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MICROBIAL ANTAGONISMS AND
ANTIBIOTIC SUBSTANCES
LONDON
HUMPHREY MILFORD
OXFORD UNIVERSITY PRESS
Microbial Antagonisms
AND
Antibiotic Substances
SELMAN A. WAKSMAN
PROFESSOR OF MICROBIOLOGY, RUTGERS
university; microbiologist, new JERSEY
AGRICULTURAL EXPERIMENT STATION
"L^ Vie em^eche la vie*'' — Pasteur
NEW YORK
THE COMMONWEALTH FUND
1945
COPYRIGHT, 1945, BY
THE COMMONWEALTH FUND
PUBLISHED BY THE COMMONWEALTH FUND
41 EAST 57TH STREET, NEW YORK 22, N.Y.
PRINTED IN THE UNITED STATES OF AMERICA
BY E. L. HILDRETH & COMPANY, INC.
This book is affectionately dedicated to
BOBILI
who has stimulated me in moments of defression^
who has been at all times an inspiration in the
search for the unknown^ my constant associate
and antagonist
PREFACE
On the basis of their relation to man, the microscopic forms of life may
be classified in two major groups: pathogenic forms that attack living
systems, especially those useful to man and to his domesticated plants
and animals J and saprophytic forms that attack inanimate matter, in-
cluding the universal scavengers and the organisms utilized in industry
and in the preparation of foodstuffs. Between true parasitism — one or-
ganism living in or upon the body of another — and true saprophytism
— one organism merely destroying the waste products and the dead
cells of another — are groups of relationships that may be designated as
antagonistic and associative. In the first of these, one organism is in-
jured or even destroyed by the other, whereas in the second, one or-
ganism assists the other and may in turn be benefited by it.
The antagonistic interrelationships among microorganisms have at-
tracted attention since the early days of bacteriology. Following the
discovery by Pasteur that microbes are responsible for certain human,
animal, and plant diseases, it was established that other organisms, later
designated as antagonists, are able to combat and even destroy the dis-
ease-producing agents. At first the soil was believed to be the natural
habitat of the bacteria that cause epidemics and disease as a whole, but
after careful study the fact was definitely established that very few of
these bacteria survive for long in the soil. On the contrary, the soil was
found to be the natural medium for the development of antagonists
chiefly responsible for the destruction of pathogens. The saprophytic
organisms that influence in various ways the disease-producing bacteria
and fungi were found to inhabit, in addition to the soil, various other
natural substrates, such as manure heaps and water basins.
The activities and potentialities of these antagonistic microbes still
present many problems. Little is known about the nature and mode of
formation of the antibiotic substances they produce, and even less about
the mode of their action. The substances vary greatly in their physical
and chemical properties. Some are soluble in water, others in ether, alco-
hol, or other solvents. Some are thermolabile, others are thermostable.
Some are sensitive to alkalies, others are not. Some are readily oxidized
viii PREFACE
and destroyed, others are not. Some are subject to destruction by spe-
cific enzymes. The substances are largely bacteriostatic in action, to a
lesser extent bactericidal j some are also fungistatic and fungicidal.
Some of the substances are highly toxic to animals. Others are either
nontoxic or of limited toxicity and are active in vivo. Some hemolyze
red blood cells, others do not. Those that are hemolytic and moderately
toxic may be useful for application to local infections. Those that are
neither hemolytic nor toxic and are active in vivo may have great im-
portance in combating certain diseases in animals and man.
Some substances are formed by only a few specific organisms, others
may be formed under proper conditions of nutrition by many different
organisms. Some antagonists produce only one type of antibiotic sub-
stance, others form two or even more chemically and biologically dif-
ferent substances.
The ability of an antagonist or its products — antibiotic substances — to
destroy a parasitic microorganism in vivo is influenced by the nature of
the host as well as by the type and degree of the infection. The manner
in which antagonists destroy or modify parasites varies greatly, depend-
ing frequently upon the nature of the antibiotic substances produced.
It is thus clear that the subject is extremely complicated, involving
numerous interrelationships among different biological systems of both
higher and lower forms of life.
In the following pages an attempt is made to present the broad inter-
relationships among microorganisms living in association, either in sim-
ple mixed cultures or in complex natural populations, with special at-
tention to the antagonistic effects. Emphasis is laid upon the significance
of these associations in natural processes and upon their relation to dis-
ease production in man and in his domesticated plants and animals. The
chemical nature of the active — antibiotic — substances produced by vari-
ous antagonists is described and the nature of the antagonistic action as
well as its utilization for practical purposes of disease control is dis-
cussed. However, because concepts of the significance of these phenom-
ena are changing so rapidly, no pretense has been made of examining
completely the practical applications of this important subject.
Due to the fact that more detailed studies have been made on the
production, nature, and utilization of penicillin, more information is
PREFACE ix
presented about this than about any of the other substances. However,
this should not be construed as desire on the author's part to emphasize
this substance.
The subject of antagonistic effects of microorganisms has been re-
viewed in both general treatises (83, 229) and special papers (134, 166,
251, 256, 354, 355, 539, 540, 584, 616, 796, 799, 800, 838) ; special at-
tention has been paid to the occurrence of such organisms in the soil
(620, 794). Advantage was taken of these reviews in the preparation of
the comprehensive bibliography presented at the end of this mono-
graph. Attention is directed also to a recent complete review of the lit-
erature on the nature and formation of penicillin, the historical develop-
ment of our knowledge of this agent, method of assaying, and clinical
application (678).
The author expresses his sincere appreciation to the members of the
staff of the Microbiology Department, New Jersey Agricultural Ex-
periment Station j to members of the Department of Research and De-
velopment of Merck & Co. and of the Merck Institute for permission
to use reproductions of their work j to Dr. G. A. Harrop of E. R, Squibb
& Sons for supplying the photograph of the penicillin-sodium crystals
used as the frontispiece to this volume j to Mrs. Herminie B. Kitchen
for her careful editing of the manuscript j and to the many investigators
in the field whose work has been freely cited both in the form of text or
tabular matter and as illustrative material.
S. A. W.
November 75, 1944
CONTENTS
1 . Soils and Water Basins as Habitats of Microorganisms i
2. Human and Animal Wastes 19
3. Interrelationships among Microorganisms in Mixed Popula-
tions 38
4. Isolation and Cultivation of Antagonistic Microorganisms}
Methods of Measuring Antibiotic Action 55
5. Bacteria as Antagonists 80
6. Actinomycetes as Antagonists 102
7. Fungi as Antagonists 124
8. Microscopic Animal Forms as Antagonists 143
9. Antagonistic Relationships between Microorganisms, Vi-
ruses, and Other Nonspecific Pathogenic Forms 152
10. Chemical Nature of Antibiotic Substances 156
11. The Nature of Antibiotic Action 189
12. Utilization of Antagonistic Microorganisms and Antibiotic
Substances for Disease Control 221
13. Microbiological Control of Soil-borne Plant Diseases 246
14. The Outlook for the Future 259
Classification of Antibiotic Substances 270
Glossary 271
Bibliography 273
Index of Microorganisms 331
General Index 339
58352
CHAPTER I
SOILS AND WATER BASINS AS HABITATS
OF MICROORGANISMS
Although microorganisms inhabit a variety of substrates, from the
dust in the atmosphere, the surface of living plants and plant residues,
and numerous foodstuffs to the living systems of plants and animals,
their natural habitations are soils and water basins.
The soil is by no means an inert mass of organic and inorganic de-
bris. On the contrary, it fairly teems with life. The organisms inhabit-
ing the soil range from those of ultramicroscopic size to those readily
recognizable with the naked eye. Many thousands of species, capable
of a great variety of activities, are represented in the soil. The physical
nature and chemical composition of the soil, the climate, the plant vege-
tation, and the topography influence greatly both the composition of
the microbiological population of the soil and its relative abundance.
One gram of soil contains hundreds, even thousands, of millions of bac-
teria, fungi, actinomycetes, protozoa, and other groups of microorgan-
isms. Under certain conditions, especially when the supply of fresh or-
ganic matter in the form of plant and animal residues is increased, the
number may be much greater. This varied microbiological population
renders the soil capable of bringing about a great variety of chemical
and biological reactions.
Through its diverse activities, the microscopic population inhabiting
soils and water basins forms one of the most important links in the chain
of life on earth. However, its great influence upon numerous phases of
human endeavor has been recognized only within recent years. All
plants and all animals, including man himself, are dependent upon
these organisms to bring about some of the processes essential to the
continuation of life. The growth of annual and perennial plants, the
supply of food for man and animals, and the provision of clothing and
shelter depend largely upon the activities of these microorganisms,
especially the transformations brought about in the state of such ele-
ments as carbon, nitrogen, sulfur, and phosphorus.
2 MICROORGANISMS IN SOILS AND WATER BASINS
Soils and water basins may be regarded as the primary reservoirs for
all living systems inhabiting this planet. Whereas the great majority of
microorganisms are saprophytic in nature, living upon inorganic ele-
ments and compounds and upon the dead residues of plant and animal
life, others have become adapted to a parasitic form of existence and
have learned to thrive upon the living tissues of plants and animals.
Many of these parasites find their way into the soil and into water basins
and may be able to survive there for long periods of time or even in-
definitely.
Although the following discussion is limited primarily to the micro-
biological population of the soil, it also applies, to a greater or lesser
extent, to the microorganisms that inhabit manures made up of animal
excreta, household wastes, and artificially prepared composts and to
those that inhabit water basins, including rivers, lakes, and seas. There
are, however, marked differences in the nature of the microbial popu-
lation of waters and of soils because of the physical and chemical differ-
ences in the composition of these two substrates. Nevertheless, some of
the underlying principles apply to all substrates. There are, for exam-
ple, marked differences in the nature and abundance of the populations
of soil and water and those of milk, sewage, and foodstuffs. Whereas
microorganisms multiply in the latter substrates at a very rapid rate,
those in the soil and in water basins are more nearly static, since the rate
of their multiplication is much slower except under very special condi-
tions, such as the addition of fresh, undecomposed plant and animal
residues or a change in the environment or in the chemical nature of
the substrate.
PHYSICAL PROPERTIES OF SOILS
The soil — the surface layer of the earth's crust — comprises three dis-
tinct phases, the gaseous, the liquid, and the solid. The last is largely
inorganic in nature, with varying concentrations of organic constituents
originating from plant and animal residues and found in the soil in dif-
ferent stages of decomposition. The organic substances together with
the living and dead cells of microorganisms that inhabit the soil make
PHYSICAL PROPERTIES OF SOILS 3
up what is known as soil organic matter or, more often, soil humus. The
soil as a medium for the development of microorganisms is thus mark-
edly different from the common artificial laboratory media, whether
these be synthetic or consist of products of animal or plant life, upon
which these organisms are grown.
The inorganic soil particles are surrounded by films of colloidal ma-
terials, which are both inorganic and organic in nature. As a rule, the
microorganisms inhabiting the soil adhere to these films, although some
move freely in the water surrounding the particles. Water and air play
essential roles in the soil system and control the nature and extent of the
soil population. The nature and size of the mineral and organic soil
fractions, as well as the phenomena of adsorption, also influence the
abundance, nature, and distribution of microorganisms in the soil. Sandy
soils are better aerated than heavy clay soils j they are, therefore, more
favorable for the growth of aerobic bacteria and fungi. However, since
such soils lack the high water-holding capacity of the heavier soils, they
are more readily subject to the process of drying out, which may result
in a reduction in microbial activities.
Oxygen, another important factor in microbial development in the
soil, becomes available to microorganisms by gaseous diffusion. The
oxygen supply diminishes with increase in depth of the soil. When an
excess of free water is present in the soil, gaseous oxygen cannot pene-
trate very deeply and soil organisms then become dependent upon the
dissolved oxygen which diffuses into the soil solution. Since the rate of
oxygen diffusion is extremely slow, waterlogged soils tend to become
depleted of oxygen. Under these conditions, there is marked change in
the microbiological population of the soil : the fungi and actinomycetes
tend to decrease, and the bacteria, especially the anaerobic types, pre-
dominate. Peat bogs are examples of soils in a perpetual anaerobic state j
the microbial population is quite distinct from that of mineral soils.
Semiarid soils, with a much greater diffusion of oxygen into the deeper
soil layers, possess a population which is largely aerobic j in these and
other mineral soils the abundance and nature of the organic matter exert
a decided influence upon the abundance and nature of the microorgan- - ^^^^
isms present. X \3\>» ^'^/^S^
4 MICROORGANISMS IN SOILS AND WATER BASINS
The microbiological populations of soils, composts, and water basins
are also influenced markedly by seasonal and temperature changes.
Certain microorganisms are capable of active life at temperatures ap-
proaching the freezing point of water j others, known as thermophilic
forms, can withstand very high temperatures, some being active even at
60° to 70° C.
The reaction of the soil is also a factor influencing the nature of the
population. Many microorganisms are active within a very limited
range of fH values j others, notably many of the fungi, are adapted to
much wider ranges of reaction. In acid soils, larger numbers of fungi
are present, because of the fact that they tolerate more readily the
more acid reactions, which limit bacterial competition. On the other
hand, actinomycetes comprise a large percentage of the microbial popu-
lation of dry and alkaline soils.
CHEMICAL COMPOSITION OF SOILS
The solid part of the upper or surface layer (20 to 30 cm.) of the soil
commonly is made up of i to 10 per cent organic matter and 90 to 99
per cent inorganic or mineral matter. The concentration of organic mat-
ter may be even less than i per cent, as in desert and poor sandy soils, or
more than 10 per cent, as in certain virgin prairie soils and, especially,
peat lands which consist of 50 to 99 per cent organic matter, on a dry
basis.
The organic matter of the soil is markedly different in chemical na-
ture from that of plant and animal materials. It contains much less cellu-
lose and hemicelluloses than the majority of plants and is higher in
lignins and proteins. It is characterized by a narrow ratio of the two
important elements carbon and nitrogen, usually about 10: 1 5 it is much
more resistant to microbial decomposition than are plant and animal
residues. It is black, is soluble to a considerable extent in alkalies, and is
partly reprecipitated by acids. These alkali-soluble constituents have
often been designated as "humic acids" or "humic bodies," thus impart-
ing the idea that soil organic matter is made up largely of these "acids"
(922).
BIOLOGICAL STATE OF THE SOIL 5
The inorganic constituents of the soil comprise largely sand, silt, clay,
and, to a more limited extent, a number of soluble and insoluble salts,
notably phosphates, sulfates, and silicates of calcium, magnesium, potas-
sium, iron, aluminum, manganese, zinc, copper, and others. Some of
the chemical elements comprise the framework of the soil and are used
to only a limited extent by plant and microbial life. Others form im-
portant nutrients (for example, C, N, S, P, H, and O) or serve as cata-
lysts for the continuation of life (Zn, Fe, Mn, Cu, Mo, B, and even K
are often considered as belonging in this category). The function of
most of these elements in the life of microorganisms is not fully under-
stood. In view of the fact that some of the elements in the latter group
have been found to form important constituents of certain enzyme sys-
tems, the difference between the two functions is not significant.
BIOLOGICAL STATE OF THE SOIL
The abundance of higher plant and animal life in and upon the sur-
face of the soil influences considerably the nature and extent of the
microbiological population. Certain plants harbor in their roots specific
microorganisms that act as true symbiontsj this is true of the root nodule
bacteria of leguminous plants and the mycorrhiza-forming fungi found
in orchids, evergreens, and many other plants (919). Higher plants
also offer a favorable environment for the growth of certain other types
of bacteria and fungi, this specific environment being designated as the
rhizosphere. The bacterial population of the rhizosphere is not very
different qualitatively from that found some distance away from the
plants, except that certain types of bacteria are more prominently repre-
sented.
The growth of plants results in the production of waste materials
and residues left in and upon the soil In the form of roots, leaves,
needles, and other products, all of which offer favorable nutrients for
microbial development. The root systems of plants also bring about bet-
ter aeration of the soil, thus making conditions more favorable for the
development of aerobic organisms. The presence of higher plants often
leads to the development of certain types of bacteria, fungi, and nema-
6 MICROORGANISMS IN SOILS AND WATER BASINS
todes that are pathogenic to the plants, such as the causative agents of
root rots, damping-off diseases, root-galls, and various others. Some of
the pathogens may become well established in the soil and may persist
there long after the specific host plants have been removed. They may
even be able to attack other hosts. Plant life thus exerts a variety of in-
fluences upon the nature and abundance of the soil-inhabiting micro-
organisms.
Higher animals also influence the soil microbiological population.
Cattle and horses on pastures contribute, through their droppings,
energy sources and various other essential nutrients for the develop-
ment of microorganisms. After death, the bodies of animals, from the
smallest insects to man, the lord of creation, also offer available nutri-
ents for the growth of numerous microorganisms. Many animals living
in the soil, such as insects and rodents, become carriers of certain bac-
teria and fungi that are destructive to their hosts j this phenomenon is
often utilized for combating injurious animals. Finally, the numerous
animals living on the surface of the soil leave waste products rich in bac-
teria, fungi, and invertebrate animals, some of which are capable of
causing serious animal diseases.
NATURE AND COMPOSITION OF THE SOIL
MICROBIOLOGICAL POPULATION
The microorganisms inhabiting the soil can be divided, on the basis
of their systematic position in the biological kingdom, into the following
eight groups: bacteria, actinomycetes, fungi, algae, protozoa, worms,
insects and other near-microscopic animals, and ultramicroscopic forms.
The last group comprises bodies that range from living systems to
products of living organisms j they possess the property of activating
similar substances and imparting to them their specific activities, as in the
case of phages and viruses.
Five methods are commonly employed for determining the abun-
dance of the various groups of microorganisms inhabiting the soilj
namely, plate culture, selective culture, direct microscopic methods,
Contact slide, and mechanical separation. Each of these has certain ad-
SOIL MICROBIOLOGICAL POPULATION 7
vantages and certain limitations. In many cases, special methods have
been devised to supplement the more common methods.
The plate method is based upon principles similar to those employed
in other branches of bacteriology. Various media are used, both organic
and synthetic. The soil microbiologist has attempted to produce media
that either allow the development of the greatest number and the great-
est variety of organisms or are particularly favorable for the growth of
certain special types of organisms. None of the media so far employed
allows the growth of the total soil population. The plate method is often
supplemented by the selective culture method, in which a great variety
of media are used in order to obtain a representative picture of the soil
population. Since the number of media required to enable all soil micro-
organisms to develop is virtually limitless, the enrichment methods can
only give a proximate idea of the nature and abundance of the micro-
biological population. Because of the development on the plate of cer-
tain organisms that exert a toxic effect upon others, the plate method
often shows excessive variation in the numbers of bacteria and fungi
(--57).
The microscopic methods have been introduced to fill this gap, since
by them the relative abundance of the various groups of organisms
found in soils, composts, or other natural substrates can be established.
Unfortunately, these methods do not allow any differentiation between
living and dead cells, nor do they permit a differentiation between the
various physiological types of microorganisms such as pathogens and
nonpathogens. A further limitation, especially of the contact slide, is
that the fast-growing forms cannot be prevented from overgrowing
the slide and repressing the slow-growing types.
The mechanical separation methods are based upon the use of special
sieves or water emulsions and are utilized for the study of the larger
forms such as insect larvae and nematodes.
The relative abundance of the different groups of microorganisms in
a given soil, as determined by any one of the foregoing methods, varies
with the nature of the soil, amount of organic matter, oxygen sup-
ply, moisture content, temperature, acidity, and buffering capacity
(Table i), as well as with the nature of the higher plants growing in
8 MICROORGANISMS IN SOILS AND WATER BASINS
the given soil (Table 2). Despite all these factors, the microbiological
population of the soil throughout the world has certain definite and
common characteristics and comprises certain well-defined, specific
TABLE I. INFLUENCE OF SOIL TREATMENT ON NUMBER
OF MICROORGANISMS
REACTION
TREATMENT OF SOIL
OF SOIL
f¥L
Unfertilized and unlimed
4.6
Lime only added
6.4
Potassium salts and phosphates
added
5-5
Salts and ammonium sulfate
added
4.1
Salts, ammonium sulfate, and
lime added
5.8
Salts and sodium nitrate added
5-5
Stable manure and salts added
5.4-
MICROORGANISMS FOUND*
Bacteria Actinomycetes Fung
3,000 1,150 60
5,410 2,410 23
5,360 1,520 38
2,690 370 112
6,990
2,520
39
7,600
2,530
4-7
8,800
2,920
73
From Waksman (925).
* In thousands per gram of soil as determined by plate method.
TABLE 2. INFLUENCE OF GROWING PLANTS ON NUMBER
OF MICROORGANISMS IN THE SOIL
SAMPLE OF
PLANT
SOIL TAKEN
MICROORGANISMS FOUND*
Bacteria
Actinomycetes
Fungi
Rye
Near roots
28,600
4,400
216
Away from roots
13,200
3,200
162
Corn
Near roots
41,000
13,400
178
Away from roots
24,300
8,800
134
Sugar beet
Near roots
57,800
15,000
222
Away from roots
32,100
12,200
176
Alfalfa
Near roots
93,800
9,000
268
Away from roots
17,800
3,300
254
From Starkey (848).
* In thousands per gram of soil.
SOIL MICROBIOLOGICAL POPULATION 9
types. The bacteria usually range in number from a few hundred thou-
sand to several hundred million per gram of soil, though many species
do not develop on the common plate. Fungi are found in the form of
mycelial filaments and as spores and may therefore constitute as large
a mass of living matter as do the bacteria j their actual number, as deter-
mined by the plate method, may vary from a few thousand to several
hundred thousand per gram of soil. The significance of these results is
not always clear, since a given colony may have originated from a
hyphal filament, a mass of mycelium, or a single spore. Determination
by the plate method of the number of actinomycetes is subject to the
same limitations j these organisms usually constitute from lo to 50 per
cent of the colonies appearing on common bacterial agar plates.
Algae are numerous in the surface layers of soil only. Protozoa are
present in the soil in an active vegetative or trophic state and in the
form of cysts. The active cells appear when excessive water is present,
even for a few hours j in dry soil, the cysts predominate. Flagellates
are represented by the largest numbers, sometimes approaching a mil-
lion individuals per gram of soil j amebae are next in abundance j cili-
ates are usually found to the extent of a few hundred to several thou-
sand per gram of soil. Nematodes, rotifers, earthworms, and larvae of
numerous insects are also abundant, often forming a large part of the
bulk of the living mass of cell substance.
By means of the selective and enrichment culture methods, several
physiological classifications of bacteria have been recognized. The fol-
lowing descriptive terms are commonly used to designate these groups :
autotrophic vs. heterotrophic, aerobic vs. anaerobic, motile vs. non-
motile, pathogenic vs. saprophytic, psychrophilic and mesophylic vs.
thermophilic, symbiotic vs. nonsymbiotic, and antagonistic vs. non-
antagonistic.
The fungi may be classified into three types: saprophytic and free-
living, mycorrhiza-producing, and plant pathogenic. The most com-
mon groups of soil fungi are found in the genera Rhizofus, Mucor^
Penkilliumy Aspergillus, Trkkoderma, Fusarium, Cladosforium, and
Cefhalosforium. The soil often harbors an abundant population of
yeasts and fleshy or mushroom fungi. The latter may produce an ex-
10 MICROORGANISMS IN SOILS AND WATER BASINS
tensive mycelium in the soil, binding the particles together and pre-
venting their falling apart.
Various bacteriolytic agents, including specific phages, have also been
demonstrated in the soil. The phage of root-nodule bacteria is of par-
ticular interest. It is readily adsorbed by the soil, but its presence can
easily be established. The repression of spore-forming bacteria and the
abundance of Pseudomonas fluorescens (139, 140) may be due to the
antagonistic action of the latter.
SOILS AND WATER BASINS AS CULTURE
MEDIA
Microorganisms require for their growth and respiration certain
energy sources and certain nutrients, as well as certain conditions favor-
able for their development. Different organisms show considerable
variation in this respect. The mineral elements required for growth and
multiplication are almost invariably present in the soil and to a large
extent also in many water basins. The available energy supply may be
limited, however, and thus usually becomes the most important factor
regulating the abundance and activities of microorganisms in natural
substrates. The autotrophic bacteria depend on the supply of oxidizable
minerals such as ammonium salts, nitrite, sulfur, iron, and manganese,
the oxidation of which makes energy available for their growth. The
heterotrophic organisms are dependent on the carbon compounds
brought into the soil in the form of plant and animal residues as well
as the bodies of many insects, earthworms, and other small animals.
The roots of plants also supply an abundance of easily available sub-
stances for microbial nutrition.
Every organic compound produced in nature finds its way, sooner or
later, into the soil or into lakes and rivers, where it serves as a source
of energy for microorganisms. This energy becomes available to some
of the organisms through anaerobic or fermentative transformation and
to others through aerobic or oxidative processes. The net change in the
energy produced by any one organism or group of organisms is accom-
panied by a loss of free energy by the system to which the culture is
SOILS AND WATER BASINS AS CULTURE MEDIA 11
confined. The synthesis of new cell material by microorganisms is ac-
companied by a gain of free energy, which must be supplied by other
chemical transformations. Ordinary soils, however, contain microbial
nutrients in concentrations sufficient to support a large number of living
cells. This can be illustrated by the fact that when a soil is sterilized and
then inoculated with a pure culture of bacterium rapid multiplication
takes place (Table 3), When fresh water taken from a lake or the sea is
kept in the laboratory for one or two days, a great increase in its bac-
terial population occurs.
There is considerable variation in the ease with which a specific or-
TABLE 3, MULTIPLICATION OF COLIFORM BACTERIA IN STERILE SOIL
BACTERIA
ORGANISM
INOCULATED*
Escherichia coli
in soil alone
2,600
Aerobacter aero genes
in soil alone
109,000
in soil and glucose
109,000
BACTERIA RECOVERED*
After 10 days After 26 days
149,000,000 138,000,000
48,000,000 42,600,000
1,660,000 240,000,000
From Waksman and WoodruflF (949).
* Per gram of soil.
ganism can be isolated from a natural substrate and consequently in
the techniques employed. Some microorganisms may be present in
abundance and can be readily isolated. Others are found only in limited
numbers and can be obtained only with considerable difficulty and by
the use of special procedures. Still others can be isolated only after the
natural substrate is treated in such a manner as to favor the multiplica-
tion of the specific organism j this can be done by enriching the soil with
a nutrient or substance which the particular organism is able to utilize,
or by changing conditions of reaction, by aeration, or by other treat-
ment that would favor the rapid development of the organism in ques-
tion. Special strains or races of microorganisms may often be developed
as a result of such treatment, which tends to favor the adaptation of the
organisms present in the soil to a particular process. ■ ^iKl C.37S.
-.,.jii«^c:
12 MICROORGANISMS IN SOILS AND WATER BASINS
NUTRITION OF MICROORGANISMS IN
NATURAL SUBSTRATES
It was at first assumed that bacteria and other microorganisms possess
a simpler type of metabolism than do higher plants and animals j al-
though some can obtain all the nutrients required for cell synthesis and
energy from simple elements and compounds, others need for their nu-
trition certain highly complicated organic substances. Recently it has
been recognized that various "growth-promoting" substances or vita-
mins play an important role in the nutrition of many microorganisms. It
has also been established that highly complicated enzyme systems are
produced by these lower forms of life, and that many interrelationships
exist among their metabolic processes, the composition of the medium,
and the environmental conditions. One thus begins to realize that the
metabolism of these microbes is also highly complicated. Most of the in-
formation on their nutrition is based upon their growth on artificial cul-
ture media. In nature, however, these organisms live in associations and
vary considerably in the degree of their interdependence. As yet no
laboratory method has been developed that duplicates these conditions.
Microorganisms vary considerably in their nutrition and energy
utilization, as well as in the breakdown and transformation of the avail-
able nutrients. Certain elements or compounds are required for cell
synthesis. In some cases, certain trace elements as well as varying con-
centrations of growth-promoting substances are also essential. Among
the nutrient elements, nitrogen occupies a prominent place. Consider-
able variation exists in the ability of microorganisms to utilize different
types of nitrogen compounds: some can obtain their nitrogen from a
wide variety of substances; others are restricted to the use of a single
group of compounds such as proteins, amino acids, urea, ammonia, or
nitrate; a few are able to use atmospheric nitrogen. The variety of or-
ganic nitrogenous bodies supplied to microorganisms in soils and in
water basins is limited only by the number of such compounds synthe-
sized by plants and animals. The complex forms of nitrogen are broken
down to simpler compounds; these may be assimilated by organisms
and again built up into complex forms, or they may be utilized only by
other organisms. Microbial activity thus regulates the state of the nitro-
GROWTH OF THE MICROBIAL CELL 13
gen in natural substrates and is responsible for the continuous stream
of ammonia and nitrate forming the available sources of nitrogen that
makes possible the growth of higher plants.
THE GROWTH OF THE MICROBIAL CELL IN
PURE CULTURE AND IN MIXED
POPULATIONS
When nutrients are available in sufficient concentration and when the
environmental conditions are favorable for the development of the
microbial cell in pure culture, growth follows a definite sigmoid-shaped
curve. Slow multiplication is followed by rapid development, until a
certain maximum number of cells within a given volume of medium is
reached J the rate of growth then diminishes. The maximum population
of Aerobacter aero genes grown in a medium containing lactose and
ammonium tartrate increases at first in proportion to the concentrations
of these nutrients but later becomes independent of them. The onset of
the stationary phase may be due to several factors: exhaustion of sub-
stances necessary for growth, change in the reaction of the medium to
one unfavorable for further development, accumulation of toxic prod-
ucts. When the nutrients in the medium are exhausted, addition will
restore growth. When an unfavorable change in reaction has taken
place, the addition of acid or alkali will render the medium again favor-
able. The production of toxic substances in the medium can be counter-
acted usually by the use of heat or by treatment with charcoal, though
some of the injurious bodies may be heat-resistant.
