermentation Technol. 35:307, 1957). 

recently isolated by Louria and Gordon 
(I960). 

Remarks: Thiamine and biotin are es- 
sential for growth; biotin also controls pig- 
mentation. Ammonium compounds, nitrates, 
and urea are not utilized as sources of 
nitrogen. Asparagine, glutamic acid, and 
peptone are good sources. 

Type cultures: IMRU 3748, 3757. 

Xonomura and Ohara (1900a) found the 
genus Waksmania (Microbispora) widely 
distributed in soils of Japan. When a par- 
ticle of soil was placed on soil-extract agar in 
a Petri dish, colonies of this group of organ- 
isms appeared around the soil particle after 
a few weeks incubation at 30°C. Five species 
and two varieties were recognized, including 
.1/. amethystogenes, .1/. amethystogenes var. 
nonreducans, M. parva, M. chro?nogencs, M. 
diastatica, and M. rosea var. nonnitritogenes. 

A system of classification of these species 
and varieties was proposed, based upon then- 
growth on different media, nitrate reduction 
to nitrite, production of soluble pigment . and 
hydrolysis of starch. Some of the strains pro- 
duced violet crystals, insoluble in water but 
soluble in benzene. Thiamine was required 
for growth of all strains, biotin to a limited 
extent. 



Chapter 11 



Thermophilic Actinomycetes 



Our knowledge of the thermophilic actino- 
mycetes dates back to the early beginnings 
of general microbiology. The first students 
of the microbiological population of soils and 
composts observed that some of the organ- 
isms found among the bacteria, actinomy- 
cetes, and fungi were able to grow at much 
higher temperatures than the great majority 
of the members of these groups. 

Globig (1888) was the first to isolate from 
the soil thermophilic actinomycetes, capable 
of growing at 52-o5°C. Rabinowitsch (1895) 
and Tsiklinsky (1903) isolated similar cul- 
tures from manure, and Xoack (1912) iso- 
lated them from hay. Numerous other iso- 
lations of thermophilic actinomycetes were 
made, from ordinary soil by Gilbert (1904), 
from desert sand, feces, air, and peat, as 
well as from human intestines and from 
sewage. It has also been observed repeatedly 
that composted manure, when it has attained 
a high temperature, or hay which has been 
allowed to heat in composts, becomes cov- 
ered with small white patches of fungus-like 
growth. Miehe (1907) remarked that the 
appearance of these patches is similar to a 
coat of lime and is due to actinomycetes. 
Similar observations of thermophilic com- 
posts have been made by various other in- 
vestigators. 

Tsiklinsky inoculated potato slices with 
soil or with manure and incubated them at 
53-55°C. Isolations were made on agar 
plates after l(i hours. Two cultures were 
thus obtained. One produced chains of spores 
and was considered to be, therefore, a true 
actinomycete of the type now designated as 



Streptomyces. The other formed round or 
ovoid spores at the end of side branches, by 
the swelling of the tips. This organism was 
believed to be widely distributed in nature 
and was named Thermoactinomyces vulgaris- 
Because of its manner of spore formation, 
this form was believed to belong to the group 
of actinomycetes designated by 0rskov 
(1923) as Micromonospora, and was, there- 
tore, classified by Waksman as Micromono- 
spora vulgaris. It grew at 48-68°C, with an 
optimum at 57°C, and no growth at 70°C. It 
remained inert for a month at 'A7°C or at 
lower temperatures, but it became active 
within 24 hours at 56-57°C. The spores were 
said not to be destroyed at 100°C, even after 
20 minutes. The organism grew readily on 
most ordinary media; it was proteolytic but 
not amylolytic. The Streptomyces, on the 
other hand, was weakly proteolytic, and the 
spores were less resistant to heat (Fig. 58). 

Gilbert cultivated from various soils 
several strains of a thermophilic actino- 
mycete, which he designated as A. thermo- 
philics. Growth on potato was much folded, 
white, later becoming gray on the surface; 
the plug" was darkened by some cultures. 
The optimum temperature was 55°C; no 
growth took place at 60°C. Most sir: 'ins 
ceased to grow at 45°C, whereas some could 
be adapted to grow on agar media at 'A7°C 
and even at 22°( 1 . The colonies on agar were, 
after 24 to 48 hours, small, folded, light 
yellow with a dark-colored center. Gelatin 
was only slowly liquefied. 

Miehe considered hot composts and not 
soils as the natural substrates of actino- 



300 



THERMOPHILIC ACTINOMYCETES 



:joi 



mycetes. The spores of these organisms were 
found to lose their vitality rapidly, especially 
on agar media, bu1 they survived on hay 
particles. One culture was designated as A. 
thermophilus Berestnew; it grew besl a1 
40-50°C, more slowly at 30°C, and not at 
all at 25°C and at 60°C. The manner of 
spore formation of this organism suggested 
its resemblance to Micromonospora. Miehe 
also reported, however, that he saw thermo- 
philic actinomycetes which formed spores 
according to the manner described by Gil- 
bert. This suggests the probability that he 
had representatives of the two different 
groups. Schutze (1908) found, in decompos- 
ing clover hay, representatives of these two 
thermophilic actinomycetes, one of which 
appeared to belong to the Streptomyces 
group and the other to the Micromonospora. 

Several methods for isolation and cultiva- 
tion of thermophilic actinomycetes have 
been described by Henssen (1957). Uridil 
and Tetrault (1959) suggested the incorpora- 
tion of colloidal silica in a highly proteinace- 
OUS medium for the growth of these organ- 
isms. 

The various thermophilic forms have often 
been classified under the common name of 
"Actinomyces thermophilus." Waksman et ol. 
(1939) demonstrated, by direct microscopic 
studies, that these organisms are very abun- 
dant in high temperature composts of stable 
manures and plant residues. Six distinct 
types or species were recognized, belonging 
to two genera, one now known as Strepto- 
myces and the other as Micromonospora. 
Two of the first group (S. thermophilus and 
S. thermofuscus) and three of the second 
(.1/. vulgaris, M. chalcea, and M. fused) were 
isolated and cultivated. Henssen ( 1957) con- 
firmed these observations and created sev- 
eral new genera and species, to include these 
and other forms (Fig. 59). 

Kosmatchev (1959) emphasized the need 
for separating the thermophilic from the 
mesophilic actinomycetes. since the former 
grow at oo°C and the latter cannot be 



"•• «k 




Figure 58. First photograph of a Thermoactino 
myces (Reproduced from: Tsiklinsky, P. Ann. 
inst. Pasteur 13: 500-505, 1899). 

adapted to grow at that temperature or 
under thermophilic conditions. The thermo- 
philic actinomycetes form a sharply defined 
ecological and widely distributed group of 
microorganisms, and retain their thermo- 
philic properties under laboratory condi- 
tions. They were believed to comprise in- 
dependent species. He suggested, however, 
that no specific thermophilic genera be 
recognized, but that they should be included 
among the mesophilic forms. 

Henssen (1957) proposed a distinct system 
for classifying thermophilic actinomycetes. 
This system is used, with certain modifica- 
tions, in this treatise. 

Classification of Thermophilic Actino- 
mycetes 

A. Substrate mycelium unseptated. Spores pic 
duced on aerial mycelium only. 

I. Aerial hyphae branched; they are almost 
completely transformed into spore chains. 
Streptomyces Waksman ami Henrici 



302 



THE ACTINOMYCETES, Vol. II 



\ 




Ago 





/ Agar 




^^: 



Figure 59. Four types of thermophilic actinomycetes (Reproduced from: Henssen, A. Arch. Mikro- 
biol. 26:379, 1957). 



II. Long sterile aerial hyphae forming single 
spores or chains of spores on side branches. 

1. Spores produced singly on simple or 
branched sporophores. 

Thermomonospora Henssen 

2. Spores produced in two's or in longer 
chains. 

Thermopolyspora Henssen 
III. Single spores or chains of spores originate 
directly from substrate mycelium, which 
sometimes emerges from the agar surface 
like an arch. 

Thermoactinomyces Tsiklinsky 
B. Substrate mycelium septated. Spores formed 
from both aerial and substrate mycelium. 
Pseudonocardia Henssen 

Genus Streptotnyces 

XVI. Series Thermophilus 

Spores produced in chains, comparable 
to the mesophilic species of Streptotnyces. 

I. Sporophores and chains of spores straight. 

a. Aerial mycelium white to light gray. 

2. Streptomyces rectus 

b. Aerial mycelium white; thermotolerant. 

1. Streptotnyces casei 
II. Sporophores and chains of spores not si raight . 



1. Spore chains spiral -shaped. 

6. Streptomyces thermovulgaris 

2. Spore chains bent or curved. 

a. Aerial mycelium white to violet-gray. 

5. Streptomyces thermoviolaceus 

b. Aerial mycelium gray to lavender. 

4. Streptomyces thermofuscus 

c. Aerial mycelium white to light gray. 

3. Streptomyces thermodiastaticus 

1. Streptomyces casei (Bernstein and Mor- 
ton, 1934) nov. comb. (Bernstein, A. and 
Morton, H. E. J. Bacterid. 27: 625, 1934). 

Morphology: Sporophores straight, 0.5 to 
0.7 p in diameter. 

Agar media: Growth colorless to white. 
Aerial mycelium white. 

Gelatin: Liquefaction rapid. 

Milk: Positive coagulation and peptoniza- 
tion. 

Starch: No hydrolysis. 

Nitrate reduction: None. 

Temperature: Optimum 40-60°C. Highly 
resistant to higher temperatures and to dis- 
infectants. Thermal death point 100°C. 

Habitat : Pasteurized cheese. 

Remarks: Krassilnikov (1949) placed this 



THERMOPHILIC A.CTINOMYCETES 



303 



culture in the same group with .1. invul- 
nerabilis (Acosta and Rossi) Berestnew, 
1897, the latter said to be even more re- 
sistant to high temperatures and to disin- 
fectants. 

2. Streptomyces rectus Henssen, L957 
(Henssen, A. Arch. Mikrobiol. 26: 373 414, 
L957). 

Not .1 . rectus Krassilnikov. 

Synonym : Streptomyces thermophilus. 

Morphology: Sporophores straight, 36 to 
60 fi long. Spores round or oval, 0.9 to 1 .2 ii. 

Sucrose nitrate agar: Growth moderate. 
No aerial mycelium. 

Glycerol-asparagine agar: Growth good. 
Aerial mycelium moderate, white to light 
gray. 

Meat-extract agar: Growth good. Aerial 
mycelium light gray. Soluble pigment brown. 

Cellulose-dextrin agar: Growth more or 
less heavy. Aerial mycelium white-gray. 

Potato: Growth colorless. Soluble pigment 
In-own. 

Starch : Slow hydrolysis. 

Nitrate reduction: Positive. 

Milk: Coagulated; not peptonized in 9 
days. 

Gelatin: Not liquefied (Waksman et al. 
I L939) obtained liquefaction). 

Habitat: Fresh horse manure. 

Remarks: Thermotolerant mesophile. 
Grows equally well under aerobic and an- 
aerobic conditions. 

The name "thermophilus" for this species 
was not recognized by Henssen (1957), since 
it was first used by Berestnew (1897) for 
another actinomycete, apparently also a 
species of Streptomyces, which was distin- 
guished from S. rectus by spiral-forming 
chains of aerial spores, gray or dark green 
aerial mycelium, and yellow to dark brown 
colonies. Noack (1912) described an or- 
ganism, under the name "thermophilus," 
which produced a soluble red pigment . Miehe 
(1907) and Schiitze (1908) also described 
organisms under this name. 



.'!. Streptomyces thermodiastaticus (Bergey, 
Mil!)) nov. comb. (Bergey, I). H. .1. Bac- 
terid. 1: 301, 1919). 

.Morphology: Sporophores form spirals. 
Spores spherical to oval, 0.9 by 0.7 or 0.8 ^. 

Synthetic agar: Growth colorless. Aerial 
mycelium well developed, white. 

Potato: Growth brownish. Aerial myce- 
lium light gray. 

( ielatin: Liquefaction slow. 

Milk: No coagulation; no peptonization. 

Starch: Strong hydrolysis. 

Cellulose: Growth good. 

Nitrate reduction: Positive. 

Sucrose: Inverted. 

Temperature: Optimum, 65°C. 

Habitat: Mouth of rabbit. Soil. 

4. Streptomyces thermofuscus (Waksman 
et al., 1939) nov. com!). (Waksman, S A., 
Umbreit, W. W., and Cordon, T. C. Soil Sci. 
47: 49, 1939). 

Morphology: Aerial mycelium gives rise 
to spiral-shaped sporophores; spores spher- 
ical. 

Sucrose nitrate agar: At 28°C, growth 
poor, deep gray, with but little aerial my- 
celium. At 50°C, growth black to violet, 
with gray to lavender aerial mycelium. Sol- 
uble pigment brown. 

Potato: Growth abundant, brown-col- 
ored. Aerial mycelium, none or a few white 
patches. Soluble pigment black. 

Gelatin: Liquefied. At 50°C, a grayish 
ring is produced and a soluble pigment is 
formed. At 28 °C, there is growth withotit 
any soluble pigment. 

Milk: Peptonized. 

Starch: Hydrolysis. 

Temperature: Good growth at 50 and 
()0°C. Will grow at 65°C. faint growth at 
28°C. 

Habitat : Horse manure. 

Remarks: This species is characterized by 
brown-colored aerial mycelium on synthetic 
media, spiral-shaped sporophores, and abil- 
ity to grow readily at i\r>°C. 



304 



THE ACTINOMYCETES, Vol. II 



5. Streptomyces thermoviolaceus Henssen, 
L957 (Henssen, A. Arch. Mikrobiol. 26: 373- 

11 1, 1957). 

Morphology: Substrate mycelium slightly 
branched. Sporophores curved, 20 to 40 m 
long. Spores oval, 1.0 to 1.2 by 1.2 to 1.0 jj.. 
Facultative aerobe. 

Synthetic agar: Growth slight. Sporo- 
phores ochre-brown. Aerial mycelium white 
to violet-gray. 

Glycerol-asparagine agar: Growth yellow. 
Aerial mycelium abundant. Soluble pigment 
violet . 

Nutrient agar: Growth good. No aerial 
mycelium. 

Cellulose-dextrin agar: Growth slight. 
Aerial mycelium produced. 

Potato: Growth good, black-violet. Aerial 
mycelium white. Soluble pigment dark 
violet. 

Starch: Rapid hydrolysis. 

Nitrate reduction: Negative. 

Milk: Coagulation rapid; peptonization 
rapid. 

Gelatin: Growth orange-yellow. Lique- 
faction positive. Soluble pigment produced. 

Habitat : Fresh horse or swine manure. 