In the presence of other microorganisms, a certain organism may
show reactions markedly different from those obtained in pure culture:
it may produce substances that are either favorable or injurious to the
other cells, it may compete with the other organisms for the available
nutrients or it may render the medium more favorable for their de-
velopment. It has been shown (936), for example, that certain bacteria
like Bacillus cereus can attack native proteins but not amino acids,
whereas others like Pseudomonas fluorescens can attack amino acids but
not proteins J when these two organisms were placed together in the
same medium, their activities supplemented one another. Numerous
14 MICROORGANISMS IN SOILS AND WATER BASINS
other instances are found in soil and water of an organism preparing
the substrate for another, ranging from distinct symbioticism, where
one organism depends absolutely for its living processes upon the ac-
tivities of another (symbiosis), to association, where one organism
merely is favored by the growth of another (metabiosis), to the injury
of one organism by another (antagonism), and finally, to the actual
destruction of one by another (parasitism).
INTRODUCTION OF DI SE ASE- PRODUCI N G
MICROORGANISMS INTO THE SOIL
Ever since higher forms of life first made their appearance on this
planet they have been subject to attack by microbes. These microscopic
organisms must have gained, at an early stage in the development of
the higher forms, the capacity of attacking them in one manner or an-
other. There is no plant or animal now living that is not subject to in-
fection by different bacteria, fungi, and protozoa. The more advanced
the animal body is in the stage of evolution, the more numerous are its
ills, most of which are caused directly or indirectly by microorganisms.
The microbial agents causing thousands of diseases of plant and ani-
mal life have now been recognized and even isolated and described. In
many cases these disease-producing agents are closely related morpho-
logically to those which lead a harmless existence in soils or water
basins J many of the saprophytes, for instance, are found to be of great
benefit to man and to his domesticated plants and animals. This sug-
gests the probability that pathogenic microorganisms represent certain
strains of soil and water-inhabiting types that have become adjusted to
a parasitic existence. During their life in the host, they multiply at a
rapid rate and produce substances toxic to the body of the host. The re-
sult is that the host is incapacitated for a certain period of time, until it
succeeds in building up resistance against the invading organisms. It
may thus overcome the injurious effect of the pathogen or it may be
killed if such resistance cannot be effected. In the first instance, a tem-
porary or permanent immunity against the specific disease-producing
microbe or its close relatives may result. The host is often able to sur-
vive the attack without being able to destroy the invading microbes j if
SAPROPHYTIC ORGANISMS IN THE SOIL 15
it again attains a normal form of life, it is designated as a carrier of the
disease-producing agent.
Pathogenic organisms pass their existence in the living body of the
plant or animal. They spread from one host to another by contact or
through a neutral medium, such as water, milk, or dust where they may
remain alive and active for varying lengths of time, or they reach the
soil or water basins in the excreta of the host. If the host is killed by
the infecting microbes, they may survive for some time upon the rem-
nants of what was once a living animal or plant and thus find their way
into the soil and water basins.
Considering the millions of years that animals and plants have ex-
isted on this planet, one can only surmise the great numbers of microbes
causing the numerous diseases of all forms of life that must have found
their way into the soil or into streams and rivers. What has become of
all these pathogenic bacteria? This question was first raised by medical
bacteriologists in the eighties of the last century. The soil was searched
for bacterial agents of infectious diseases. It was soon found that, with
very few exceptions, organisms pathogenic to man and animals do not
survive very long. This was at first believed to be due to the filtration
effect of the soil upon the bacteria (32). It came to be recognized, how-
ever, that certain biological agents are responsible for the destruction
of the pathogenic organisms. These investigations led to the conclusion
that the soil can hardly be considered as a carrier of most of the infec-
tious diseases of man and animals. The fact that many pathogens can
grow readily in sterilized soil but do not survive long in normal fresh
soil tends to add weight to the theory of the destructive effect upon
pathogens of the microbiological population in normal soil.
INTRODUCTION OF SAPROPHYTIC ORGANISMS
INTO THE SOIL
It often becomes necessary to inoculate the soil with organisms not
usually found there. The common practice of inoculating soil with bac-
teria capable of forming root nodules on leguminous plants is a case in
point. It is essential, therefore, to know how long these organisms will
survive. The survival period is influenced greatly by the presence of a
16 MICROORGANISMS IN SOILS AND WATER BASINS
host plant that protects the specific bacteria from attack by antagonistic
organisms. In the absence of the host plant, the bacteria seem to disap-
pear gradually, and reinoculation becomes advisable when the host is
again planted in the given soil. It has been observed also that specific
strains of bacteria tend to deteriorate in the soil, and that it is necessary
to reinoculate the soil with more vigorous strains of the organisms in
question.
The ability of other bacteria, notably members of the Azotobacter
group, to fix nitrogen independently of host plants and the fact that
these organisms are absent in many soils led to the suggestion that such
soils might benefit from inoculation. It has been found, however, that
when soils and peats are inoculated with Azotobacter chroococcum
large-scale destruction of the latter often occurs isil^)- This is believed
to be due to the presence in the soil of antagonistic organisms as well as
toxic substances (477, 648, 951).
Certain fungi are unable to grow in fresh nonsterilized soil but are
capable of growing in heated soil. This was found to be due to the fact
that normal soils contain certain substances that render the growth of
the fungus impossible j these substances are destroyed by heating. An
extract of fresh soil was found to act injuriously upon the growth of the
fungus Pyronema; the injurious effect was partly removed on boiling
(500).
The survival of microorganisms added to soil or water is thus influ-
enced by the nature of the native soil or water population, the organ-
isms added, the composition of the substrate, and various environ-
mental conditions.
SAPROPHYTIC AND PATHOGENIC NATURE OF
CERTAIN SOIL MICROORGANISMS
Various fungi and actinomycetes causing animal diseases, notably
skin infections, appear to resemble very closely the corresponding soil
saprophytes. It was therefore suggested that many of the dermato-
phytic fungi normally lead a saprophytic existence in the soil but are
also capable of developing on epidermal tissue, and bringing about in-
fection of the tissues. This was found to be true especially of species of
SAPROPHYTIC AND PATHOGENIC MICROORGANISMS 17
SforotrichufUy various actinomycetes such as those causing lumpy jaw of
cattle, and certain other organisms. Henrici (396) divided fungus in-
fections of animals into two groups: first, superficial mycoses, compris-
ing moniliases and dermatomycoses, that are caused by a variety of
fungi widely distributed in nature j and, second, deep-seated infections,
namely, aspergillosis, sporotrichosis, and blastomycosis, with a marked
tendency to restricted distribution. The latter were said to be caused
primarily by saprophytic forms, including varieties capable of chance
survival and of multiplication when accidentally introduced into ani-
mal tissues.
Walker (952) suggested that the partly acid-fast coccoid, diph-
theroid, and actinomycoid organisms that have been cultivated repeat-
edly from leprosy are merely different stages in the life cycle of the
same form. The causative agent of leprosy, like certain pathogenic
actinomycetes, is believed to be a facultatively parasitic soil organism,
probably of wide but irregular distribution. Leprosy was thus looked
upon primarily as a soil infection, brought about presumably through
wounds J a secondary means of infection by contagion was not excluded.
A comparison of cultures obtained from rat leprosy, human leprosy,
and bacteria of soil origin led to the conclusion that the strains from all
three sources were identical ; human and rat leprosy were said to have
the same etiology and endemiology, finding a normal habitat in the soil.
An interesting relationship has been shown to exist between Texas
fever and the capacity of cattle tick {Boofhilus bovis)^ the parasite car-
rier, to persist in the soil (836). The causative agent is an organism
with protozoan characteristics. It persists in southern pastures where
the carriers survive from one season to the next and keep the cattle con-
tinuously infected. The disease is of little importance in northern re-
gions, the ticks being destroyed during the winter. When northern cat-
tle are moved to southern pastures, they become subject to the disease.
Pathogenic microorganisms capable of surviving in the soil have pre-
sented important economic problems to farmers raising hogs, cattle,
poultry, and other domestic animals, but disease incidence through this
source has been greatly diminished by the proper practice of sanitation.
The rotation of crops has been utilized for the purpose of overcoming
these conditions, several years usually being required to render infected
18 MICROORGANISMS IN SOILS AND WATER BASINS
pastures safe for use. The fact that most pathogenic organisms rapidly
disappear when added to the soil makes this problem rather simple j the
prevention of infectious diseases would have presented far more diffi-
cult problems were the infecting agents to remain indefinitely virulent
in the soil. The few disease-producing agents that are capable of per-
sisting, such as anthrax, blackleg, and coccidiosis, have been the cause,
however, of considerable damage to animals.
Of greater economic importance than the survival in the soil of hu-
man and animal pathogenic agents is the fact that the soil harbors a
number of plant pathogens, including not only fungi, bacteria, and
actinomycetes, but also nematodes and insects. Fortunately, the con-
tinued development of these organisms in the soil also leads to the ac-
cumulation of saprophytic organisms destructive to them.
The extent to which virus diseases persist in the soil is still a matter
for speculation. It has been demonstrated that the phage of legume
bacteria may persist and become responsible for a condition designated
as "alfalfa-sick soils" and "clover-sick soils" (169, 474). In order to
overcome this condition, the breeding of resistant varieties of plants has
been recommended.
CHAPTER 2
HUMAN AND ANIMAL WASTES
A7td a place shalt thou have without the camf, whither thou shalt
go forth abroad: and a spade shalt thou have with thy weapons ;
and it shall he, when thou sittest abroad, thou shalt dig therewith,
and sh-alt afterward cover that which cometh from thee. —
Deuteronomy 2^:1^ and 14.
Human and animal excreta and other waste products, which are or fre-
quently become both offensive and dangerous to public health, sooner
or later find their way into the soil and water basins. The soil also re-
ceives the many residues of growing crops that are annually left on the
land, together with the waste materials of the farm and the home (439,
922). These wastes contain substances partly digested by man and ani-
mals, and their metabolic waste products, as well as freshly synthesized
material in the form of microbial cells. The microbial population of
such waste materials comprises agents of digestion, some microbes that
are present accidentally, and some that possess the capacity of causing
human, animal, and plant diseases.
These waste materials do not remain long in an unaltered form and
do not accumulate in or on the surface of the soil or in water basins j
otherwise both soil and water long ago would have been rendered un-
sightly, disagreeable bodies, which man would not dare to tread upon
or enter. On the contrary, the soil and the water are capable of di-
gesting all these cast-off materials and of completely destroying their
undesirable characteristics. Through all past ages, the waste products of
plant and animal life have disappeared, whereas the soil and the water
in the rivers, lakes, and seas have remained essentially the same, except
under very special conditions such as those that brought about the pro-
duction of peat in water-saturated basins and, in past geological ages,
the formation of coal. The capacity of soil and water to destroy these of-
fensive wastes is due entirely to the microorganisms that inhabit the
substrates. The important ultimate products of destruction are am-
monia, carbon dioxide, and water j often hydrogen and methane are
20 HUMAN AND ANIMAL WASTES
produced J various mineral compounds, such as phosphates, sulfates, and
potassium salts are also liberated. These mineralized substances are es-
sential for the continuation of plant and animal life on this earth.
Largely because of the activities of the microorganisms inhabiting
soils and water systems, man does not need to worry about the disposal
of plant and animal wastes. These activities need only be regulated, in
order to accomplish the breakdown of complex substances with the
greatest efficiency and the least loss of valuable nutrient elements. The
following principal objectives are usually to be attained: first, the de-
struction of plant and animal pathogens, including pathogenic bacteria
and fungi and disease-producing protozoa, worms, and insects j second,
the liberation of the essential elements required for plant nutrition in
available forms, especially carbon, nitrogen, and phosphorus j and,
third, the formation of certain resistant organic substances, known col-
lectively as humus, which are essential for the improvement of the
physical, chemical, and biological condition of the soil.
STABLE MANURES AND FECAL RESIDUES
Microbial Population
Fresh excreta of animals and man are rich in fecal bacteria, consisting,
on the average, of 5 to 20 per cent bacterial cells (802). Lissauer (533)
calculated that the bacterial substance of feces ranges from 2.5 to 15.7
per cent of the dry weight, with an average of 9 per cent. Bacteria were
reported to make up as much as 9 to 42 per cent of the bulk of animal
stools, the number depending on the composition of the foodstuffs, the
nature of the animal and its condition of health, and other factors (364).
Since i mg. of dry bacterial substance contains about 4 billion bacterial
cells, the number of these organisms in fecal excreta can be seen to be
very large, although many, if not most, of the cells are no longer in a
living state. Osborne and Mendel (659) removed from the feces of
white rats the residual food material and found that the bacterial cells
made up 23 to 41 per cent of the total material j the nitrogen content of
these cells varied from 10.7 to 12.2 per cent. Since the removal of the
residual foodstuffs consisted in treatment with ether, alcohol, and acid,
some of the bacterial cell constituents were also removed j the actual
STABLE MANURES AND FECAL RESIDUES 21
concentration of bacterial substance may, therefore, have been even
greater.
It has been reported (441 ) that i ml. of the intestinal contents of cat-
tle contains 10 billion cells capable of development. By suitable methods
of cultivation, human feces were found (588) to contain 18 billion
bacteria per gram. Determination (542) of the number of bacteria in
stable manure gave 1 1.6 billion cells per gram of material by the plate
method, and, by the dilution method, 5 billion peptone-decomposers,
100 million urea-decomposers, and 2.5 million cellulose-decomposers.
About 100 billion bacteria may be produced daily in the human in-
testine. Human feces are made up, on an average, of 32,4 per cent bac-
terial cells amounting to 2,410 millions of bacteria per milligram of
moist material. Feces of healthy persons were shown (300) to contain
8.2 to 24.2 per cent bacterial cells j in those of persons suffering from
intestinal disturbances the percentages were 20.1 to 40.2. With the de-
velopment of the microscopic technique for counting bacteria, much
larger numbers of cells were shown to be present than could be deter-
mined by the plate method.
The urine of healthy persons is sterile or very low in bacteria. Be-
cause of the ability of many bacteria to utilize the chemical constituents
of urine, rapid bacterial multiplication takes place in fresh urine, espe-
cially when mixed with animal feces and bedding (775).
The microbiological population of animal excreta is characteristic. In
addition to the common fecal bacteria, it contains fungi, thermophilic
bacteria, and, in herbivorous animals, anaerobic cellulose-decomposing
bacteria (543).
Various methods have been developed for permitting the prefer-
ential development of certain types of bacteria. Gram-negative bacteria
in the feces can be repressed by certain reagents (839) j gram-positive
bacteria can be repressed by the addition of antibiotic substances such as
actinomycin (Table 4).
The bacterial population of fresh cow manure was found (796) to
consist of 47.5 per cent streptococci {Streftococcus -pyogenesy Sarcina sp.,
and Micrococcus candkans) ,21,2 per cent coli-like colonies {Escherichia
coli,A. aero genes y and S. sefticemiae), and many dark colony-forming
types. Other groups represented were BacteroideSy Flavobacteriuniy
22
HUMAN AND ANIMAL WASTES
TABLE 4. EFFECT OF ACTINOMYCIN ON THE MICROBIOLOGICAL
POPULATION OF CERTAIN NATURAL SUBSTRATES
MILLIGRAMS
DILU-
OF ACTINO-
TION
MYCIN PER
FOR
10 MILLILITERS
COLONIES
TYPES OF BACTERIA
SUBSTRATE
PLATING
OF AGAR
ON PLATE
ON PLATE
Air-dry soil
1,000
0
Numerous
Largely gram-positive,
many spore formers
1,000
O.OI
Fewer
Gram-negative
1,000
O.IO
96
Gram-negative
1,000
1. 00
0
None
Fresh soil
1,000
0
Numerous
Largely gram-positive
1,000
O.OI
Fewer
Gram-negative
1,000
O.IO
Few
Gram-negative
1,000
1. 00
0
None
Fresh milk
100
0
790
Gram-positive and
gram-negative
100
O.OI
346
Gram-negative
100
O.IO
251
Gram-negative
100
1. 00
I
Gram-negative
Fresh sewage
1,000
0
1,248
Mostly gram-negative
1,000
O.OI
1,172
Gram-negative
1,000
O.IO
1,131
Gram-negative
1,000
1. 00
121
Gram-negative
From Waksman and Woodruflf (945).
PseudomonaSy Bacillus^ various anaerobic bacteria, Oidium, and many
others. When the manure was allowed to decompose, yellow rods,
fluorescent bacteria, and mesentericus types took the place of the strep-
tococci.
The following heterotrophic bacteria have been demonstrated (775)
in manure : Bacillus subtilisj Bacillus mesentericus y Bacillus cereus. Ba-
cillus tumescenSy Bacillus fetasiteSy Pseudomonas fiuorescenSy Pseudo-
monas futiday Salmonella enteritidisy Escherichia coliy Proteus vul-
garis y Micrococcus luteusy Micrococcus candicansy Staphylococcus alhusy
Sarcina jiavay Streptococcus -pyogeneSy and others. Anaerobic bacteria
are also abundant (329).
STABLE MANURES AND FECAL RESIDUES 23
Pathogenic bacteria may also occur frequently in human feces and
in stable manure j Alycobacter'min tuberculosis and various hemolytic
streptococci (830), as well as pathogenic anaerobes including Clos-
tridium voelchiiy Clostridium se-pticum^ Clostridium^ oedematis y and
Clostridium jallax have been found (468).
The protozoa capable of developing in manure and in urine include
not only saprophytic forms but also certain parasites, such as Tricho-
mastric and Trichomonas^ capable of living and even of multiplying
in excreta. The coprophilic protozoa comprise various flagellates, cer-
tain amebae, and ciliates. The liquid part of the manure is considerably
richer than the solid in total number of protozoa as well as in species,
including Polytoma uvella^ Cryftochilum nigricans y and Tetramitus
rostratus. These protozoa nearly all feed upon bacteria. The infusoria
may feed upon smaller protozoa, so that forms like Colpidium may not
destroy bacteria at all.
Human and animal excreta also contain a large population of fungi,
chiefly in a spore state. Schmidt (801) divided the manure-inhabiting
fungi into three groups :
Those found only in manure ; their spores are swallowed with the feed,
and they pass unchanged through the digestive tract, though they
are favorably influenced toward germination by the body heat and
digestive fluids of the animal. Their natural multiplication by spores
is impossible without the physiological action of the digestive proc-
esses.
Those that do not have to pass through the digestive tract of an animal in
order to germinate and develop. The representatives of this group
occur in nature only in manure, although some are able to grow also
on other substrates. They can be cultivated both on manure and on
other media, mostly at ordinary temperatures.
Organisms found both in manure and on other substrates. They grow
readily at room temperature on a number of media.
Composition and Decomposition
The chemical composition of human and animal excreta, and of
stable manures in general, varies considerably, depending on the nature
of the animal, its age, mode of nutrition, and composition of food-
24
HUMAN AND ANIMAL WASTES
stuffs (454). As soon as voided, manure begins to undergo rapid de-
composition. This results in the formation of ammonia (140) and vari-
ous other nitrogenous degradation products (290). These give rise to
offensive smells, which are controlled by the conditions of decomposi-
tion. From a sanitary point of view, it is essential that decomposition
should be accompanied by the destruction of the injurious organisms
present in the manure. The fecal organisms gradually disappear and
their place is taken by a population concerned in the decomposition of
cellulose, hemicelluloses, and proteins (922).
The decomposition of complex plant and animal residues leads to a
rapid reduction in carbohydrates and is accompanied by the evolution
of considerable heat, the temperature of the compost reaching as high
as 80° C, as shown in Figure i.
In order to hasten the decomposition of manure, conditions must be
favorable to the activities of microorganisms. It must be properly
15 20 25 30 35 40 45
COMPOSTING PER.IOD IN DAYS
50 55 60 65
Figure I. Influence of straw bedding upon temperature changes in the
composting of manure. Circles indicate times of turning composts. From
Waksman and Nissen (940)
SEWAGE 25
aerated and well moistened but not saturated with water. By placing the
manure, together with the waste materials of the farm and the home,
in heaps, designated as composts, the decomposition processes can be
controlled so as to lead to heat liberation j this results in the destruction
of the injurious organisms and the conservation of the plant nutrient
elements. When not properly regulated, the decomposition processes
may be wasteful, unsanitary, and unsightly, and may even become a
source of infection to man and his domesticated animals.
SEWAGE
Disposal of sewage and other home wastes is one of the important
sanitary problems of men living in industrial and residential centers.
Haphazard methods of disposing of sewage not only lead to conditions
most unpleasant to human habitation but they are dangerous from the
standpoint of infectious diseases.
Sewage abounds in microorganisms that originate not only from hu-
man excreta but also from other household and industrial wastes. The
various saprophytic bacteria present in sewage rapidly attack the or-
ganic constituents and bring about their gradual mineralization. The
destructive action of saprophytic organisms greatly reduces the number
of pathogens (334). Activated sludge, for example, has been shown
(853) to possess a definite and consistent bactericidal action against the
colon bacteria. In addition to antagonistic organisms, active bacterio-
phages against nearly all types of intestinal bacteria are present in sew-
age. The destruction of pathogens by bacteriolysis thus readily finds a
place in the activated-sludge method of sewage purification.
Dissolved oxygen is generally present when sewage is diluted with
water. As the destruction of the organic matter proceeds rapidly, the
oxygen becomes depleted, so that none is left after a few hours. The
predominant bacterial flora of the water may then become anaerobic,
with the result that the chemical processes of decomposition are com-
pletely changed J hydrogen sulfide, mercaptans, and other foul-smell-
ing substances are then formed. This is accompanied by a typical
anaerobic breakdown of carbohydrates, leading to the formation of vari-
26 HUMAN AND ANIMAL WASTES
ous organic acids, carbon dioxide, hydrogen, and methane. The nitro-
gen in the protein and urea is transformed to ammonia and various
amines. When sewage is aerated, the anaerobic processes gradually give
way to aerobic processes, as the oxygen diffuses into the liquids or as the
sewage is diluted with water containing dissolved oxygen.
When sewage is freed from solids by sedimentation before discharge,
or when it is aerated sufficiently to maintain the concentration of dis-
solved oxygen, decomposition proceeds rapidly without the production
of the bad odors usually associated with the anaerobic breakdown. The
destruction of the pathogenic bacteria results largely through the ac-
tivities of the saprophytes (772, 980). For the purpose of promoting
the development of aerobic bacteria, processes employing the use of
intermittent sand filters, broad irrigation, contact beds, trickling filters,
and activated sludge are applied.
The modern methods of sewage purification are based on the long-
known fact that the soil is a destroyer of offensive wastes. In early days,
in fact, the soil handled all sewage problems. Sewage disposal plants in
modern cities are so operated that microorganisms found to be so effi-
cient in the soil are able to act under optimum conditions, resulting in
rapid purification. Sewage freed from most of its organic constituents
can be discharged into a stream and will not deplete the water of its dis-
solved oxygen. Chlorine is frequently employed in the final treatment
to assure the complete destruction of the pathogens.
GARBAGE
The processes involved in the disposal of garbage from the home
are similar to those utilized in the disposal of stable manure rather than
of sewage. At present, garbage usually is destroyed by burning, which
results in great economic waste, or is dumped outside cities, thus creat-
ing centers of infection and unpleasant appearance. More logical and
less wasteful processes are based upon the principle of composting. Sev-
eral of these processes are now utilized in India and China, where eco-
nomic pressure is greatest. By proper handling, a product is formed that
is free from injurious insects, parasitic worms, and bacteria, and that has
conserved all the valuable elements essential for plant growth.
SURVIVAL OF PATHOGENS IN SOIL AND WATER 27
DESTRUCTION OF INJURIOUS MICRO-
ORGANISMS
Improper methods of disposal of human and animal wastes were
responsible, in the early history of mankind, for many epidemics of
cholera, typhoid, plague, and other diseases. Only in recent years, after
man learned the nature of the spread of these diseases, were proper
methods developed for disposing of human wastes. According to Win-
field (990), fecal-borne diseases rank with venereal disease and tuber-
culosis as the most important infectious diseases of China, because the
people do not maintain proper sanitation and because human excreta are
used as fertilizers. Any successful system for the control of these dis-
eases must be sanitary and at the same time profitable. The composting
method can meet these requirements.
To illustrate this point, it is sufficient to consider an analysis (990)
of the occurrence of Ascaris lumbricoides, its transmission, and its rela-
tion to Entamoeba histolytica. Of 1,190 persons examined, 81 per cent
were positive for ascaris, with an average egg count of 14,000 per cubic
centimeter. Children had a higher count than adults, and females a
higher count than males. The life habits of the Chinese people are
highly favorable for the spread of ascaris. By a special process of com-
posting of feces, sufficient heat was produced to destroy disease-produc-
ing organisms and their reproductive bodies. The compost thus pro-
duced is highly effective as a fertilizer.
Many other natural substrates, like saliva (704), possess antibacterial
properties due to the antagonistic action of their own bacterial popula-
tions. Though antagonistic microorganisms may persist in soil or in
other natural substrates, substances toxic to bacteria soon tend to be
destroyed (444).
SURVIVAL OF HUMAN AND ANIMAL PATHOGENS
IN SOIL AND WATER
During the period 1878 to 1890 following the brilliant work of
Pasteur, when bacteriology was still in its infancy, medical bacteriolo-
gists took much interest in soil microbes. This was due largely to the
belief that causative agents of disease that find their way into the soil
28 HUMAN AND ANIMAL WASTES
may survive there and thus become a constant and important source of
infection. The introduction by Koch, in 1881, of the gelatin plate
method placed in the hands of the investigator a convenient procedure
for measuring the abundance of the soil population and determining the
survival in the soil of agents causing serious human diseases. In spite of
the fact that this method revealed only a very small part of the soil
population, it enabled the medical bacteriologist to establish beyond
doubt that such organisms tend to disappear in the soil. This resulted in
definite conviction on the part of the public health and medical world
that the soil is seldom a source of infection. It was soon demonstrated
that disease-producing agents die out in the soil at a rather rapid rate,
depending on the nature of the organisms, the soil, climate, and other
conditions.
Organisms that Survive for Long Periods
Only a few disease-producing microorganisms are able to survive in
the soil for any considerable periods of time. These few include the or-
ganisms causing tetanus, gas gangrene, anthrax, certain skin infections,
actinomycosis in cattle, coccidiosis in poultry, hookworm infections,
trichinosis, enteric disorders in man, blackleg in cattle, and Texas fever.
To these may be added the botulinus organism and others producing
toxic substances, as well as bacteria, actinomycetes, and fungi that cause
plant diseases such as potato scab, root rots, take-all disease of cereals,
and damping-off diseases.
Anthrax, a scourge of cattle and sheep, is a persistent survivor in
soil j spores of this organism are known to retain their vitality and viru-
lence for fifteen years. Anthrax survives particularly well in damp re-
gions, especially in soils rich in decomposing organic matter 5 the hay
and feed from these lands may transmit the disease to animals. The fact
that certain fields carry anthrax infection was recognized in Europe long
before the nature of the disease was known. Human infection results
from contact with diseased animals or animal products.
The anaerobic, spore-forming bacteria that cause gas gangrene are
widely distributed in nature. They are found extensively in soils and in
decomposing plant and animal residues. The causation of disease by
SURVIVAL OF PATHOGENS IN SOIL AND WATER 29
these organisms received particular attention during the first world
war, which was fought chiefly in trenches (957).
Another important pathogenic anaerobe able to survive in soil for
long periods of time is Clostridium chauvoeiy the causative agent of
blackleg in cattle j southern pastures are said to be better carriers of
blackleg than northern pastures.
Clostridium tetani is also widely distributed in the soil and appears to
be associated with the use of stable manures, Nicolaier (638, 639)
found, in 1884, that tetanus could be produced in experimental animals
by the injection of soil samples, 69 positive results being obtained from
140 inoculations. This organism is believed to occur in the soil in the
form of spores ; its mode of survival, however, is not sufficiently known
because of a lack of careful study.
The botulinus organism not only may remain alive in the soil for a
long time (602), but it may also produce there a potent toxin that
causes much loss of water fowl and other wild life. Aeration of the soil
results in the destruction of this toxin by aerobic bacteria (710),
Thus we see that pathogenic spore-forming bacteria are always found
in the soil. Other pathogens are able to survive in the soil only for lim-
ited periods of time. They are eliminated sooner or later from the soil,
either because of their inability to compete with the soil population or
because of their actual destruction by the latter. Although the patho-
gens seem to possess considerable resistance toward unfavorable soil con-
ditions, they are unable to multiply at rates permitting their indefinite
survival in the soiL The anthrax bacillus and certain other parasites in-
festing domesticated and wild animals belong to this group. Certain in-
sect and animal carriers make possible the survival and spread of many
pathogens in the soil.
Organisms that Survive for Brief Periods
The great majority of disease-producing bacteria, however, are able
to survive only for very brief periods outside their respective hosts,
especially in soil and water. It is sufficient to cite the fact that typhoid
and dysentery bacteria, which are known to contaminate watersheds
and water supplies, disappear sooner or later. It has been estimated, for
30 HUMAN AND ANIMAL WASTES
example (997), that in a sewage sludge free to undergo normal diges-
tion, typhoid bacteria probably survive for less than 7 days. It was sug-
gested, therefore, that sludge held in a digestion tank for about 10 days
might be applied to the soil for fertilizer purposes without detriment to
public health.
The gram-negative bacteria of the typhoid-dysentery group die out
rapidly in septic material j the typhoid bacteria survive for about 5
days, the Flexner type of dysentery for about 3 days, and the Shiga
bacillus dies out even in a shorter period. If decomposition in the tank
has not advanced far enough, as shown by low alkalinity, the organisms
may survive for a much longer period. The efficiency of ripe tank ef-
fluent to destroy bacteria is believed to be due to both the alkaline re-
action and the presence of antagonistic metabolic products. The destruc-
tion of typhoid and dysentery bacteria in the soil depends on a number
of factors, chief among which are the moisture content and reaction,
and the nature and abundance of the microbiological population. In
moist or dry soils, most of the pathogenic bacteria were found to die
within 10 days (488).
Numerous other pathogenic agents, including those causing some of
the most deadly human and animal scourges — tuberculosis, leprosy,
diphtheria, pneumonia, bubonic plague, cholera, influenza, mastitis and
abortion in cattle, the many poxes — constantly find their way into the
soil in large numbers. They disappear sooner or later, and no one now
ever raises the question concerning the role of the soil as the carrier of
these disease-producing agents or as the cause of severe or even minor
epidemics.
This rapid disappearance of disease-producing bacteria in the soil may
be due to a number of factors: (a) unfavorable environment j (b) lack
of sufficient or proper food supply j (c) destruction by predacious agents
such as protozoa and other animals j (d) destruction by various sapro-
phytic bacteria and fungi considered as antagonists} (e) formation by
these antagonists of specific toxic or antibiotic substances destructive to
the pathogens} (f ) in the case of some organisms at least, increase of the
bacteriophage content of the soil resulting in the lysis of some bacteria,
especially certain spore-formers (49).
The course of survival of only a few disease-producing organisms
SURVIVAL OF PATHOGENS IN SOIL AND WATER
31
outside the host has been studied in detail. Sufficient information has
been accumulated, however, to justify certain general conclusions.
When E. coli is added to sterile soil, it multiplies at a rapid rate, but
when added to fresh, nonsterile soil it tends to die out quickly (Table 5 ) .
The rate of its disappearance is independent of the reaction of the soil
and of the temperature of incubation, but a marked increase in the num-
ber of soil organisms antagonistic to E. coli accompanies the disappear-
ance (Table 6).
TABLE 5. SURVIVAL OF BACTERIA ADDED TO SOIL AND THEIR EFFECT
UPON THE SOIL MICROBIOLOGICAL POPULATION
INOCULUM
INCUBATION
ORGANISMS RECOVERED*
Number
Tem-
Coliform
of days
perature
Total
bacteria
Control soil
5
28° c.
21,400
<200
E. coli addedf
5
28° c.
25,600
6,800
E. coli addedt
5
28° c.
39,700
3>5oo
E. coli added
5
37° C.
22,800
4,700
Control soil
33
28° C.
5,900
<io
E. coli added
33
28° c.
22,100
130
E. coli added!
33
28'^ c.
17,600
140
E. coli added
33
37° C.
23,000
<I0
From Waksman and Woodruff (951).
* In thousands per gram of soil.
t Washed suspension of E. coli cells added at start and after 5 days.
i CaCOs added to soil.
TABLE 6. INFLUENCE OF ENRICHMENT OF SOIL WITH ESCHERICHIA COLI
ON NUMBER OF ANTAGONISTIC MICROORGANISMS
Control soil
Enriched soil
Enriched soil + CaCOgf
TOTAL MICRO-
ORGANISMS PER
GRAM OF SOIL
ANTAGONISTS* PER GRAM OF SOIL
After 65 days After 1 1 7 days After 1 1 7 days
500,000
4,000,000
6,000,000
1,150,000
5,700,000
4,700,000
9,100,000
40,000,000
36,300,000
From Waksman and Woodruff (949).
* An antagonistic colony is one surrounded by a halo on the E. coli plate.
t This container received fewer enrichments with E. coli than the one without CaCOj
32 HUMAN AND ANIMAL WASTES
In order to illustrate the fate of certain important disease-producing
bacteria which find their way into the soil or into natural water basins, it
is sufficient to draw attention to reports of experiments made on a few
typical pathogens.
The Colon-Tyfhoid Grouf of Bacteria
Frankland (295, 296) was the first to establish that Eberthella ty-
-phosa may survive in sterilized polluted water or in pure deep-well
water for 20 to 5 1 days although it died out in 9 to 1 3 days in unsterile
surface water. In other studies (464) it was found that the typhoid or-
ganism is able to survive in sterilized tap water for 15 to 25 days, as
against 4 to 7 days in fresh water j the bacteria died off even more rap-
idly in raw river or canal water, the survival time being reduced to i to
4 days. The degree of survival of the typhoid organism in water was
found to be in inverse ratio to the degree of contamination of the water,
the saprophytic bacteria in the water being apparently responsible for
the destruction of the pathogen. These conclusions were later confirmed
(777). Freshly isolated cultures of E. tyfhosa survived a shorter time
than laboratory cultures, higher temperatures (37° C.) being more de-
structive than lower ones (438). Although some investigators (1007)
claimed that E. coli may survive in soil for 4 years, others (789) found
that it disappeared rapidly. Viable typhoid bacteria were recovered
(774) from polluted soil after lOO days in unsterilized soil, and after
16 months in sterilized soil. Sedgwick and Winslow (811) reported
that cells of E. coli rapidly die out in the soil, 99 per cent destruction
occurring in dry soil in 2 weeks, with a longer survival in moist soil.
In general E. tyfhosa is able to survive only a short time in unsteri-
lized soil, but much longer in sterile soil (831). S. Martin (586), for
example, observed that typhoid bacteria survived and grew readily in
sterile soilj however, when added to a well-moistened and cultivated
soil they were rapidly destroyed. The same phenomenon occurred
when the pathogens were added to a culture of a soil organism in a
nutrient medium. Only in certain soils were conditions favorable for
the prolonged survival of the pathogen. The conclusion was reached
that the typhoid organism is destroyed by the products of decomposi-
tion taking place in the soil. It was further concluded that an antagonis-
SURVIVAL OF PATHOGENS IN SOIL AND WATER 33
tic relation appeared to exist in some soils but not in others and that this
was due to the action of specific antagonistic bacteria present in the par-
ticular soils.
Frost (302) also reported that typhoid bacteria are rapidly destroyed
when added to the soil. In 6 days, 98 per cent of the cells were killed,
and in the course of a few more days all the cells tended to disappear
entirely from the soil. Under conditions less favorable to the growth of
antagonists, the typhoid organism survived not only for many days, but
even for months. The conclusion was reached that when soil bacteria are
given a chance to develop by-products, there results a marked destruc-
tion of typhoid organisms brought into contact with them.
The survival of typhoid and dysentery bacteria in soil has been the
subject of many other investigations (256, 577, 685).
Among the factors responsible for the disappearance of E. typhosa
in water, the presence of certain water bacteria was found to be of spe-
cial importance (899). Rochaix and Vieux (760) demonstrated that
when an achromogenic strain of Pseudomonas aeruginosa was present in
drinking water, it was not accompanied by any other bacteria. Media
inoculated with this organism and E. coU gave, after 13 days' incubation,
only cultures of the former. That the two organisms could coexist, how-
ever, was shown by inoculation into sterilized water. Only the actual
development of the antagonist led to the repression of the fecal organ-
ism. The oxygen supply of the water is important (975). E. tyfhosa
added to activated sludge increased within the first 4 to 6 hours ; this was
followed by a reduction in 24 hours, and a 99 per cent destruction in sev-
eral days (411). The survival period was shorter in sewage-polluted
than in unpolluted waters, especially when the sewage was aerated.
About 80 per cent reduction of typhoid bacteria was obtained in the
Netherlands East Indies by the passage of sewage through Imhoff
tanks. Digestion of sludge reduced the number further but did not
eliminate the bacteria completely j after the sludge was dried no typhoid
bacteria could be found (613).
A study of microorganisms antagonistic to E. coli resulted in the iso-
lation of organisms from 5 of 44 samples of well water, i of 1 2 sam-
ples of spring water, and 6 of 1 6 samples of surface water. The antag-
onists included 3 strains of Pseudomonas, i each of Sarcina, Micro-
34 HUMAN AND ANIMAL WASTES
coccus, Flavobacterium, and yeast, 2 actinomycetes, and 3 unidentified
nonspore-forming, gram-negative rods (445).
The survival of E. tyfhosa in manure and in soil is known to be re-
pressed decidedly by various saprophytic bacteria. When a carrier was
induced to urinate on a soil, E. tyfhosa could be recovered within
6 hours from the washings of the soilj however, after 30 hours the or-
ganism could no longer be demonstrated, although the soil was still
moist with the urine (616). In the absence of sunlight, the organism
was recovered after 24 hours but not later. When the urine was allowed
to dry on towels, the bacterial cells survived for 10 days because sapro-
phytic microorganisms failed to develop on the dry towels. Other evi-
dence was submitted that E. tyfhosa is destroyed by bacteria grown in
association with it (382). Moisture was found (785) to be the most
important factor influencing the longevity of typhoid bacteria in the
soil J 50 per cent of the bacteria died during the first 48 hours, the sur-
vival of the remainder extending over a period of months.
E. coli was rapidly crowded out by other organisms in manure
piles (623). The addition of 9 million cells of E. coli and 13 mil-
lion cells oi A. aerogenes to a soil resulted, in 106 days, in reductions to
6,000 and 25,000 respectively} in 248 days, both organisms had com-
pletely disappeared (828). The occurrence of coliform bacteria in soil
depends entirely on the degree of pollution ; soil relatively free from
pollution contains no coliform bacteria or only a small number. No evi-
dence of multiplication of these bacteria in the soil could be detected
(873).
Sea water, as well, appears to have a bactericidal effect upon organ-
isms added to it (937, 1014). This is believed to be due to the presence
of some substance other than salt. Dysentery and typhoid organisms
were found to disappear from sea water in 1 2 and 1 6 hours, whereas
paratyphoid organisms survived for 21 and 23 days (887). Protozoa
were found to be at least partly responsible for the destruction of the
typhoid organism added to water systems (250, 440, 709, 815).
Mycobacterium tuberculosis
The fate outside the hosts of the bacteria causing tuberculosis in man
and in animals has also been studied extensively. Considerable diffi-
SURVIVAL OF PATHOGENS IN SOIL AND WATER 3 5
culty has often been encountered, however, in demonstrating the pres-
ence of this pathogen, which must be detected usually by guinea pig
inoculation methods (894). The organism was found to be alive in a
dark room after 157 to 170 days, but not after 172 to 188 daysj in dif-
fused light, the longevity was only 124 days 5 in the incubator, the or-
ganism retained its virulence for 33 days, but not for 100 daysj on ice,
virulence was still evident after 102 days but not after 153 days (618).
Pure cultures of the bovine organism mixed with cow manure and ex-
posed in a 2-inch layer in a pasture remained virulent for 2 months in
sunlight and longer in the shade. Tubercle bacteria were still alive in a
garden soil on the 213th day and dead on the 230th day. They were
alive in buried tuberculous guinea pigs on the 71st day, and dead on
the 99th day. In running water, they survived for more than a year
(85). Mycobacterium tuberculosis survived for 309 days in sputum
kept in darkness, even when completely desiccated j in decomposing
sputum, living organisms could be isolated after 20 but not after 25
days (792, 842). Under conditions prevailing in southern England, it
was found (987) that the tubercle organism may remain alive and viru-
lent in cow's feces exposed on pasture land for at least 5 months dur-
ing winter, 2 months during spring, and 4 months during autumn j in
summer, no living organisms were demonstrated even after 2 months.
Under protection from direct sunlight, the survival period was longer.
Feces protected from earthworms yielded viable cells even after 5
months. Virulent bacteria were still present in stored liquid manure at
least 4 months after infection, though during this time a gradual reduc-
tion in virulence of the organism was observed.
The addition of manure to soil was found to favor the survival of the
tubercle bacteria, as indicated by a higher proportion of test animals
becoming tuberculous when the amount of manure added to the soil was
increased (574). Positive tests were obtained for soil and manure after
178 days, but not later. The organism survived on grass for at least 49
days. Samples of milk of tuberculous cows, kept frozen and examined
periodically, gave positive tests even after 2 years and 8 months (551 ).
Rhines (746) found that M. tuberculosis multiplied in sterile soil as
well as in the presence of certain pure cultures of bacteria 5 however, a
fungus was found to check the development of the pathogen, especially
36 HUMAN AND ANIMAL WASTES
in manured soil. In nonsterile soil, the pathogen was slowly destroyed,
the plate count being reduced to about one sixth of the original in
I month. In a study of the survival of avian tubercle bacteria in sewage
and in stream water, there was a reduction, in 73 days, from 48,000 to
1,400 per milliliter in sewage and to 4,200 in water (745).
Other Disease-froducing Microor ganisms
A study of the viability of Brucella melltensls in soil and in water in
Malta brought out the fact that the organism survived in sterile tap
water 42 days and in unsterile tap water only 7 days. It survived 25
days in soil and 69 days in dry sterile soil, but only 20 days in unsterile
manured soil, 28 days in dry natural road dust, 20 days in dry sterile
sand, and 80 days on dry cloth (326, 430, 431, 432).
The rapid destruction of cholera bacteria added to soil was first
pointed out by Houston (437). Similar rapid destruction of the diph-
theria organism was also noted. Serratia, however, retained its vitality
for 158 days. Vibrio comma also survived for a short time only in feces
(358), different strains showing considerable variability; temperature
was an important factor. During the hot season in Calcutta, the viable
period was somewhat longer than a day, as compared to 7 or 8 days
during the cold season ; the critical cholera months were found to fol-
low directly the cool months. The organism did not survive very long
in fresh water, although the time appeared to be long enough to cause
occasional serious epidemics. It remained alive for 47 days in sea water
(450). The conclusion was reached that although the organism is ordi-
narily destroyed rapidly in water as a result of competition with other
microbes, it may survive in certain instances for some time.
THE SOIL POPULATION
The nature of the soil population can thus be considered as more or
less dynamic, its modification being controlled by the addition of or-
ganic matter and by soil treatments. The introduction of foreign organ-
isms tends not to change the nature of the population, but merely to
stimulate the development of such members as are capable of destroying
the foreign organisms. The production of antibiotic substances by mem-
THE SOIL POPULATION 37
bers of the soil population may also be directly responsible for the rapid
destruction of the added organisms. It has been shown (635), for ex-
ample, that subsurface soils contain microbiological populations that
are smaller, less versatile, and less adaptable than surface soil popula-
tions. Some factor in the subsurface soils was believed to cause the pre-
vention of the rapid development of the introduced organisms. Anti-
biotic or inhibitory substances were said to be responsible for this effect,
these substances being of microbial origin. Aqueous extracts of soil did
not adversely affect the growth of soil bacteria in vitro, but alcohol ex-
tracts reduced their activity in the soil and in artificial culture media.
CHAPTER 3
INTERRELATIONSHIPS AMONG MICROORGAN-
ISMS IN MIXED POPULATIONS
It must not be forgotten that there are extremes in another di-
rectiony where one of the two associated organisms is injuring
the other, as exemflified by m,any farasites, but these cases I
leave out of account here. This state of affairs has been termed
antibiosis. — H. M. Ward.
SYMBIOSIS AND ANTIBIOSIS
Microbes grow and bring about many metabolic reactions in natural
substrates, such as soils and water basins, in a manner quite different
from those in pure cultures where they are not influenced by the
growth of other organisms. In artificial and natural media, whether
these be synthetic materials, complex organic mashes and infusions used
for the preparation of industrially essential products, or the bodies of
plants and animals, pure cultures of microbes are free from the asso-
ciative and competitive effects of other microbes found in natural sub-
strates. In mixed populations, a number of reactions that do not com-
monly take place in pure cultures are involved. Even in the case of
mixed infections, a pathogen may be preceded or followed by one or
more saprophytes, whereby the processes of destruction brought about
in the living animal or plant body are alleviated or hastened. In the
mixed populations found in natural substrates, the ecological relation-
ships are largely responsible for many of the essential differences in
the behavior and metabolism of the microbes, as compared with the
same organisms growing in pure culture.
Almost all microorganisms inhabiting a natural milieu, such as soil
or water, are subject to numerous antagonistic as well as associative, or
even symbiotic, interrelations. Every organism is influenced, directly
or indirectly, by one or more of the other constituent members of the
complex population. These influences were at first visualized as due
primarily to competition for nutrients {S"^^)- This was well expressed
SYMBIOSIS AND ANTIBIOSIS 39
by Pfeffer (684), who said that "the entire world and all the friendly
and antagonistic relationships of different organisms are primarily
regulated by the necessity of obtaining food." It was soon recognized,
however, that this explanation does not account fully for all the com-
plex interrelations among microorganisms in nature.
De Bary (165) was the first to emphasize, in 1879, the significance
of the antagonistic interrelations among microorganisms j when two or-
ganisms are grown on the same substrate, sooner or later one overcomes
the other and even kills it. This phenomenon was designated "anti-
biosis" (42, 953). Symbiotic, or mutualistic, and antagonistic relation-
ships among microorganisms indicate whether advantages or disadvan-
tages will result to the organisms from the particular association; the
first are beneficial and the second are injurious and may even be para-
sitic. Kruse (508) asserted that, when two organisms are capable of
utilizing the same nutrients but are diflFerently affected by environ-
mental conditions such as reaction, air supply, and temperature, the one
that finds conditions more suitable for its development will grow more
rapidly and in time be able to suppress the other. According to Porter
(695), the effects produced by fungi in mixed culture are due either to
exhaustion of nutrients or to the formation of detrimental or beneficial
products. E. F. Smith (829) emphasized that when two or more or-
ganisms live in close proximity they may exert antagonistic, indifferent,
or favorable effects upon one another. These potentialities were later
enlarged (loii) to include stimulating, inhibiting, overgrowing, and
noninfluencing effects. After considerable experimentation and specula-
tion, Lasseur (513, 514) came to the conclusion that antagonism is a
very complex phenomenon and is a result of numerous and often little-
known activities. Antagonism influences the morphology of the organ-
isms, their capacity of pigment production, and other physiological
processes.
No sharp lines of demarcation can be drawn between associative and
antagonistic effects. Well-defined effects of two symbionts may change
during the various stages of their life cycles or as a result of changes in
the environment. It is often difficult to separate strictly symbiotic phe-
nomena from associations of less intimate nature, frequently desig-
40 INTERRELATIONSHIPS AMONG MICROORGANISMS
nated as commensalisms. The various stages of transition from obligate
parasitism to true saprophytism can be represented as follows:
Obligate parasitism (cer- Facultative parasitism (spe- Modified parasitism;
tain bacteria, smut fungi) — > cies of Fusarium, Rhizoc- — > hosts may derive some — >
tonia, and Actinomyces^ benefit (certain mycor-
rhiza)
Balanced parasitism (vari- True symbiosis (root- True saprophytism (auto-
ous mycorrhiza) — > nodule bacteria, lichen — » trophic and heterotrophic
formations) bacteria and fungi).
The phenomena of antagonism do not fit exactly into the above
scheme but are parallel with it: the injurious effects of one organism
upon another range from antagonism of varying degrees of intensity
to the actual living or preying of one organism upon another. The lat-
ter may be classified with the phenomena of parasitism and disease pro-
duction.
Microorganisms inhabiting the soil live in a state of equilibrium
(943). Any disturbance of this equilibrium results in a number of
changes in the microbial population, both qualitative and quantitative.
The ecological nature of this population found under certain specific
conditions, as well as the resulting activities, can be understood only
when the particular interrelationships among the microorganisms are
recognized. Because of its complexity, the soil population cannot be
treated as a whole, but some of the processes as well as some of the
interrelations of specific groups of organisms can be examined as sepa-
rate entities. Some have received particular attention, as the relations
between the nonspore-forming bacteria and the spore-formers, the ac-
tinomycetes and the bacteria, the bacteria and the fungi, the protozoa
and the bacteria, and the relations of the bacteria and the fungi to the
insects.
The term "synergism" has been used to designate the living together
of two organisms, resulting in a change that could not be brought about
by either organism alone (425). Microbes living in association fre-
quently develop characteristics which they do not possess when living
in pure culture. Schiller (797, 798), for example, found that when beer
yeasts are placed together with tubercle bacteria in a sugar-containing
but nitrogen-free medium, the yeasts develop antagonistic properties
THE NATURE OF A MIXED MICROBIAL POPULATION 41
toward the bacteria and use the latter as a source of nitrogen j the yeasts
secrete a bacteriolytic substance that is also active outside their cells.
Various bacteria are able to kill yeasts when they are inoculated into
suspensions of the latter in distilled water. The destruction of the
fungus Ofhiobolus, the causative agent of the take-all disease of cereals,
by soil organisms was believed (312) to be a result of the need of a
source of nitrogen by the latter.
The term "autoantibiosis" has been used (670) to designate the
phenomenon of self-inhibition or "staling" of medium as a result of the
previous growth of the organism in this medium.
THE NATURE OF A MIXED MICROBIAL
POPULATION
A mixed microbial population is made up of a great variety of bac-
teria, and often also of fungi, actinomycetes, and protozoa j to these are
added, under certain conditions, various algae, diatoms, nematodes and
other worms, and insects. The specific nature and relative abundance of
the various microorganisms making up a complex population in either a
natural or an artificial environment depend upon a number of factors,
which can be briefly summarized as follows:
The physical nature of the medium in which the population lives: soil,
compost, or manure pile; river, lake, or ocean; sewage system; or
peat bog.
The nature, concentration, and availability of the chemical constituents
of the medium used by the microbes as nutrients, especially the ma-
terials used as sources of energy and for the building of cell sub-
stance. Various organic and inorganic substances, whether complex
or simple in chemical composition, favor the development of specific
groups of microorganisms capable of utilizing them. For example,
sulfur favors the development of specific sulfur bacteria, and cellu-
lose favors such organisms as are capable of attacking this complex
carbohydrate as a source of energy. In many instances there is con-
siderable competition for the available food material. Organisms that
possess a greater capacity of attacking the particular compound, or
are capable of preventing the development of other organisms by the
formation of substances injurious to the latter, usually become pre-
42 INTERRELATIONSHIPS AMONG MICROORGANISMS
dominant. Proteins, starches, and sugars can be acted upon by a
great variety of microorganisms. The predominance of one group
may depend not only upon the chance presence of the particular or-
ganism or its capacity for more rapid growth, but also upon its ability
to form alcohols, acids, and other products that influence the growth
of other organisms.
Environmental conditions favorable or unfavorable to the development
of specific organisms. Of particular importance in this connection
are temperature (thermophilic vs. mesophilic organisms), oxygen
supply (aerobic vs. anaerobic organisms), moisture content (bac-
teria and fungi vs. actinomycetes), reaction (acid-sensitive vs. acid-
tolerant forms), as well as the physical conditions of the substrate as
a whole.
The presence and abundance of organisms that produce substances having
a favorable and stimulating or an injurious and toxic effect upon
other organisms, or that may compete for the available nutrients.
The equilibrium in the microbiological population in a natural me-
dium such as soil or water may be upset by the introduction of spe-
cific nutrients, as well as by treatment with chemical and physical
agents whereby certain organisms are destroyed and others stimu-
lated.
The presence of specific microorganisms in a natural medium may be con-
siderably influenced by the presence of certain parasitic or phagocytic
agents. The role of protozoa in controlling bacterial activities by
consuming the cells of the bacteria has been a subject of much specu-
lation. The presence of bacteria, fungi, and nematodes capable of
destroying insects is of great importance in human economy. Many
other relationships, such as the presence of phages against specific
organisms, are often found greatly to influence the nature and com-
position of a specific population.
ASSOCIATIVE INTERRELATIONSHIPS
Numerous instances of associative interrelationships among micro-
organisms are found in nature. These may be grouped as follows :
Preparation or modification of the substrate by one organism whereby it
is rendered more favorable or more readily available for the growth
of another organism. As an illustration one may cite the breakdown
Large root u ith bacterial cells and
filaments of actin()m\cetes
V
^■«*^
Root hair with rod-shaped bacteria in
colonies and short chains
■.-'i
Terminal portion of root hair show-
intj bacteria in form of mantle
Root hair undergoing attack by
bacteria and actinomycetes
i ^
"4:^?:
^' ■^^t^ '^'^
'■■•'£ii « 'w?< - ■•■■ %
Masses of coccoid bacteria growing
< about a funijus filament
■ vt , • ■ >
Dense colonies of bacteria sur
rounding root hairs
Figure 2. Relationships between microorganisms and root systems of higher plants.
From Starkey (848).
ASSOCIATIVE INTERRELATIONSHIPS 43
of cellulose by specific bacteria, thereby making the particular en-
ergy source available to noncellulose-decomposing organisms, in-
cluding not only certain bacteria and fungi but also higher forms of
life such as ruminant animals (herbivores) and insects (termites,
cockroaches), which carry an extensive cellulose-decomposing micro-
biological population in their digestive systems. Another illustration
is the breakdown of complex proteins by proteolytic bacteria, result-
ing in the formation of amino acids and polypeptides, which form
favorable substrates for peptolytic bacteria. The ammonia liberated
from proteins and amino acids supplies a source of energy for nitrify-
ing bacteria and a source of nitrogen for many fungi. The ability of
bacteria to concentrate in solution those nutrients that are present
only in mere traces enables animal forms (protozoa) to exist at the
expense of the bacteria (102).
Influence upon the oxygen concentration available for respiration. This
involves the phenomenon first observed by Pasteur (673) of con-
sumption of oxygen by aerobic bacteria, thus making conditions fa-
vorable for the development of anaerobes (650—652, 843).
Symbiotic interrelationships, where both organisms benefit from the asso-
ciation. The three most important examples found in nature are:
(a) the phenomenon of symbiosis between root-nodule bacteria and
leguminous plants; (b) mycorrhiza formations between certain
fungi and higher plants; (c) lichen formation between algae and
fungi. Certain other interrelations are not strictly symbiotic, but are
found to fall between groups a and c; here belong nitrate reduction
accompanied by cellulose decomposition and nitrogen-fixation with
cellulose decomposition, carried out in each case by two specific
groups of organisms.
Production by one organism of growth-promoting substances that favor
the development of other organisms. The formation of riboflavin by
anaerobic bacteria in the digestive tract of herbivorous animals is an
interesting and highly important phenomenon in the nutrition of such
animals. The production of bacterial growth stimulants by yeasts
and the beneficial action of mixed populations upon nitrogen-fixation
by Azotobacter are other illustrations of this general phenomenon.
The presence of specific bacteria has been found necessary for the
sporulation of certain yeasts and for the formation of perithecia by
( various Aspergilli (612, 787 ). Various other processes of association
have also been recognized (920).
44
INTERRELATIONSHIPS AMONG MICROORGANISMS
Destruction by one microorganism of toxic substances produced by an-
other, thereby enabling the continued development of various mem-
bers of the microbiological population.
Modification of the physiology of one organism by another. In the presence
of certain bacteria, Clostridium granulobacter-fect'movorum forms
lactic acid instead of butyl alcohol (845). The presence of Clos-
trid'tum acetobutyVicum in cultures of bacteria producing dextro-lactic
acid and laevo-lactic acid causes such bacteria to form the inactive lac-
tic acid (870) ; intimate contact of the bacteria is essential, the use of
membranes preventing this effect. The presence of A. aero genes
modifies the optimum temperature for nitrogen-fixation by Axoto-
bacter (749). Pigment formation by P^. aeruginosa may be weak-
ened when the latter is grown together with other organisms. E. coli
may lose the property of fermenting sugars when grown in the
presence of paratyphoid organisms (453).
Some associations of microorganisms are not so simple. The complex
system of animal infection by more than one organism, with the result-
ing complex reactions in the animal body, is a case in point.
The effect of one organism upon the activities of another can be illus-
trated by the results of the decomposition of complex plant material by
pure and mixed cultures of microbes (Table 7). Trichoderma, a fungus
TABLE 7. DECOMPOSITION OF ALFALFA BY PURE AND MIXED
CULTURES OF MICROORGANISMS
TOTAL
HEMICELLU-
CELLU-
ALFALFA DE-
LOSES DE-
LOSE DE-
NH3-N
ORGANISM
COMPOSED
COMPOSED
COMPOSED
PRODUCED
Per cent
Per cent
Per cent
mgm.
Trichoderma
9-3
4.7
0
61
Rhizofus
6.6
12.8
2.9
53
Trichoderma + Rhizofus
13-7
22.6
10.6
63
Trichoderma + Cunningharnella i 5 .0
15.4
5-7
47
Trichoderma + Ps. jluorescens
10.5
14.5
6.4
32
Streftomyces 3065
16.6
43-0
23.2
52
Trichoderma + Streftomyces
3065
12.5
14.6
4.8
56
Soil infusion
28.4
40.9
50.8
21
From Waksman and Hutchings (938).
COMPETITIVE INTERRELATIONSHIPS 45
known to be an active cellulose-decomposing organism, did not attack
at all the cellulose of alfalfa and decomposed the hemicelluloses only to
a limited extent j however, the organism utilized the proteins rapidly,
as illustrated by the amount of ammonia liberated. Rhizofus^ a non-
cellulose-decomposing fungus, attacked largely the hemicelluloses in
the alfalfa and some of the protein j a small reduction in cellulose was
recorded, probably because of an analytical error. When Trichoderma
was combined with Rhizofus, the former attacked readily both the cel-
lulose and the hemicelluloses. The same effect upon the activity of
Trichoderma was exerted by other noncellulose-decomposing organ-
isms, such as the fungus Cunnmghamella and the bacterium Ps. fuores-
cens. On the other hand, when Trichoderma was combined with a cellu-
lose-decomposing Streftomyces, there was considerable reduction in the
decomposition of the total plant material as well as of the cellulose and
hemicelluloses. These results further emphasize the fact that two or-
ganisms may either supplement and stimulate each other or exert an-
tagonistic effects. The total soil population is far more active than any
of the simple combinations of microorganisms.