Remarks: This species was divided by 
Henssen into two subspecies: (a) pingens, 
producing a violet pigment on potato; (b) 
apingens, producing no pigment on potato. 

(i. Streptomyces thermovulgaris Henssen, 
1957 (Henssen, A. Arch. Mikrobiol. 26: 373- 
414, 1957). 

Morphology: Mycelium shows little 
branching. Sporophores produce spirals, 20 
to 40 /x long. Spores oval, 1 .0 to 1 .2 by 1.1 to 
1.5 fi. Facultative aerobe. 

Sucrose nitrate agar: Growth slight. 
Aerial mycelium present or absent. 

Glycerol-asparagine agar: Growth mod- 
erate to good. Aerial mycelium white to 
black-violet. 

Nutrient agar: Growth good, colorless. 
Aerial mycelium violet-gray. No soluble 
pigment. 



Cellulose-dextrin agar: Growth good. 
Aerial mycelium produced. 

Potato: Growth good in places. Aerial 
mycelium white to gray in spots. Soluble 
pigment often black. 

Starch: Rapid hydrolysis. 

Nitrate reduction: Positive. 

Milk: Rapidly coagulated and peptonized. 

Gelatin: Liquefaction varies. 

Temperature: Grows well at 40-50°C, 
somewhat better under anaerobic than 
aerobic conditions. At 28°C growth slight ; 
at ()0°C growth moderate. No aerial myce- 
lium at 28°C; slight aerial mycelium at 60°C. 

Habitat: Fresh compost and fresh horse 
manure. 

Genus Thermomonospora 

The type species is Thermomonospora fusca 
(Waksman et al.) Henssen. 

These organisms produce colorless or 
yellow growth on agar media. Substrate 
mycelium nonseptated. The aerial myce- 
lium is sharply limited and white. Aerial 
hyphae are simple or branched, developing 
as side or terminal branches on the substrate 
hyphae. The spores are formed singly on 
simple or branched sporophores on the un- 
branched aerial hyphae. Spore formation is 
acropetal. Gram-positive. Not acid-fast. 
Thermophilic, facultative aerobes. 

I. Spore-masses produced in form of a head or 
a hunch of grapes. 

2. Thermomonospora fusca 
II. Spore-masses spiked or entangled. 

3. Thermomonospora lineata 

III. Spores mostly single on simple or branched 
sporophores. 

1. Thermomonospora curvala 

1 . Thermomonospora curvaia Henssen, 
1957 (Henssen, A. Arch. Mikrobiol. 26: 373- 
114, 1957). 

Morphology: Aerial hyphae 30 to 50 n 
long. Mass of spores clumped or hairy. 
Spores round, 1.2 to 1.8 y.. 

Agar media: Colonies colorless or yellow. 



THERMOPHILIC A.CTINOMYCETES 



305 



Aerial mycelium chalk-white. Cultures are 
thermophilic, facultatively aerobic. Aerial 
spores produced in 3 to 4 days in hanging 
drops. Spores formed on simple or branched 
sporophores. Spores oval, later round. 

Sucrose nitrate agar: Growth colorless. 
Aerial mycelium moderate. 

Glycerol-asparagine agar: Growth abun- 
dant, yellow. Aerial mycelium produced. 

Nutrient agar: Growth good, yellow. 
Aerial mycelium thick. 

Potato: Growth yellow, partly covered 
with aerial mycelium. Soluble pigment light 
brown. 

Starch: No hydrolysis. 

Nitrate reduction: Weak. 

Milk: Unchanged in 16 days. 

Gelatin: Not liquefied. 

Temperature: Optimum growth at .">()('. 
Limited growth at 28 and 65°C. 

Habitat: Fresh cow manure and manure 
compost. 

2. Thermomonospora fusca (Waksman et 
ill., 1939) Henssen (Henssen, A. Arch. 
Mikrobiol. 26: 373-414, 1957). 

Morphology: Aerial hyphae 20 to 30 /j, 
long. Spores round 1.5 to 2.0 p.. Since the 
Spores are not produced on the substrate 
mycelium as in Micromonospora, but ex- 
clusively on the aerial mycelium, the species 
was transferred from Micromonospora fusca 
to Thermomonospora fusca. The branching 
of the substrate mycelium is monopodial. 
The hyphae are long, straight, and form 
straight side branches. The branching is so 
characteristic, as compared to all the other 
thermophilic species, that this species can 
easily l>e recognized. The aerial mycelium 
is colored brown (Fig. 60). 

Sucrose nitrate agar: Growth at 28°C 
deep gray; at 50°C, growth is dark brown to 
violet. Aerial mycelium gray to lavender. 
Soluble pigment brown. 

( relatin: Liquefied. 

Potato: Growth brown-colored. No aerial 
mycelium. Soluble pigment black. 




] 


[Guee (>(). Thermo 


monosport 


fusca 


i Etepro- 


due 


id from: Henssen, 


A. Arch 


Mi km 


biol. 2<>: 


401 


L957). 









Milk: No coagulation; slight peptoniza- 
tion. 

Habitat: Horse manure. 

Starch: Hydrolysis. 

Nitrate reduction: Slight. 

Cellulose: Growth good. 

Temperature: Growth and aerial myce- 
lium formation are good at 50 65°C. 

3. Thermomonospora lineata Henssen, 
1957 (Henssen, A. Arch. Mikrobiol. 26: 373- 
414, 1957). 

Morphology: Sporophores straight, 50 to 
SO p. long; spores round, 1.5 to 2.0 //• Spore 
chains hairy or clumpy (Pig. 61). 

Culture was isolated on nutrient agar but 
was not obtained in pure state. Aerial hy- 
phae mostly branched. Spores produced on 
simple or branched sporophores. Substrate 
hyphae monopodial and branched. Hyphae 
are not as long as in the case of T. fusca. 
Side branches are more compact. 

Optimum growth: 50-60°C. 

Habitat : Composted slice]) manure. 

Genus Thermopolyspora 

( irowth on agar media colorless to yellow. 
Substrate mycelium not septated. Aerial 
mycelium limited, white. Aerial hyphae not 
branched, developing in the form of side or 
terminal branches of the substrate hyphae. 
Spores produced in short chains on un- 
branched sporophores. Spore formation 
acropetal. Spore chains unbranched, straight, 



306 



THE ACTINOMYCETES, Vol. II 




Figure 61. Thermomonospora lineata (Repro- 
duced from: Henssen, A. Arch. Mikrobiol, 26: 
401, 1957). 

bent or spiral-shaped. Gram-positive, non- 
acid-fast, thermophilic, facultatively aerobic 

This genus comprises two species: T. 
bispora with chains made up of double 
spores, and T. polyspora with more Than 
two spores. 

Usually only the side branches of the 
substrate mycelium grow into aerial hyphae. 
The chains of spores are produced from the 
unbranched sporophores around the aerial 
hyphae. 

Type species: Thermopolyspora bispora 
Henssen. 

1. Thermopolyspora bispora Henssen, 1057 
(Henssen, A. Arch. Mikrobiol. 26: 373-414, 
L957). 



Morphology: Aerial hyphae 20 to 30 m 
long, with 20 to 40 spore chains. Spores are 
double, round, 0.0 to 1.3 p.. 

Cultural characters: Colonies mostly col- 
orless. Aerial mycelium chalk-white. Organ- 
ism thermophilic, facultatively aerobic. 

The following properties are discussed on 
the Uisis of anaerobic growth at G0°C. 

Sucrose nitrate agar: Sparse growth. 

( ilycerol-asparagine agar: Growth slight 
or abundant. Xo aerial mycelium. 

Nutrient agar: Good growth. Aerial my- 
celium thick. 

Starch agar: Xo hydrolysis. 

( relatin: Unchanged. 

Potato: Individual colonies without aerial 
mycelium. 

Nitrate reduction: Negative. 

Milk: Xot changed after 7 days. 

Habitat: Fresh cow and swine manure or 
composted sheep manure. 

Type culture: IMRU 3750. 

2. Thermopolyspora polyspora Henssen, 
1957 (Henssen, A. Arch. Mikrobiol. 26: 373- 
414, 1057). 

Morphology: Aerial hyphae 45 to 50 /j. 
long, 1.0 to 1.3 fi thick. Sporophores straight 
to spiral-forming, individual or in groups. 
Spores round, 3 to 10 by 1.1 to 1.8 /x. 

Physiological properties of spore-free cul- 
tures, at 00°C under aerobic conditions: 
Colonies yellow, aerial mycelium dirty 
white. 

(Ilycerol-asparagine agar: Growth orange- 
yellow. 

Cellulose-dextrin agar: Growth colorless. 

Nutrient agar: Growth yellow. 

Potato: Growth yellow. 

Remarks: These cultures resemble, 
through their colored compact colonies, the 
spore-free cultures of Micromonospora. Spiral 
formal ion has resulted in the confusion of 
T. polyspora with species of Streptomyces. 

Genus Thermoactinomyces 

The genus Thermoactinomyces is similar in 
some respects to the genus Micromonospora; 



THERMOPHILIC ACTINOMYCETES 



307 



it is distinct from it, however, in the forma- 
tion of a typical aerial mycelium. 

Henssen emended this genus as follows: 
Colonics on agar colorless or yellow to 
orange. Substrate mycelium no1 septated. 
Aerial mycelium not sharply delimited; 
white or bluish-green. Aerial hyphae are 
simple or branched, formed as terminal or 
side branches; they may also be curved; 
they grow upwards from the substrate my- 
celium. Spores, single or in chains, remain 
on the unbranched aerial hyphae. The or- 
ganisms are thermophilic, capable of grow- 
ing at 50-65°C. They are aerobic or faculta- 
tively aerobic. Some have their optimum at 
60°C. 

Type species: Thermoactinomyces thalpo- 
philus Waksman and Corke. 

The genus can be classified as follows: 

1 . Aerial mycelium unite. 
a. No soluble pigment . 

is. Thermoactinomyces vulgaris 
1). Soluble wine-colored to rose pigment in 
certain media. 

3. Thermoactinomyct s thalpophilus 
II. Aerial mycelium grayish-green. 

2. Tin rmoactinomyces monosporus 
III. Aerial mycelium scant . white to bluish-green. 

a. No soluble pigment. 

1. Thermoactinomyces glaucus 

b. Soluble pigment green. 

5. Thermoactinomyces viridis 
L\". Aet'ial mycelium white to dark gray. 

4. Thermoactinomyces thei mophilus 

1. Thermoactinomyces glaucus Henssen, 
l ( .).->7 (Henssen, A. Arch. Mikrobiol. 26: 
157:5-414, 1957). 

Morphology: Spore chains simple or 
branched, about 7 ^ long, containing 4 to 
10 spores. The chains are straight or bent. 
Spores round or oval, 0.9 to 1.4 by 0.5 to 
L.9/Z- 

Cultural properties: Growth almost color- 
less; aerial mycelium white to bluish-green. 
Facultative aerobe. 

Sucrose nitrate agar: Growth moderate. 
Aerial mycelium moderate. 



Glycerol-asparagine agar: Growth moder- 
ate. Aerial mycelium scant. 

Nutrient agar: Growth sparse. 

Cellulose-dextrin agar: Growth good. 
Aerial mycelium abundant, white to green. 
Cellulose decomposed very actively. 

Gelatin: Unchanged. 

Potato: Individual colonies without aerial 
mycelium. 

Standi: Slow hydrolysis. 

Nitrate reduction: Positive. 

.Milk: Slowly coagulated and peptonized. 

Habitat: Composted sheep manure. 

2. Thermoactinomyces monosporus (Leh- 
mann and Schiitze) Waksman (Schiitze, H. 
Arch. Hyg. 67: 35, 1008; after Krassilnikov, 
1<)41). 

Morphology: Substrate hyphae about 1.0 
/x in diameter. Oval spores 1.5 to 1.8 by 
1.0 to 1.4 /j. produced singly. 

Agar media: Growth yellowish, compact, 
smooth or lichenoid. Aerial mycelium gray- 
ish-green. Good sporulation of hay infusion- 
peptone agar; somewhat less on glycerol- 
peptone and lactose-peptone agar; none on 
peptone-glucose agar. 

Potato: No growth. 

Gelatin: Liquefaction positive. 

Milk: No coagulation or peptonization. 

Blood serum: Good, smooth growth: lique- 
faction positive. 

Temperature: Optimum 37-55°C; grows 
poorly at 27°C and not at all at 60°C. 

Habitat: Self-heated hay. 

Remarks: Henssen (1957) considers this 
species as more closely related to the genus 
Thermomonospora. 

:!. Thermoactinomyces thalpophilus Waks- 
man and Corke, 1'.).").'; (Waksman, S. A. and 
Corke, C. .1. Bacteriol. 66: :',77, 1953). 

Morphology: Spores, produced singly or 
in short chains, are round, 0.S to 1.2 fi. 

Agar media: Grows equally well on a 
variety of media under aerobic and anaero- 
bic conditions. Colonies colorless to orange. 



308 



THK ACTINOMYCETES, Vol. II 



Aerial mycelium white. Soluble wine-red 
pigment produced on sugar-containing salt 
media. 

Sucrose nitrate agar: Very little growth. 

Glycerol-asparagine agar: Growth abun- 
dant. Aerial mycelium formed, orange. 

Nutrient agar: Growth yellow. Aerial 
mycelium limited. 

( ielatin: Some liquefaction. 

Potato: Growth limited. Aerial mycelium 
limited. 

Starch: Rapidly hydrolyzed. 

Nitrate reduction: Positive. 

Milk: Coagulation rapid; peptonization 
rapid. 

Temperature: Grows well at 50-60°C, 
lesser growth at 40°C; no growth at 28 and 
65°C. 

Habitat: Manure compost. 

4. Thermoactinomyces theimophilus (Be- 
restnew, 1897) now comb. 

Morphology: Sporophores straight (No- 
ark, Waksman et al.) or spiral-shaped 
( Krassilnikov). Spores spherical. 

Cultural properties: Substrate growth 
yellow-brown. Soluble pigment brown. Aerial 
mycelium white to dark gray. 

Synthetic agar: Growth colorless. Aerial 
mycelium thin white. No soluble pigment . 

Potato: Growth yellowish. No aerial my- 
celium. Soluble pigment brown. 

Gelatin: Liquefaction rapid. No pigmen- 
tation. 

Milk: Coagulation and peptonization. 

Starch agar: Growth yellowish. Aerial 
mycelium powdery, white-gray. Starch 
rapidly hydrolyzed. 

Nitrate reduction: Rapid. 

Cellulose: Slight growth. 

Temperature: 35-55°C. Optimum 50°C. 