COMPETITIVE INTERRELATIONSHIPS
The following competitive relations among the microscopic forms of
life inhabiting the sea have been recognized (i8) :
Competition among chlorophyol-bearing diatoms for the available nutri-
ent elements in the water
Competition among the copepods for the available particulate food mate-
rials, notably the diatoms
Competition between individuals belonging to one species and individuals
belonging to another
Competition between young, growing, and reproducing cells and older,
respiring cells
Food competition and space competition
Competition between transitory and permanent populations
Competition between sedentary or sessile organisms and free-moving forms
This list has been enlarged (924) to include other factors that are par-
ticularly prominent in nonaquatic environments :
46 INTERRELATIONSHIPS AMONG MICROORGANISMS
Degree of tolerance of the immune or resistant varieties and of the less re-
sistant or more sensitive forms to attack by disease-producing or-
ganisms
Fitness for survival of microbes that are able to adapt to a symbiotic form
of life, such as leguminous plants or mycorrhiza-producing plants,
and those that are not so adapted
Survival of parasitic forms that require living hosts for their development,
as contrasted with saprophytes that obtain their nutrients from min-
eral elements or from dead plant, animal, and microbial residues
Various special types of competition, for example, competition between
strains of root-nodule bacteria (Rhizobium), whereby one strain
checks completely the multiplication of other strains, even outside the
plant, the dominant strain then becoming responsible for all the
nodules produced, as shown by Nicol and Thornton (637).
These phenomena of competition are found not only in natural sub-
strates, such as soil and water, but also in artificial media. When several
microbes are growing in the same culture medium, some will be re-
pressed in course of time whereas others will survive and take their
place. This is due to the fact that these microbes compete for the use of
the same nutrients or that conditions, such as reaction, oxygen supply,
and temperature, are more favorable to some organisms than to others.
Another phenomenon may also be involved, that some organisms may
produce toxic substances that repress the growth of others. In artificial
media, slowly growing tubercle bacteria, diphtheria organisms, and
others will be repressed by the rapidly growing saprophytes. Under
aerobic conditions, aerobic bacteria and fungi will repress yeasts and
anaerobic bacteria, whereas under anaerobic conditions the reverse will
take place. An alkaline reaction will favor the development of bacteria,
an acid reaction will favor the growth of fungi.
ANTAGONISTIC INTERRELATIONSHIPS
When two or more organisms live together, one may become antag-
onistic to the others. The composition of the medium and the conditions
of growth influence the nature and the action of the antagonist 5 its
metabolism and cell structure may become modified or the cell itself
may be destroyed (174). In urine, for example, staphylococci may be-
ANTAGONISTIC INTERRELATIONSHIPS 47
come antagonistic to E. coli or vice versa, depending on the initial num-
bers of the two groups, on the formation of metabolic products, or on
the exhaustion of nutrients (247). The toxic substances produced by
the antagonists comprise a variety of compounds, ranging from simple
organic acids and alcohols to highly complex bodies of protein or poly-
peptide nature.
Various types of antagonism are recognized. Nakhimovskaia (627)
concluded that all phenomena of antagonism among microorganisms
can be conveniently classified into four groups of reactions:
1. Antagonism in vivo vs. antagonism in vitro. According to some inves-
tigators (513, 514), only the inhibitive forms of antagonism {in
vitro) may be designated as true antagonisms; the in vivo forms
were designated as phenomena of antibiosis. Usually, however, this
differentiation is not recognized.
2. Repressive, bactericidal, and lytic forms of antagonism. One may fur-
ther distinguish between bacteriostatic and bactericidal, fungistatic
and fungicidal forms of antagonism, as well as between antagonism
of function and antagonism of growth.
3. Direct, indirect, and true antagonism.
4. One-sided and two-sided antagonism; antagonism between strains of
the same species and antagonism among strains of different species
(228).
Duclaux (208) was the first to demonstrate that the growth of a
fungus upon a certain medium renders the medium unfavorable for the
further growth of the same organism. Kiister (509) has shown that
culture solutions in which fungi have grown are not suitable for the
germination of freshly inoculated spores but are improved by boiling.
This effect was observed as a result of the growth not only of the same
organism but also of other species. Similar observations were made for
bacteria: Marmorek (583) reported, in 1902, that the growth of
hemolytic streptococci in broth rendered the medium unsuitable for
subsequent growth of the same organism. The production of spores by
bacteria was believed to be caused by the formation of toxic, thermola-
bile organic substances; upon the destruction of these by boiling, the
rnedium was made again favorable for the growth of bacteria and bac-
terial spores were able to germinate again. Some of the toxic substances
48 INTERRELATIONSHIPS AMONG MICROORGANISMS
appeared to be thermostable j Nadson and Adamovic (625) showed
that certain metabolic products of microorganisms, even when heated to
120° C, may have a strong influence upon the subsequent growth of
the organisms.
Fungi are capable of producing not only growth-inhibiting but also
growth-promoting substances (509, 547). By means of certain proce-
dures, it was found possible to separate the two (654). The tendency of
fungus hyphae to turn away from the region in which other hyphae of
the same fungus were growing was explained as a negative reaction to
chemical substances produced by the growing fungus (304). This nega-
tive chemotropism was shown to be due to thermolabile staling sub-
stances (352). The phenomenon of staling was often spoken of as vacci-
nation of medium (45), and was ascribed to the action of protein degra-
dation products.
These and other experiments led to the conclusion that many micro-
organisms are capable of producing substances that are injurious to their
own development (iso-antagonistic) or, and sometimes much more so,
to other organisms growing close to them (hetero-antagonistic). The
growth of certain fungi and bacteria in practically pure culture, even in
a nonsterile environment, was believed to be due to this phenomenon.
It is sufficient to mention the lactic and butyric acid bacteria, the citric
acid-producing species of Asfergillus, the lactic and fumaric acid-
producing species of Rhizofus, and the alcohol-producing yeasts. The
chemical substances produced by these organisms in natural substrates
may be looked upon as protective metabolic products of microorgan-
isms in their struggle for existence. Such products play a highly sig-
nificant part in the metabolism of various organisms, especially those
that grow parasitically upon living plant and animal bodies.
Among the various types of antagonism, the one resulting in the pro-
duction of active substances that can be isolated and purified has re-
ceived the greatest consideration recently. These substances have been
designated as toxins, poisons, antagonistic agents, bacteriostatics, and
antibiotics. The chemical nature of some has been elucidated, but that
of many others is still unknown. Some of these substances are destroyed
by boiling, by exposure to light, or by filtration, whereas others are re-
sistant to heat and to ultraviolet raysj some are readily adsorbed by
ANTAGONISTIC INTERRELATIONSHIPS 49
certain filters, from which they can be removed by the use of special
solvents such as ether, alcohol, chloroform, and acetone. The concen-
tration of the antagonistic substance produced by many fungi and bac-
teria was found (240, 641 ) to be greatly influenced by the energy and
nitrogen sources in the medium and by environmental conditions, such
as temperature and aeration.
The three important types of antagonism are (a) the repressive, in-
hibitive, or bacteriostatic, (b) the bactericidal, and (c) the bacteriolytic.
When one bacterium is inoculated into the filtrate of another, the
growth of the first is slower than that of the control (299). Certain
types of antagonism express themselves in the destruction by the an-
tagonist of the other organisms present in the mixed culture, with
or without producing a lytic effect, B. mesenterkus^ for example, is
capable not only of depressing but also of killing the cells of diphtheria
and pseudodiphtheria (1016), The lytic form of antagonism is illus-
trated by the action of Ps. aeruginosa^ Bacillus hrevis, and certain other
antagonists upon micrococci and various spore-forming bacteria.
In differentiating between "direct antagonism" and "passive antag-
onism," attention was directed (627) to the fact that the latter depends
not upon the direct action of the antagonist but upon the changed con-
ditions of culture under the influence of the antagonist's growth. This
may comprise a change in ^H and rH of medium or an impoverish-
ment of some of the nutrient constituents, "Direct antagonism" was
often distinguished (634) from "indirect antagonism," the first being
limited to those phenomena in which the antagonistic action Is con-
nected with the direct action of the living cell, whereas in the second the
metabolic products produced by one organism are Injurious to others.
Intestinal bacteria were found (365, 367) to repress the anthrax organ-
ism only when the former were in an active living state. Other Investi-
gators (407) designated the action of the living cell itself as "true
antagonism."
Bail (31) suggested that for every bacterium there is a typical
constant number of cells capable of living In a given space. When this
concentration (M) Is reached, multiplication comes to a standstill, in-
ciependent of exhaustion of the nutrients or formation of toxic sub-
stances. The same phenomenon was believed to hold true when two
50 INTERRELATIONSHIPS AMONG MICROORGANISMS
bacteria live together (983, 984): if the limiting cell-in-space concentra-
tions are different for the two organisms, the one with a higher M value
represses the other j however, the weaker species may check the stronger
when planted in sufficient excess (244). It has been suggested (368)
that certain physiological properties of the individual organisms, desig-
nated as "biological activity" and "competitive capacity," must also be
taken into consideration in evaluating this relationship (634, 983, 984).
Brown (90) explained the fact that the number of yeast cells reaches a
maximum independent of the initial number of cells added or the con-
centration of nutrients in a given volume of medium by the amount of
oxygen originally present.
Garre (311) deserves the credit for having first noted that antago-
nism may be either one-sided or two-sided. In the first case, one organ-
ism represses another that is not antagonistic to itj in the second case,
both organisms repress each other. A one-sided antagonism may become
two-sided under certain conditions of culture. E. coU is antagonistic to
E. tyfhosa; however, if the latter is inoculated into a medium some-
what earlier than the former, E. tyfhosa becomes antagonistic to E. colt
(324,915).
Although the most common antagonisms are between organisms of
different species, there are numerous instances where one strain of a
species may be antagonistic toward another strain of the same species
(52, 368, 611). Certain strains may develop antagonistic properties in
the presence of other strains (77). Nonflagellated variants of typhoid
bacteria were repressed by a flagellated form, smooth variants of para-
typhoid bacteria by rough forms, and so on. The fact that all bacterial
cultures stop growing after a certain period of time has been interpreted
to be a result of the antagonistic action of some cells upon others. When
the filtrates of such cultures are added to fresh nutrient media they may
stop the growth of the same species as well as that of other species.
Certain organisms produce pigments in the presence of others j these
pigments are believed to be in some way associated with the phenome-
non of antagonism. In the presence of S. lutea^ V. comma forms a
dark violet pigment that is accompanied by an increase in agglutination
and in virulence (627). The destruction of Dktyostelium muco-
roides by a red-pigment-forming bacterium was accompanied by an in-
NATURE OF ANTAGONISTIC ACTION 51
crease in intensity of the pigment (690); the blue pigment of Bac-
terium violaceum, however, only delayed the growth of the fungus. Ac-
cording to Doebelt (177), Pemc'illlum ajricanum produces a more in-
tense pigment in contact with other fungi such as Aspergillus niger; this
pigment accumulates in the mycelium of the latter, which may thereby
be killed. Nadson (626) demonstrated that some fungi {Penicillium
luteum and Spcaria furfuro genes) produce a pigment that is used not
only for purposes of protection, but also for attack upon other organ-
isms, whereby the latter are killed and stained.
DISTRIBUTION OF ANTAGONISTIC PROPERTIES
Numerous microbes found among the bacteria, fungi, actinomycetes,
and protozoa possess the capacity of bringing about injurious or de-
structive effects upon other microorganisms belonging to their own
groups or to others. In some instances, the antagonistic effects are ob-
tained only in the presence of the antagonizing organism; in many
other cases, excretion products consisting of definite chemical com-
pounds are produced by the antagonist. A few of these products have
been isolated and have been found to be effective against certain few
specific organisms or able to act upon a great variety of organisms
(920). The wide distribution of antagonistic properties among micro-
organisms is brought out in subsequent tables.
THEORIES OF THE NATURE OF
ANTAGONISTIC ACTION
The various theories proposed to explain the mechanism of antago-
nistic effects of microorganisms may be summarized under the follow-
ing processes :
Exhaustion of nutrients
Physicochemical changes in medium
Enzyme action, either directly by the antagonist or as a result of cell
autolysis, under the influence of the antagonist
Production and liberation of toxic substances
Pigment action
' Action at a distance
Space antagonism
52 INTERRELATIONSHIPS AMONG MICROORGANISMS
Pasteur (672, 674, 675) ascribed the antagonistic effect of aerobic bac-
teria upon the anthrax organism to the consumption of the oxygen by
the former j the unfavorable influence of normal blood upon the growth
of anthrax was believed to be due to competition for the oxygen by the
red blood corpuscles. Freudenreich (299) considered the antagonism
between Ps. aeruginosa and Bacillus anthracis as due to exhaustion of nu-
trients by the former. These studies were soon followed by numerous
other investigations in which the exhaustion of nutrients in the media
was believed to be responsible for the phenomenon of antagonism j the
onset of the stationary phase in bacterial growth was believed (539) to
belong here. The change in -pW of medium and the accumulation of
toxic products were also found to become limiting factors. Palevici
(667) added fruit juice to a stale medium and brought about improve-
ment in bacterial growth, thus suggesting the exhaustion of growth-
promoting substances as the cause of staling. Broom (89) emphasized,
however, that the effect was due to addition of nutrients, including
glucose.
It thus became apparent, even in the early days of bacteriology, that
certain changes are produced by microbes in the medium in which they
grow which render it unfit for the growth of other organisms. It also
was soon recognized that the problem is more complicated than the
mere exhaustion of nutrients. The relationships produced by changes in
surface tension, in oxidation-reduction potential, in reaction, and in os-
motic pressure were suggested as explanations (24, 627, 827). Among
the classical examples of the effect of reaction upon the growth of other
organisms is the acidification of milk by lactic acid bacteria. Metchnikov
emphasized the fact that Lactobacillus bulgaricus acts antagonistically
not only by means of the lactic acid that it produces but also by the
formation of special substances. The production by bacteria of alkali-
reaction products that have an injurious effect upon the further growth
of the organisms has also been demonstrated (334). These substances
were found to correspond to amino compounds, liberated in the process
of cellular disintegration. Numerous other physical and physicochemi-
cal factors influence the growth of an organism in an artificial medium.
It is to be recalled that the rate of survival of bacterial cells in water or in
NATURE OF ANTAGONISTIC ACTION 53
salt solution is markedly influenced by the colloids present (991), the
concentration of electrolytes (816), the reaction (897), and the tem-
perature (36).
Microbial antagonism was thus looked upon (496) largely as a re-
sult of a series of physical factors, including various radiations such as
mytogenetic rays (9, 679, 814), fH. changes, conductivity, electric
charge, and surface tension.
Most antagonisms, however, can be explained by the production of
toxic substances by the antagonists. Because of their thermolability,
sensitivity to chemical reagents, or adsorption on bacterial filters, con-
siderable difficulty has been experienced in isolating the active sub-
stances. Many of these substances have been found to be iso-antagonistic
(autotoxins [141]), whereas others are able to act upon different bac-
teria. Most of them have been found to be thermostable.
The first antibiotic substance recognized as such was pyocyanase
(235), soluble in alcohol, ether, and chloroform. Somewhat similar
substances appear to be produced by Serratia marcescens ( 230) , Ps. fluo-
rescens {s2S)jB. mesentencus ( 10 1 6) , and Bacillus mycoides. Whereas
Emmerich and Low (236) considered pyocyanase to be a proteolytic
enzyme, others (370, 410, 668, 679) found it to be a lipoid. Since that
early work and especially during the last five years, many new agents
have been isolated or demonstrated. These will be discussed in detail
later. It is sufficient to mention gramicidin and tyrocidine, produced by
B. brevis, which are polypeptides 5 citrinin and fumigatin, which are
quinone-like in nature j actinomycin, aspergillic acid, and iodinin, which
are nitrogenous ring compounds j gliotoxin, which is a sulfur-bearing
compound J streptothricin, streptomycin, and proactinomycin, which are
nitrogenous bases. Some of the most important compounds (penicillin)
have not as yet been sufficiently elucidated. Certain microbial pigments
(pyocyanin, hemipyocyanin, prodigiosin) have also received considera-
tion as bacteriostatic and fungistatic agents.
The production of antibiotic substances by various microorganisms is
greatly influenced by reaction, temperature, and aeration of substrate,
as well as by the presence of other organisms. Evidence is still lacking
as to whether these substances may accumulate in the soil and in water
54 INTERRELATIONSHIPS AMONG MICROORGANISMS
(361), whether the organisms thereby affected are able to overcome
their effect, and whether they are destroyed by other members of the
soil or water microbiological population (947, 951).
Different organisms possess different degrees as well as different
mechanisms of antagonism. Often one organism may completely check
the growth of another j later, growth may be resumed, although it will
not be quite normal. Antagonism stimulates spore-production and
brings about deformed growth of the mycelium in fungi or the forma-
tion of gigantic cells in bacteria. Distortions were found to be produced
in Alternar'ia (231) and in HelTninthosforium (695) by a bacterial
antagonist. The morphological effects produced by the antagonists com-
prise changes in form, size, and structure of hyphae, direction of growth,
and complete cessation of growth and abbreviation of hyphal segments.
In surveying the phenomena of antagonism among microorganisms,
Porter (695) reached the conclusion that, among bacteria, the spore-
formers are strong inhibitors. Actinomycetes also exhibit strong inhibi-
tory action against most filamentous fungi. Phycomycetes usually
neither cause inhibition nor are inhibited j the Basidiomycetes contain
very few organisms possessing antagonistic properties. Ascomycetes
and Fungi Imperfecti vary greatly in their ability to produce antibac-
terial substances; some yeasts are strong inhibitors. Certain algae, no-
tably species of Chlorella, produce a substance (chlorellin) that inhibits
the growth of various gram-positive and gram-negative bacteria (701).
CHAPTER 4
ISOLATION AND CULTIVATION OF
ANTAGONISTIC MICROORGANISMS^ METHODS
OF MEASURING ANTIBIOTIC ACTION
In nearly all the earlier work and even In many recent investigations
on the antagonistic properties of microorganisms and the production
of antibiotic substances, two procedures were employed: indiscriminate
testing of pure cultures of bacteria and fungi, commonly taken from
culture collections, for antagonistic effects against one another or against
certain specific or test organisms j and isolation of occasional antagonistic
organisms from old plate cultures, as air contaminants, or from mixed
infections. These studies were carried out either by medical bacteri-
ologists interested in agents capable of suppressing bacterial pathogens
or by plant pathologists interested in organisms capable of inhibiting
the growth of fungi, principally those concerned in the causation of
plant disease. They resulted in the accumulation of considerable infor-
mation concerning antagonistic organisms, the nature of the phenome-
non of antagonism, and, to a more limited extent, the mechanisms in-
volved. Neither of these methods, however, is suitable for a systematic
study of antagonism as a natural process.
The last decade has witnessed a number of systematic attempts to de-
termine the distribution of antagonists in nature, to isolate specific or-
ganisms capable of bringing about the desired reactions, and to estab-
lish the mechanism involved in these reactions. These studies were
undertaken by a group of Russian investigators interested largely in
fungi and actinomycetes as agents antagonistic to other microorganisms
chiefly causing plant diseases, and by American and British investigators
interested in agents active against bacterial pathogens of man.
The early significant, but unrecognized, investigations of Schiller
(797? 798) on forced antagonisms and the studies of Gratia and his as-
sociates (349, 350) on mycolysates were in direct line of the more re-
cent studies of Dubos (190), who made a systematic attempt to isolate
from specially enriched soils bacteria capable of destroying specific
56 ISOLATION AND CULTIVATION OF ANTAGONISTS
pathogenic organisms. Although it had been previously established that
many spore-forming bacteria are capable of producing substances that
have antibacterial properties, as shown by the work of Pringsheim
(705), Much (621), and others, Dubos was the first to succeed in iso-
lating in crystalline form the active substances involved and in demon-
strating their chemical nature. He utilized for the isolation of the or-
ganisms the enrichment culture method. This consisted in adding
repeatedly various pathogenic bacteria to a soil which, as a result, be-
came enriched with antagonistic organisms capable of destroying the
bacteria added j these organisms were then isolated by appropriate pro-
cedures. The isolation of the specific substances will be described later
(page 156).
These investigations, as well as the work of Fleming (265) and
other British investigators (3, 7, 8, 113) on the antibacterial properties
of molds belonging to the PenicilUum notatum group, served as the di-
rect stimulus to numerous studies that followed. The entire series of
studies led to the development of simple methods for the systematic iso-
lation of microorganisms capable of inhibiting the growth of fungi and
bacteria, both pathogenic and saprophytic (857, 934), and for separat-
ing many of the antibiotic substances produced by these organisms.
METHODS OF ISOLATING ANTAGONISTIC
MICROORGANISMS
Four, and possibly five, methods are now available for the isolation
of antagonistic microorganisms from natural substrates such as soil,
stable manure, composts, sewage, water, and food products. These
methods are different in nature, but they are all based on the same prin-
ciple, that of bringing a living culture of a bacterium or fungus into
close contact with a mixed natural population, thereby allowing certain
members of this population to develop at the expense of the added
culture.
Soil Enrichment "Method
By this method a soil Is enriched with known living pathogenic bac-
teria. Fresh garden or field soil is placed in glass beakers or pots, and
METHODS OF ISOLATION 57
the moisture of the soil is adjusted to optimum for the growth of aerobic
bacteria, which is about 6s per cent of the water-holding capacity of the
soil (20 to 50 per cent of the moist soil)j the containers are covered
with glass plates and placed in an incubator at 28° or 37° C. Washed
suspensions of living bacteria are added to the soil at frequent intervals,
care being taken to avoid puddling it with an excess of the fluid, so con-
ditions will not be made anaerobic. Samples of the enriched soil are
removed at intervals and tested for the presence of organisms antag-
onistic to the bacteria added. Fresh washed suspensions of the living-
bacteria are inoculated with the enriched soil as soon as the presence of
antagonistic organisms is demonstrated j this results in the development
of the antagonistic organisms and the destruction of the bacteria in sus-
pension. Transfers are then made to fresh suspensions of the bacteria,
resulting in an enrichment of the antagonist, which can finally be iso-
lated in pure culture (427).
Bacterial Agar Plate Method
This method was first used by Gratia and Dath (350) for the isola-
tion of antagonistic agents, actinomycetes having been found readily
by it.
To isolate antagonistic bacteria, agar (1.5 per cent) is washed in dis-
tilled water, then dissolved in water supplemented by i per cent glucose
and 0.2 per cent K^HPO^. Ten-milliliter portions of the sugar-
phosphate agar are placed in glass tubes and sterilized. The sterile agar
is melted, and the tubes are placed in a water bath kept at 42° C. A
washed, centrifuged suspension of living bacteria, grown on solid or in
liquid media, is then added and thoroughly mixed with the agar. This
"bacterial agar" is poured into a series of Petri plates containing one-
milliliter portions of fresh or enriched soil, diluted i : lOO to i : 10,000
times with sterile water. The contents of the plates are thoroughly
mixed in order to distribute the diluted soil suspension in the bacterial
agar. The plates are inverted and incubated at 28° or 37° C.
After I to 10 days' incubation, depending on the nature of the or-
ganism used for the preparation of the plates, the presence of antago-
nists is manifested by the formation of clear zones surrounding their
colonies (Figure 3). The organisms are isolated from these colonies
. V
" L I IS R A R V
lA-S^i-
58 ISOLATION AND CULTIVATION OF ANTAGONISTS
and are retested for antagonistic properties, either by transfer to fresh
bacterial agar plates or by inoculating solidified agar plates and cross-
streaking with test organisms (934, 949).
In the isolation of antagonistic fungi the same method is followed,
except that it is preferable to make the bacterial agar acid by using
KH2PO4 in place of K0HPO4. The resulting acidity (pH 4.5) inhibits
the growth of bacteria and actinomycetes. Since the soil contains fewer
fungi than bacteria, lower dilutions of soil are employed for this pur-
pose (i: loto i: 1,000).
By the use of the soil enrichment and bacterial agar plate methods,
it is possible to demonstrate that ordinary soils contain a large popula-
tion of microorganisms that are antagonistic to bacteria, including both
gram-negative and gram-positive forms. The number of antagonists can
be greatly increased when the soil is enriched with living cells of
bacteria.
Crowded Plate Method
Ordinary field or garden soil is plated out on common nutrient (beef-
peptone) agar, very low dilutions (1:10 to 1:1,000) being used to
enable a large number of bacterial colonies to grow on the plate. The
resultant crowding of these colonies allows the development on the
plate of potential antagonists that are normally present in the soil. The
production of antibacterial substances by these antagonists inhibits the
growth of bacteria in close proximity to them and, in consequence, clear
zones are formed around the colonies (Figure 4). It is possible, by
means of this method, to demonstrate that many strains of spore-form-
ing bacteria possessing antagonistic properties are present in the soil and
can readily be isolated from it (857).
Direct Soil Inoculation Method
Nutrient agar plates are inoculated with the bacteria or fungi for
which antagonists are to be found, and the plates are incubated for 24
to 48 hours at 28° or 37° C. Particles of fresh or enriched soil placed
on the surface of the bacterial or fungus growth on the plate will give
rise to antagonistic organisms. These organisms will bring about the
killing or even the lysis of the original culture. By this method, or-
Figure 3. Development of antagonistic fungi on hacterial-agar plate. From
Waksman and Horning (934).
Figure 4. Bacterial plates made from soil, showing clear zones surround-
ing colonies of antagonistic organisms. From Stokes and Woodward (857).
Antagonistic action of i\ (Uiti-
hioticiis upon S. lutea
Antagonistic action of 5. ayit'i-
hiot'icus upon B. rn^co'idcs
Bacteriostatic action of actin(
m)'cin upon iS\ luttui
I^actcnostatic action of acti
m\'cin upon R. rnxcoidn
Figure 5. Antagonistic effects of living organisms and their products. P'rom
Waksman and Woodruff (945).
METHODS OF ISOLATION 59
ganisms antagonistic to many bacteria and fungi causing plant and ani-
mal diseases have been isolated (644, 646).
For the isolation of bacteria antagonistic to fungi, the latter are
grown on potato agars until they have spread over the plate j particles
of moist soil are then placed on the surface of the mycelium, and the
plates are incubated in a moist chamber. Bacteria lysogenic to the fungi
grow out of the soil and gradually dissolve the mycelium until the en-
tire surface of the plate becomes free of the hyphae of the fungus. By
transferring some of the material from the lysed spots, pure cultures of
bacteria have been obtained that are capable of producing destructive
effects upon the fungi, similar to the action of the particles of soil.
To these four methods may be added the "forced antagonism"
method of Schiller (798), previously referred to, which consists in feed-
ing a culture of an organism with another one, thereby forcing the sec-
ond to develop the capacity of destroying the first.
Isolation of Antagonistic Microorganisms from Soil
By means of the foregoing methods, as well as various modifications
of them, it was possible to demonstrate that soils, composts, and water
basins contain an extensive population of microorganisms possessing
antibacterial and antifungal properties. When E. coli was used as the
test organism, it was found that although this organism was capable not
only of surviving but actually of multiplying in sterile soil, it died off
very rapidly when added to fresh soil. The rate of its destruction was
greatly increased with every subsequent addition of washed bacterial
cells to the soil. This was accompanied by the development of certain
antagonistic microbes capable of destroying E. coli in pure culture.
A large number of fungi, actinomycetes, and bacteria possessing an-
tagonistic properties have thus been isolated. The nature of the test or-
ganism was found to be of great importance in this connection. When
Stafhylococcus aureus y S. lutea, and B. subtilis were used, a large num-
ber of antagonists could readily be isolated. With E. coli, however, a
much smaller number of microbes thus isolated possessed antagonistic
properties. Certain other gram-negative bacteria, like Brucella abortus,
were more sensitive than E. coli, whereas certain gram-positive bac-
60 ISOLATION AND CULTIVATION OF ANTAGONISTS
teria, like B. mycoides and B. cereus, were less sensitive than B. subtilis
(934, 936).
Bacteria destructive to fungi, or possessing fungistatic and fungicidal
properties, have also been isolated from soils as well as from the surface
of plants, such as flax, by transferring small sections of soil or plant
stem to plates of fungi growing on potato agarj transfers made from
the lytic spots yielded antagonistic bacteria (647). By the use of this
method, Chudiakov (130) isolated various bacteria antagonistic to
Fusanum. The antagonists were found abundantly in cultivated soils,
but not in flax-sick soils rich in Fusanum. Bamberg (37) demonstrated,
in the soil, bacteria capable of bringing about in 10 days complete de-
struction of Ustiliago zeae and other fungi. Myxobacterium was also
found (457) capable of bringing about the disintegration of fungus
mycelium. Nonspore-forming bacteria, similar to the cultures of
Chudiakov, were isolated and shown to be able to attack a number of
fungi, including species of Fusanum , Sclerotinia^ Gleos-porium, Acro-
stalagmus, Alternarla^ and Zygorhynchus (695).
METHODS OF TESTING THE ANTAGONISTIC
ACTION OF MICROORGANISMS
Once antagonistic organisms have been isolated, it is essential to es-
tablish their bacteriostatic spectrum — that is, their ability to inhibit the
growth of various specific microorganisms. Usually these antagonists
do not affect alike all bacteria and fungi, some acting primarily against
gram-positive bacteria and against only a few gram-negative forms
(mostly cocci), others acting upon certain bacteria within each of these
two groups.
A considerable number of methods have been developed for meas-
uring these antagonistic effects. They measure the selective nature of
the antagonistic action and they can also give quantitative information
concerning the intensity of this activity. Because of the great differences
in the degree of sensitivity of bacteria to the action of the antagonists,
the proper selection of one or more test organisms is highly essential.
S. aureus has been employed most commonly, different strains of this
organism having been found to vary greatly in their sensitivity even to
METHODS OF TESTING ANTAGONISTIC ACTION 61
the same substance. Streftococcus viridans, B. subtilis, Micrococcus ly-
sodeikticus, S. lutea, E. coU, and E. tyfhosa are other organisms that
are frequently employed for testing the activity of antagonists. Al-
though for purposes of concentration and purification of a known sub-
stance a single test organism is sufficient, it has been found advisable
during the isolation of antagonistic organisms and the study of the na-
ture of the antibiotic substance or substances that they produce to use
more than one test bacterium, including one or more gram-positive and
one or more gram-negative bacteria.
Most of the methods for testing antagonistic action are based upon
the growth of the test organisms in the presence of the living antago-
nists or of the antibiotic substances produced by them in liquid and on
solid nutrient media (302, 627). Only a few of these methods are now
utilized, most of them being chiefly of historical interest.
Liquid Media
Several methods using liquid media have been proposed for testing
the antagonistic activities of microorganisms:
Simultaneous inoculation of the medium with the antagonist and the test
organism.
Inoculation of the medium with the antagonist first, followed after 6 to
48 hours by inoculation with the test organism.
Inoculation of the medium with the test organism first, followed, after a
certain interval, by the antagonist.
Effect of the metabolic products of the antagonist upon various micro-
organisms. In 1888, Freudenreich (299) first filtered the culture
through a Chamberland candle and inoculated the filtrate with the
test organisms. The culture filtrate is usually added to the fresh me-
dium, either previously inoculated with the test organism for the
purpose of establishing the lytic effect of the filtrate, or followed by
the test organism, whereby the bacteriostatic action is measured.
Placing a porcelain filter or cellophane membrane between the cultures
of the antagonist and of the test organism. Frankland and Ward
(297) used a filter of the Pasteur-Chamberland type partly filled
with broth and placed in a beaker containing the same kind of broth ;
the antagonist and test organism were inoculated into the two lots of
broth, and the effect of each upon the growth of the other was de-
62 ISOLATION AND CULTIVATION OF ANTAGONISTS
termined. Frost (302) emphasized, however, that, although theo-
retically this is an ideal method, it is open to criticism since motile
bacteria are usually able to grow through the filter after a certain
lapse of time.
Collodion sac method. Collodion sacs, prepared by means of test tubes
from which the bottoms have been cut out, are partly filled with
broth and placed in a flask containing the same kind of broth. The
test organism is inoculated into the medium inside the sac, and the
antagonist into the flask (302).
Solid Media
Solid media have also been used extensively for testing the action of
antagonists. These media offer certain advantages over liquid media.
The following methods are most commonly used :
Simultaneous inoculation of antagonist and test organism. This method,
introduced by Garre (311) in 1887, consists in streaking the an-
tagonist and the test organism on the surface of a solidified agar or
gelatin medium. The streaks are alternate and may be parallel, radi-
ating from a common center, or intersecting at right angles (Fig-
ure 5). If the active substance produced by the antagonist does not
diffuse for any considerable distance into the medium, the method is
not satisfactory. Frost (302) modified this method by inoculating
the whole medium with the test organism and, when the medium
had hardened, streaking the antagonist across the surface. The first
of these came to be known as the anaxogramic method; the second
is often spoken of as the implantation method (705). The spotting
of the two organisms on the plate is illustrated in Figure 6.
Successive inoculation of the test organism, after the antagonist has al-
ready made some growth, so as to enable the active substance to dif-
fuse. This method, suggested by Garre (311), consists in allowing
the antagonist to produce a good growth on the surface of the me-
dium; the mass of growth is then removed, and the test organism
inoculated into the same medium.
Double plate methods (302). A Petri dish is divided into two parts by
means of a small glass tube or rod. After sterilization, one tube of
molten agar is heavily inoculated with the antagonist and poured
into one half of the plate. When the agar has hardened, another tube
of sterile agar is poured into the other half of the plate. Both sides are
METHODS OF TESTING ANTAGONISTIC ACTION
63
Hel7ninthosforln7n (A and B) in- Pestaloz-zm (A) inhibited by one
hibited by Fusarium (C) species of Pcnicillium (C) but not
by another (B)
Helminthosforiuni (A) inhibited Helminthosforium (A and B) in-
by a bacterium (C) hibited by a white yeast (C)
Figure 6. Inhibition of fungus development by antagonists. From Porter
(695).
64 ISOLATION AND CULTIVATION OF ANTAGONISTS
then streaked with the test organism, each side being equally inocu-
lated by separate streaking. This can be done by using a loop bent at
nearly right angles; the charged loop is moved from the circumfer-
ence toward the glass rod. The loop is then sterilized, recharged with
the test culture, and the streak continued on the other side of the
plate. The inoculation with the test organism may be made soon
after the plate is poured, or the antagonist may be given an opportu-
nity to develop before the test organism is streaked thus making the
antagonistic effect more striking. This method has also been used
(261, 267, 270) for testing the antibiotic properties of fungus
cultures.
Mixed culture inoculation. The cultures of the antagonist and the test or-
ganism are mixed and inoculated upon the surface of the solidified
agar or before the molten agar has been added to the plate. The colo-
nies of the antagonist will be surrounded by clear sterile zones, free
from any growth of the test organism.
Spot inoculation of the antagonist upon an actively growing culture of a
bacterium or fungus on an agar plate (844). This method is particu-
larly convenient for detecting antagonists that possess lytic prop-
erties.
A layer of molten sterile agar is used to cover the surface of an antagonist
that has made some growth in a plate, and the surface of the agar
layer is then inoculated with the test organism. The active substance
produced by the antagonist will difiFuse through the agar and reduce
the growth of the test bacterium (571).
Semisolid media are used for testing the action of antagonists against
motility of bacteria (173).
METHODS OF GROWING ANTAGONISTIC
ORGANISMS FOR THE PRODUCTION
OF ANTIBIOTIC SUBSTANCES
Once the antagonistic action of any organism has been established,
the next step is to determine the nature of the antibiotic substance pro-
duced by the antagonist and to measure quantitatively this antibiotic
action. Before this can be done, however, the organism must be grown
upon suitable media and suitable conditions must be established for the
favorable production of the antibiotic substance.
METHODS OF GROWING ANTAGONISTIC ORGANISMS 65
The media used for the production of antibiotic substances can be
classified into two groups: synthetic media and complex organic media.
The first contain a source of carbon, usually glucose (2 to 6 per cent) j
a source of nitrogen, usually nitrate (0.2 to 0.6 per cent), as well as sev-
eral salts, namely, K0HPO4 or KH2PO4 (o.i to 0.2 per cent),
MgSO^.yH.O (0.05 per cent), KCl (0.05 per cent), and FeS04.7H.O
(0.00 1 per cent) 5 certain supplementary materials, such as yeast ex-
tract, meat extract, or corn steep, and other salts, such as NaCl (0.05
to 0.5 per cent), ZnS04, MnS04, or CUSO4 ( i to 2 ppm.) may also be
added. The organic media contain a complex form of nitrogen, such as
tryptone, peptone, casein digest j either no other source of carbon is used
or a carbohydrate is added in the form of glucose, starch, brown sugar,
molasses, or similar products as well as several salts similar to those
listed above. Some media are supplemented with CaCO.j, and others
are not, depending upon the extent of acidity produced by the organism.
The medium may be solid or liquid, but the latter type is more com-
mon. Agar and bran are used as solid media. Several types of culture
vessels are used, depending on the condition of aeration. Since so far as
is known all the microorganisms capable of producing antibiotic sub-
stances are aerobic, either shallow layers of medium (1.5 to 2 cm. in
depth) are placed in stationary vessels (flasks or trays), or deep vessels
(tanks) are filled with the medium and properly aerated by forced draft
with sterilized and filtered air.
For the production of penicillin, a constant-flow apparatus similar to
the quick-vinegar process has been suggested (134), the medium trick-
ling over a column of wood shavings. The establishment and operation
of large-scale production of penicillin under submerged conditions have
been described in detail by Callaham (103).
The optimum temperature required for the growth of the antagonis-
tic organisms and production of the antibiotic substances ranges be-
tween 20° and 30° C. The length of incubation varies from 2 to 6 days
for submerged cultures and from 3 to 20 days for stationary cultures.
A knowledge of the preliminary treatment of the inoculum or spore
material is essential. For the growth of spore-forming bacteria, the use
of a pasteurized spore suspension is advisable in order to avoid the vari-
able factor due to vegetative cells. Actinomycetes and fungi are grown
66 ANTIBIOTIC ACTION OF ANTAGONISTS
on agar slants in order to obtain abundant spore material for the inocu-
lation of stationary cultures. For submerged cultures, special spore sus-
pensions are produced by growing the organisms in shaken cultures.
The cultures must be tested carefully in order to establish the opti-
mum activity when the culture filtrate is cooled and extraction of active
substance is started.
METHODS OF MEASURING THE ACTIVITY OF
ANTIBIOTIC SUBSTANCES
It has long been recognized that the evaluation of bacteriostatic and
bactericidal substances is controlled to a considerable extent by the
methods employed. These methods are based upon the following fac-
tors: (a) proper selection of the test organism, (b) composition of the
medium used for testing activity, (c) time of action, (d) conditions of
carrying out the test, and (e) nature of the active substance. The results
obtained in a comparison of substances containing the same active prin-
ciple may not be very reliable when different agents are compared, since
these vary greatly in their specific action upon different bacteria. This is
especially true of antibiotic agents.
In most of the work on chemical disinfectants, which are primarily
bactericidal agents, the death rate of the viable cells has been used as a
basis for evaluation. Different substances have been compared with a
standard, ordinarily phenol. Since antibiotic and chemotherapeutic
substances are primarily bacteriostatic in action, the inhibition of the
growth and multiplication of the test organism is commonly used as a
basis for their evaluation.
In any attempt to select a single standard method for measuring
quantitatively the activity or potency of an antibiotic substance, it is es-
sential to recognize several pertinent facts, which may be briefly sum-
marized as follows :
Antibiotic (antibacterial, antimicrobial) substances are primarily bac-
teriostatic (or fungistatic) in their action; they are bactericidal (or
fungicidal) only to a limited extent, although some substances may
possess marked bactericidal properties.
Antibiotic substances are selective in their action; they are able to inhibit
METHODS OF MEASURING ANTIBIOTIC ACTIVITY 67
the growth of some bacteria in very low concentrations, whereas
much larger amounts are required to affect other bacteria and some
organisms may not be inhibited at all by the particular substance even
in very high concentrations.
Conditions for the bacteriostatic activity of different antibiotic substances
vary greatly. Some substances are not active at all, or their activity
is greatly reduced in some media because of the neutralizing effect of
certain constituents of the media, such as peptone or /"-amino-benzoic
acid. Other agents require the presence in the medium of specific
constituents for their activity to become effective. The activity of
some is reduced at an acid reaction (287, 1002), whereas that of
others is not affected.
The mechanism of the action of different antibiotic agents is different.
Some agents interfere with bacterial cell division, others with bac-
terial respiration, and still others with utilization by the bacteria of
essential metabolites.
Many antagonistic organisms produce more than one antibiotic substance.
Ps. aeruginosa produces pyocyanase and pyocyanin ; B. brevis, grami-
cidin and tyrocidine; Streftomyces antibioticus, actinomycin A and
B ; P. notatuniy penicillin and notatin ; Asfergillus fumigatus, spinu-
losin, fumigatin, fumigacin, and gliotoxin; Asfergillus flavus, asper-
gillic acid and flavicin. The culture filtrate of an antagonistic or-
ganism often differs, therefore, in its activity from that of the
isolated active substance.
The course of production of antibiotic substances by two typical antago-
nistic organisms is illustrated in Figures 7 and 8.
In view of the bacteriostatic nature of antibiotic substances, few of
the methods commonly used for testing the efficiency of antiseptics and
germicides can be employed. This is particularly true of the "phenol
coefficient test," which measures the germicidal action of phenol upon
E. tyfhosa. The limitations of this method, based on the bactericidal ac-
tion of a single substance on a single organism, even as applied to chemi-
cal antiseptics have long been recognized (735, 773).
A number of methods have been developed for determining the ac-
tivity of antibiotic substances. They vary greatly, each having its limita-
tions and advantages. Because of lack of uniformity in the methods, the
results obtained by one are not always comparable with those obtained
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METHODS OF MEASURING ANTIBIOTIC ACTIVITY
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by another. The most important methods at present in use are briefly
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The Agar Plate-Dilution Method
If an unknown antibiotic substance is tested, it is essential to employ
several test organisms in order to throw light upon the selective activity
of the substance on different bacteria. Nutrient agar media have usually
been employed. Sterility is not absolutely essential for this method, al-
though it is desirable. The unknown substance is diluted to various con-
centrations (i, 0.3, 0.1, etc.j or I, 0.5, 0.25, etc.); these dilutions are
added and thoroughly mixed with definite volumes (lo ml.) of sterile
agar medium, melted and cooled to 42° to 45° C. The agar is allowed
to solidify, and is streaked with three or four test bacteria, among the
most common of which are E. coli, E. ty-phosa, Br. abortus, B. subtilis,
S. aureus, M. lysodeikticus, and S. lutea. The age of the cultures ( 1 6 to
24 hours) is important. The plates are incubated at 28° or 37° C. for
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METHODS OF MEASURING ANTIBIOTIC ACTIVITY 71
1 6 to 24 hours, and readings are made. The highest dilution at which
the test organism fails to grow is taken as the end point. Activity is ex-
pressed in units, using the ratio between the volume of the medium and
the end point of growth or the dilution at which growth is inhibited
(948).
The bacteriostatic spectra of a group of antibiotic substances com-
pared with certain chemical agents are shown in Table 8. Different bac-
teria show different degrees of sensitivity to the different substances. A
comparison with the action of phenol can result in what may become
known as the "bacteriostatic phenol coefficient" for each active sub-
stance.
Serial Dilution Method
Once a substance is characterized as regards its selective action upon
specific bacteria, its activity or concentration can be measured more ac-
curately by the liquid dilution or titration method. One test organism is
selected, usually a strain of S. aureus. Different strains may vary in their
action. In some cases, Streftococcus hemolyticusy B. subtilis, and others
have been used for measuring the activity of a substance against gram-
positive bacteria, and E. coli for gram-negative bacteria. Definite vol-
umes of the test medium are placed in test tubes and sterilized (sterility
is essential in this method), and various dilutions of the active sub-
stance are added. The dilutions can range in order of 3 : i , 2 : i, or even
narrower, namely in series of i .2 : i , i .5 : i , etc. The tubes are inoculated
with the test organism and incubated for 16 to 24 hours. In some cases
the medium is inoculated before it is distributed into the tubes. The
highest dilution of the antibiotic substance giving complete inhibition of
growth, as expressed by a lack of turbidity of medium, is taken as the
end point. Activity is expressed in units as above (804).
The dilution method has two disadvantages (276) : first, every assay
takes much time j second, during chemical fractionation, the substance
may become contaminated with bacteria not sensitive to the active sub-
stances.
One modification of the method has been adapted for measuring the
actjivity of penicillin. Several dilutions of the active agent are prepared
and 0.5 ml. portions added to 4.5 cm. quantities of liquid medium in
72 ANTIBIOTIC ACTION OF ANTAGONISTS
TABLE 9. BACTERIOSTATIC SPECTRUM OF PENICILLIN
DILUTIONS AT WHICH INHIBITORY
ORGANISM AFFECTED EFFECTS WERE OBSERVED
Complete Partial None
l^l . gonorrhoeae* 2,000,000 >2,000,000 )>2,ooo,ooo
N . meningitidis 1, 000,000 2,000,000 4,000,000
S. aureus 1,000,000 2,000,000 4,000,000
S. -pyogenes 1,000,000 2,000,000 4,000,000
B.anthracis 1,000,000 2,000,000 4,000,000
A.bovis 1,000,000 2,000,000 4,000,000
CI. tetaniif i ,000,000
CI. zoelchii 1,500,000
CL sefticum 300,000 1,500,000 7,500,000
Cl.oedematiens 300,000 1,500,000
5. viridans% 625,000 3,125,000
Pneumococcus\ 250,000 500,000 1,000,000
C. difhtheriae {mitis) 125,000 625,000
C. difhtheriae {graz'is) 32,000 64,000 128,000
5". gartneri 20,000 40,000 8o,000
S.tyfhi 10,000 30,000 90,000
PneumococcusX 9,000 27,000
Anaerobic streptococcuslj! 4,000 8,000 16,000
P. vulgaris 4,000 32,000 60,000
S.viridansX 4,000 8,000 16,000
P.festis 1,000 100,000 500,000
S. tyfhimurium < 1,000 8,000 16,000
5. faratyfhi B < i ,000 5 ,000 I o,000
5^. dysenteriae 2,000 4,000 8,000
Br. abortus 2,000 4,000 8,000
Br. melitensis <i,000 2,500 I0,000
Anaerobic streptococcus <^4,ooo <(4,ooo 4,000
V. comma < 1,000 1,000 2,000
E.coli <i,ooo <i,ooo 1,000
K. fneuTnoniae <( i ,000 <^ i ,000 i ,000
Ps. aeruginosa <^ i ,000 < i ,000 i ,000
M. tuberculosis ^ i ,000 < i ,000 1 ,000
L. icterohoemorrhagioe < 3,600 <( 3,600 3j6oo
From Abraham et al. (7).
* Another strain was inhibited only up to 32,000.
t Grown in Lemco broth; in beef broth complete inhibition reached only 100,000.
X In Pneumococcus, S. viridans, and anaerobic streptococci, different strains appear at different
levels in the table.
METHODS OF MEASURING ANTIBIOTIC ACTIVITY 73
test tubes. These are inoculated with a standard drop (0.04 ml.) of a
24-hour culture of the test organisms. Complete or partial inhibition is
shown by the absence of turbidity after 24 hours of incubation at 37° C.
Dilutions higher than those required for complete or partial inhibition
gave, after 24 hours of incubation, only a retarding effect (2, 7) j a nii-
croscopic examination (308) indicated defective fission of the bacteria,
even though the macroscopic appearance of the culture did not show any
inhibition. Pneumococci and S. viridans show marked strain differences
by this method. In one experiment with Salmonella tyfhi, partial in-
hibition was obtained in a dilution of i : 10,000 j however, elongation
of the cells was detected in a dilution of i : 6o,000, a concentration
which was considered as a therapeutic possibility (Table 9).
The Agar Diffusion or "Agar Cuf" Method (7, 284, 285, 385)
This method, first employed by Reddish (735) and by Ruehle (773)
largely for qualitative purposes, was later developed (7, 385) for
quantitative use. A suitable agar medium is inoculated with the test or-
ganisms {S. aureus or B. subtilis), the active agent being placed upon
the agar, within a groove or in a special small glass cup with an open
bottom from which the substance diffuses into the medium. The rate of
diffusion of the active substance is parallel to its concentration. By meas-
uring the zone of inhibition and comparing it with that of a known
standard preparation, the potency of the active substances can be calcu-
lated. This method has the advantage of simplicity and convenience,
since it does not require sterile material and several preparations or
duplicates can be tested on the same plate. The method also possesses
certain disadvantages, however, since it cannot be used for comparing
different substances but is limited to the measurement of activity of only
one type of substance ; it cannot be used for the study of unknowns until
a standard has been established for each unknown.
Nutrient agar containing 5 gm. NaCl, 3 gm. meat extract, 5 gm.
peptone, 15 gm. agar, 1,000 ml. tap water, and adjusted to ^H 6.8, is
poured into plates to a depth of 3 to 5 mm. The plates are seeded thor-
oughly with the test organism (S. aureus) by flooding with i: 10 or
I : so dilution of i6-to-24-hour-old broth culture in sterile water. The
excess fluid may be removed with a pipette. The surface of the agar is
74 ANTIBIOTIC ACTION OF ANTAGONISTS
allowed to dry somewhat in the 37° C. incubator for i to 2 hours, the
lids of the plates being raised about i cm. above the bottoms of the
dishes. Sterile short glass cylinders (5 mm. inside diameter) are placed
on the agar, the lower edge of the cylinder sinking into the agar, and are
filled with the test solution. Several cylinders may be placed in one dish.
For measuring the activity of penicillin, the plates are incubated for
12 to 16 hours at 37° C. The diameter of the zone around the cylinder
is measured to the nearest 0.5 mm. by means of pointed dividers. The
relation of concentration of penicillin in the solution to the zone of in-
hibition, or the "assay value," is expressed by a curve which is obtained
with standard solutions. This curve tends to flatten out above 2 units of
penicillin per milliliter. The assay value is not influenced by the fH of
the test material, the thickness of the agar, or the sterility of the ma-
terial.
The "Oxford unit," as determined by this method, is the amount of
penicillin that will just inhibit completely the growth of the test strain
of 5. aureus in 50 ml. of medium. Thus, a preparation containing one
unit of penicillin per milligram of material just inhibits the growth of
the test organism in a dilution of 1:50,000 (7, 273, 385).
One of the modifications of this method (285) consists in using a
spore suspension of B. subtilis as the test organism. It is grown for sev-
eral days under forced aeration, and the cultures are pasteurized in or-
der to destroy the vegetative cells. The spore suspension is stored in the
cold and used as the stock inoculum j it is titrated in order to determine
the optimum amount for seeding purposes. The lowest level (usually
0.1 to 0.2 ml. per 100 milliliters of agar) that gives a dense, continuous
growth of the organism under the assay conditions is selected as the
optimum.
This method is also very convenient for measuring the activity of
streptothricin. A standard curve is obtained by filling the cups in quad-
ruplicate with dilutions of the standard containing 10, 20, 40, 60, 80,
and 100 streptothricin units per milliliter. The dilution of the unknown
contains about 50 units per milliliter. After overnight incubation at
30° C, the inhibition zones around the cups are measured and plotted
to give a standard curve. The units of the unknowns are read off this
curve by projecting the value of the inhibition zones.
METHODS OF MEASURING ANTIBIOTIC ACTIVITY
75
The agar cup method has also been utilized (869) for comparing
the disinfectant action of chemical antiseptics. S. aureus and B. suhtilu
were found to be most sensitive to the action of aliphatic alcohols. Vari-
ous modifications of this method have been introduced, including the
use of paper discs treated with known dilutions of the active preparation
(818,917,985).
Turbidimetric Method
End-point methods have long been recognized as having many limi-
tations. Since it is difficult to determine accurately the end point and
since it takes a relatively much larger amount of an antibiotic substance
to inhibit completely the growth of the test organism as compared with
only 50 or 99 per cent inhibition, the suggestion has been made that
partial inhibition of growth be measured and, from this, the concentra-
tion of the active substance be calculated in a manner similar to the
24
2.8
^^
»
0
• \
"ao
- V,
_
2.4^
<0
*
0
lU
V
u
•,
0
5i6
-
\
^^
( -
2.0 3
a
\*
UJ
Oi
UJ
1-
w 8
-
»^
-
1
cj
1.6 ^
>-
1-
1.25
2
0
0
2
-
^^
-
0.8
<
0
"■"-l-..^
Q
1
1 1 1 t 1 "'^
n /I
0
.05
.10 .15 .20 .25 .30 .35 .40" 1
PENICILLIN IN 5 PER. MILLILITER
Figure 9. Relation between penicillin concentration and inhibition of
Staphylococcus aureus. The penicillin preparation contained 42 Oxford units
per milligram, and the incubation period was sixteen hours at 37° C. From
Foster (281).
76 ANTIBIOTIC ACTION OF ANTAGONISTS
measurement of the potency of bactericidal agents. Partial inhibition
can be determined by plating for the number of viable bacteria, as com-
pared with the control, or it can be measured by a convenient turbi-
dimeter. The results obtained by this method are more nearly quantita-
tive in nature than those obtained by other methods (281, 520), as
shown in Figure 9. By proper modifications, the length of time re-
quired to obtain a satisfactory reading can be reduced to four hours
(4^5) 572)5 or even to 90 minutes (281, 520).
S fecial Methods
Certain methods were found to be specific for measuring the action of
certain substances. The ability of tyrothricin to hemolyze red blood
cells served as the basis for measuring the potency of this substance
( 1 72) : the tyrothricin content is calculated from the amount of hemoly-
sis by the unknown and is read from a standard curve. The inhibition
of growth of a |3-hemolytic streptococcus, group A, as measured by
hemolysin production has been used for assaying the potency of peni-
cillin (715, 989). Penicillin can also be estimated by its inhibition of
nitrite production by S. aureus cultures (343). The antiluminescent
test has been utilized (716, 717) not only for measuring the activity of
certain substances but also for determining their possible usefulness.
The results of a comparative study of a number of antibiotic substances
by this and the dilution methods are brought out in Table 10.
Various other methods have been suggested for measuring the activ-
ity of antibiotic substances. Some are based upon interference with a
given physiological function of the test organism such as dehydrogenase
activity and respiration, others upon the prevention of growth of the
test organism (pneumococcus) in semi-solid tissue culture medium
(387). Although only a single method is usually employed in the con-
centration and standardization of a given antibiotic substance such as
penicillin, it is often advisable to check the results by another method,
especially where several test organisms are used, in order to ascertain
that one is still dealing with the same type of chemical compound.
Some of the above methods can also be adapted to the determination
of the concentration of antibiotic agents in the body fluids and exudates
(719).
METHODS OF MEASURING BACTERICIDAL ACTIVITY 77
TABLE 10. ANTILUMINESCENT AND ANTIBACTERIAL ACTIVITIES
OF VARIOUS ANTIBIOTIC SUBSTANCES
SMALLEST AMOUNT SHOWING ACTIVITY, IN MICROGRAMS
al/ab ratio
Antiluminescent test
Antibacterial test*
Tolu-p-quinone
O.II
Gramicidin
.002
Tolu-p-quinone
. .002
Pyocyanase
3
Tyrothricin
.008
Pyocyanase
.07
Clavacin I
"
Penicillin II
Penicillin I
Flavatin
.0156
.06
.256
Clavacin I
.18
Aspergillic acid
15
Gramidinic acid
.23
Sodium clavacinate .18
Gliotoxin
17
AP2lt
•31
Clavacin II
.19
Clavacin II
22
Actinomycin A
•54
Sulfanilamide
Phenol
<-56
•5
Pyocyanin
47
Aspergillic acid
2.0
Pyocyanin
1-7
Actinomycin A
54
Gliotoxin
2.1
Lauryl sulfate
4.6
Streptothricin
56
Streptothricin
2.8
Aspergillic acid
7-5
Sodium clavacinate
94
Fumigacin
13.0
Gliotoxin
8.0
Flavatin
256
Fumigacin
273
Pyocyanin
27.0
Streptothricin
20.0
Lauryl sulfate
273
Pyocyanase
42.0
Fumigacin
21.0
Phenol
II 70
Tolu-p-quinone
55.0
Actinomycin A
Flavatin
1 00.0
1 000.0
Penicillin I
1650
Lauryl sulfate
59.0
AP2lt
>i630
Sulfanilamide
3940
Clavacin I
63.0
Gramidinic acid
>2i75
Gramicidin
>5oo
Clavacin II
II 3.0
Penicillin I
27,500
Gramidinic acid
>500
Sodium
clavacinate
500.0
Tyrothricin
>62,5oo
Tyrothricin
>500
Phenol
2300.0
Gramicidin
>250,000
AP2it
>500
Sulfanilamide >7000.0
Penicillin II
>325,ooo
Penicillin II
>50oo
From Rake, Jones, and McKee (716).
* Streptococcus pyogenes used as test organism.
t A tyrothricin-Iike preparation.
METHODS OF MEASURING BACTERICIDAL
ACTION OF ANTIBIOTIC SUBSTANCES
Several methods are commonly employed for measuring bactericidal
action of antibiotic substances. A suspension of washed bacterial cells in
saline or other suitable solution, or a 5-to-i2-hour-old broth culture of
the test organism, is treated with various dilutions or concentrations of
78 ANTIBIOTIC ACTION OF ANTAGONISTS
the active substance. After incubation at 37° C. for i to 24 hours, the
number of living cells is determined. If the active substance has lytic
properties or if the test organism undergoes lysis readily, the readings
are simplified. If no lysis occurs, the treated bacterial suspension or cul-
ture is streaked or plated out. The streaking procedure gives only a
relative idea of the extent of bactericidal action. If 50 to 90 per cent
killing of cells is to be taken as a unit of measurement, the culture is
plated on a suitable medium and the actual number of surviving cells
determined.
Various modifications of this method have been developed. In one
such modification (607), the bacterial cells are suspended for 15 to 30
minutes in various dilutions of the active substance j the cells are then
centrifuged oflF, washed, and cultured. This procedure can be utilized
for substances that have a rapid bactericidal action. Its value is limited,
however, by the fact that most antibiotic substances do not kill bacteria
so rapidly as do chemical antiseptics (p. 189).
Some of the foregoing methods can also be utilized for measuring
the fungistatic and fungicidal properties of antibiotic substances. Pro-
tective fungicides may first function as fungistatic agents, others func-
tion better either as fungicidal or as fungistatic agents, and still others
show either a high or a low for both (550). The growth of Ceratosto-
mella ulmi was inhibited by several substances, comprising actinomycin,
clavacin, and hemipyocyanin in concentration of i: 100,000 (803).
METHODS OF TESTING THE IN VIVO ACTIVITIES
OF ANTIBIOTIC SUBSTANCES
Ordinary pharmacological, bacteriological, and pathological proce-
dures are used for testing the toxicity and activity of antibiotic sub-
stances in the animal body. Some of the results obtained are presented
in subsequent chapters.
ISOLATION AND UTILIZATION OF
ANTIBIOTIC SUBSTANCES
The isolation of antagonistic microorganisms from natural sub-
strates, the determination of the nature of the antibiotic substances pro-
UTILIZATION OF ANTIBIOTIC SUBSTANCES 79
duced by them, and the utilization of such substances for chemothcra-
peutic purposes involve ten distinct steps which may be briefly sum-
marized as follows:
1. Enrichment of soil or water with specific organisms against which an-
tagonists are to be obtained.