Remarks: Noack described, under this 
name, organisms of a bright red color, with 
a red soluble pigment. Optimum 40-59°C. 
Miche and Schutze also described organisms 
under this name Figure 62. Pseudonocardia thermophila (Repro- 

duced from: Henssen, A. Arch. Mikrobiol. 26: 

.">. Thermoactinomyces viridis Schuurmans 40 9 , 1957). 



et al., 1956 (Schuurmans, D. M., Olson, B. 
H., and San Clemente, C. L. Appl. Micro- 
biol. 4: 61-66, 1950). 

Morphology: Spores borne singly; oval 
(about 1.0 by 1.3 n). Diameter of hyphae 
approximately 0.5 n. 

Glucose-asparagine agar. No growth. 

Calcium malate agar: Colonies colorless, 
1 to 2 mm in diameter; a few colonies with 
blue-green aerial mycelium. 

Nutrient agar: ( Irowth wrinkled, colorless, 
and close to agar surface. Aerial mycelium 
blue-green. Soluble pigment emerald-green. 

Glucose-peptone agar: Colonies colorless, 
1 to 2 mm in diameter. No aerial mycelium. 

Nutrient broth: Flocculent cream-colored 
submerged growth; surface growth blue- 
green. Soluble pigment green. 

Gelatin: Liquefied. 

Potato: No growth. 

Milk: Coagulation positive, followed by 
peptonization. 

Nitrate reduction: Negative. 

Starch: Hydrolysis. 

Temperature: Minimum, 37°C; optimum, 
55°C; maximum, 60°C. 

Antagonistic properties: Produces the 
antibiotic thermoviridin, active primarily 
against gram-positive bacteria. 

Habitat: Composted manure pile. 

Remarks: Description of growth and bio- 
chemical reactions after 14 days' incubation 




THKRMol'HII.lC A.CTINOMYCETES 



309 



at 45°C. The organism in many ways re- 
sembles Tkermoactinomyces monosporus. The 
points of difference, however, arc considered 
significant, the latter possessing hyphae 
with a diameter of about 1 m, an optimum 
growth range of 37 to 55°C, and failing to 
coagulate milk. 

(i. Thermoactinomyces vulgaris Tsiklinsky, 
L899 (Tsiklinsky, P. Ann. inst. Pasteur Pi: 
500, 1899). 

Synonym: Micromonospora vulgaris Waks- 
man et aL, 1939; Erikson, 1953. 

Morphology: Substrate mycelium fine, 0.5 
ft in diameter. Spherical and oval spores are 
borne singly at the ends of short branches, 
from which they are easily broken. They 
often appear to sit directly on mycelium. 

Sucrose nitrate agar: Growth colorless. 
Aerial mycelium white. 

Nutrient agar: Growth good. Aerial my- 
celium white. 

Potato: ( Irowth good. 

Gelatin: Liquefaction positive. 

Milk: Coagulation and peptonization. 

Starch: Hydrolysis positive. 

Cellulose: No decomposition. 

Nitrate reduction: Negative. 

Sucrose: Not inverted. 

Temperature: Grows at 18 68°C; opti- 
mum at 57°C. 

Source: Human and animal excreta, high 
temperature composts, self-heated hay, soil. 

Remarks: Resembles mesophilic members 
of the genus Micromonospora, except that it 
produces an aerial mycelium which forms 
single spherical spores. The aerial phase of 
the development of this organism is believed 
to be intimately associated with its thermo- 
philic nature (Erikson, 1952, 1953, 1955a). 
Oxygen concentration has an important 
effect upon the growth of the aerial mycelium 
of this organism (Webley, 1954). The effect 
of composition of medium and the growth- 
temperature relationships of this organism 
were sttidied recently by Tendler (1959). 



Pseudonocardia 



Substrate mycelium septated. Spores pro- 
duced in substrate and in aerial mycelium. 

On the basis of its morphology, Pst udo- 
nocardia should be placed between Strepto- 
myces and Norcardia. Along with Nocardia, 
it has the common property of septal ion of 
the substrate mycelium, but no fragmenta- 
tion. In common with Streptomyces, it pro- 
duces aerial mycelium which is thicker than 
the substrate mycelium, and which changes 
into long spore chains. It differs from Strep- 
tomyces in being unable to hydrolyze gelatin 
or starch. 

Type species: Pseudonocardia thermophila 
Henssen. 

Pseudonocardia thermophila Henssen, 1957 
(Henssen, A. Arch. Mikrobiol. 26: 373-414. 

1957). 

Morphology: Substrate mycelium sep- 
tated. Spore formation in substrate myce- 
lium. Aerial hyphae unbranched, formed as 
side branches of the substrate hyphae. 
Spores basipetal. Substrate spores 2.5 by 
1.5 to I.S fji. Aerial spores produced in chains 
at the tip of the hyphae are 2.5 n long; spores 
produced at the base of the chain are 5 ju 
long by 1.5 to l.S ^ wide. Thermophilic, 
facultative aerobe (Fig. 62). 

Sucrose nitrate agar: Growth yellow. 
Aerial mycelium limited, while. 

Glycerol-asparagine agar: Growth moder- 
ate, yellow. Aerial mycelium produced, 
white. 

Nutrient agar: Growth good, yellow. 
Aerial mycelium in thick colonies. 

Potato: Colonies individual, yellow. No 
aerial mycelium. Soluble pigment yellow. 

( ielatin : Not liquefied. 

Starch: No hydrolysis. 

Nitrate reduction : Positive. 

Milk: Unchanged in Hi days. 

Habitat : Fresh horse manure. 



C h a p t e 



Actinoplanaceae 



Substrate mycelium usually inconspicu- 
ous, formed in water, on a variety of plant 
and animal materials. Aerial mycelium 
usually lacking; only certain species pro- 
duce such mycelium, thus resembling 
Streptomyces. Reproduction by spores formed 
in sporangia. The spores in Actinoplanes 
possess flagella and are motile. The spores 
of Streptosporangium are without flagella 
and are nonmotile. Many species produce 
aerial spores. These organisms can be culti- 
vated on a variety of artificial media; they 
will then resemble in their growth other 
actinomycetes. The family is widely dis- 
tributed in soil and in fresh water. The 
Actinoplanaceae can be classified as follows: 

1. Aerial mycelium usually not formed; 
coiled conidiophores lacking; sporangio- 
spores motile. 

Genus I. Actinoplanes 

II. Aerial mycelium abundant; coiled co- 
nidiophores as well as sporangia an 1 
formed in some species; sporangiospores 
nonmotile. 

Genus II. Streptosporangium 

Genus iclinoplaiies Couch 

(Couch, J. N. J. Elisha Mitchell Sci. Soc. 
66, 87, 1950; 71, 48, 1955. Trans. X. Y. 
Acad. Sci. 16, 315, 1954). 

Occur on sterilized leaves in water, form- 
ing a very inconspicuous mycelium which 
branches throughout the leaf tissue. The 
externa] hyphae are scattered or in tufts on 



the leaf surface and form a fringe around 
the edge of the leaf. Aerial mycelium is 
lacking or sparingly formed; usually pinkish 
to reddish, sometimes hyaline; frequently 
decolorizes the green leaf and gives it a 
pinkish or reddish color. Hyphae slightly to 
considerably branched, irregularly coiled, 
twisted or straight, sparingly septate, 0.2 to 
2.6 m in diameter. Sporangia usually abun- 
dant on leaves, formed only when the leaf is 
at or close to the surface of the water, i.e. 
formed typically only in air, and appearing 
black under the low power of the microscope, 
owing to refraction; of varied sizes and 
shapes. Spores in coils, nearly straight 
chains, or irregularly arranged, in sporangia; 
1 to 1.5 ju in diameter, globose or subglobose, 
usually slightly angular, with one to several 
shiny bodies, with several polar flagella, and 
motile; germination by a minute germ tube 
which branches to form a mycelium. Spo- 
rangial wall evanescenl or persistent (Fig. 63). 
The organisms form on various nutrient 
agars a brilliantly colored, tough to pasty 
growth. Surface very variable: smooth and 
even with the agar or elevated bumpy, 
convoluted, ridged, folded, cracked, etc., 
usually moist and shiny, rarely pulverulent. 
Hyphae of two more or less distinct forms, 
the submerged and the surface hyphae, the 
latter usually more or less vertical and in 
some species forming a compact "palisade." 
Sporangia abundant on souk 1 agars, usually 
formed at the surface. Spores formed in 
some species. ( )n certain agars, the mycelium 
of some species breaks up, when crushed, 



310 



ACTINOPLANACEAE 



311 



into irregular pieces of hyphae, rods and 
coccoid bodies. 

Aerobic, gram-positive, and acid-fast. 

Occur saprophytically in soils and infresh 
water, and are world-wide in distribution. 
Over L20 cultures were isolated. 

The genus Actinoplanes is readily dis- 
tinguished from Streptosporangium. On 
leaves, the latter produces a conspicuous 
aerial mycelium which resembles that in 
most species of Streptomyces, whereas no 
such mycelium is usually found in Actino- 
planes. The isolates of the latter grow much 
more vigorously on agar than do those of 
Streptosporangium. due most striking dif- 
ference is that in Actinoplanes the sporangio- 
spores are motile, whereas in Streptosporan- 
gium they are nonmotile. 

Under certain conditions of culture, some 
species of Actinoplanes resemble Micro- 
monospora. A nonsporangial strain of Ac- 
tinoplanes might easily be confused with 
certain micromonosporas. The spores of 
Micromonospora, however, are formed singly 
or in grape-like clusters hut never in chains, 
whereas in Actinoplanes they are formed 



"•> 










• 



< 



§ 



Figure 63. Actinoplanes (Prepared by H. 
Lechevalier, Institute of Microbiology). 



singly and also in chains but not in grape- 
like clusters. In most species of Micromono- 
spora, on certain agars, the sporulating 
surface t urns black, whereas this change does 
noi occur in Actinoplanes. In general, the 
species of Micromonospora are less vigorous 
in growth than those of Actinoplanes. 

Several species of Actinoplanes, when 
grown on potato-glucose and certain other 
agars, will form a small pasty culture which, 
when mounted and crushed under a cover- 
slip, breaks up into minute spheres, irregular 
rods, and short, branched, hyphal segments, 
much as in Nocardia. Such growth, however, 
is not the normal condition for any species 
of Actinoplanes. None of the 25 species 
of Nocardia examined by Couch formed 
sporangia when grown either on any of the 
agars most favorable for sporangia] forma- 
tion or on Paspalum leaves in water. 

Gaertner (1955) isolated cultures of Ac- 
tinoplanes from soil and found them capable 
of decomposing keratin. 

The type species is Actinoplanes philip- 
pinensis Couch. 

Actinoplanes philippinensis Couch, 1950 
(Couch, J. X. J. Elisha Mitchell Sci. Soc. 
66, 87, 1950). 

Morphology: Produces a very dedicate, 
hyaline to pinkish-buff internal mycelium 
and an inconspicuous external fringe of 
threads around the entire edge of the leaf of 
sterile Paspalum grass in water. Sometimes a 
compact mound or tufts of hyphae are 
scattered over the top surface, giving the 
leaf a speckled or finely powdered appear- 
ance. Hyphae are 0.5 to 1.5 m thick, 
branched, sparingly septate. Sporangia, 
usually formed abundantly on grass after 
about 10 days, on long unbranched stalks, 
mostly spherical when mature, S.4 to 22 fi. 
Spores arranged, at maturity, in coils or 
irregularly in the sporangium, about 1 to 
1.2 /J. They are discharged through a pore 
or by the partial dissolution of the sporangia] 
wall, and swim vigorously. 



112 



THE ACTINOMYCETES, Vol. II 



Sucrose nitrate agar: Growth at room 
temperature poor to fair, flat or slightly 
elevated. Margin smooth or scalloped, sec- 
toring frequent. Color pale buff to tawny, 
changing in some old cultures to brown with 
a lighter margin. Forms a compact surface 
layer, made up mostly of distinct palisades, 
and a submerged region of loosely arranged 
hyphae. Surface region frequently stratose in 
old cultures, with narrow, orange-colored 
layers. Sporangia fairly abundant in some 
cultures, not formed in others; spherical to 
irregular; frequently beneath the surface in 



old cultures, owing to overgrowth by pali- 
sade hyphae. Sometimes a new layer of 
sporangia forms over the first layer. Odor 
slightly fragrant. Usually colors the agar 
pale yellow. 

Glucose-asparagine agar: Growth good to 
very good, consisting of a central area of 
elevated, fine convolutions, radial ridges or 
bumps, and a smooth area with radial 
grooves gradually sloping into the submerged 
margin. Surface moist -appearing and glossy. 
Color of center apricot-orange to brown, 
surrounded by an ochraceous-salmon or 




Figuke 64. Streptosporangium roseum (Reproduced from: Couch, J. N. J. Elisha Mitchell Sci 
Soc. 71: 152, 1955). 



ACTINOPLANACEAE 



313 



light ochraceous-salmon margin. Sporangia 
usually on the smooth areas, none on the 
elevated parts; formed on palisade hyphae. 

Potato-glucose agar: Growth good to 
very good. Central area with coarse con- 
volutions or large bumps and irregular 
ridges separated by radial grooves which 
slope to the smooth distinct margin. Surface 
glossy. Color apricot-orange to russet, be- 
coming gray in old cultures. Soluble pigment 
darkens the agar. Sporangia formed on the 
margin of some cultures, absent in most. 
Palisades formed. 

Nutrient agar: Growth fail-. Center 
slightly elevated and with a wide flat margin. 
Color ochraceous-orange to cinnamon-rufous. 
Sporangia very rarely formed. Palisade 
hyphae usually not distinct. 

( iclatin: Liquefied. 

Habitat: Soil from Philippine Islands; 
also found in African soils and in marshland 
soils in ( iormany. 

Remarks: This species is characterized 
by the predominantly spherical sporangia 
usually on long unbranched stalks, the 
rather poor and usually fiat growth on 
synthetic agar, and the very distinct palisade 
hyphae on this medium. The dark brown 
diffusible pigment on potato-glucose agar is 
also characteristic. 

Genus Streptosporangium Couch 

Occurs on sterilized leaves of Paspalum 
grass in water, forming an inconspicuous 
mycelium which overgrows the leaves, and 
an aerial mycelium which grows in scattered 
or concentrically arranged tufts. The aerial 
mycelium is white to pinkish on the leaves; 
hyphae are much branched, sparingly sep- 
tate, and about 0.5 to 1.2 id in diameter. On 
some media, sporangia are formed abun- 
dantly in the aerial mycelium. Spores are 
abundant in the sporangia, without flagella, 
and are nonmotile. Growth poor to good on 
a variety of semisolid media (Fig. 64). 

lour cultures, representing three distinct 



species, were found 
Two of the species 
by the soil dilution 
from dog manure. 

The type specie 
rost uni ( 'ouch. 



ompnse t bis genus. 
1 isolated from soil 
hod and the third 

Streptosporangium 



Streptosporangium r<>s<uin ('ouch, 1!).").") 
(Couch, J. X. J.Elisha Mitchell Sci. Soc. 71, 
148, 1955). 

.Morphology: Grows on sterile leaves, 
either in soil water or on damp sterile soil, 
forming a substrate mycelium which spreads 
over the surface of the leaf, not penetrating 
or decolorizing it; it also spreads over the 
soil. Aerial mycelium white at first, changing 
to pale pink; it appears as single hyphae or 
as minute tufts which grow to form mounds 
up to 2 mm across, arranged more or less in 
concentric circles. Sporangia first appeal- on 
scattered single hyphae, apical on the main 
thread or on short, lateral branches, a fvw 
to many sporangia on one hypha. The 
sporangia are white in small groups, pink in 
large masses; spherical, 7 to 1!) p. in diameter. 
Shortly after their formation, spores are 
visible as a single coil in each sporangium; 
when completely formed, they are irreg- 
ularly arranged. Immersion of the mature 




Figi be 65. Streptosporangium isolated from 
forest litter (Reproduced from: Van Brummelen, 
J. and Went. .]. V. Labor. Microb. Univ. Amster 
dam 2;?: 391, 1957 , 



:;i 1 



THE ACTINOMYCETES, Vol. II 



sporangium in water brings about the swell- 
ing of an intersporal substance, causing 
the wall and the spores to push out on one 
side, forming a cone-shaped projection 
about half as long as the diameter of the 
sporangium. The spores are forcibly ejected 
through an opening in the cone. They are 
noninotile, spherical, 1.8 to 2.0 n in diameter, 
with one shiny globule. Sporangia] wall is 
persistent for several hours after spore 
discharge. In addition to sporangia, spores 
are also formed in coils somewhat as in 
Streptomyces, though the coils are much less 
conspicuous (Fig. 65). 

Sucrose nitrate agar: Colony usually flat, 
level with agar surface; concentric zonation 
distinct or absent. Surface glossy or powdery. 
Color usually white, sometimes pinkish- 
buff or cream-buff. Sporangia absent to 
fairly abundant, always formed some dis- 
tance above the surface of the agar. In some 
cultures, coils form which break up into 
spores as in Streptomyces. 

Glucose-asparagine agar: Growth poor, 
slightly elevated and minutely ridged, slop- 
ing to the fimbriate margin. Surface of 
central region powdery with white aerial 
hyphae. Sporangia absent. 

Potato-glucose agar: Growth usually good, 
center elevated with irregular bumps and 



ridges; margin flat and even with surface of 
agar. Color of colony at first creamy, be- 
coming tawny and then brown, after which 
white floccose spots of hyphae appear, 
usually spreading to cover the entire culture. 
Sporangia usually formed in vast numbers, 
the white areas becoming rosy pink as the 
sporangia mature; the pinkish areas are 
frequently minutely pocked. Surface moist 
at first, appearing dry and floccose as aerial 
hyphae and sporangia are formed. Agar 
colored reddish-brown with a vinaceous 
tinge. 

Nutrient agar: Growth fair, color usually 
cream-buff, rarely buff-brown. Surface usu- 
ally glossy, sometimes powdery with aerial 
hyphae which may be united to form many 
upright fascicles. Sporangia absent. 

Habitat: Garden soil in North Carolina 
and forest litter in Holland and Denmark 
(Van Brummelen and Went, 1957). 

Remarks: Nonomura and Ohara (1960b) 
found the genus Streptosporangium widely 
distributed in the soils of Japan. In addition 
to the original S. roseum Couch, four new 
species were isolated: S. a/hum, S. iiridial- 
bwn, S. amethystogenes, and S. vulgare. These 
were classified on the basis of the color of 
the aerial mycelium and formation of soluble 
pigment on oatmeal agar media. 



C h a p 1 e r I 3 



Incompletely Described Species 
of Actinomycetes 



Numerous isolates 
mycetes listed in i 
variety of differem 

names, could not b 
due to insufficient 
reported here as "ii 
The naming of such < 



of cultures of actino- 
le literature, under a 
generic and specific 
■ identified at present, 
lescriptions. They are 
completely described." 
ultures was based either 
on a casual observation or on the assumed 
occurrence of such an organism in a certain 
disease condition. Frequently the particular 
organism was not even obtained in pure 
culture, but was given a name, often for 
the mere purpose of obtaining for the author 
credit for the particular isolation or observa- 
tion. In other cases, it is fairly certain that 
the culture said to have been isolated from 
a disease condition was nothing more than a 
dusl contamination. 

In the preparation of this list, the author 
has used freely the carefully collected records 
of Brumpt (1939), Dodge (1935), Baldacci 
(1944), and Krassilnikov (1949). Very few 
of these descriptions were complete enough 
de the recorded cultures among the 
identifiable species. This is partic- 
ruo, even in recent years 



win 



) posit 



to mc 
readih 

ularly 

tnedia of unknown con 

for descriptive purposes. 

Xot all the synonyms are recorded here. 
For further detail, the reader is referred to 
the reviews of Chalmers and Christ opherson 
( 1!)1(»), and Dodge ( L935),* especially for the 

*Some of these compilations may be designated, 
quite properly, in the words of Erikson l 1940), as 



pathogenic or largely would-be pathogenic 
organisms, and to Brumpt (1939) and 
Krassilnikov (194!)) for the nonpathogenic 
forms. Only those synonyms that would 
tend to throw light upon the systematic 
position of the culture are listed here. 

Since the name "Actinomyces" has been 
largely used for the incompletely described 
cultures, it is left as such, and the list is 
recorded in the order of species. Where the 
name Nocardia was originally used, it is 
either reported as a synonym of "Actino- 
myces," or under Nocardia. The same prin- 
ciple was applied to Micromonospora and 
other well-recognized genera. 

Xo serious attempt was made to record 
all the other listings of cultures believed to 
be actinomycetes, especially those that have 
been insufficiently described under a variety 
of different names, such as Cladothrix, 
Discomijccs, etc. Most of these names appear 
to be synonyms of those listed above. ( >nly a 
few of the names listed under Streptothrix 
and Oospora are included under the "Ac- 
tinomyces." Xo effort was made to list cul- 
tures for which only a generic name was 
given without any specific designation, or 
which were recorded by a number only. 

A large number of species have recently 
been listed under the genus Streptomyces 
without any description at all or with a 
totally insufficient description. Frequently 

"veritable mausoleums wherein the errors of the 
past .-tre indiscriminately embalmed." 



815 



316 



THE ACTINOMYCI-TKS. Vol. II 



the description is credited to a company and 
not to an individual scientist. This was 
done primarily as an effort to establish 
priority for an antibiotic isolated from such 
a culture, or for patent purposes. These 
designations are placed in a separate cate- 
gory, with emphasis on the antibiotic. A 
group of cultures described by Gause et al. 
(1957) has also been placed in a separate 
category, since no decision can be reached 
as yet concerning their synonymity with 
previously described species. 

For more complete lists of names of 
actinomycetes, comprising both genera and 
species, the reader is referred to Buchanan 
and Lessel (195!)), Lessel (1960), and es- 
pecially to their forthcoming treatise "Index 
Bergey an a." 

According to Buchanan and Lessel (1959), 
there are now about 3000 names given to 
different strains of bacteria that are recog- 
nized as belonging to one or another of the 
genera of the order Actinomycetales. They 
emphasize, however, that "this is not to be 
interpreted as meaning that there are three 
thousand species, for a large proportion of the 
names (probably two-thirds) are not available 
for use because they were not validly pub- 
lished, or are homonyms, or synonyms, or 
are not binary combinations, or were pro- 
posed as hypothetical names, or were in- 
sufficiently described and are to be regarded 
as naked names (nomina nuda) or as 
doubtful names (nomina dubia) or are 
officially rejected names, or because 
illegitimate as contravening some other 
Qomenclatural rule." 

An attempt has been made to present in 
the following lists some of the incompletely 
described forms of actinomycetes. Many 
additional names are found in the previous 
chapters as synonyms of well described 
organisms. 

The first list comprises the forms described 
under the names Actinomyces (.1), Str< />!<>- 
thrix (St.), or Oospora (0). 



The name Actinomyces used in this list is 
not to be confused with the genus Actino- 
myces recognized at present, although some 
of the cultures so designated here would no 
doubt be considered as members of this 
genus. 

A. acidoresistans, a culture obtained from 

Pribram collection in Vienna. IMRU 

3049. 
A. actinoides (Smith, 1918) Bergey, 1923. 
A. actinomorphus (Gray and Thornton, 

1928) Bergey, 1930. 
St. actinomyces Rossi-Doria, 1891. 
St. aetinomycotica Foulerton, 1899. 
A. aerugineus Wollenweber, 1920. 
A. agrestis (Gray and Thornton, 1928) 

Bergey, 1930. 
St. albido Chester, 1901. 
A. albidoflava (Rossi-Doria, 1891) Ford, 1927 
A. albidofuscus Berestnew, 1897. 
A. albido-fuscus Neukirch, 1902. 
A. alboatrus Waksman and Curtis, 1916. 
A. albopurpureus Duche, 1934. 
A. albus-acidus Neukirch, 1902. 
A. albus var. acidus Nannizzi Pollacci, 1934. 
.1. albus var. alfa Ciferri, 1!)27. 
A. albus asporogencs Berestnew, 1897. 
A. albus chlamydosporus Krassilnikov, 1949. 
A. albus vulgaris Krassilnikov, 1941. 
St. alpha Price-Jones, 1900. 
.1. allenbachi Sartory and Meyer, L932. 
A. almquisti Duche, 1934. 
A. americanus (Cohnistreptothrix americana 

Chalmers and Christopherson, L916) 

Dodge, L935. 
A. anaerobies (Plant, L920; Oospora anae- 

robies Sartory, L923) Dodge, L935. 
A. anaerobicus (Plant, L920) Ford. 1927. 
St. aquatilis Johan-Olsen, 1893. 
A. aquatilis Salimowokaja, 1928. Krassil- 
nikov states that it is related to A. glaucus. 
A. arborescens (Edington, 1887) Gasperini, 

L894. 
A. aromaticus Krassilnikov, 1941. 
A. asteroides var. serratus Sartory and 

Meyer, L930. 



INCOMPLETELY DESCRIBED SPECIES OF ACTINOMYCETES 317 

A. atypica pseudoturberculosa Hamm and Syn. 0. buccalis Roger et al., 1009. 

Keller, L909. X. buccali* Castellani and Chalmers, 

St. aurea (Saint-Severin, L895) Ford, L927. L913. 

.1. aureus Lachner-Sandoval, 1898. .1. cameli (Mason, L919) Ford, L927. 

Syn. N. anna Castellani and Chalmers, A . caminiti Ford, L927. 

L913. St. Candida (Petruschky, L898) Ford, 1927. 

A. avadi Dodge, 1935. Syn. N. Candida Castellani and Chalmers, 

A. baarnensis Duche, 1934. 1913. 

Syn. of A. viridis Duche, L934. A. ranis (Rabe, 1888) Gasperini, 1894. 

A. bahiensis (daSilva, L919) Brumpt, 1 1)27. A. canis familiaris Rivolta, 1884. 

A. bellisari Dodge, 1935. St. caprae (Silberschmidt, 1899) Ford, 1927. 

A. berestneffi (Chalmers and Christopherson, -4. carnea (Rossi-Doria, 1891; Gasperini, 

1916) Brumpt, 1939. 1894; Kruse, 1896) lord, 1927. 

Syn. A', berestneffi Chalmers and Christ o- Syn. AT", carnea Castellani and Chalmers, 

pherson, 1916. 1913. 

A. berardinisi (Namyslowsky, 1912) Brumpt, A. carougeaui (Gougerot, 1909) Brumpt, 

L939. 1939. 

St. beta Price-Jones, 1900. Syn. N. carougeaui Castellani and Chalm- 

.4. bicolor Trolldenier, 1903. ers, 1913. 

Syn. X. bicolor deMello and Fernandes, .4. catarrhalis (Bailly, 1921) Brumpt, 1939. 

1919. .4. rati (Rivolta, 1878) Gasperini, 1894. 

A. bolognesii-chiurcoi (Vuillemin) Dodge, A. caviae (Snijders, 1924) Erikson, 1935. 

1935. .4. cerebriformis (Namyslowsky, 1910) 

A. bostroemi Baldacci, 1937. Brumpt, 1939. 

.4. boris albus Gasperini, 1894. .4. cereus Lieske, 1921. 

St. bovis communis Foulerton and Price- A. chalmersi (deMello and Fernandes, 1919) 

Jones, 1901. Dodge, 1935. 

.4. bovis far cinicus Gasperini, 1894. Syn. X. chalmersi deMello and Fernandes, 

A. ban's luteoroseus Gasperini, 1894. 1919. 

A. bovis sulfureus Gasperini, 1894. A. christopherson i (deMello and Fernandes, 

A. boris var. nigerianus Erikson, 1935. 1919) Dodge, 1935. 

A. bronchialis (Sartory and Lasseur, 1914) Syn. N. christopherson i deMello and 

Brumpt, 1939. Fernandes, 1919. 

A. bronchiticus (Castellani et al., 1921) A - chromogenes (Gasperini, 1S91; Kruse, 

Brumpt, 1939. 1896) Ford, 1927. 

Syn. Anaeromyces bronchitica Castellani °' chromo 9^" Lehmann and Neumann, 

, i 1.101 1896. 
et al., 1921. 

, , ,. , u ,. .. , ,,., , . . A. cinereo-mger Lieske, 1921. 

A. brumpt/ (Bordioski and Milocheviteh, „_, . . ' 

,„„., ,. . '. )M <v. cuureonigeraromaUcus (Berestnew, 1897) 

19.;.)) Brumpt, 1939. v , . , 1ftAO 

. ; • ,ni i i ™. ■ . Neukirch, 1902. 

A. brum (Chalmers and Christopherson, A. a> e a (Gasperini, 1894) Ford, 1927. 

1916) Brumpt, 1939. A citroiremeus Nannizzi Pollacci, 1934. 

Syn. A . brunt Chalmers and Christopher- A . cloaca* Brussoff, 1919. 

son > 1( • ,,(, • A. coccocidus Krassilnikov, 1955. 

-4. buccalis (Roger et al, 1909) Brumpt, A. colorata Sanborn, 1926. 