2. Plating of the enriched or unenriched soil or water upon special bac-
terial agar.
3. Isolation of the antagonistic organisms from the plates or other culture
media.
4. Testing of the isolated culture for bacteriostatic and fungistatic proper-
ties against a variety of bacteria or fungi.
5. Growing the antagonist in suitable media and testing the filtrate or
extract of the culture for the presence of the antibiotic substance.
6. Separation and concentration of the antibiotic substance from the cul-
ture medium, and determinations of its bacteriostatic spectrum.
7. Chemical isolation of the antibiotic substance.
8. Determination of the chemical nature of the active substance.
9. Testing of the bacteriostatic and bactericidal properties of the sub-
stance in intra.
10. Animal experimentation and practical application; study of toxicity, in
vivo activity, and therapeutic action.
CHAPTER 5
BACTERIA AS ANTAGONISTS
Following the early work of Pasteur (674) on the antagonistic ef-
fects of bacteria against the anthrax organism, considerable attention
has been centered upon bacteria as agents possessing antibacterial prop-
erties. A systematic study of this phenomenon was first made by Babes
in 1885 (150), followed by Garre in 1887 and Freudenreich in 1888.
Freudenreich (299) demonstrated that when certain bacteria were
grown in a liquid medium, the filtrate obtained by passing the culture
through a porcelain candle supported the growth of the typhoid or-
ganism not at all or only very feebly. Garre (311) observed that Ps.
-putida inhibited the growth of S. aureus, E. tyfhosa, and Bacillus muco-
sus-cafsulatus but not of B. anthrac'ts and other bacteria. It was soon
reported (524), however, that B. anthracis was also killed by the
Pseudomonas antagonist, whereas the growth of 5. aureus and V .
comma was only retarded j no effect at all was exerted upon E. tyfhosa
and E. coli. In consequence, the antagonist was claimed to be active
against B. anthracis but not against other bacteria. Olitsky {6ss) con-
cluded that Ps. fuorescens inhibited the growth not only of E. tyfhosa
but also of B. anthracis, V . comma, S. marcescens, and S. aureus. These
and other apparently contradictory results were undoubtedly due to
diflFerences in the specific nature of the strains of the organisms used by
the various investigators and to different methods of cultivation.
The presence of Ps. fiuorescens in sewage was found (517) to reduce
greatly the period of survival of the typhoid organism. The latter did
not develop even in gelatin upon which Ps. fluorescens had previously
grown (326, 430, 431, 432), and it could not be detected in sterile sew-
age in which the antagonist was present for seven days. According to
Frost (302), E. tyfhosa can be antagonized by a number of different
soil bacteria, of which Ps. fluorescens exhibits the strongest effect. He
observed that although P. vulgaris acted more rapidly, the active sub-
stance did not diffuse to so great a distance in the medium, thus point-
BACTERIA AS ANTAGONISTS 81
ing to a different inhibition mechanism. Mixed cultures showed greater
activity than pure cultures, either because the latter lose their antibiotic
property when grown for a long time on artificial media or because
mixed cultures comprise two or more species with a greater combined
action. The antagonistic substances produced by these bacteria were ac-
tive at 37° C, whereas at ice-chest temperature the action was delayed
so that the pathogen had an opportunity to develop. This was believed
to offer a possible explanation for the fact that when water supplies
become contaminated in cold weather, their power of producing infec-
tion is retained for a longer time than when the contamination takes
place in warm weather.
Frost concluded that the phenomenon of antagonism results in
checking the growth of E. tyfhosa as well as in killing the pathogen.
Evidence that antagonistic substances exist in an active state in the soil
or in water appeared to be lacking j rather, the results suggested that
formation of such substances depends on the actual development of
specific antagonistic organisms. Changes in environment, such as tem-
perature, oxygen supply and reaction of the medium, and nature and
concentration of nutrients, were believed to have little or no influence
on the production of the antibiotic substances j these were produced
under conditions favoring growth of the antagonists.
The activity of the influenza organism was found (993) to be largely
dependent on the presence of accompanying bacteria. Some of these,
especially micrococci, are favorable to the growth of this organism
whereas others, such as Ps. aeruginosa and B. subtilis, are injurious.
According to Lewis (525), luxuriant growth of Ps. fluorescens in
manured soil and in protein solution containing B. cereus is due to an-
tagonistic action of the former organism against the latter. The former
also inhibits the growth of B. anthracis, Bacillus megatherium^ V .
commay Chromobacterium violaceum, and Rhodococcus. Other species
of the genera Bacillus, Eberthella, Sarcina, Neisseria, and Phytomonas
are somewhat more resistant to the action of Ps. -fluorescens. Salmonella
species are less sensitive, whereas E. coli, A. aerogenes, and S. marces-
cens are highly resistant. Ps. fuorescens produces a thermostable sub-
stance which is toxic to all bacteria except the green fluorescent forms
82 BACTERIA AS ANTAGONISTS
and which is active against actinomycetes but not against fungi. This
substance is water-soluble and dialyzable through collodion and other
membranes.
In addition to the aforementioned bacteria, numerous other groups
were found to contain strains which had strong antagonistic properties
toward bacteria as well as fungi. Some of the antagonists were highly
specific, as in the case of those acting upon the various types of pneumo-
cocci (819, 821, 822) J others were less selective, as in the case of certain
soil bacteria that can bring about the lysis of living staphylococci and
inhibit the growth of various gram-positive and gram-negative bacteria
(820). S. marcescens was shown (61) to be antagonistic to B. subtilis,
B. mycoidesy and B. megatherium. These spore-formers, in turn, were
antagonistic to sarcinae, bringing about their lysis, to V. comma, and to
various other bacteria. It was further found that the antagonists modi-
fied the physiology of the antagonized organism. When two bacteria
were planted, for example, in the same medium, metabolic products
were formed that were not produced in the culture of either organism
alone, whereas certain decomposition processes were either hastened or
retarded (632).
The various antagonistic bacteria can be divided into several groups,
on the basis of their morphological properties.
SPORE-FORMING BACTERIA
Many strains of aerobic spore-forming bacteria possessing antagonis-
tic properties and differing in morphological, cultural, and physiologi-
cal characteristics have been isolated from a great variety of sources,
such as soil, sewage, manure, and cheese. Among these bacteria, B. sub-
tilisy B. mycoides, B. mesentericus, and B. brevis occupy a prominent
place, as shown in Table 11. It was established (205) that some of
these bacteria produce in peptone media an alcohol-soluble, water-
insoluble substance endowed with bactericidal properties.
Duclaux (208) was among the first to isolate and describe antagonis-
tic spore-forming bacteria. Cantal cheese was their origin, and the or-
ganisms were designated as Tyrothrix. NicoUe (640) isolated from the
dust in Constantinople a strain of B. subtilis that had decided bacterio-
SPORE-FORMING BACTERIA
83
lytic properties, especially against members of the pneumococcus group
as well as against various other bacteria such as the typhoid, anthrax,
and Shiga organisms. E. coli, V. comma, and staphylococci were less
affected, and Bacillus suifestifer was least acted upon. The filtrate of
the organism grown in peptone broth had strong antibiotic properties}
TABLE II. SPORE-FORMING BACTERIA ANTAGONISTIC TO OTHER BACTERIA
ANTAGONIST
B. ant hr acts
B. brevis
B. mesentericus
B. mesentericus
B. mesentericus
vulgatus
B. mycoides
B. mycoides
B. mycoides,
var. cytolyticus
B. subtilis
B. subtilis
B. subtilis
B. subtilis-
mesentericus
B. therm-ofhilus
ORGANISM AFFECTED
Anthrax, typhoid, and
lactic acid bacteria
Gram-positive bacteria
Many bacteria
Diphtheria bacteria
C difhtheriae
7 to 20 species of
bacteria
M. tuberculosis
Most pathogens and
many nonpathogens
Various bacteria
Various bacteria, espe-
cially certain plant
pathogens
M. tuberculosis, E.
tyfhosa, etc.
Mostly living gram-
positive bacteria and
dead gram-negative
bacteria
5". lutea
KNOWN PROPERTY REFERENCES
299, 781, 827
Produces tyrothricin 190,191,203
Bacteriolytic 408
Bactericidal 28, 956
Substance thermola- 705
bile, nonfilterable
Lytic 61,620,621
Thermostable sub- 482, 483
stance produced,
precipitated by
tungstic acid
294
Bacteriolytic 61,640
442
903
Lytic 768, 770
Suppression of 835
growth
84 BACTERIA AS ANTAGONISTS
it liquefied gelatin and hemolyzed red blood corpuscles. When various
bacteria cultivated on a solid medium were suspended in physiological
salt solution and seeded with the antagonist, the latter developed abun-
dantly and the bacterial suspensions became clarified. The lysed solu-
tions of pneumococcus prepared by the use of the filtrate of B. subtilis
could be used for purposes of vaccination. In this connection, Nicolle
spoke of the work of Metchnikoff who had proved, in 1897, that or-
ganisms belonging to the B. subtilis group are capable of destroying
various bacterial toxins. Humfeld and Feustel (442) recently demon-
strated that an acid extract of B. subtilis cultures has a very high activity
against certain plant pathogens. This substance was designated as sub-
tilin.
Rosenthal (768) isolated, from soil and from fecal matter, facultative
thermophilic antagonistic bacteria belonging to the B. mesentericus
group capable of dissolving both living and dead bacteria. The simul-
taneous growth of the antagonist with V . comma and other bacteria
brought about the clarification of the culture of the latter in about 5 or
6 days. These bacteriolytic organisms were designated as "lysobacteria."
It was recognized that the action of antagonists is different from that of
phage in several respects: (a) the filtrate of the antagonist is active
against other bacteria 5 (b) both living and dead cultures of bacteria are
dissolved J (c) antagonistic action is not so specific as that of phage j (d)
races of E. colt resistant to phage are dissolved by the filtrate of the an-
tagonist. The active substance was believed to be of the nature of an
enzyme. Friedlander's bacillus was not acted upon, possibly because of
the formation of a pellicle by the bacillus. The active substance was
formed in 4 to 5 days but increased in activity after 2 to 3 weeks. It was
essential that a surface pellicle of the organism be maintained. Sub-
merged growth was less favorable. Fresh filtrates had the greatest ac-
tivity, the property being lost after storage for 3 months. The substance
was thermolabile, activity being destroyed at 70° C. The filtrate of an
organism dissolved by the action of the antagonist proved to be as ac-
tive as the filtrate of the culture of the antagonist. It acted injuriously
upon intestinal bacteria not only in vitro but also in vivo.
Much and associates (620) isolated several strains of B. mycoides
that possessed strong antagonistic properties. The active strains were
SPORE-FORMING BACTERIA 85
said to be found only rarely in nature. They gave a mesentericus-like
growth, producing a pellicle and no turbidity in bouillon. One strain
was able to lyse 20 species of bacteria, another acted upon 1 8, a third on
12, and a fourth on only 7. Marked differences were shown (621) to
exist in the degree of antagonistic activity of the different strains.
P. vulgaris, E. ty-phosa, and V. comma were lysed in 24-hour bouillon
cultures as a result of adding pieces of agar containing colonies of the
antagonist. A lytic effect was also exerted upon staphylococci (786) and
M. tuberculosis (482). The active substance (483) was precipitated by
10 per cent tungstic acid and lead acetate and was thermostable.
Much and Sartorius (621) came to the conclusion that B. mycoides
Flugge comprises two groups of organisms. One produces branching
colonies on agar and forms no pellicle in meat broth, the flaky growth
dropping to the bottom and the medium remaining more or less clear.
The second group forms flat surface growth similar to that of B. mes-
entericus on agar and a pellicle on the surface of liquid media. Many
of the pellicle-forming strains have the capacity, in varying degrees, of
dissolving various cultures of bacteria. This is not due to their proteo-
lytic activity, since members of the first group may be more actively
proteolytic. The culture filtrate of the antagonist dissolves the bacteria
but does not destroy their antigenic properties. The lytic substance,
designated as Much-lysin, was said to have a double effect: one, bound
to the living cells of the organism, had nothing to do with phage, and
the other, found in the bacteria-free filtrate, had an apparent similarity
to phage but was distinct from it.
The idea that in the case of bacterial antagonists one is dealing with
specific strains rather than with distinct species was further emphasized
by Franke and Ismet (294). Various strains of B. mycoides, desig-
nated as cytoliticusy were shown to be able to lyse many pathogenic and
nonpathogenic bacteria but not their own cells j the same action was
exerted by the culture filtrate (Table 12). The lytic action of strains of
B. subtilis upon different bacteria, including M. tuberculosis (903),
pneumococci, typhoid, diphtheria (62), and other organisms has also
been definitely established.
Pringsheim (705) isolated a strain of B. mesentericus-vulgatus that
had a decided inhibiting effect upon a variety of bacteria, particularly
86
BACTERIA AS ANTAGONISTS
C orynehacterium difhtherlae. On agar plates the antagonist produced
a circular zone of inhibition, just beyond which was a ring of larger
colonies, indicating a stimulating effect. It was suggested that the an-
tagonist produced a toxin that was stimulating in small doses and in-
jurious in larger concentrations. The active substance was thermolabile
and nonfilterable. The antagonistic properties appeared to be inherent
in the particular strain of an organism and were not increased by serial
passage (1016). The action of filtrates of B. mesenterkus against diph-
theria organisms was considered (956) as highly specific. Other strains
of this organism were reported to be active against Pasteurella pestis
(246). Living gram-positive bacteria were found (768) to be more
susceptible than gram-negative organisms to the antagonistic action of
spore-forming aerobes j in the case of dead organisms, the reverse was
true. Plates were heavily seeded with the test bacteria and the centers
TABLE 12. LYSIS OF PATHOGENIC BACTERIA BY VARIOUS STRAINS OF A
SPORE-FORMING ANTAGONIST (CYTOLYTICUS)
ORGANISM LYSED
STRAIN NUMBER OF CYTOLYTICUS
I
II
III
VI
VII
VIII
IV
E. ty-phosa
+++
-H-f
-H-
+++
0
0
Paratyfhoid A
+
-hH-
+
-H-
-H-f
-K-l-
Paratyfhoid B
0
+
-F
++
-1^
0
Shigella
-H-
4-f
-H-
+
++
+f
0
Y bacillus
-1-
^H-
4-
++
0
-h
E. coli
-H-+
4-H-
-H-
+-H-
0
0
C. difhtheriae
-t-H-
++
+
-H-
+
Ps. fyocyaneus
0
-H-
-1-
-H-+
0
S. aureus
+
0
0
H^-l-
-f-F
S. alius
-h
-1-
0
-H-+
-f-
S. citreus
4-f+
0
HH-+
-f++
S. viridis
+
+
-f-H-
-f-H-
S. Ivaemolyticus
-H-
-f-
-F+
4^+
S. mucosus
^
-t-
-H-
+f+
P. vulgaris (Weil-Felix)
++
-1-
+
-1-
^H-
Pneumococcus
-H-f
+++
+^
0
0
From Franke and Ismet (294).
0 no clearing.
+ trace but no true clearing.
++ clearing,
-t-H- clearing
slight sediment,
without sediment.
SPORE-FORMING BACTERIA 87
of the plates inoculated with the antagonist. Inhibition of growth and
lysis were used as measures of antagonistic action.
Hettche and Weber (408) isolated 41 strains of B. mesentericus
from 25 samples of soil. These were streaked on blood agar, and the
diphtheria organism was used for testing their effect. In 1 8 strains the
antagonistic action was detected in 24 hours j there was no parallelism
between inhibition and hemolysis. Of the 18 active strains, 1 1 lost the
property after two transfers and 2 were exceedingly active.
Dubos (190) isolated from a soil enriched with various living bac-
teria a gram-negative, spore-bearing bacillus {B. brevis) that had a
marked lytic effect against gram-positive bacteria, including staphylo-
cocci and pneumococci. The antagonist was grown for 3 to 4 days in
shallow layers of peptone media at 37° C. The bacterial cells were re-
moved by centrifuging, and the filtrate was acidified, giving a precipi-
tate from which a highly active substance (tyrothricin) was isolated.
Dubos and Hotchkiss (205) soon demonstrated the presence in natural
substrates, such as soil, sewage, manure, and cheese, of various spore-
forming bacteria that have marked antagonistic properties against vari-
ous gram-positive and gram-negative bacteria.
Hoogerheide (427) isolated from the soil an aerobic, spore-forming
bacterium that produced a highly active bactericidal substance j it also
prevented the formation of capsules by Friedlander's bacterium. This
substance appeared to be similar to gramicidin (885).
It has thus been definitely established (857) that strains of spore-
forming bacteria possessing antagonistic properties are widely distrib-
uted in the soil and possess certain physiological characteristics that
differentiate them from the inactive strains. This is brought out in
Table 13.
Spore-forming bacteria are also able to produce substances antagonis-
tic to fungi (231, 695, 734, 738). Cordon and Haenseler (149) iso-
lated an organism {B. simplex) that was antagonistic to Rhizoctonia
solani, an important plant pathogen. The antagonist produced a thermo-
stable substance that inhibited the growth and even caused the death of
the fungus. When the substance was added to the soil it controlled to
some extent seed decay and damping-off disease of cucumbers and peas.
Christensen and Davies (128) found that a strain of B. mesentericus
88 BACTERIA AS ANTAGONISTS
produced on artificial media an active substance that suppressed the
growth of Helminthosforium sativum. It increased sporulation of the
fungus, inhibited or retarded spore germination, caused abnormal
hyphal development, and induced mutations in certain strains of the
fungus. The substance was thermostable and diffusible. It passed
through a Berkfeld filter, was absorbed by infusorial earth, withstood
freezing and desiccation, and did not deteriorate readily. It was de-
stroyed by alkalies but not by acids. It was inactivated or destroyed,
however, by certain fungi and bacteria.
TABLE 13. BIOCHEMICAL CHARACTERISTICS OF ACTIVE AND INACTIVE
STRAINS OF SPORE-FORMING SOIL BACTERIA
lique-
HY-
pro-
fac-
DROLY-
STRAIN ACID PRODUCTION
FROM
duction
tion OF
SIS OF
GRAM
Dextrose
Lactose
Sucrose
OF HoS
gelatin
STARCH
STAIN
Active Strains
A-2
-
-
+
+
-
-
A-5
-
-
+
+
-
-
A-io
-
-
+
+
-
-
A-2 I
-
-
+
+
-
-
A-23
-
-
+
+
-
-
A.27
-
+
+
-
-
-
A-34
-
-
+
+
-
-
Inactive Strains
A-15
-
+
-
-
+
+
A-31
+
+
-
+
+
+
A-32 +
+
+
-
+
+
+
From Stokes and Woodward (857).
— reaction becoming alkal!
ine.
+ acid produced.
NONSPORE-FORMING BACTERIA: PS. AERUGINOSA,
PS. FLUORESCENS, AND S. MARCESCENS
Among the bacteria, those belonging to the fluorescent, green-
pigment and red-pigment producing groups have probably received
the greatest attention as antagonists. Bouchard ( 8 1 ) first reported, in
1888, that Ps. aeruginosa was antagonistic to B. anthracis; the presence
NONSPORE-FORMING BACTERIA
89
of the antagonist was shown (62, 1 20) to reduce considerably the action
of the pathogen. When grown on artificial media the pyocyaneus or-
ganism was found (299) to be strongly antagonistic to a number of
bacteria, including E. tyfhosa, Pjeijferella mallei, V. comma, and Bac-
terium tyrogenes. The growth of staphylococci, micrococci, diplococci,
and spore-forming rods was also reduced. The antagonist inhibited its
own growth as well.
These early observations were amply substantiated (Table 14). Ps.
aeruginosa was shown to be active against E. coU, M. tuberculosis (62,
81, 120, 760, 999), and a variety of other bacteria (522). The addition
of top minnows {Gambusia) to water polluted with E. coli caused the
disappearance of the bacteria j this was shown to be due to the inhibit-
ing effect of the pyocyaneus organism present in the intestinal flora of
Gambusia. The presence of this antagonist in water renders the colon
index of the water an unreliable guide to pollution (384). When a mix-
ture of the antagonist and the colon organism was incubated, the former
tended to outgrow the latter after 24 hours {2)S(>)- Even after steriliza-
TABLE 14. NONSPORE-FORMING BACTERIA AS ANTAGONISTS TO BACTERIA
ANTAGONIST
Ps. aei-uginosa
Ps. aeruginosa
Ps. fluorescens
ORGANISMS AFFECTED
B. anthraciSy E. typhosa, V .
comma, etc.
Gram-negative bacteria, M.
tuberculosis, and yeasts
E. coli, S. marcescens, C.
difhtheriae, B. ant hr acts,
etc.
Ps. fluorescens Actinomycetes
5. marcescens CI. chauvoei, B. anthracts,
staphylococci, micrococci
5. marcescens
Gram-positive but not gram-
negative bacteria
KNOWN PROPERTY
Thermostable, filter-
able substance
Depresses growth
Thermostable, filter-
able substance
Lytic action
Colorless, thermo-
stable, lytic sub-
stance
Alcohol-soluble
pigment
REFERENCES
62, 81, 120, 235,
236, 299
62, 81, 760, 762,
763,999
246, 302, 311, 326,
406, 407, 409, 410,
430-432, 524, 525,
656, 827
50, 209, 230, 743,
764
409
90
BACTERIA AS ANTAGONISTS
TABLE 14 {continued)
ANTAGONIST
E. coli
E. coli
E. coli
ORGANISMS AFFECTED
Typhoid, paratyphoid, diph-
theria, staphylococci, and
proteolytic bacteria
Other E. coli strains
M. tuberculosis and spore-
forming bacteria
KNOWN PROPERTY
Growth-inhibiting
REFERENCES
52, 54,61,93, 121,
366,491,643, 736,
759, 876, 886, 954
643
105,336,365,367,
448, 469, 781, 823,
A . aerogenes
E. tyfhosa
S. faratyfhi
Streptococci
Streptococci
Staphylococci
Staphylococci
Micrococci
Diplococci and
pneumococci
K. fneumoniae
P. vulgaris
P. avicida
Myxobacteria
Anaerobic bac-
teria
B. anthracis, P. festis
E. tyfhosa, Ps. fiuorescens,
E. coli, B. anthracis
E. coli, B. anthracis, P. festis
B. afithracis, C. difhtheriae
B. anthracis, Ph. tumefaciens,
S. lactis, P. festis, L. bul-
garicus
Dead cells of various bacteria
Gram-positive bacteria, C,
difhtheriae, P. festis
V . com?na, M. tuberculosis,
E. tyfhosa, Br. melitensis
Various bacteria
B. anthracis, C. difhtheriae,
P. festis
B. anthracis, P. festis, CI.
sforogenes
B. anthracis, E. tyfhosa
Plant-discase-producing
bacteria
M. tuberculosis. B. anthracis
Activity not associ-
ated with hemoly-
sis or virulence
Thermostable, non-
filterable substance
Thermolabile sub-
stance
Active filtrate
Thermostable lytic
substance
246,365, 367
224, 311, 347,898,
246, 453, 823, 898
52, 105, 131, 178,
233, 302,627,676,
799, 800
69, 246, 765, 979
351,994,996
52, 150, 213, 214,
246, 248, 861
21 1, 212, 541, 627
21 1, 212, 244, 246,
368, 540, 541,634,
669,733
52, 54, 246, 302,
634,676, 823
40, 246, 426, 649,
898,958
299, 672
841
425,671
NONSPORE-FORMING BACTERIA 91
tion, media in which Ps. aeruginosa had grown depressed the growth
of other microorganisms including S. marcescens, Ps. fluorescens, and
Saccharomyces cereviseae; spore formation by the last was favored
(762).
The specific antagonistic action of Ps. aeruginosa upon various bac-
teria was found by early investigators to be due to the production of an
active heat-resistant substance (120, 299). By filtering the culture
through a Berkfeld, evaporating to a small volume, dialyzing through
a parchment membrane, precipitating with alcohol, and drying over
sulfuric acid, Emmerich and Low (236) obtained a preparation which
was designated as pyocyanase. It was soluble in water and highly bac-
teriolytic.
Pyocyanase was at first looked upon as an enzyme belonging to the
class of nucleases. It was found to have, even in very low concentrations,
a marked destructive effect upon diphtheria, cholera, typhus, and
plague organisms, as well as on pyogenic streptococci and staphylococci.
It rapidly dissolved V . comma cells and in a few seconds rendered in-
active such bacterial toxins as that of diphtheria. Since the bacteriolytic
action of pyocyanase was in direct proportion to the time of its action
and concentration, and in inverse proportion to the numbers of bacteria
acted upon, its enzymatic nature was believed to be substantiated. The
preparation withstood heating in flowing steam for 2 hours. Other
proteolytic systems of bacteria are known to be resistant to high tem-
peratures and to remain active even when kept in a moist state for 1 5 to
30 minutes at 100° C. ( i ). Pyocyanase was believed to be transformed
in the bodies of animals into high molecular proteins which still re-
tained the bacteriolytic action of the free enzyme.
Since the early work of Emmerich and Low, an extensive literature
has accumulated on the nature of pyocyanase. Its lytic effect has been
established against diphtheria (52), streptococci (237), meningococci,
the typhoid organism, pneumococci (yb(i)^P. festis ( 246) , Vibrio metch-
nikovi (501), V. comma (692), and many others (794). There has been
considerable disagreement, however, concerning the chemical nature
and therapeutic action of pyocyanase (234, 322), due largely to the
variation in the nature of the preparations obtained (540). Kramer
(501), for example, has shown that the activity of the substance de-
pends on three factors: nature of strain, not all strains being equally
92 BACTERIA AS ANTAGONISTS
effective j composition of medium, glycerol-containing media being
most favorable J and method of extraction of active substance from cul-
ture media.
The enzymatic nature of pyocyanase was not universally accepted,
largely because of the thermostability of the substance (489). Dietrich
(171) ascribed the action of pyocyanase to a change in osmotic pressure,
Raubitchek and Russ (733) emphasized that the solubility of the sub-
stance in ether, chloroform, or benzol is not indicative of an enzyme,
nor is the fact that temperatures of o to 37° C. fail to influence its ac-
tivity {55, 185,409,669,919).
Ps, aeruginosa was found to produce (409), in addition to pyocya-
nase, a blue pigment, pyocyanin. Both substances possess lytic proper-
ties, 1:1,000 dilution of the pigment being able to lyse E. coli in
6 hours (366). The pigment was believed (501) to act only on gram-
positive bacteria. Pyocyanin was said (407) to be more effective in
younger cultures, and pyocyanase in older.
In order to test the action of Ps. aeruginosa upon other bacteria,
Kramer (501) placed a drop of a suspension of this organism upon a plate
inoculated with M. tuberculosis or with V. metchnikovi. In 24 hours, a
sterile zone surrounded the colony of the antagonist, the width of the
zone depending upon the moisture content of the medium, the degree
of diffusion of the active substance, its concentration, and the resistance
of the test bacteria. When the two pathogens were inoculated into liquid
media and the antagonist was introduced simultaneously or within 24
hours, the latter had a decided bactericidal effect. No bactericidal fil-
trate could be obtained. These results were confirmed (634, 919), the
conclusion being reached that the active molecules do not pass through
the ultrafilter (949). Pyocyanin had a bactericidal action also upon
S. hemolyticus, S. albus, S. aureus, C. difhtheriae, M. tuberculosis, V.
metchnikovi, and the Y-Ruhr bacillus, but not upon P. vulgaris, E. coli,
or the typhoid organism. In general, gram-positive bacteria were
largely affected.
More recently, Schoental (809) succeeded in isolating three anti-
bacterial substances from the chloroform extracts of cultures of Ps.
aeruginosa: (a) a blue pigment, pyocyanin j (b) a yellow pigment,
tf-oxyphenazine, a derivative of pyocyanin j and (c) an almost colorless
NONSPORE-FORMING BACTERIA 93
bacteriolytic substance most readily found in old culture media. None
of these substances was enzymatic in nature. Schoental found that pyo-
cyanin had a strong bactericidal activity, but its high toxicity and insta-
bility made it unpromising for therapeutic purposes. The action of
^-oxyphenazine against many bacteria made it comparable to the fla-
vinesj however, it was less toxic and non-irritant. The third antibacterial
substance had a marked lytic action on vibrios, being bactericidal in a
concentration of i : 1 0,000 and bacteriostatic in i : 100,000.
No less extensive is the literature on the antagonistic action of the
fluorescent group of bacteria, first established by Garre (311) in 1887
and later by others (50, 246, 302, 326, 430-43^, 462, 524, 525, 6s 5,
827). Hettche and Vogel (407) described the inhibiting effect of
strains of this organism on the growth of various gram-negative and
gram-positive bacteria (Table 15). The active substance is thermo-
stable, dialyzes through a membrane, and passes through Seitz and
Berkfeld filters (525). It is said (407) to be soluble in chloroform.
Aerobic culture conditions are favorable to its accumulation. Members
of this chromogenic group of bacteria were also found to be able to
bring about the lysis of infusoria (123). Rahn (711) observed the
phenomenon of iso-antagonism, which is associated with the formation
of a thermolabile substance that does not pass through a filter. Certain
bacteria were found (228, 711) to be favored by their own metabolic
products, whereas others had an adverse effect j the products of the
first group were usually thermostable and nonfilterable, and the second
were thermolabile (at 60° to 100° C.) and were destroyed by light.