1939. A. congolensis (Baerts, 1925) Brumpt, 1939. 



318 



THE ACTINOMYCETES, Vol. II 



.1 . convolutus (( Ihalmers and ( Ihristopherson, 

L916) Brumpt, L939. 

Syn. N. convoluta Chalmers and Christo- 

pherson. According to Gonzalez 

Ochoa and Sandoval (1956), this is a 

synonym of N. asteroides. 

A. coremiales Krassilnikov, 1949. 

A. cretaceus (Kriiger, 1905) Wollenweber, 

1920, who considered it as a variety of A. 

albus. 

Syn. 0. cretacea Kriiger, 1905. 
A. cruoris (Macfie and Ingram, 1921) 

Brumpt, 1927. 

Syn. N. cruoris Macfie and Ingram, 1921. 
A. crystallophagus (Gray and Thornton, 

192S) Bergey, 1930. 
A. cuniculi (Schmorl) Gasperini, 1894. 

Syn. N. cuniculi deMello and Fernandes, 
1919. 
>S7. cuniculi Foulerton and Price-Jones, 1901. 
.1. cylindraeeus (deKorte, 1918) Brumpt, 

1939. 

Syn. .V. cylindracea deKorte, 1918. 
0. ci/lindracea Sartory, 1923. 
A. dassorwillei (Brocq - Rousseu, 1907) 

Brumpt, 1939. 

Syn. N. dassorwillei Liegard and Landrieu, 
1911. 
A. decussatus (Langeron and Chevallier, 

1912) Brumpt, 1939. 

Syn. Discomyces decussatus Langeron and 
Chevalier, 1912 

N. decussata Castellani and Chalm- 
ers, 1913. 

0. decussata Sartory, 1923. 
A. denitrificans (Nikolaeva, 1914) Krassil- 
nikov, 1949. 
A. dermatonomus Bull, 1929 (Polysepta 

dermatonomus Thompson and Bisset, 

1957). 
.1. dispar (Vidal, 1882) Brumpt, 1939. 
.1. donnae Dodge, 1935. 
.1. dori (deBeurmann and (iougerot, 190(1) 

Brumpt, 1939. 
0. doriae Sauvageau and Radais, 1892. 



A. egypti Gohar el al., 1954 (Ettlinger el al., 
1958). 

A. claeagni Roberg, 1934. Produces tubers 
on the roots of the oleander plant. 

A. elephantis primigenii Omeliansky, 1909. 

A. enteritidis (Pottien, 1902) Brumpt, 1927. 
Syn. N. enteritidis Castellani and Chalmers, 
1913. 

St. eppingeri (Kossi-Doria, 1891) Namy- 
slowsky, 1912. 

.4. equi (Chalmers and Christopherson, 
1916) Brumpt, 1939. 

Syn. N. equi (Dean, 1900) Chalmers and 
Christopherson, 1916. 

.1. erysipeloides (Neumann and Lehmann, 
1895) Lachner-Sandoval, 1898. 
Syn. N. rosenbachi (Gouge rot, 1913). 

St. erythrea Foulerton, 1902. 

St. farcinica (Trevisan, 1889) Rossi-Doria, 
1891. 

.4. ferruginous (Naunyn) Krassilnikov, 1949. 
Syn. N . ferruginea de Toni and Trevisan. 

A.flava (Sanfelice, 1904) Ford, 1927. 

A.flavus (Chester, 1901) Dodge, 1935. 

A. fluorcscens Krassilnikov, 1955. 

St. Foersteri Cohn, 1875. Observed in con- 
cretions in the lachrymal canal. A faculta- 
tive anaerobe, not pathogenic for labora- 
tory animals. Numerous synonyms of 
this name are found in the literature. It is 
sufficient to list Leptothrix oculorum 
Sorokin, 1881; CI. foersteri Winter, 1884; 
CI. dichotomei Mace, 1888; N. forsteri 
Trevisan, 1889; 0. forsteri Sauvageau et 
Radais, 1892; St. forsteri Kruse, 1896; 
N. aurea Saint-Severin, 1902; Cohn- 
istreptoihrix silberschmidti Chalmers and 
Christopherson, 1916. 

A. foulertoni (Chalmers and Christopherson, 
1916) Brumpt, 1939. 

Syn. N. foulertoni Chalmers and Christo- 
pherson, 1916. 

St. freeri Musgrave and Clegg, 1907. 
Syn. A. freeri Bergey, 192:!. 

Pr. freeri Krassilnikov, 1949. Defi- 
nitely a Nocardia. 



INCOMPLETELY DESCRIBED SPECIES OF ACTIXOMYCETES 



319 



A. fusca SohngeD and Fol, L914. 

A. fuscus (Karwacki, 1911) Brumpt, L939. 

Syn. A', fusca Castellani and Chalmers, 
1913. 
A. gabritschevski (Berestnew, L898) Krassil- 

nikov, 1941. 
.1 . garteni Brumpt, 1 ( .)27. 

Syn. .V. garteni Gougerot, 1913. 
A. gedanensis (Lohlein, 1909) Bergey, 1923. 
A. gedanensis (Scheele and Petruschky, 

1897) Ford, 1927. 

Syn. A*, gedanensis Chalmers and Christo- 
pherson, 1916. 
St. gelatinosus Johan-Olsen, 1897. 
A. genesii (Fr6es, 1930) Dodge, 1935. 

Syn. A*, genesii Fr6es, 1930. 
A. gibsoni Dodge, 1935. 
A. goensis (deMello and Fernandes, L919) 

Dodge, 1935. 

Syn. A*, goensis deMello and Fernandes, 
1919. 
A. gonadiformis Bergey, 1930. 
A. graminearus (Berestnew, 1S97) Krassil- 

nikov, 1949. 
A. graminis (Bostroem, 1891) Topley and 

Wilson, 1929, 1946. 
A. griseo-viridis Nikolaeva, 1914. 
A. i/ris, us variabilis Krassilnikov, 1949. 
.4. gruberi (Terni, 1S94) Sanfelice, 1904. 

Syn. N. gruberi Blanchard (In Bouchard, 
1896). 
.4. guegueni (Brumpt, 1921) Brumpt, 1939. 

Syn. D. lingualis Guegen, 1908. 

A', lingualis Castellani and Chalmers, 
1913. 
.1. guerrai (Langeron, 19*29) Brumpt, 1939. 
A. guignardi (Sauvageau and Radais, 1892) 

Ford, 1927. 
.1. t/i/psoides Henrici and Gardner, 1921. 
A. halotricus ZoBell and Upham, 1944. 
A. heimi Duehe, 1934. 
A. hobnesi (Gedoelst, 1902) Nannizzi Pollacci, 

1934. 
A. hofmanni (Gruber, 1891) Gasperini, 

1894. 
A. hominis Bostroem, 1S90. 



St. hominis II, III, [V (Foulerton, 1906 
1910). 

St. hominis ( Berestnew, 1897). 
Xot A. hominis Waksman, L919. 

St. humifica Johan-Olsen, 1897. 

A. incanescens Wollenweber, 1922. 

A. indicus (Kanthack, 1893) Brumpt, 1939 
Syn. N. indica var. flava Kanthack, 1893 

A . innominatus Baldacci, 1939. 

A. interproximalis (Fennel, 1918) Ford, 1927. 

A. invulnerabilis (Aeosta and Rossi, 1893; 
Kruse, 1896; Lachner-Sandoval, 1898) 
Ford, 1927. Isolated as laboratory con- 
tamination, and also from water. With- 
stands heating at 100-120°C. Grows in 
media containing 0.5 per cent copper sul- 
fate, 0.5 per cent phenol, 1 percent boric 
acid, and 0.01 per cent mercuric chloride. 

St. isracli Kruse, 1896. 

A. japonica (Petruschky, 1913) Ford, 1927. 

.4. japonic us (Aoyama and Miyamoto, 1901 ) 
Brumpt, 1939. 

A.jollyi (Vuillemin, 1920) Brumpt, 1927. 

A. keratolyticus (Acton and McGuire, 1931) 
Brumpt, 1939. 

A. hrainskii Duehe, 1934. 

A.krausei (Chester, 1901) Brumpt, 1927; 
Ford, 1927. 

Syn. N. krausei Chalmers and Christopher- 
son, 1916. 

.4. lacertae (Terni, 189(5) Foulerton, 1912. 
Syn. .1. sulfureus lacertae Berestnew, 1897. 

A. lanfranchii Sani, 1916. 

Syn. N. lanfranchii deMello and Pais, 
1918. 

A. lasserei (Verdun, 1912) Brumpt, 1939. 
Syn. N. lasserei (Verdun) Castellani and 
Chalmers, 1913. 

St. lathridii (Petruschky, 1898) Ford, 1927. 

A. leishmani (Chalmers and Christopherson, 
1916) Brumpt, 1939. 

A. lepromatis (deSouza-Araujo, 1929) 
Brumpt, 1939. 

St. leucea Foulerton, 1902. 

St. leucea saprophytica Foulerton, 1912. 

.1 . levyi Dodge, 1935. 



320 



THE ACTINOMVCETES, Vol. II 



.1. lieskei Duche, L934. 

Actinobacillus ligniersi (Brumpt, 1910) 

Brumpt, 1!):!!). 
A. liguire (Urizer, 1904) Nannizzi Pollacci, 

19.34. 
A. lingualis (Weibel, 1888; non Gueguen, 

1908) Brumpt, 1939. 
A. liquefaciens (Hesse, 1892) Brumpt, 1939. 

Syn. N. liquefaciens Castellani and Chal- 
mers, 1913. 
A. liquefaciens (Garten, 1895) Ford, 1927. 
A. londinensis (Chalmers and Christopher- 
son, 1916) Brumpt, 1939. 

Syn. N . londinensis Chalmers and Christo- 
pherson, 1916. 
.4. longisporus Krassilnikov, 1941. 
.4. longisporus ruber Krassilnikov, 1941. 
.4. longisporus griseus Krassilnikov, 1941. 
.4. longissimus Krassilnikov, 1941. 
A. luteolus (Foulerton and Jones, 1910) 

Brumpt, 1939. 
A. luteo-roseus Gasperini, 1894. 
A. macrodipodidarum (Fox, 1923) Dodge, 

1935. 

Syn. N. macrodipodidara Fox, 1923. 
.4. malenconi Duche, 1934. 
.4. matruchoti (Mendel, 1919) Brumpt, 1939. 
A. melanogenes Rubentschik, 1928. 
A. melanoroseus Issatschenko, 1!)27. 
A. melanosporeus Krainsky, 1914. 
St. melanotica Price-Jones, 1903. 
A. metchnikovi (Sauvageau and Radais, 

1892) Ford, 1927. 
.4. mexicanus (Boyd and Crutchfield, 1921) 

Brumpt, 1939. 

Syn. N. mexicana Ota, 1928. 
A. micetomae (Greco, 1916) Dodge, 1935. 

Syn. 0. micetomae Sartory, 1923. 
.1. mshagiensis Salimowskaj a, 1928. 
A. microfloras Krainsky, 1914. 
4 . mihi ( laminiti, 1907. 
A. mineaceus (Kruse, 1896) Lachner- 

Sandoval, 1898. 
A. minimus ( LeCalve and Malherbe, 1900) 

Dodge, 1935. 
A. minutissimus (Burchard) Brumpt, 1927. 



Syn. N. minutissima (Verdun, 1912) Cas- 
tellani and Chalmers, 1913. 
A. mucosas Basu, 1943. 
.4. multijidus Krassilnikov, 1941. 
A. muris-ratti (Schottnuiller, 1914) Ford, 

1927. 
A. musculorum Hertwig, 1886. 
A. mutabilis Masumoto, 1943; Cause et al., 

1957. 
.4. myricae Peklo, 1910. 
St. necrophora Wilhelm, 1902. 
A. necrophorus (Fliigge, 1886) Lehmann and 

Neumann, 1926. 
.4. neddeni (Xamyslowsky, 1912) Brumpt, 

1939. 
.4. neschczadimenki (Chalmers and Christ o- 

pherson, 1916) Dodge, 1935. 
.4. nicollri (Delanoe, 1928) Brumpt, 1939. 

Syn. N. nicollei Delanoe, 1928. 
.4. nigcr (Rossi-Doria, 1891) Krassilnikov, 

1949. 
A: niger aromaticus Berestnew, 1897. 
St. nigra (Rossi-Doria, 1891) Sanfelice, 1904. 
.4. nigricans Killian and Feher, 1935. 
A. nigrificans (Kriiger, 1905) Wollenweber, 

1920. 
St. nigrescens Foulerton, 1901. 
A. nitrcgenes Sartory et al., 1936. 
.4. nocardii (Foulerton, 1901) Buchanan, 

1911. 
.4. nodosus (Beveridge, 1941) Hagan, 1943. 
.4. nondiastaticus Bergey, 1919. Grows at 

65°C; differs from S. thermodiastaticus in 

not decomposing starch. 
.4. non-fluorescens Krassilnikov, W^^. 
A. ochraceus Xeukirch, 1902. 
.4. ochroleucus Xeukirch, 1902. A culture 

described under same name was isolated 

from diseased potatoes by Wollenweber 

(1922) who considered it as a variety of 

A. albus. 
A. odoratus Krassilnikov, 1941. 
.1 . odorifi r Koelz, 1936. 
St. oidioformis Johan-Olsen, 1893. 
A. oligocarbophilus (Beijerinck and Van 

Delden, 1903) Lantzseh, 1922. 



INCOMPLETELY DESCRIBED SPECIES OF A.CTINOMYCETES 



321 



St. orangica Berestnew, L897. 

.1. orangico-niger Lieske, 1921. 

A. orangicus (Rossi-Doria) Lieske, 1021. 

.4. panginensis (deMello and Fernandes 
L919) Dodge, 1935. 

Syn. .V. panginensis deMello and Fernan- 
des, L919. 

St. paulotrophus Beijerinck, 1914. 

.1 . pelogenes Sawjalow, L913. 

A. penicilloides Sartory and Meyer, L936. 

.4. phagocidus Krassilnikov, L955. 

A. phenotoleransWerkman&nd Patrick. 1932. 
Isolated from granuloma in man. Grows 
well in phenol-containing media. Gon- 
zalez Ochoa and Sandoval (1956) con- 
sidered it a synonym of N. asteroides. 

A. pijperi (Castellani and Chalmers, L919) 
Brumpt, 1939. 

Syn. .V. pijperi Castellani and Chalmers, 
1919. 

A. pinoyi (deMello and Fernandes, 1919) 
Dodge, 1935. 

Syn. X. pinoyi deMello and Fernandes, 
1919. 

A. plurichromogenus (Caminiti, 1907) Dodge, 
L935. 
Syn. N. plurichromogena Caminiti, 1907. 

A. pluricolor Gasperini, 1S94. 

Syn. .V. pluricolor Terni, 1894; N. pluri- 
color deMello and Fernandes, 191!). 

.4. pluricolor diffundens (Berestnew, L897) 
Lieske, 1921. 

.4. polychromogenus Dodge, L935. 

A. ponceti (Verdun, 1912) Brumpt, L939. 
Syn. N. ponceti (Verdun, 1912) Castellani 
and Chalmers, L919. 

A. pretorianus (Pijper and Pullinger, 1927) 
Brumpt, L939. 

Syn. .V. pretoriana (Pijper and Pullinger, 
1927). 

A. protea (Schurmayer, L900) lord, 1927. 

A. pseudonecrophorus Harris and Brown, 
1927. 

A. pseudotuberculoscn (Flexner, L898) 
Brumpt, L939. 

A. pseudotuberculosis (Keller) Dodge, 1!):!."). 



Syn. X. pseudotuberculosis deMello and 
Fernandes, L919. 

pseudotuberculosus (Flexner, L898) Leh- 
mann and Neumann, L912. 

pulmonalis Burnett, 1909; (Roger et al., 

L909) Brumpt, L939. 

puntonii Lopez Ortega, 1!).'!4. 

purpurogenus Waksmanand Curtis, L916. 

purpureus (Orloff, 1913) Brumpt, L939. 
. putorii Dick and Tunnicliff, 1918. 

putridogenes (Vezspremi, 1907) Nannizzi 
Pollacci, L934. 

putrificus Nikolaeva, 1914. 

pyocyaneus Rullmann, L895. 
. pyogenes Caminiti, 1907. 

pyogenes (Chalmers and Christopherson, 
1916) Dodge, 1935. 

radiatus (Namyslowsky, 1912) Brumpt, 

l!):;!). 

. ratti Schottmiiller, 1914. 

, ribeyroi Dodge, 1935. 

rivierei (Verdun, L912) Brumpt, L939. 

rodellae Dodge, 1935. 
, rogersii Brumpt, L939. 
Syn. N. rogersi deMello, 1919. 
, rosaceus Lieske, 1921 . 

rosella Kriiger, 1905. 

rosenbachi (Kruse, L896) Holland, 1920. 

roseolus Nadson, 1903. 

rubea Wilbert, 1908. 

. rubea Chalmers and Christopherson, 1910. 
, ruber (Kruse, L896) Sanfelice, 1904. 
. rubra (Kruse, 1X90) Ford, 1927. 
. rubidaureus Lachner-Sandoval, 1S98. 
Syn. A. mordore" Thiry, 1897. 
0. mordore Sartory, 1923. 
A", thiryei deMello and Pais, 191S. 
A. sabrazes (Ferre and Faguet, L895) Dodge, 

1935. 
.1 . saharae Killian and Feher, 1935. 
A. salvati Langeron, L922; Fontoynont and 

Salvat, L922. 
A. sanfelicei (Redaelli, L928) Nannizzi 
Pollacci, L934. 

Syn. N. sanfelicei Redaelli, 1928. 
.1 . sanguinis Basu, 1937. 



THE ACTINOMYCETES, Vol. II 



St. sanninii ( liferri, L>22. 

.4. saprophyticus Gasperini, 1892. Lieske, 

L921. 
.4. saprophyticus var. chromogenes Gasperini, 

1892. 
,4. sartoryi Dodge, 1935. 
A. scabies var. anglica Baldacci and Spalla, 

1956. 
.4. sendaiensis (Ping-Ting-Huang, 1933) 

Brumpt, 1939. 
A. septicus MacNeal and Blevins, 1945. 
.1 . serratus (Sartory et al., 1930) Dodge, 1936. 
A. silberschmidti (Chalmers and Christo- 

pherson, 1916) Dodge, 1936. 

Syn. N. silberschmidti deMello and 
Ferriandes, 1919. 
A. somaliensis (Brumpt, 1906) St. John- 
Brooks, 1931. 
.4. sommeri (Greco, 1910) Brumpt, 1939. 
A. spinae Velieh, 1929. 
A. spinosporus (Spini) Verlich, 1914. 
St. spirilloides Johan-Olsen, 1893. 
.4. spitzi (Lignieres and Spitz, 1904) Dodge, 

1935. 

Syn. 0. spitzi Sartory, 1923. 
.4. splenicus (Gibson, 1930) Brumpt, 1939. 

Syn. N. splenica Gibson, 1930. 
A. spumalis (Sartory, 1923) Dodge, 1935. 

Appears to be a Nocardia. 
St. taraxeri cepapi (Schottmiiller, 1914) 

Ford, 1927. 
A. tarozzii (Miescher, 1917) Dodge, 1935. 
St. tartari Sanfelice, 1904. 
O. tenax Kriiger, 1905. 
,1. tenuis (Castellani, 1911) Dodge, 1935. 

Syn. .V. tenuis Castellani, 1911. 
S. termitum Duehe et al., 1951. 
.1. thermotolerans Lieske, 1921. 
.1. thibiergei (Ravaut and Pinoy, 1909) 

Brumpt, 1939. 

Syn. .V. thibiergei ( Jastellani and Chalmers, 
I'll 3. 
A. thjottae (Thj0tta and Gundersen, 1925) 

Dodge, 1!):;:.. 
A. thuillieri (deToni and Trevisan, 1889) 

Brumpt, 193!). 

Syn. .V. thuillieri Vuillemin, 1931. 



A. tossicus (Rossi, 1905) Dodge, 1935. 

A. totschidlowslditicrhmoY, 1925. 

.1. toxicus Krassilnikov, 1955. 

A. transvalensis (Pijper and Puilinger, 1927) 

Brumpt, 1939. 
A. tricolor Wollenweber, 1922. 
.1. tyrosinaticus Beijerinck, 1 ( .)14. 
.1. urethritidis (Rocek, 1920) Brumpt, 1939. 
.1. urinarius (Pijper, 1918) Brumpt, 1939. 
A. valvulae (deMello and Pais, 1918) Nan- 

nizzi Pollacci, 1934. 

Syn. .V. valvulae deMello and Pais, 1918. 
A. valvularis (Luginger, 1904) Ford, 1927. 

A. valvulas destruens bovis Luginger, 1904. 
.1. verrucosus Nadson 1903; (Miescher, 1917) 

Brumpt, 1939. 
.4. violacea (Rossi-Doria, 1891) Ford, 1927. 
A . waksmanii Bergey, 1930. 
.1. xanthostromus Wollenweber, 1922. 
St. zopji (Casagrandi) Caminiti, 1907. 

The above is only a partial list of incom- 
pletely described cultures, most of which 
would now be included in the genus Strepto- 
myces. Some cultures were listed as Dis- 
comyces, such as D. pleuriticus Vachetta 
(1882) and D. pleuriticus canis familiaris 
Rivolta. Various others were given under 
the generic names Bacillus, such as B. 
actinoides Smith, 1918; Bacterium, such as 

B. actinocladothrix Afanassiev, 1888; Myco- 
bacterium, such as M. paraffinicum (Davis 
et al., 1956), also under the anaerobic genus 
Actinobacterium, including A. meyeri, A. 
abscensus, A. cellulitis (Linhard, 1949); and 
as Actinobacillus, such as Act. ligniersi 
Brumpt, 1910. 

A large number of cultures were simply 
given numbers, as done by Drechsler, 
Lieske, and many others. Finally, some were 
mentioned under a generic name, without 
even the affixation of a uumber, such as 
Actinomyces sp., Streptomyces sp., etc. All of 
these, with very few exceptions, need not be 
considered any further here. 

Many of the above cultures have been 
isolated from excretions of man and labora- 



[NCOMPLETELy DESCRIBED SPECIES OF ACTINOMYCETES 



323 



tory animals. Some were found to be 
pathogenic to experimental animals. Mos1 
of them were not tested, however, for their 
pathogenicity. 

Organisms Belonging to the Genus Vo- 
cardia 

In addition to many of the forms listed 
previously, certain other incompletely de- 
scribed forms which probably belong to the 
genus Nocardia may be mentioned. These 
have been described under the generic names 
of Nocardia (X.), Asteroides (As.), and 
Proactinomyces I Pr. >. 

A', actinomyces Trevisan, 1889. 

N. albida Chalmers and Christopherson, 

1916. 
N. albosporea Chalmers and Christopherson, 

1916. 
.V. appendicis Chalmers and Christopherson, 

1916. 
Pr. aquosus Turfitt, 1944. 
Pr. asteroides var. crateriformis Baldacci, 

1937. 
Pr. asteroides var. decolor Baldacci, 19.47. 
N. bifida (B. bifidus Tissier, L901). 
A', bovis ( rougerol et al., 1934. 
.V. cuniculi Snijders, 1924. 
Pr. cyaneus (Beijerinck, 1914) Krassilnikov, 

1941. 
Pr. cyaneus antibioticus Gause, 1940. 
A', erythropolis (Gray and Thornton, 1928) 

Waksman and Henrici, 1948. 
A'. Jiliformis (Boas, 1897) Vuillemin, 1931. 
N. krainskii Chalmers and Christopherson, 

1917. 
A", lignieresi (Brumpt, L910) Chalmers and 

Christopherson, 1910. 
As. liskeyi Puntoni and Leonardi, 19:!."). 
A', minima (Jensen, 1931) Waksman and 

Henrici, 1948. 
A', pluricolor Namyslowsky, 1912. 
As. pseudocarneus Puntoni and Leonardi, 

i9:;.->. 
Pr. psevdomadurae Baldacci, 194.'!. 
A*, ramosa (B. ramosus Veillon and Zuber, 

1898). 



J'r. restrictus Turfitt, L944. 
A', ripens (Eklund, 1883) Vuillemin, 1931. 
A', saprophytica Chalmers and Christopher- 
son, 1919. 
A', syhodorifera Castellani, 1911. 

Various o1 her specific names for organisms 
probably belonging to the genus Nocardia 
have been listed under several other genera, 
such as Cladothriz (CI. actinomyces Rossi- 
Doria, 1891; Mace, 1897); Cohnistreptoihrix 
I ( 'o. a/nericana Chalmers and ( ihristopherson, 
1916); Discomyces (J). asteroides Eppinger, 
L891; Godoelst, 1902); Flavobacterium (/•'. 
salmonicolor den Dooren de Jong, 1927; 
Bergey, 1930); Mycobacterium (M. albuvia- 
lum Bergey, 1923); Serratia (S. corralina 
Hefferan, 1904; Bergey, 192:!), and others. 
Krassilnikov (1949) listed or described 
numerous other forms belonging to this 
genus under the names of Nocardia, Proac- 
tinomyces, Mycobacterium, Bacillus, Bac- 
terium, Brevistreptothrix, Cladothrix, Cohn- 
istreptoihrix, Discomyces, and various others. 
Thompson and Bissel (1957) suggested a 
new generic name Polysepta. 

Descriptions Incomplete or Needed for 
Actinomycetes Producing Specific 
Antibiotics and Vitamins 

Because of the growing importance of 
actinomycetes as producers of antibiotics 
and because of the desire to claim priority 
for a new antibiotic, many names have been 
introduced for species of Streptomyces and 
Nocardia. Often these names are mentioned, 
with incomplete descriptions or with no 
descriptions, in the patent literature or even 
in trade journals. An attempt was made to 
colled these. The list (Table 27) is far from 
complete, however. 

A number of new species of actinomycetes 
belonging to the genus Streptomyces have 
been created by Cause et al. (1957-1959), 
who justified this by the fact that there was 
a great need for describing more organisms 
capable of producing antibiotics. Only a 
limited attempt was made in these descrip- 



324 



THE ACTINOMYCETES, Vol. 11 



Table 27 
Incompletely describe! antibiotic producing species of Nocardia and Streptomyces 



Organism 



Antibiotic or vitamin 



Nocardia 

N. acidophilus 
Pr. actinoides 
N. lurida 
N. narasinoensis 
Streptomyces 
S. albicans 
S. albidofuscus 
S. albulus 

S. aminophilus 
S. arabicus 
S. bacillaris 

S. bad/us 

S. blastmyceticus 

S. caiusiae 

S. carcinomyceticus 

S. cellostaticus 

S. chattanoogensis 

S. chibaensis 

S. chrestomyceticus 

S. cinnamonensis 

A. circulatus var. 

monomycin} 
A. coeruleus antibio- 

ticus 
S. colombiensis 

S. chim crisis 

S. fasciculatus 

S. flavofungini 

S. fluorescens 

A . fluorescens 

A .fradiae var. spiralis 

S. ganmycicus 

S. ganmyceticus 

S. globisporus tundra- 

mycini 
S. graminofaciens 

S. griseoplanus 

S. grisinus 

S. arise us vn r. farino 

sus 
S. griseus var. spiralis 
S. hepaticus 



J. Bacteriol. 54: 281, 1947 

Antibiotiki 2(5): 44. 1957 

Antibiotics Ann. 1956-1957, 687, 693, 699 

J. Antibiotics (Japan) 7A: 1, 1954; 8B: 253, 1955 

Med. Parasitol. USSR 4, 1947 

J. Antibiotics (Japan) 6A: 140, 1953 

Absl r. Papers 134th Ann. Meet. Am. Chem. Soc. 

Chicago, 1958, 22. 
Antibiotics Ann. 1955-56, 236 
J. Antibiotics (Japan) 9B: 62, 1956 
Folia Biol. 4: 260, 1958 
J. Antibiotics (Japan) 3: 582, 1949 
J. Antibiotics (Japan) 10A: 40, 1957 
J. Sci. Ind. Res. India 16c: 76-81, 1957 
Chemotherapy (Tokyo) 3: 129, 1955 
Tohoku J. Exptl. Med. 67: 173, 1958. 
Antibiotics & Chemotherapy 9: 398, 1959 
J. Antibiotics (Japan) 114:81, 1958 
Giorn. Microbiol. 7: 242, 1959 
Ann. Soc. Biol. Pernambuco 13: 3, 1955; II: 9, 

1956 
Antibiotiki 5(4): 3, 1960. 

Antibiotiki 2(1): 25, 1957 

U. S. 2,595,499, May 6, 1952 

J. Antibiotics (Japan) 7B: 168, 1954 

Antibiotics & Chemotherapy 3: 718, 1953 

Nature L81: 90S, 1958 

Folia Biol. 1: 259, 1958 

Antibiotiki 5(1): 25, I960 

Antibiotiki I: 4, L956 

.J. Antibiotics (Japan) 9A: S, 113. 1956; 91$: 160, 

1956 
.1. Antibiotics (Japan) <U : ('., 9, 113, 1956 
Bull. Moscow Soc. Nat. Sci. Biol. 62(2): 79, 1957. 