5. marcescens is known to exert antagonistic effects against a number
of bacteria, including diphtheria, gonococci, anthrax (743, 764), and CI.
chauvoei (209), as well as fungi causing insect diseases (587). The for-
mation of antibiotic substances by this organism has been demonstrated
by various investigators. These substances are active not only in vitro
but also in vivo ( 743 ) . Their activity increases with the age of the cul-
ture. Their formation was believed not to be associated with the pro-
duction of the pigment by the organism. They were also of a nonlipoid
nature. Hettche (409), however, asserted that the bactericidal action
of Serratia is closely related to pigment production. The pigment was
extracted with alcohol and was found capable of dissolving dead gram-
94 BACTERIA AS ANTAGONISTS
TABLE 15. ANTAGONISTIC ACTION OF PS. FLUORESCENS UPON
VARIOUS MICROORGANISMS
ORGANISM
PERCENTAGE OF AGED MEDIUM IN THE AGAR
0.5
I.O
2.5
5.0 10 15 20 30 40 50
B. cereus
-
-
+
B. mycoides
-
-
+
B. anthracis
-
+
B. vulgatus
-
-
+
B. subtilis
-
-
+
B. megat/ierium
-
+
R. cinnebareus
-
+
R. roseus
-
-
+
M. -flavus
-
-
-
+
N . catarrhal is
-
-
-
+
Ps. aeruginosa
-
-
-
Ps. fiuorescens
-
-
-
S. lutea
-
-
-
+
S. marcescens
-
-
-
- - +
5. albus
-
-
+
S. aureus
-
-
-
+
S. citreus
-
-
+
K. pneumoniae
-
-
-
+
V. comma
-
+
Ch. violaceum
-
+
E. tyfhi
-
-
+
Sh. faradysenteriae
-
-
4-
S. enteritidis
-
-
-
4-
S. suisfestifer
-
-
-
4-
S. fullorum
-
-
-
4
.E. coli
-
-
-
_ _ _ +
A . aero genes
-
-
-
_ _ _ +
Ph. b owl e sit
-
-
+
Sac. marianus
-
-
-
Sac. ellifsoideus
-
-
-
- - - - +
Sac. fastorianus
-
-
-
----- +
Xygosac. friorianus
-
-
-
- - - - +
Torula sfhaerica
-
-
-
A . niger
-
-
-
From Lewis (525).
+ denotes complete inhibition.
COLON-TYPHOID BACTERIA 95
positive bacteria but not gram-negative organisms. Eisler and Jacobsohn
(230) ascribed the antagonistic action of Serratia not to the pigment but
to certain water-soluble, thermostable (70° C. for 30 minutes) lytic
substances.
THE COLON-TYPHOID BACTERIA
Members of the colon-typhoid group are not typical soil inhabitants,
although they find their way continuously into the soil and into water
basins. Various organisms belonging to this group have been said to
possess antagonistic properties (425). Bienstock (54) reported, in 1899,
that proteolytic bacteria are repressed by the presence of E. coU and
A. aerogenes. Tissier and Martelly (886) emphasized that this phe-
nomenon occurs only in the presence of sugar, the effect being due to
the fermentation of the sugar by E. colt, resulting in the production
of acid.
Wathelet (954) observed in 1895 that in mixed culture the colon
bacterium gradually replaces the typhoid organism and this was later
fully confirmed (141, 383, 491, 619, 643, 799, 800, 876). The occur-
rence of slowly growing lactose-fermenting strains of E. coli in stools
has been ascribed to the phenomenon of antagonism (453), and the
inhibitory action upon E. tyfhosa added to certain stools was also as-
cribed to the antagonistic action of E. coli (643), Different strains of
E. coli repress the typhoid organism to a different extent. The ratio of
the two organisms developing on agar was designated as the antagonis-
tic index 5 an index of 100:20 means that for every 100 colonies of the
colon organism, 20 colonies of typhoid developed. Manteufel (581),
however, ascribed this antagonistic action of E. coli to the exhaustion of
nutrients in the medium.
Active colon strains were found to be inhibitive to other strains of
the same organism. The existence of strong and weak antagonistic
strains has been questioned frequently (1005). Nissle (642) ascribed
to many of these strains a strong antagonistic action against the patho-
genic intestinal flora. These results were contested, however (98, 512).
The action of E. coli of different origin varies (750), freshly isolated
strains being more active than stock cultures (837). It has also been
96 BACTERIA AS ANTAGONISTS
reported that fresh, actively growing cultures of E. tyfhosa inhibited
the growth of E. coli, older cultures not being antagonistic (915). The
antagonistic properties of E. coli were often believed to be associated
with the formation of unstable, thermolabile lytic substances, that
would not pass through a filter (365, 367, 580).
A bacteriophage was found (532) to develop as a result of the an-
tagonistic action of E. coli against the Shiga bacillus and was said to
occur in the intestines where antagonistic conditions are always present.
A similar effect was observed by Fabry (245), due to the antagonistic
stimulus of E. coli by a strain of S. albus. Gratia (348) found that
the filtrates of one race of E. coli inhibited another race and caused an
agglutination of the latter in fluid media. According to Hashimoto
(383), the weakest antagonists belong to the paracolon group, the
strains of medium activity to the colon group, and the strongest an-
tagonists to the colon-immobilis type. Whenever the feces were found
to contain large numbers of E. coli, no typhoid organisms were present.
The resistance of certain persons to intestinal diseases was, therefore,
ascribed to the high antagonistic colon index. By utilizing the principle
of antagonism of some strains of E. coli against others, two types of
E. coli resistant to the antagonistic substance were isolated ( 168) : one
produced giant colonies, the other small punctiform, translucent
colonies.
E. coli exerts an antagonistic action also upon Salmonella schottmUl-
leri (305), C. difhtheriae (52, 905), staphylococci (366, 491), M. tu-
berculosis (6So,6Si)yB.anthracis (105,365-367,781, 823, 898), vari-
ous spore-forming soil bacteria (469), and putrefactive water bacteria
(759). The action against anthrax was said to be only temporary (336).
It was also suggested (448) that only living cultures of E. coli are ac-
tive. The simultaneous inoculation of S. aureus and E. coli was found
(736) to be injurious to the first and not to the second organism j this
effect was increased by an increase in the number of E. coli cells in the
inoculum. Gundel and Himstedt (366) have shown that E. coli, but
not A. aero genes y is antagonistic to S. aureus and S. albus.
The term autophage has been used (334) to designate the process of
clearing a water emulsion of dead cells by a culture of an antagonist
such as E. coli. This clearing effect was said to be due to the fact that the
COCCI 97
dead cells are used as nutrients by the living organism. The mechanism
of the action was variously explained by a change in the fH value of
the medium or in the oxidation-reduction potential or by a direct enzy-
matic effect. In some cases thermolabile, filterable substances were dem-
onstrated (141, 26Sy 36^, 580). These substances have been considered
either as autotoxins (141) or as proteolytic enzymes (683). According
to Schilling and Califano (799, 800), the filtrate of E. colt depressed
only the dysentery organism of Shiga. From a bouillon culture of E.
coliy Gundel (370) isolated thermostable lipoids capable of bringing
about the lysis of the colon bacteria and other bacteria. The antagonistic
relations between E. coli and V. comma have been well established, the
cholera organism also possessing antagonistic properties (307, 480).
The typhoid organism is also capable of exerting an antagonistic ac-
tion against itself, as well as against Ps. fuorescens (311) and E. coli
(323). Similar antagonistic effects (224) have been obtained against
various other bacteria, including B. anthracis (823, 898). The nature
of the action is not clearly understood. Salmonella -paratyphi possesses
antagonistic properties against E. coli (453), B. anthracis (898), P.
pestis (246), and various other bacteria.
It may be of interest to record here that E. coli isolated from persons
affected by rectal cancer are able to convert substances related to bile
acids to carcenogenic bodies. The possibility of synthesis of such sub-
stances has also been suggested (186).
COCCI
Numerous cocci have been found to possess antagonistic properties
against other bacteria. Doehle (178) first demonstrated in 1889 that
streptococci are able to antagonize B. anthracis y especially on solid
media. Similar action was exerted against diphtheria bacteria (52, 71,
131? 799j 800), a phenomenon apparently not correlated with the
hemolytic properties or the virulence of the antagonist. Further studies
established the effect of various streptococci against anthrax (233, ^dG^
676). According to Cantani ( 105) this effect is more pronounced in liq-
uid than in solid media, and is highly specific as regards the strain. S.
pyogenes was shown to be antagonistic, in vivo, to B. anthracis and to
98 BACTERIA AS ANTAGONISTS
Phytomonas tumefacienSy even to the extent of suppressing vegetative
malformations brought about by the latter (69). Streftococcus cremoris
was active against Streftococcus lactis (979), Streftococcus mastidis
against S. lactis and Lactobacillus acidophilus^ and Streftococcus muco-
sus against P. festis (246). Rogers (765) reported an antagonistic effect
of S. lactis against L. bulgaricus; the active substance was thermostable
and was unable to pass through a bacterial filter (150, 178, 669, 676).
Freudenreich (299) first emphasized the antagonistic action of
staphylococci against various bacteria. The list was later enlarged to in-
clude gram-positive acid-resisting forms (211), corynebacteria (52,
213, 214), and the plague organism (246). Some of these antagonists
were found to be able to lyse the dead cells of their own kind (351, 994-
996) as well as those of various other organisms. Gundel (370) isolated
from staphylococci an active lipoid which had bactericidal properties. A
water-soluble, alcohol-insoluble substance, said to be an enzyme capable
of bringing about the lysis of corynebacteria, was isolated from a strain
of staphylococcus (213, 214).
Various micrococci possess strong antagonistic properties. Lode
(541 ) isolated a micrococcus which affected a variety of microorganisms
three or more centimeters away, the active substances being dialyzable.
An organism related to Micrococcus tetragenus and described as Micro-
coccus antibioticus (211) was found to possess a strong antagonistic ac-
tion against V . comma^ M. tuberculosisy E. tyfhosa. Ph. tumejacienSy
Br. melitensisy various spore-forming bacteria, numerous cocci, and
others (627). Diplococci exerted an antagonistic action against various
bacteria (541 ), including pyogenic staphylococci and streptococci in the
sputum (363), spore-formers, and gram-negative bacteria (447). They
produced, under aerobic conditions only, a filterable substance that was
heat resistant.
The antagonistic action of pneumococci has definitely been estab-
lished (212, 244, 246, 368, 571, 634, 669, 733). The active substance
of these organisms was said (541) to be thermolabile, since it was de-
stroyed at 80° to 85° C. } it was produced only under aerobic conditions.
In reviewing the literature on the longevity of streptococci in symbiosis,
Holman (425) observed that many chances of error are inherent in
mixed cultures, particularly with closely similar organisms j pneumo-
OTHER BACTERIA 99
cocci, for example, were found to be able to live for long periods to-
gether with nonhemolytic streptococci. Peculiar antagonistic relations
between pneumococci and staphylococci were also reported (15). Adap-
tive alterations could be expected in the growth of bacteria in mixed
cultures (31). Which of the two organisms antagonizes the other was
believed to depend frequently upon the numerical abundance of one or
the other (244).
OTHER AEROBIC AND ANAEROBIC BACTERIA
The antagonistic action of Klebsiella pneumoniae against B. anthracis
has been reported (216, 676, 823). Freudenreich (299) found that the
filtrate of this antagonist repressed the growth of a number of bacteria
including the diphtheria (52, 634) and plague (246) organisms.
Other aerobic bacteria were found capable of exerting antagonistic
effects against one or more organisms, these effects varying considerably
in nature and intensity. It is sufficient to mention the action of P. vul-
garis against B. anthracis and P. festis (246, 898)5 of Ps. aviseftica
against B. anthracis (672) and E. tyfhosa (299) j of Bacterium lactis
aerogenes against B. anthracis (365, 367) and P. festis (246). B. an-
thracis is capable of iso-antagonism (781, 827) and of antagonizing cer-
tain other organisms, including E. tyfhosa and Bacterium acidi lactici
(299, 827). Certain Myxobacteriales have been shown (841) to be ca-
pable of bringing about the lysis of various plant-disease-producing bac-
teria j a thermostable lytic substance, passing through cellophane but
not through a Seitz filter, was obtained. Although certain bacteria like
Achromohacter lifolyticum were found capable of reducing the patho-
genicity of M. tuberculosis^ no active cell-free extract could be ob-
tained (82).
The morphology of one bacterium may be considerably modified by
the presence of another. Living cultures of L. bulgaricus influenced the
variation of E. coli from the "S" to the "R" phase, inhibited develop-
ment of the organism, and even brought about its lysis. No active sub-
stance could be demonstrated j the lactic acid itself had only a limited
effect (11). Korolev (499) has shown that when a yellow sarcina was
added to solid media a stimulating effect was exerted on the growth of
100 BACTERIA AS ANTAGONISTS
species of Brucella {Br. melitensis, Br. abortus, Br. suis) ; in liquid
media, however, the activities of these species were repressed, the sar-
cina thus acting as an antagonist. A white staphylococcus exerted an an-
tagonistic action on Brucella species both in liquid and on solid media.
Certain acid-producing aerobes were found capable of inhibiting toxin
production by Clostridium hotulinum in glucose but not in noncarbo-
hydrate media (372). Since acid itself cannot bring about this effect,
Holman (426) suggested that the acid must be active in a nascent state.
A mixture of a Clostridium sf or 0 genes and CI. hotulinum also inter-
fered with the development of the toxin ; it was even thought possible
that the first anaerobe might cause the disappearance of toxin already
produced (158, 1 59, 463 ) . 5. aureus, E. coli, P. vulgaris, and other bac-
teria permitted the growth of CI. hotulinum in aerobic cultures, accom-
panied by toxin production (291 ). However, Streftococcus thermofhi-
lus inhibited the growth of CI. hotulinum, the toxin of the latter being
gradually destroyed (478).
Passini (671) claimed that Bacillus futrificus verrucosus destroyed
M. tuberculosis in nine days. The effect of other anaerobes on the sur-
vival of anthrax spores in dead animals has been extensively studied
(425). Novy (649) reported that the injection into guinea pigs of P.
vulgaris and Clostridium oedematiens resulted in rapid death of the
animals and extensive growth of the anaerobe in the animal bodies j
however, the simultaneous inoculation of CI. sforogenes and P. vul-
garis did not result in putrid lesions (426). According to Barrieu (40),
the presence of P. vulgaris and certain nonpathogenic spore-bearing
aerobes in wounds favors, through their proteolytic activity, the viru-
lence of pathogenic bacteria. Pringsheim (705) grew CI. welchii with
Alkali genes fecalis for ten generations on agar slants and could easily
detect in the growth of the latter the opaque colonies of the anaerobe.
A liquefying sarcina allowed CI. welchii and Clostridium butyricum to
grow in open tubes. Weinberg and Otelesco (958) believed that many
war-wound infections are due to an association of P. vulgaris with
anaerobes, since the former increased the virulence of Clostridium fer-
fringens and others.
The antagonistic effects of lactic acid bacteria of the L. hulgaricus and
L. acidofhilus groups have received considerable attention (76), espe-
OTHER BACTERIA 101
dally in regard to their action against intestinal bacteria. This was be-
lieved to be due to the production of acid by the bacteria rather than to
the formation of specific antagonistic substances (590). This phenome-
non aroused particular interest because of the function of some of these
organisms in replacing bacterial inhabitants of the human digestive
system (497).
CHAPTER 6
ACTINOMYCETES AS ANTAGONISTS
AcTiNOMYCETES are found in large numbers in many natural sub-
strates. They occur abundantly in soils, composts, river and lake bot-
toms, in dust particles, and upon plant surfaces. Certain species are
capable of causing serious animal and plant diseases.
Actinomycetes, like fungi, produce a mycelium, but they are largely
unicellular organisms of dimensions similar to those of bacteria. Some
of the constituent groups are closely related to the bacteria, others to
the fungi. On the basis of their morphology, the order Actinomycetales
has been divided into three families, Mycobacteriaceaey Actinomy-
cetaceaey and Streftomycetaceaey comprising the genera Mycobacte-
riuniy Actinomyces y NocardiUy StreftomyceSy and IVLicromonosfora.
These genera are represented in nature by many thousands of species,
of which several hundreds have been described. A few are shown in
Figure lO.
Comparatively little is known of the physiology of actinomycetes.
Some produce certain organic acids from carbohydrates (287, 1002)^
others prefer proteins and amino acids as sources of energy, many spe-
cies being strongly proteolytic. Some are able to attack starch, with the
production of dextrins and sugar, accompanied by the formation of
diastatic enzymes. Many reduce nitrates to nitrites. Some attack sucrose
and form the enzyme invertasej others, however, do not. Certain spe-
cies are able to utilize such resistant compounds as rubber and lignin.
Synthetic media are favorable for the production of a characteristic
growth and pigmentation. Among the pigments, the melanins have re-
ceived particular attention. They range from the characteristic brown
to various shades of black and deep green and are formed in protein-
containing and in some cases also in protein-free media. The other pig-
ments range from blue, yellow, and orange to various shades of grey.
According to Beijerinck (43), the process of pigment production by
actinomycetes in gelatin media is associated with the formation of a
quinone, which turns brown at an alkaline reaction and in the presence
of oxygen. The action of quinone in the presence of iron was found to
S. antthioticus, important antagonist.
From Waksman and Woodruff (945)
S. lavendulaCy important antagonist
Submerged growth of S. iavendulae.
From Woodruff and Foster (1002)
\.
Thermophilic Streftomyces. From
Waksman, Umbreit, and
Cordon (944a)
'X
\>r
M. vulgaris. From Waksman,
Cordon, and Hulpoi (931)
Streftomyces 3042, showing close spiral
type of branching. Prepared by Starkey
Figure 10. Types of actinomycetes.
ACTINOMYCETES AS ANTAGONISTS 103
be similar to that of the enzyme tyrosinase. Since an excess of oxygen is
required for the formation of quinone, only limited amounts are found
in deep cultures. The quinone is believed to be formed from the pep-
tone in the medium j although good growth was produced on media
containing asparagine, KNO;., and ammonium sulfate as sources of ni-
trogen, only traces of quinone, if any, were found. The tyrosinase reac-
tion is not involved in the production of all black pigments by actinomy-
cetes (945) i some species produce such pigments in purely synthetic
media, in the complete absence of peptone.
Actinomycetes grow in liquid media in the form of flakes or small
colonies, usually distributed either on the bottom and walls of the con-
tainer or throughout the liquid j often a ring is formed on the surface
of the medium around the wall of the vessel. In many cases, a full sur-
face pellicle is produced, which may be covered with aerial mycelium.
As a rule, the liquid medium does not become turbid, even in the pres-
ence of abundant growth. When grown on solid media, actinomycetes
form small, compact, soft to leathery colonies j a heavy lichen-shaped
mat is produced that may become covered by an aerial mycelium. The
addition of a small amount of agar (0.25 per cent) to a liquid medium is
highly favorable to growth, especially in large stationary containers.
Actinomycetes can also be grown in liquid media in a submerged con-
dition, with suitable agitation and aeration in order to supply oxygen j
the medium may also be kept in shaken state (287, 926, lOOi, 1002).
Growth occurs in the form of a homogeneous suspension of discrete
colonies and mycelial fragments throughout the liquid. Responses in
growth and biochemical activities as a result of treatments may thus be
obtained under more homogeneous physiological conditions.
Although most actinomycetes are aerobic, some are anaerobic, and
many can grow at a reduced oxygen tension. The aerobic actinomycetes
commonly found on grasses and in soil are said (490) never to have
been isolated from animal infections. Mixed infections consisting of
anaerobes growing at body temperature together with aerobes have
often been demonstrated. Certain aerobic species also are capable of
causing infections in man and other animals, and certain plant diseases
(potato scab, sweet potato pox) are caused by aerobic species of actino-
mycetes.
104 ACTINOMYCETES AS ANTAGONISTS
ANTAGONISTIC PROPERTIES
Many actinomycetes have the ability to antagonize the growth of
other microorganisms, notably bacteria, fungi, and other actinomycetes j
this is brought out in Tables 1 6 and 1 7. The antagonistic species are not
limited to any one genus but are found among three genera, NocardiUy
StreftomyceSy and Micromonosfora.
Gasperini (317) first demonstrated, in 1890, that certain species of
the genus Streftomyces had a marked lytic effect upon other micro-
organisms. He emphasized that "Streftothrix develops habitually in a
spontaneous manner upon the surface of bacteria and fungi, upon which
it lives to a limited extent in the form of a parasite, due to the faculty
that its mycelium possesses to digest the membrane from these lower
fungi." Greig-Smith (360, 361) found that soil actinomycetes are able
to antagonize not only bacteria but also certain fungi j since actinomy-
cetes grow abundantly in normal soils, it was suggested that they may
become an important factor limiting bacterial development. Lieske
(527) demonstrated that specific actinomycetes are able to bring about
the lysis of many dead and living bacterial cells j they are selective in
their action, affecting only certain bacteria such as S. aureus and S. -pyo-
genes, but not S. lutea, S. marcescens, or Ps. aeruginosa.
Rosenthal (767) isolated from the air an actinomyces species which
he designated as the true biological antagonist of the diphtheria or-
ganism. He inoculated the surface of an agar plate with an emulsion of
the bacteria and inoculated the actinomyces into several spots. At the
end of two days, the plate was covered with the diphtheria organisms,
but the colonies of the actinomyces were surrounded by large trans-
parent zones. In another method utilized, agar was mixed with an emul-
sion of the diphtheria bacteria killed by heat, and the mixture was
poured into plates. After solidification of the medium, the antagonist
was inoculated in several spots on the plates. Its colonies gradually be-
came surrounded by clear zones, thus proving that it produced a lytic
substance that diffused through the agar and dissolved the diphtheria
cells.
Gratia and Dath (350) suspended dead cells of staphylococci and
other bacteria in 2 per cent agar and exposed the plates to the air. A cul-
ANTAGONISTIC PROPERTIES
105
TABLE I 6. ANTAGONISTIC PROPERTIES OF VARIOUS ACTINOMYCETES
ANTAGONIST
ORGANISMS AFFECTED
KNOWN PROPERTY
REFERENCES
S. alius
Pneumococci, strepto-
Thermolabile sub-
347,350,
cocci, staphylococci,
stance, causes lysis
971-973
Ps. aeruginosa, etc.
of dead cells
S. albus
Various fungi
Protein, enzyme,
causes lysis of dead
and certain living
bacteria
12-14
S. antibiottcus
All bacteria and fungi,
Thermostable sub-
947
especially gram-posi-
stance, bacterio-
tive types
static
S. lavendulae
Various gram-positive
Organic base, water-
950
and gram-negative
soluble
bacteria
S. fraecox
S. scabies
604, 605
Streftomyces sp.
Bacteria and fungi
Lytic action
317
Strefiomyces sp.
Diphtheria
Growth inhibition
767
Streftomyces sp.
B. mycoides, proactino-
Bactericidal action.
80, 504
mycetes, mycobacteria
with or without
lysis
Streftomyces sp.
Fusarium
Lytic action
595
N. gardneri
Gram-positive bacteria
Bacteriostatic action
309, 936
Micromonosfora
Gram-positive bacteria
Thermostable active
substance produced
936
Actinomycetes
Dead and living bacteria
Lysis
527
Actinomycetes
Spore-forming bacteria
Repression of growth
360, 970
Actinomycetes
Gram-positive bacteria
Thermostable sub-
stance, produced on
synthetic media,
resembles lysozyme
507, 628
Actinomycetes
Pythium
Thermostable sub-
stance
884
106
ACTINOMYCETES AS ANTAGONISTS
ture of a white actinomyces developed on the plates, each colony being
surrounded by a clear zone of dissolved bacterial cells. By transferring
this culture to a suspension of dead staphylococci in sterile saline, a
characteristic flaky growth was produced, the bacterial suspension be-
TABLE 17. ANTIBACTERIAL SPECTRUM OF CERTAIN ANTAGONISTIC
ACTINOMYCETES
TEST ORGANISM
ZONE OF INHIBITION,
, IN MILLIMETERS
S. violaceus
5. aurantiacus
5. griseus
5. globisporus
A'', rubra
35
32
0
0
N. corallina
4-0
45
22
10
N. alba
4-0
25
0
0
M. rubrum
40
33
10
0
M. citreum
38
37
0
0
M. tuberculosis
8
10
0
0
M. smegmae
10
8
0
0
M.fhlei
20
25
0
0
Corynebacterium sp.
12
10
0
0
E. coli
0
0
0
0
S. aureus
25
19
0
0
M. ruber
35
28
0
0
B. mycoides
30
10
0
0
B. megatherium
33
5
0
0
B. mesentericus
30
2
0
0
B. subtilis
23
I
0
0
B. tumescens
22
0
0
0
Ps. fiuorescens
0
0
0
0
Ps. aeruginosa
0
0
0
0
P. vulgaris
0
0
0
0
S. marcescens
0
0
0
0
M. luieus
30
25
0
0
M. candicans
37
22
0
0
M. roseus
42
27
0
0
M. lysodeikticus
38
33
0
0
S. lutea
30
27
0
0
Az. vinelandii
3
0
0
0
Az. chroococcum
5
0
0
0
Rh. leguvmiosarum
0
0
0
0
Radiobacter
0
0
0
0
From Krassilnikov and Koreniako (504).
ANTAGONISTIC PROPERTIES 107
coming clarified in 36 hours. When the lysed emulsion was filtered, the
filtrate could again dissolve a fresh suspension of dead staphylococci.
This culture was found able to attack all staphylococci tested as well as
certain other gram-negative bacteria, such as Ps. aeruginosa; however,
it was inactive toward M. tuberculosis and E. coli. Some antagonistic
strains could also attack E. coli, though this property was readily lost.
This type of antagonism was believed to be widely distributed in na-
ture and to be directed against many bacteria, pathogenic and sapro-
phytic. The culture of the antagonist in bouillon gave a very active
agent, whereas the lysed bacterial suspension was weaker in its action.
The active substance was present extensively in old cultures and was
fairly stable. The material obtained by lysing the suspension of bacteria
by means of an antagonist was designated as "mycolysate." It did not
possess the toxicity of the nonlysed suspension but it preserved its anti-
genic properties (349). Gratia (347) also reported that actinomycetes
were able to attack living cells of bacteria, except E. coli and E. tyfhosa
which had to be first killed by heat before they could be dissolved.
Welsch (972, 973) made a detailed study of the lytic activity of an
actinomyces culture, presumably identical with the one employed by
Gratia and later described as Actinomyces alhus. The culture was grown
in different media, the best results being obtained in very shallow layers
of ordinary bouillon. The active substance present in the filtrate was
designated as "actinomycetin." It was able to dissolve, at least partly,
all dead bacteria, whether killed by heat or by chemicals, gram-positive
or gram-negative, though gram-negative bacteria were, as a rule, more
susceptible. The growing culture of the antagonist brought about better
clarification (lysis) of the bacterial suspension than the filtrate. The
solubilizing properties of the active agent, its susceptibility to heat and
to ultraviolet rays, its size as measured by ultrafiltration, suggested its
protein nature. The kinetics of its action pointed to its being an enzyme
(971). It was precipitated by acetone, alcohol, and ammonium sulfate.
Most of the gram-negative bacteria were not attacked either by actino-
mycetin or by the living culture of the antagonist. Only a few of the
gram-positive bacteria, including certain pneumococci and streptococci,
could be dissolved by sterile actinomycetin, A definite parallelism in
the activity of the preparation against dead bacteria and of the living
108 ACTINOMYCETES AS ANTAGONISTS
culture against living bacteria suggested that the same substance is con-
cerned in both cases. The bacteria were therefore divided (970), on
the basis of their relation to actinomycetin, into three groups:
Bacteria that were lysed by the culture filtrate; these included pneumo-
cocci and hemolytic streptococci
Bacteria that were not dissolved even by the most active soluble sub-
stance, but which were depressed by the mycelium of the living ac-
tinomyces; these comprised various sarcinae and fluorescens types
Bacteria that were not acted upon by either the living culture or the
actinomycetin preparation ; these included the colon-typhoid and the
pyocyaneus groups, though when the latter were killed by heat or
inactivated by radium emanations, as in the case of E. colt, or were
placed under conditions unfavorable to multiplication, they were dis-
solved by the lytic substance.
The first detailed survey of the distribution of antagonistic organisms
among actinomycetes was made by a group of Russian investigators.
According to Borodulina (80), actinomycetes are able to antagonize
various spore-forming bacteria and to bring about the lysis of their liv-
ing cells. A thermostable substance was produced on agar media. The
activity of this substance was greatly reduced at an alkaline reaction,
whereas an acid reaction favored it. When B. mycoides and an antago-
nist were inoculated simultaneously into peptone media, no inhibitive
effect was obtained, because the bacterium changed the reaction of the
medium to alkaline, thereby making conditions unfavorable for the
production of the antibiotic substance by the antagonist. When the an-
tagonist was first allowed to develop in the medium, before the bac-
terium was inoculated, a strong antagonistic effect resulted, which led
to the elongation of the vegetative cells of B. mycoides; this was due to
a delay in fission and was accompanied by the suppression of spore
formation.
Krassilnikov and Koreniako (504) found that many species of actino-
mycetes belonging to the genus Streftomyces but not Nocardia pro-
duced a substance that possessed a strong bactericidal action against a
large number of microorganisms. This substance was particularly active
against nocardias, mycobacteria, and micrococci j it was less active upon
ANTAGONISTIC PROPERTIES 109
spore-bearing bacteria and had no action at all on nonspore-forming
bacteria, as illustrated in Table 17. Under the influence of the anti-
biotic factor, the microbial cells were either entirely lysed or killed with-
out subsequent lysis. The action upon spore-bearing bacteria was bac-
teriostatic but not bactericidal. The nonspore-forming bacteria, includ-
ing species of Rhizobium and Azotobacter, not only were not inhibited
but were actually able to develop in filtrates of the antagonists.