Antibiotics it Chemotherapy 3: 
Antibiotics Ann. 1953-1954, 171 
Antibiotics Ann. 1956-1957, 730 
Folia Biol. 4: 263, 1958 
Bacteriol. Proc. p. 18, 1951 

Antibiotics Ann. 1959-1960, 191. 
Brit. Pat. 730,341, May 18, 1955 



M \comycin 
Actinoidin 
Ristocetin 
Nocardorubin 

Actinolysin 

Pyridomvcin 
Antitumor substances 

Antibiotic 19(58 
Croceomycin 

Various antibiotics 

St reptothricin 

Blastmycin 

Antibiotic X 

Carcinomycin 

Cellostatin 

Tennecetin 

Cellocidin 

Aminocidin 

St roptothricin like 

antibiotic 
Monomycin 

An antibiotic 

\ it amin B12 

( 'andimycin 

Amicetin 

Flavofungin 

Fluorin 

Actinomvcin 

Colimycin; probably 
neomycin 

( ranmycin + Carcino- 
mycin 

Carzinocidin 

Tundramycin 



1283, 1953; Streptogramii 

Alazopeptin 
( rrisin 
Streptolin 

Aspartocin 



INCOMPLETELY DESCRIBED SPECIES OF ACTINOMYCETES 



325 



Tabi.k 27 Continual 



Organism 



Reference 



.1 . jucous 
S. levoris 

S. leydt in matis 

S. I Had mix 

S. longisporus 

S. luteochromogenes 

S. I nil olutescens 

S. hi id iter ranei 

S. mi lanochromogt nes 

S. melanosporus (mel 

anosporofacii ns I 
S. n 1 1 tn 1 1 nsis 
S. orckidaceus 

S. /miirisporogenes 
S. pluii iifiuii lis 

S. phoenix 

S. pleofaciens 

S. plicatus 

S. jiluricolorescens 

S. primycini 

,S. racemochromo- 

l/l II us 

S. raffinosus 
S. recifei 



S. rutgersensis var. 

casteranse 
S. sakaiensis 
S. salmonicida 
S. sindenensis 
S. subtropicus 

S. toxicus 

S. iiiijorai tixis 

S. vendargus 

S. verticil I us 

S. vinaceus-drappus 

A . violareus-cristallo- 

iii hi in 
S. viridifaciens 

S. viridosporus 
S. vulgaris 

S. xanthochromog< m s 
S. zaomyceticus 



Krassilnikov, 1955 

Folia Biol. 1:260, 1958; resembles S. grist us var. 

candicidinus 
Trans. Am. Microscop. Soc. 5:370, 1953. 
.1. Antibiotics (Japan) «>H: si. L956 
Folia Biol. 1: 263, 1958 
.J. Antibiotics (Japan) 6A: 183, 1953 
Kurilowicz in Udientzev et at., p. 151, 1959 
Antibiotics Ann. 1959-1960, 262. 
Kurilowicz in LTdientzev it "/., 1959 
Giorn. Microbiol. 7: 207, 1959 

Antibiotics Ann. 1957-1958,878 

Antibiotics tv. Chemotherapy 5: 204, 1955; 

British Patent 7CN.007, February 13, 1957 

Ann. Pharm. Franc. 16: 585, 1958 

Japan. Med. J. 5: 327, 1952 

Antibiotics & Chemotherapy 3: 788, 1953 

Antibiotics Ann. 1954-1955, 806 

Brit. Pat. Spec. 707,332, Apr. 14, 1954 

J. Antibiotics (Japan) 9A: 75, 1956 

Pharmazie II: 304, 1950 

J. Antibiotics (Japan) «>B: 170, 1950; 11B: 277, 

1958. 
Folia Biol. 4: 259, 1958 
(Lima et al.) Morais, Lima and Maia, Syn. A . 

recifei Lima et nl. (An. Soc. Biol. Pernambuco 

15: 239, 1957) 
Rev. Inv. Agr. Buenos Aires 8: 203, 1954 

Ann. Rept. Takeda Research Lab. 14:8, 1955 
J. Bacterid. 58: 059. 1949 

Ann. Rept. Takeda Research Lab. 13: 41, 1954 
Brit. Med. J. Nov. 12, 1955, Doklady Akad. 

Xauk. SSSE 99:827. 1954 
Folia Biol. 4: 259, 1958 
Japanese Pat. 32-3049, 1957 
Australian Pal. 3985, Oct. 20, 1954 

J. Antibiotics (Japan) 124:285, 1959 
Brit. Pat. Spec. 708,686, May 5. 1954 
Antibiotiki 2(5): 58, 1957 

Q.S. 2,712,517, July 5, 1955; Brit. Pat. Spec 

770.005, Mar. 13. 1957 
Brit. Pat. Spec. 712,547, July 28. 1954 
Folia Biol. I: 200. 1958 
Bull. Agr. Chem. Soc. Japan 30:409, 1950 
J. Antibiotics (Japan) 7\: 134. 1954 



Bionivcin 
Levorin 



( lladomycin 
Longisporin 
Pht hiomycin 
Antit amor agent 
Rifomycin 
Antitumor agent 
Melanosporin and 

Elaiophylin 
Pimaricin 

Cycloserine 



Phaeot'acin 
Rhodocidin 
Pleomycin 
Antibiotics C and D 
Pluramycin 

Uacemomycin 

? 
? 



Camphomycin 

Monilin 

? 

Allomycin 
Albomycin (grisein) 

Xecrot in 
Toyocamycin 
( )xyte1 racycline 

Vengicide 
Pleomycin 
Amicetin 
( Jrystallomvcin 



md 



Tetracycline and/or 

chlortet racycline 
Sistomycosin 
Pneumocin 

X.-mt lii<'in 
Zaomycin 



:;2G 



THE ACTINOMYCETES, Vol. II 



Table 28 

Incompletely described Streptomyces species of 

Go use et al. (1957) 

.!. abikocnsinu var. spiralis 

A . acrimycini 

A. acrimycini var. globosus 

A. albidus var. invcrtens 

A . alborubidus 

A . albovinaceus 

A . atroolivaceus 

A . aurentiogriseus 

A . a mini 

A . badius 

A. bicolor 

A . biverticillatus 

A. candidus var. alboroseus 

A . chromofuscus 

A . cinnabarinus 

A. cinnamonensis var. proteolytics 

A. coelicolor var. achrous 

A. coelicolor var. flavus 

A . coeruleofuscus 

A. coeruleorubidus 

A . coerulescens 

A. coerulescens var. longisporus 

A . cremeus 

A . cyanofuscatus 

A . daghestanicus 

A. flaveolus var. rectus 

A . flavotricini 

A . fiavidovirens 

A. fiavidovirens var. fuscus 

A . f ratline var. spiralis 

A . fa man us 

A . glaucescens 

A. glaucescens var. badius 

A. globisporus var. caucasicus 

A. globisporus var. flavofuscus 

A . gobitricini 

A . griseoloalbus 

A . griseoincarnatus 

A . griseomycini 

A . griseorubens 

A . griseoruber 

A . griseorubiginosus 

A. griseorubiginosus var. spiralis 

A. griseostramineus 

. I . ircrini 

A . kurssanovii 

A. later ih us 

A . litmocidini 

I , malachiticus 
A. mutabilis 

1 . nigrescens 
A , (iliraceiiririilis 



Table 



-Continued 



A . prunicolor 

A . roseofulvus 

A . roseolilacinus 

A. roseolas 

A . roseoviolaceus 

. I . roseoviridis 

A . rubiginosohelvolus 



A . rubiginosus 

A. syringini 

A. toxytricini 

A. umbrinus 

A . variabilis 

A . variabilis var. roseolus 

A. venezuelae var. spiralis 

A . violaceorectus 

A. violaccus var. rubescens 

A. violasct ns 

A . viridoviolaceus 



tions of "new" organisms to emphasize the 
ability of the cultures to produce melanin 
pigments, an important and characteristic 
property for distinguishing and identifying 
species of Streptomyces, as brought out in 
the various editions of Bergey's Manual. In 
spite of the fact that these descriptions of 
"new" organisms had been undertaken for 
the purpose of identifying antibiotic-pro- 
ducing cultures, no attempt was miulc to 
list any antibiotic for the various "new" 
species; all one finds are such expressions 
as: "strong repression," "weak activity," 
"no activity." Even the antibiotic "al- 
bomycin" that has been greatly publicized 
by the senior author is not listed in this 
I real ise; neither is the species .4. subtropicus, 
which produces this antibiotic, described. 
Very little effort has been made in creating 
these species to compare the newly isolated 
cultures with those previously described and 
well known in the literature. Many of the 
new descriptions remind one of known organ- 
isms. 

For these reasons, this collection of "new" 
species is listed here (Table 28) as incom- 
pletely described, or at least as requiring 
further information for proper identification. 



Appendices 



Two appendices are given here, each of 
which is of special significance. 

I. Since the colors of the substrate growth, 
the aerial mycelium, and the spores, as well 
as the pigmenl dissolved in the medium, are 
highly importanl in classifying actinomycetes, 
a well -recognized and universally available 
standard must he used for color evaluation. 
Unfortunately, some of the besl standard 
charts and volumes are not generally ac- 
cessible. Hence, a simplified system, readily 
understood, must he used. Such a system has 
been proposed by Lindenbein (1952). It is 
presented here, with certain modifications 
and in terms of English equivalents, 
as Appendix I. Several color charts are 
available for this purpose. The following 
standards may he consulted: Ridgeway 
(1912), Sacchardo (1912), Maerz and Paul 
(1930), and Munsell. 

II. A knowledge of the chemical com- 
position of the media used for the growth of 
actinomycetes is essential for characteri- 
zation of species and varieties. Suitable 
media also are essential for good sporula- 
tion and for the production of importanl 
biochemical products, notably antibiotics 
and vitamins. Numerous media have been 
proposed for the growth of actinomycetes, 
to serve one purpose or another. To list 
them all here is hardly necessary. Only a 
few have been selected. These include syn- 
thetic, artificial organic, and complex natural 
media. 

For microscopic examination of the 
culture, it is desirable to make stained 
preparations. Some of the general principles 
underlying light microscopic and electron- 
microscopic preparations have been dis- 
cussed in Chapter 2 of Volume I. 



Appendix I 

< 'olor Designations for Describing 
Actinomycetes ( Lindenbein) 

/. White: (a) snow-white (niveus), (b) 
glossy white {candidus), (c) silver-white 
(argenteus), (d) milk-white (lacteus), (e) 
chalk-white (cretaceus), (f) gray-white (far- 
inaceus); also cream, egg-shell, and ivory. 

2. Violet: (a) bluish-violet (violaceus), 
(b) reddish-violet (lilaceus), also lavender, 
mauve, purple. 

3. Blue: (a) dark blue (caeruh us), (b) 
cornflower blue (cyaneus), (c) sky blue 
(azureus), (d) gray-blue (caesius), (e) 
yellowish-blue (lividus). 

4. Green: (a) grass-green (viridis), (b) 
emerald green (smaragdin us), (c) blue- 
green (glaucus), (d) forest-green (prasinus), 

(e) olive-green (olivaceus). 

5. Yellow: (a) light yellow (flavus), (b) 
deep yellow (luteus), (c) citron-yellow 
(citrinus), (d) golden yellow (aureus), (e) 
sulfur-yellow (sulfureus), (f) white-yellow 
(stramineus) , (g) brownish-yellow (gilvus), 
(h) egg-yolk yellow (vitellinus) , (i) pale 
yellow (luridus), (k) greenish-yellow (galbus). 

6. Orange: (a) light orange (aurantiacus) , 
(b) dark reddish-orange (croceus). 

7. Red: (a) dark red (ruber), (b) carmine- 
red (puniceus), (c) scarlet red (coccineus), 
(d) fire-red (igneus), (e) pale carmine (roseus), 

(f) flesh-red (carneus, incarnatus), (g) 
purple-red (purpureus), (h) cinnabar-red 
(cinnabarinus) , (i) lead-red (miniatus), (k) 
brick-red (lateritius) , (1) blood-red (san- 
guineus), (m) brownish-copper red (cupreus), 
(n) light yellow-red (rutilus); also pink, 
coral-pink, rose, and wine-colored (vinaceus). 

8. Brown: (a) light brown (brunneus), 



327 



328 



THE ACTINOMYCETES, Vol. II 



(h) dark brown (umbrinus) (c) chestnut 
brown (badius), (d) reddish-brown (fuscus), 
(e) yellow-rusty -brown (ferrugineus) , (f) 
greenish-brown (hepaticus), (g) cinnamon- 
brown (cinnamomeus) ; also beige, tan, and 
ocher. 

9. Gray: (a) greenish-gray (griseus), (b) 
ash-gray (cinercus), (c) white-gray (incanus), 

(d) brownish-gray (fumigatus), (e) reddish- 
gray (murinus), (f) bluish-gray (plumbeus). 

10. Black: (a) gray-black (niger), (b) 
coal black (ater), (c) brownish-pitch-black 
(piceus), (d) greenish-jet-black (coracinus), 

(e) blue-black (atramentarius) . 

Appendix II 

Certain Important Media for the Study 
of Actinomycetes 

Krainsky (1914) and Waksman and Curtis 
(1916) were the first to report on the sig- 
nificance of simple synthetic media in the 
study of the morphological and cultural 
properties of actinomycetes. Conn (1021) 
made a careful comparison of the growth of 
7.") cultures of actinomycetes on a large 
tiumber of media. He came to the conclu- 
sion that "extreme variation in chromo- 
genesis is possible with some of the cultures 
studied, according to the composition of 
i he medium, and some cultures even vary 
greatly when studied at different times on 
the same medium. The appearance of one 
of these organisms on any medium should 
not be described until it has been cultivated 
on several lots of this medium at different 
times. No culture, moreover, can be con- 
sidered nonchromogenic until it has been 
studied on a great variety of different pro- 
tein-free media." 

The following have been selected as rep- 
resenting the more important media recom- 
mended for the study of actinomycetes. All 
constituents are reported in grams per liter. 
/. Sucrose nitrate agar: 

Sucrose 30.0 gm 

NaN0 3 2.0 gm 



K 2 HP0 4 i.Ogm 

MgS0 4 -7H 2 0.5 gm 

KC1 0.5 gm 

FeS0 4 0.01 gm 

Agar 15.0 gm 

Distilled water 1000.0 ml 

pH 7.0 to 7.:; 
This medium is frequently also known as 
"Czapek's agar," or as "Czapek's solution 
agar." Glycerol or glucose may besubstituted 
for sucrose, giving glycerol-, or glucose-ni- 
trate agar. Ammonium chloride may be sub- 
stituted tor NaN0 3 , giving sucrose-ammo- 
nium agar. 
2. Glucose-asparaginc agar: 

Glucose 10.0 gm 

Asparagine 0.5 gm 

K 2 HP0 4 0.5 gm 

Agar 15.0 gm 

Distilled water 1000.0 ml 

pH 6.8 
Meat extract (2.0 gm) may be added to this 
medium. Tap water may be used. 
■ !. Glycerol-asparagine agar: 

Glycerol 10.0 gm 

Asparagine 1.0 gm 

KoHPO, 1.0 gm 

Agar 20.0 gm 

Tap water 1000.0 ml 

Adjust to pll 7.0 with NaOH. 

4. Glycerol-asparaginate agar I: 

Glycerol 35 . gm 

Ammonium lactate (5.5 gm 

Sodium asparaginate 3.5 gm 

K2HPO4 2.5 gm 

NaCl 5.0 gm 

CaCl 2 0.1 gm 

MgSO, 0.8 gm 

Agar 20.0 gm 

Distilled water 1000.0 nil 

This medium is often spoken of as Ushin- 
sky's, especially when used as a solution, 
without agar. 

5. Glycerol-asparaginate agar II: 

( rlycerol 10.0 gm 

Sodium asparaginate 1.0 gm 



APPENDICES 



329 



K2HPO4 1 .0 gm 

Agar 1 5.0 gm 

Distilled water 1000. ml 

This medium is often known as Conn's. 

6. Glycerol-glydm agar: 

Glycerol 20.0 gm 

Glycine 2.0 gm 

K 2 HP0 4 1.0 gm 

NaCl 2.0 gm 

MgSOwHoO 0.5 gm 

FeS0 4 0.1 gm 

CaC0 3 0.2 gm 

Agar 18.0 gm 

Distilled water 1000.0 ml 

Adjust to pi I 7.2 with NaOH 
This medium is often known as Plotho's. 

7. Glycerol-calcium malate agar: 

Glycerol 10.0 gm 

Calcium malate 10.0 gm 

NH4CI 0.5 gm 

K..HPO4 0.5 gm 

Agar 15.0 gm 

Distilled water 1000.0 ml 

Glucose or mannitol may be used (20 gm) 
to replace the glycerol. Calcium citrate may 
be used to replace the malate. 

8. Glucose-ammonium salt agar: 

Glucose 10.0 gm 

(NH 4 ) 2 HP0 4 4.0 gm 

NaCl 5.0 gm 

K,HP() 4 2.0 gm 

MgS0 4 -7H 2 1.0 gm 

CaCl, 0.4 gm 

FeS0 4 -7H 2 0.02 gm 

MnS() r 7H,() 0.01 gm 

Agar 15.0 gm 

Distilled water 1000.0 ml 

9. Glycerol-ammonium salt agar: 

< Glycerol 10.0 gm 

NH 4 C1 1.0 gm 

K 2 HP0 4 1.0 gm 

MgS0 4 -7H 2 0.5gm 

NaCl 0.5 gm 

CaC0 3 1.0 gm 

Agar 15.0 gm 



Distilled water 1000.0 ml 

pll 7.0 
The ammonium salt may be replaced by 
KNO3 . This medium was proposed by 
Masumoto (1952). 
10. Glycerol-urea agar: 

( rlycerol 15.0 gm 

Urea 2.0 &m 

KTIPO4 0.5 gm 

MgS0 4 0.5 gm 

NaCl 0.5 gm 

FeS0 4 0.1 gm 

Agar 15.0 gm 

Distilled water 1000.0 ml 

//. Glucose-tyrosine agar: 

Glucose 10.0 gm 

Tyrosine 1.0 gm 

(NH 4 ) 2 S0 4 0.5 gm 

IvdIPO, 0.5 gm 

Agar 15.0 gm 

Distilled water 

Reaction made neutral with Na< )H. 
12. Glucose-peptone agar: 

Glucose, crude 40 gm 

Peptone 10 gm 

Agar, powdered 20 gm 

Distilled water 1000.0 ml 

pH 5.6 
Dissolve glucose and peptone in 500 ml dis- 
tilled water. Dissolve agar, by heating a1 
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 
M0 min, allow clot to settle, filter, distribute 
in lubes, and autoclave at 1 15°C for 10 min. 
This medium, also known as Sabouraud's 
agar, is fairly acid. 
IS. Tyrosine-casein-nitrate agar: 

Tyrosine 1.0 gm 

Sodium casemate 25.0 gm 

Sodium nitrate 10.0 gm 

Agar 15.0 uin 

Tap water 1000.0 ml 

This medium has been recommended (Men- 
zies and Dade, 1959) for the isolation of 
Streptomyces scabies from potato scab lesions 



330 



THE ACTINOMYCETES, 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. 
/ /. Peptone-beef extract or nutrient agar: 

Peptone 5.0 gm 

Beef extract 5.0 gm 

NaCl 5.0 gm 

Agar 15 to 20 gm 

Distilled water 1000.0 ml 

pH 7.2 to 7.4 
Frequently 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. 
to. Peptone-beef -salt agar: 

Glucose 10.0 gm 

Peptone 5.0 gm 

Beef extract 5.0 gm 

K 2 HP0 4 1.0 gm 

MgS() r 7H,() 0.5 gm 

KC1 0.5 gm 

Agar 15.0 gm 

Distilled water 1000. ml 

The organic nutrients may be reduced to 
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: 

Glycerol 20.0 gm 

Peptone 5.0 gm 

Beef extract 3.0 gm 

Agar 15.0 gm 

Distilled water 1000.0 ml 

pH 7.0 

17. Tryptone-yeast agar: 

Tryptone 1.0 gm 

Yeast extract 1.0 gm 



NaCl 8.5 gm 

Agar 17.0 gm 

Tap water 1000.0 ml 

18. Glucose-yeast-ammonium agar: 

Glucose 10.0 gm 

Yeast extract 1.0 gm 

(XH 4 ) 2 HP0 4 1.0 gm 

KC1 1.0 gm 

MgS0 4 -7H 2 0.2 gm 

Agar 15.0 gm 

Distilled water 1000.0 ml 

pH 7.0 

19. Gelatin media: 

Gelatin 100-200 gm 

Tap water 1000.0 ml 

Adjust to pH 7.4 

Sterilize 30 minutes at 110°C 
This medium may be supplemented with 

glucose (20.0 gm) and peptone (5.0 gm) to 
give glucose-peptone gelatin. 

20. Gelatin agar medium: 

Peptone 5.0 gm 

Beef extract 3.0 gm 

Gelatin 4.0 gm 

Agar 15.0 gm 

Distilled water 1000.0 ml 

pH 7.0 

21. Starch agar: 

Soluble starch 10.0 gm 

XaXO:; 1.0 gm 

KoHPO, 0.3 gm 

XaGl 0.5 gm 

MgG<) :! 1.0 gm 

Agar 1 5 . gm 

Distilled water 1000.0 ml 

CaC0 3 (3 gin) and MgS( ), ■ 7II,< ) (1 gm) may 

replace the MgC0 3 . (XII,),.S( ), (2 gm) or 
asparagine (0.05 gm) may be used to replace 
the nitrate. 

22. Starch-casein agar: 

Soluble starch 10.0 gm 

Casein (dissolved in XaOH) 1.0 gm 

Iv.IIPO, 0.5 gm 

Agar 1 5 . gm 

Water 1000.0 ml 

pH 7.0 to 7.5 



APPENDICES 



331 



23. Starch-peptone-beef -agar: 

Starch 10.0 gm 

Peptone 5.0 gm 

Meat extract 3.0 gm 

Agar L5.0 gm 

Distilled water 1000.0 ml 

pH7.0 
34- Glycerol-starch-glutamaU agar: 

Glycerol 10.0 gm 

Starch 10.0 gm 

Sodium glutamate 1.0 gm 

NaN0 3 0.5 gm 

Proline 0.25 gm 

VitamiD B 0.01 gm 

K2HPO4 0.25 gm 

MgS0 4 0.25 gm 

FeS0 4 0.01 gm 

Agar 15.0 gm 

Distilled water 1000.0 ml 

pH 7.0 to 7.2 

Shinobu (1958b) considered this one of the 

best synthetic media for growth of actino- 
mycetes, especially for production of aerial 
mycelium. 

25. Egg-albumin agar: 

( rlucose 10.0 gm 

Egg albumin 0. 15 gm 

K2HPO4 0.5 gm 

.MgSO r 7II,() 0.2 gm 

Fe 2 (S0 4 ) 3 Trace 

Agar 15.0 gm 

Distilled water 1000.0 ml 

Egg albumin is first dissolved in water and 

made neutral to phenolphthalein with X 10 
NaOH. 

26. Potato-glucose agar: 

Peeled potatoes 200.0 gm 

( rlucose 20.0 gm 

CaC() 3 0.2 gm 

MgS() r 7H 2 0.2 gm 

Agar 15. gm 

Tap or distilled water 1000.0 ml 

pH 6.8 to 7.2 

27. Potato-peptone-glycerol agar: 

Potato 100.0 gm 

Peptone 2.0 gm 



( rlycerol 5.0 gm 

MgSO, 0.5 gm 

K2HPO4 0.5 gm 

XaCl 0.5 gm 

TcSO, 0.01 gm 

Agar 15.0 gm 

Tap water 1000.0 ml 

The potatoes are cut into small cubes to 
which 100 ml of water is added and the 
whole steamed for three quarters of an hour. 
The extract is strained through fine muslin 
without squeezing the pulp. The other nu- 
trients are dissolved in 500 ml of water which 
is then added to the potato extract, and the 
whole steamed for three quarters of an hour. 
The mixture is then made up to bulk, stand- 
ardized and filtered, after which the agar is 
added. 

28. Glucose-yeast extract-beef -peptom <t(j<ir: 

Glucose 10.0 gm 

Yeast extract 10.0 gm 

Beef extract 4.0 gm 

Peptone 1.0 gm 

XaCl 2.5 gm 

Distilled water 1000.0 ml 

This medium is often known as Emerson's. 
Various modifications of this medium are 
used. The concentration of the first two 
constituents may be reduced to 1.0 gm per 
liter; the XaCl may be left out. 

29. Glucose- yeast extract agar: 

Glucose 10.0 gm 

Yeast extract 10.0 gm 

Agar 15.0 gm 

Tap water 1000.0 ml 

pH 6.8 

This medium may also contain 0.5 gm 

K.I1PO,. 

80. Glucose-casein digest-yeast-beef agar: 

( rluCOSe 10.0 gm 

Yeast ext ract 1.0 gm 

Beef extract 1.0 gm 

X-Z-Amine A 2.0 gm 

Agar 15.0 gm 

Distilled water 1000.0 ml 

pll 7.:5 



332 



THE ACTINOMYCETES, Vol. II 



This medium is usually known as Bennett's 
agar. 

31. Glucose-yeast-malt agar: 

Glucose 4.0 gm 

Yeast extract 4.0 gm 

Malt extract 10.0 gm 

Agar 20.0 gm 

Distilled water 1000.0 ml 

pH 7.3 

32. Dextrin-casein digest agar: 

Dextrin 10.0 gm 

Yc-ist extract 1 .0 gm 

Beef extract 1.0 gm 

( 'asein digest ( X-Z-Amine A) . 2.0 gm 

CoCl 2 -7H,0 0.02 gm 

Agar 20.0 gm 

Distilled water 1000.0 ml 

Adjust to pH 7.3 
This medium is often known as Hickey and 
Tresner's agar. 
34- Oatmeal agar: 

Rolled oats 20 to 65 gm 

Tap water 1000.0 ml 

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: 

Heinz baby oatmeal food. . . . 20.0 gm 

Tomato paste 20.0 gm 

Tap water 500.0 ml 

Add these two ingredients to the 500 ml of 
boiling tap water. Do not adjust pH. 

Difco agar 15.0 gm 

Tap water 500.0 ml 

Melt by steaming at 100°C for 15 to 20 min. 
Do not adjust pH. Mix the two solutions, 
steam at L00°C for 10 min, dispense, and 
sterilize for 15 min at 121 °(\ 
35. Soil extract agar: 

Beef extract 3.0 gm 

Peptone 5.0 gm 

Agar 15.0 gm 

Soil extract 1000. ml 

pH 7.0 



The soil extract is prepared by treating 1 kg 
garden soil with 2.5 liters of tap water for 1 
hour in autoclave at 15 lb pressure. Filter 
hot. Add talc for clarification if necessary. 
.;'/. Carbon nutrition medium: 

Carbon source 10.0 gm 

(NH 4 ) 2 S0 4 2.04 gm 

KH 2 P0 4 2.38 gm 

K,HP0 4 5 . 65 gm 

MgSCV7H,0 1.00 gm 

CuS0 4 -5H 2 0.0064 gm 

FeS0 4 -7H,0 0.001 gm 

MnCl 2 -4H 2 0.0070 gm 

ZnS0 4 -7H 2 0.0015 gm 

Agar 15.0 gm 

Distilled water 1000.0 ml 

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: 

Peptone 5.0 gm 

Meat extract 3.0 gm 

KN0 3 1.0 gm 

Distilled water 1000.0 ml 

pH 7.0 

38. Medium for hydrogen sulfide production: 
Peptone-iron agar (Difco) . . 36.0 gm 

Yeast extract 1.0 gm 

Distilled water 1000.0 ml 

39. ( 'ellulose 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 
Kiister (1955). 
', / . Milk or litmus milk: 

Skim milk powder is used. Litmus or 
brom-cresol may be added. 



APPENDICES 



333 




Control (No Carbon) 1% Glucose 1% Sucrose 0.15%NaOAc 

+ © 

Streptomyces Sp. 10 Days a1 28 C 

Figure 6f>. Growth responses of streptomyceteSj on chemically defined medium, to various 
carbon sources (Courtesy of T. (1. Pridham). 

)!. Melanin formation medium: Use 4 to 5 ml portions in tesl tubes. Incubate 

Yeast extract 1.0 gm at 27°C and read after 1 to 2 days and after 

L-Tyrosine 1.0 gm 4 days. 

NaCl 8.5 gm 48. Other media: 

Agar 16.0 gm A variety of oilier media are frequently 

Tap water 1000.0 ml used, often depending upon the nature of the 



334 THE ACTINOMYCETES, Vol. II 

organism and the problem. This is true par- complex media may be listed certain egg 

ticularly of the pathogenic forms, of organ- media (Dorset's), blood agar, blood serum, 

isms not growing readily upon ordinary carrot plug, and others (Waksman, 1919; 

media, and of those used for special purposes, Levine and Schoenlein, 1930 ; Pridham et al, 

such as antibiotic production. Among the 1956). 



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