Nakhimovskaia (628) found that antagonistic actinomycetes are
widely distributed in nature. Of 80 cultures isolated from different
soils, 47 possessed antagonistic properties, but only 27 secreted anti-
biotic substances into the medium (Table 18). These agents were ca-
pable of inhibiting the growth of gram-positive but not of gram-nega-
tive bacteria or fungi. The nature of the action of the various antagonists
TABLE 18. OCCURRENCE OF ANTAGONISTIC ACTINOMYCETES IN
DIFFERENT SOILS
TOTAL STRAINS
NUMBER OF
STRAINS WHICH
OF ACTINOMY-
ANTAGONISTIC
LIBERATED TOXIC
NATURE OF SOIL
CETES TESTED
STRAINS
SUBSTANCES
Chernozem
24
10
9
Podzol
II
7
Solonets
4
4
High altitude soil
9
6
Sandy soil
6
5
Dry desert soil
5
4
River bank meadow
14
7
Cultivated soil
7
4
2
Total
80
47
27
From Nakhimovskaia (628).
was found not to be identical. Some secreted water-soluble substances
into the medium, others did not. All the antibiotic agents were thermo-
stable, since heating for 30 minutes at 1.5 atmospheres only reduced
somewhat their activity. For those antagonists which did not excrete
any substance into the medium, the presence of the growing antagonist
was essential in order to bring about an inhibition of bacterial develop-
110 ACTINOMYCETES AS ANTAGONISTS
ment. On the basis of their sensitivity to the antibiotic substance of
actinomycetes, mycobacteria could be differentiated from nonspore-
forming, especially nodule-forming, bacteria. The production of the
antibiotic substance was highest in synthetic media and was rather weak
or even totally absent in media that contained proteins. The substance
was filterable and was able to resist the effect of radiation.
It was further reported (628) that the antagonistic effects of actino-
mycetes were manifested not only in artificial media, but also in soil, the
interrelations here being much more complex. Some of those strains
that produced antagonistic effects in artificial nutrient media were inef-
fective under soil conditions. The antagonistic action was more intense
in light podzol soils and was greatly reduced in heavy or chernozem
soils. One of the factors that resulted in a decrease in the antagonistic
properties of actinomycetes in the heavy soils was apparently the high
content of organic matter. By adding peptone to a light soil, the antago-
nistic action of the actinomycetes was greatly weakened. When actino-
mycetes were allowed to multiply in the soil before inoculation with
B. mycoidesy the antagonistic effect was highly pronounced even in the
presence of high concentrations of peptone.
An attempt to isolate an antibiotic substance from some of the soil
actinomycetes was made by Kriss (507). On the basis of its properties,
he was led to conclude that this substance could be classified definitely
with lysozyme. It was insoluble in ether, petroleum ether, benzol, and
chloroform, and was resistant to the effects of light, air, and high tem-
peratures. The optimum reaction for the production of this substance by
Streftomyces violaceus was found to be f¥L 7.1 to 7.8, the activity not
being increased by selective cultivation. On the basis of its properties,
this substance could hardly be classified with egg-white lysozyme. It
must be concluded also that the differences in the antibiotic properties
of the various antagonistic actinomycetes isolated by the Russian investi-
gators definitely point to the fact that more than one antibiotic substance
was involved.
In a more recent survey (936) of the distribution of antagonistic ac-
tinomycetes in soils and in composts, it was found that of 244 cultures
isolated at random from different soils, 49, or 20 per cent, of the cultures
were actively antagonistic j 57, or 23 per cent, showed some antagonistic
ANTAGONISTIC PROPERTIES
111
properties} and 138, or 57 per cent, possessed no antagonistic action at
all (Table 19). A somewhat similar distribution of antagonistic prop-
erties was observed among a group of well-identified species taken from
a type culture collection, embracing 161 pure strains. Only one of the
TABLE 19. ISOLATION OF ANTAGONISTIC ACTINOMYCETES
FROM VARIOUS SUBSTRATES
GROUP I
GROUP II
GROUP III
GROUP IV
TOTAL
Percent-
Percent-
Percent-
Percent-
SOURCE OF CULTURES
Cul-
age of
Cul-
age of
Cul-
age of
Cul-
age of
ORGANISMS
ISOLATED
tures
total
tures
total
tures
total
tures
total
Fertile, ma-
-
nured, and
limed soil
74
20
27.0
5
6.8
I
1-3
48
64.9
Infertile, un-
manured.
limed soil
75
I I
14.7
8
10.7
4
5.2
52
69-3
Potted soil
13
I
7-7
I
7-7
0
0
II
84.6
Potted soil, en-
riched with
E. coli
21
I
4.8
4
19.0
4
19.0
12
57.2
Potted soil, en-
riched with
mixtures of
bacteria
15
12
80.0
2
13-3
0
0
I
6.7
Lake mud
9
3
33-3
4
44.4
0
0
2
22.2
Stable-manure
compost
37
'
2.7
20
54.0
4
10.8
12
324
Total
244
49
20.1
44
18.0
13
5-3
138
56.6
From Waksman, Horning, Welsch, and Woodruff (936).
Note. The organisms in group I were the most active antagonists, those in groups II and III had more limited
antagonistic properties, and those in group IV showed no antibacterial effects with the methods used.
members of the genus Nocardia proved to be antagonistic j only one of
the Micromonospora forms was active. Most of the antagonists were
found among the members of the genus Streftomyces. These cultures
were also examined for bacteriolytic properties, living S. aureus being
used as the test organism. On this basis, 87 cultures (53.1 per cent)
112 ACTINOMYCETES AS ANTAGONISTS
were found to be inactive, 53 cultures (32.3 per cent) were moderately
active, and 24 cultures (14.6 per cent) were highly active. The conclu-
sion was reached (970) that bacteriolytic activities against killed bac-
teria and living gram-positive bacteria are widely distributed among
the actinomycetes. Growth-inhibiting properties of actinomycetes were
found to be significantly associated with bacteriolytic action upon living
gram-positive bacteria (Table 20).
TABLE 20. BACTERIOLYTIC AND BACTERIOSTATIC PROPERTIES OF
VARIOUS SPECIES OF ACTINOMYCETES
BACTERIOSTASIS
BACTERIOLYSIS
bacteriolysis
OF B. SUBTILIS
BY LIVING
BY BROTH
BY AQUEOUS
ORGANISMS
ORGANISMS*
FILTRATEf
EXTRACTS
Group I. Actinom
VCETES Hi
[GHLY Bacteriostatic to B. subtilis
S. antibiodcus
0
-
-H-
S. californicus
+
C
0
S. candidus
-H-
c,s
++
S. cellulosae
+
c
0
S. griseus (3326b)
+
c
0
S. lavendulae
+
c
++
S. reticuli
+
c
0
S. roseus
+
C
+
S. ruber
+
-
0
S. sap-ophyticus
-H-
c,s
0
S. scabies (3031)
-1-
c
0
Strefiomyces s^. (3069)
-H-
c
0
5. albus (G)
-H-
c,s
0
Streftomyces sp. (33
187)
-K-
c,s
0
N. gardneri
0
c
-H-
Micromonosfora sp.
0
-
-H-
Group II. Actinomycetes Moderately Bacteriostatic to B. subtilis
S. albus {^T,()\) -H- C, S o
S. cretaceus + c O
5. albus, var, ochraleuceus -H- C, S O
5. annulatus + — O
S. aureus -h CO
5. bovis -J- CO
S.f radii -H- C, S o
ANTAGONISTIC PROPERTIES
TABLE 20 {continued)
113
BACTERIOSTASIS
BACTERIOLYSIS
BACTERIOLYSIS
OF B. SUBTILIS
BY LIVING
BY BROTH
BY AQUEOUS
ORGANISMS
ORGANISMS*
FILTRATE t
EXTRACTS
Group II {contmued)
S. griseus
++
C,S
0
S. halstedii
+
C
0
S. hominis
++
C,S
0
S. lifmanii
+
c
0
S. mtcroflavus
+
c
0
S. odortfer
++
-
0
S. fraecox
+
c
0
S. rutgersensis
-H-
C,S
0
S. samfsonii
-H-
C,S
0
S. scabies (3352)
+
-
0
5". scabies (302 1)
-hH
c
0
5. setonii
-H-
c,s
0
S. tetanusemus
++
c,s
0
S. coelicolor (3033)
+
Not tested
Not tested
Streftomyces^'p. (Lleske,
No.
23) ++
c,s
0
Streftomyces sp. (Lieske,
No.
25a) 4-f
c
0
From Waksman, Horning, Welsch, and Woodruff (936).
* No activity against 5. aureus is indicated by o, moderate activity by +, high activity by ++.
t Lysis of heat-killed E. coli is indicated by C (high activity) and c (moderate activity); lysis of
living 5. aureus is indicated by S ; — indicates no activity.
Actinomycetes also show antagonistic activities against fungi (12-
14, 844). S. albusy for example, was capable of inhibiting the growth
of all species of fungi tested, an effect shown to be due to the production
of an active substance. By the use of a culture of Colletotrichum gloe-
osforioidesy the antagonistic activities of 80 type cultures of actino-
mycetes were measured. The antagonist was allowed to grow for 5 days
on maltose agar, at f¥L 7.4, and the fungus was then inoculated. The
cultures of actinomycetes were divided, on this basis, into three groups:
strong, weak, and noninhibitors. The first group comprised 17.5 per
cent of the cultures} the second, 38.8 per cent; and the third, 43.7 per
cent. These results are surprisingly similar to those reported for the
distribution of actinomycetes possessing antibacterial properties, includ-
114 ACTINOMYCETES AS ANTAGONISTS
ing those that were isolated at random from the soil and those taken
from a culture collection.
Meredith (595) made a survey of the distribution of organisms an-
tagonistic to Fusarium oxys forum cubense in Jamaica soils j most of
these antagonists belong to the actinomycetes. The antagonists were not
evenly distributed in the various soil samples, 10 of the (iG samples giv-
ing 44 per cent of the antagonistic organisms. Those actinomycetes that
were antagonistic to Fusarium when grown in their own soil-solution
agar were not always antagonistic when tested in soil-solution agar pre-
pared from other soil. A culture of actinomyces isolated from a compost
produced lysis of the Fusarium,. When spores of both organisms were
mixed in an agar medium, the fungus developed normally for two days
but began to undergo lysis on the fifth day, large sections of the my-
celium disappearing. On the seventh day only chlamydospores were ob-
served. In 9 days the fungus completely disappeared, the actinomyces
making a normal growth.
NATURE OF ANTIBIOTIC SUBSTANCES
It has already been established that antagonistic actinomycetes read-
ily produce a variety of different types of antibiotic substances. Some of
these have been isolated and even crystallized and information has been
gained concerning their chemical nature. Others have been obtained in
the form of crude but highly active preparations. Still others are known
but they have not been isolated as yet and have, therefore, been rather
insufficiently studied. So far, six substances have been definitely recog-
nized j namely, actinomycetin, actinomycin, streptothricin, streptomy-
cin, proactinomycin, and micromonosporin.
Among the various antagonistic actinomycetes, five species have
been studied in detail and, therefore, deserve particular attention,
namely, S. antibiotkus (945), Streftomyces lavendulae (973), Stref-
tomyces griseus (795), Nocardia gardneri (309), and 5. albus (970).
S. antibiotkus produces two highly active antibiotic substances that
have been isolated and described as actinomycin A and B. The first of
these has been studied in greater detail. It was shown to be antagonistic
to all species of bacteria tested as well as to many fungi, as brought out
NATURE OF ANTIBIOTIC SUBSTANCES 115
in Table 2 1 . The organism produces dark-brown to black pigments on
media containing protein and peptone. It is not affected by heat. It is
soluble in ether and in alcohol as well as in other solvents, but in water
only in very high dilutions. It is highly toxic to animals. Actinomycin
TABLE 21. BACTERIOSTATIC SPECTRUM OF ACTINOMYCIN
ACTINOMYCIN
ADDED, MILLIGRAMS
ORGANISM
GRAM STAIN
PER LITER OF MEDIUM
O.I
I.O
10
I GO
5. marcescens
-
3
3
3
A . aero genes
-
3
3
3*
E. coli (intermediate)
-
3
3
3*
E. coli
-
3
3
I*
Ps. aeruginosa
-
3
3
0
Ps. -fluorescens
-
3
3
0
Br. abortus
-
3
3
0
N. catarrhalis
-
3
2
0
E. carotovora
-
3
2
0
Sh. gallinarum
-
2
2
o
A . stutzeri
-
2
I
0
H. fertussis
-
3
0
0
Az. vinelandii
-
0
0
0
S. cellulosae
+
2
I
0
S. calif ornicus
+
3
2
0
M. tuberculosis
+
3
0
0
CI. welchii
+
0
0
0
B. macerans
+
3
o
0
B. megatherium
+
0
0
0
B. folymyxa
+
0
0
o
B. mycoides
+
o
0
0
B. mesentericus
+
o
0
o
B. cereus
+
o
0
0
B. subtilis I
+
o
o
0
0
B. subtilis II
+
o
0
0
0
G. tetragena
+
0
0
0
o
S. lutea
+
o
0
o
0
Streptococci and staphylococci
+
0
0
o
0
From Waksman and Woodruff (946).
Note, o indicates no growth; I, trace of growth; 2, fair growth; 3, good growth.
* rf 200 mg. per liter were added the results were usually as follows: for A. aerogenes, fair; for
E. coli (intermediate), trace; for E. coli, no growth.
16
ACTINOMYCETES AS ANTAGONISTS
is produced in both organic and synthetic media, the addition of a small
amount of agar increasing considerably the growth of the organism and
the production of the active substance. The addition to the medium of a
small amount of starch, as well as of phosphate and sodium chloride, was
also found to be favorable. S. antibioticus is strictly aerobic, and is able to
produce the active substance only under aerobic conditions that can be
brought about by growing it either in very shallow layers or in aerated
or agitated submerged growth. Actinomycin is extracted directly from
the medium by means of ether j the ether is then evaporated and the
substance taken up in alcohol. Further purification is accomplished by
means of petrol ether and passage through a chromatographic column,
as shown later (p. 171).
Streftomyces lavendulae is capable of inhibiting the growth of many
gram-negative bacteria as well as of various gram-positive forms. The
antibiotic substance produced by this organism was designated as strep-
tothricin. The organism is grown in a tap-water medium containing i .0
per cent glucose, 0.5 per cent tryptone, 0.2 per cent K0HPO4, 0.2 per
cent NaCl, 0.00 1 per cent FeS04, and 0.25 per cent agar. The glucose
TABLE 22. COMPARATIVE ACTIVITY OF TWO STRAINS OF S. LAVENDULAE
TREAT-
DAYS
GROWTH
IN MG.
ACTIVITY
IN UNITS
STRAIN
SOURCE OF
MENT OF
OF INCU-
PER 100 ML.
E.
B. sub-
NUMBER
NITROGEN
CULTURE
BATION
OF MEDIUM
colt
tilts
8
Tryptone
Shaken
2
346
150
1,000
H
Tryptone
Shaken
2
361
150
750
8
Tryptone
Shaken
5
253
100
1,000
H
Tryptone
Shaken
5
296
100
500
8
Glycine
Shaken
2
162
30
30
14
Glycine
Shaken
2
146
30
30
8
Glycine
Shaken
5
266
100
500
14
Glycine
Shaken
5
271
30
150
8
Tryptone
Stationary
8
245
20
200
14
Tryptone
Stationary
8
-
75
300
8
Glycine
Stationary
8
239
25
150
14
Glycine
Stationary
8
-
75
200
From Waksman (926).
Note. The organism was grown in i per cent starch medium.
NATURE OF ANTIBIOTIC SUBSTANCES 117
can be replaced by starch, in which case the presence of agar is unneces-
sary. When grown in submerged or agitated cultures, the agar is left
out. The tryptone can be replaced by a variety of simple nitrogenous
compounds, such as glycine, alanine, aspartic acid, asparagine, and glu-
tamic acid (Table 22)} the carbohydrate may be left out completely,
with only limited reduction in activity. No growth of the organism is
obtained on tryptophane, phenyl alanine, and certain other forms of
nitrogen. With ammonium sulfate or sodium nitrate good growth may
be obtained but the production of the active substance is limited unless
the organism is grown under submerged conditions. Iron appears to
play an essential role in the production of the active substance. An in-
crease in growth as a result of an increase in carbohydrate concentration
does not result in an increase in streptothricin content, but an increase
in growth as a result of an increase in the amino-acid concentration, with
the same amount of carbohydrate, causes an increase in the production
of streptothricin. When the medium contains one amino acid as the only
source of carbon and nitrogen, there is a gradual increase in the alka-
linity of the medium, resulting in the destruction of the streptothricin.
Neither the growth of the organism nor the production of the strepto-
thricin, however, is influenced by the reaction of the medium, within
certain limits, even between /)H 4.4 and 8.0 (lOOi). The metabolism
of S. lavendulae and the course of production of streptothricin under
stationary and submerged conditions are illustrated in Figure 1 1 . Meth-
ods of isolation of streptothricin and its chemical properties are de-
scribed later (p. 173). Its bacteriostatic spectrum or action against vari-
ous bacteria is shown in Table 23. It has only limited toxicity to animals
and is active in vivo against both gram-positive and gram-negative bac-
teria (755).
Certain strains of Streftomyces griseus produce an antibiotic sub-
stance, designated as streptomycin, that is also active against both gram-
positive and gram-negative bacteria. It is similar in its solubility and
various chemical properties to streptothricin j however, it acts readily
against B. mycoides and is more active than the latter against certain
gram-negative bacteria, such as Ps. aeruginosa. The organism grows
well in stationary liquid media containing meat extract or corn steep.
Streptomycin is active in vivo against a variety of bacteria, some of
1
7 ~l
zl
UJ,
1
:i:
1
■2.
<
1
O
I
J
/cf
o
/<
D
/ "
/^
Q
O
q;
/
/P
Q.
/
//? "
z
f
ho
o
W^
4
Q/
''»>^
\
I
>-^
\
1-
o
\\
1-
» \
a
UJ
v\
q;
\ \
(-
10
1 1 1
1 1
D
O
CM Z
asmnniiAj 2i3d s±iNn
3siniinD JO s^BinniiPN 99 2i3d si^vyonim
TABLE 23. INHIBITORY EFFECT OF STREPTOTHRICIN UPON GROWTH
OF VARIOUS BACTERIA
CRUDE STREPTOTHRICIN ADDED,
ORGANISM
MILLIGRAMS PER 10 CUBIC CENTIMETERS AGAR
3
I
0.3
O.I
0.03
O.OI
B. subtilis
0
0
0
0
0
I
B. mycoides
2
2
2
2
2
2
B. macerans
2
2
2
2
2
2
B. megatherium
0
0
0
0
I
2
B. folymyxa
0
0
2
2
2
2
B. cereus
2
2
2
2
2
2
M. lysodeikticus
0
0
0
I
2
2
S. muscae
0
0
0
I
2
2
S. lutea
0
0
0
0
I
2
A . aerogenes*
0
0
I
2
2
2
A . aero genes
0
0
0
Tr
2
2
E. coli-\
0
0
0
0
2
2
E. colt (4348)
0
0
Tr
I
2
2
5. marcescens
0
I
2
2
2
2
S. marcescens
I
I
2
2
2
2
Ps. fluorescensX
2
2
2
2
2
2
Sh. gallinarum
0
0
0
0
I
2
P. fseudotuberculosis
0
0
0
Tr
2
2
Br. abortus
0
0
0
0
2
2
S. cholerasuis
0
0
0
Tr
2
2
S. schottmillleri
0
0
0
I
2
2
S. abortivoequina
0
0
0
Tr
2
2
S. tyfhimurium
0
0
0
2
2
2
H. suis
0
0
0
2
2
2
H. influenzae
0
0
0
0
0
I
Br. abortus
0
0
0
0
2
2
Az. agile
0
0
0
0
0
2
Az. vinelandii
0
0
0
0
0
2
Az. chroococcum
0
0
0
Tr
2
2
Az. indicum
0
0
0
2
2
2
M. fhlei
0
0
0
I
2
2
CI. butyricum\
2
2
2
2
2
2
L. casei^
0
0
0
2
2
2
S. albus
0
0
0
I
2
2
S. violaceus-ruber
0
0
5. lavendulae
0
I
2
2
2
2
From Waksman and Woodruff (950).
Note, o indicates no growth; i, limited growth; 2, good growth; Tr, trace of growth.
* Representing 3 distinct strains.
t Representing 5 strains of £. co// obtained from different sources.
J Representing 4 strains.
§ Cultured anaerobically.
120 ACTINOMYCETES AS ANTAGONISTS
which, like Ps. aeruginosa, are rather resistant to streptothricin (460a,
795)-
A^. gardneri produces an active bacteriostatic substance which has been
designated as proactinomycin (309). Its bacteriostatic spectrum is
shown in Table 24. It is produced both on synthetic and organic media.
Its action is largely directed against gram-positive bacteria, although to
a more limited extent than that of actinomycin. Its isolation and chemi-
cal nature are brought out later (p. 175).
TABLE 24. BACTERIOSTATIC EFFECT OF PROACTINOMYCIN
APPROXIMATE DILUTION OF
MATERIAL IN MILLILITERS
ORGANISM GIVING HIGHEST EFFECT
D . fneumoniae 1,500,000
S.fyogenes 500,000
S. aureus 500,000
A^. meningitidis 500,000
B. anthracis 500,000
F. cholerae 6,000
5. tyfhiy S. paratyphi B, Shigella, E. coli 2,000
From Gardner and Chain (309).
S. alhus produces a bacteriolytic substance designated as actinomyce-
tinj it has been described on page 107. This substance is a protein and
is enzymatic in nature. It has not yet been isolated in a pure state. Its
lytic action was visualized by Welsch (971 ) as a two-step reaction: first,
the susceptible cells are killed by the selectively bactericidal lipoid j sec-
ond, those dead cells are dissolved by the bacteriolytic enzyme, which
alone is responsible for the lysis of heat-killed bacteria. The phenome-
non does not take place in complex culture-media, since the bactericidal
action of the lipoid is greatly impaired under those conditions j the pres-
ence of living actinomyces is generally necessary, since free lipoid
should be secreted in the susceptible suspension.
Wieringa and Wiebols (981) observed that certain actinomycetes
can produce lytic agents that are capable of exerting a lytic effect not
only upon the actinomycetes themselves but also upon other organisms.
ACTION AGAINST PLANT DISEASES 121
The formation of an autolytic substance by a thermophilic actinomyces
was also demonstrated (477, 502). The filtrates of such lysed cultures
were said to offer promise in the treatment of actinomycosis caused by
Actinomyces bovis (175).
Despite a seeming similarity in their growth characteristics, and de-
spite the fact that some investigators (349, 973) assumed that all ac-
tinomycetes are able to act as antagonists, it is now definitely established
(504, 945) that one is dealing here with highly specific types or even
strains. For example, an examination of many species for an active sub-
stance similar to actinomycin brought out the fact that only S. antibioti-
cus was capable of producing this substance. Although many other
forms yielded an ether-soluble substance that had some bacteriostatic
activity, it could not be compared in chemical nature and in biological
action with actinomycin (944, 946).
ANTAGONISTIC EFFECTS OF ACTI N O M YCETES
AGAINST AGENTS PRODUCING
PLANT DISEASES
Various species of Streftomyces are also strongly antagonistic against
bacteria causing plant diseases, such as Bacterium solanacearum (414).
According to McCormack (552), aerobic conditions are necessary for
the development of the antagonistic properties of actinomycetes j those
requiring less oxidized conditions are themselves antagonized. B. mega-
theriumy for example, was said to be antagonistic to certain species but
was antagonized by others. Ps. fluorescenSy however, was antagonistic
to actinomycetes as a whole, causing their lysis.
Actinomycetes possess antagonistic properties not only against bac-
teria but also against other actinomycetes (552), The more aerobic spe-
cies are antagonistic to the less aerobic types. Millard (604) believed
that he succeeded in controlling potato scab caused by Streftomyces
scabies by the use of green manures such as grass cuttings. The develop-
ment of scab on potatoes grown in sterilized soil and inoculated with
S. scabies was reduced by the simultaneous inoculation of the soil with
Streftomyces fraecoXy an obligate saprophyte ( 605 ) . By increasing the
proportion of the latter organism to the pathogen, the degree of scab-
122 ACTINOMYCETES AS ANTAGONISTS
bing on the test potatoes was reduced from lOO per cent to nil. The
sterilized soil provided sufficient nutrients for the development of the
antagonist and only a small increase in the control was obtained when
grass cuttings were added and sterilized along with the soil.
Sanford (782) was unable, however, to control potato scab by the
inoculation, with S. scabies and S. -praecoXy of both steam-sterilized and
natural soil containing different amounts of green plant materials.
These organisms were perfectly compatible on potato dextrose agar, as
well as in a steam-sterilized soil medium. The control of scab (605),
therefore, was said to have been due not to the direct action of S. fraecox
but to certain other undetermined microorganisms favored by the pres-
ence of the green manure. S. scabies was found (782) to be very sensi-
tive to various products of fungi and bacteria. When grown in close
proximity to various bacteria, the acid production of the latter inhibited
S. scabies to a considerable degree. Its complete inhibition was not due
to the acid reaction alone, however, since a certain bacterium was iso-
lated from the soil which definitely inhibited the growth of this plant
pathogen.
Goss (342) observed that the severity of scab is dependent on the
amount of S. scabies present in the soil, which was believed to be con-
trolled by the soil microflora. No evidence was obtained as to whether
the effect of the soil flora on S. scabies was due to specific organisms.
Kieszling (481 ) isolated two cultures of bacteria which were antagonis-
tic to S. scabies; when added to the soil, these bacteria prevented the
development of scab on potatoes.
The ability of antibiotic substances produced by actinomycetes to
exert a marked inhibiting effect upon plant pathogenic bacteria has been
established (930).
IN VIVO ACTIVITY OF SUBSTANCES PRODUCED
BY ACTINOMYCETES
Just as the chemical nature of the antibiotic agents produced by ac-
tinomycetes varies, so does the action of these agents in the animal body.
Some, like actinomycin, are very toxic, whereas others, like streptothri-
cin and streptomycin, have low toxicity and give great promise of prac-
IN VIVO ACTIVITY 123
tical application. Because of the activity of streptothricin and strepto-
mycin against gram-negative bacteria (460a, 597, 752) and because of
the lack of reliable chemotherapeutic agents active against these bac-
teria, the utilization of these substances in the treatment of certain dis-
eases caused by such bacteria becomes very significant (p. 243). Some
preparations, like actinomycetin, have been utilized in the preparation
of a bacterial hydrolysate (mycolysate) for vaccination purposes.
CHAPTER 7
FUNGI AS ANTAGONISTS
The antagonistic interrelationships in which fungi are involved com-
prise the following reactions: (a) the antibacterial activities of fungi j
(b) the antagonistic effects of fungi upon fungi; (c) the effects of bac-
teria and actinomycetes upon fungi j (d) the action of fungi upon in-
sects and other animal forms. From the point of view of practical utiliza-
tion, two aspects deserve special consideration: (a) the utilization of
fungi for combating human and animal diseases; (b) the antagonistic
interrelationships of fungi with other organisms, since fungi comprise
the most important group of microorganisms that cause plant diseases.
ANTIBACTERIAL EFFECTS OF FUNGI
Duchesne (207) was the first to report, in 1897, that certain green
Penicillia are capable of repressing the growth of various bacteria or of
bringing about their attenuation. Vaudremer (912) demonstrated in
19 13 that the presence oi A.fumigatus results in the attenuation of the
cells of M. tuberculosis.
Since these early studies a number of fungi have been found to pos-
sess antibacterial properties; this phenomenon has sometimes been
spoken of as mycophagy (914). Several fungi have been studied in de-
tail, and in some cases one or more antibiotic substances have been iso-
lated (Figure 12). The property of inhibiting the growth of bacteria is
not characteristic of any one genus or even species, but of certain strains
within a given species. Some organisms produce more than one active
substance. Two genera, Penicillium and Aspergillus, have been found
to comprise a large number of antagonistic forms. Several other genera
are also known to contain organisms that possess antibacterial proper-
ties; very few of these, however, were ever found among the Phycomy-
cetes and Basidiomycetes. The known fungi capable of producing anti-
biotic substances may be divided (934) into the following ten groups:
Aspergillus clavatus A . fumigatus
A. jiavus-ory'z.ae Penicilliwrn cyclofium-clavijorme
p. 7iofatu7fi, sporulating bodies
(X530). Prepared by Foster
P. notdtumy submerged growth
(X530). Prepared by Foster
! \ / t ~-~'!i'
W
P. c'ltrinum. From Bioiirge
(54a)
fft
^
% /■
/u. »
/ ;/// f
41
, A. clavatus. From Wehmer
(955)
P. chrysogenum. From Bfourgf
(54a)
A. fumigatus. From
Wehmer (955)
Figure 12. Some typical fungi producing antibiotic substances.
TABLE 25, ANTAGONISTIC EFFECTS OF SOME REPRESENTATIVE
FUNGI AGAINST BACTERIA
ANTAGONIST
ORGANISMS AFFECTED
ACTIVE SUBSTANCE
REFERENCES
A . clavatus
Gram-negative and gram-
Clavacin, highly
935>942, 982
positive bacteria
bactericidal
A . flavus
Streptococci, staphylo-
cocci, and certain gram-
positive bacteria
Aspergillic acid
461, 708,978
A . flaz'us
Mostly gram-positive
Flavicin, similar to,
100, 565, 929
bacteria
if not identical
with, penicillin
A . fumigatus
Gram-positive bacteria
Fumigacin, glio-
toxin
95,933>935
A . fumigatus
Various bacteria
Fumigatin, spinu-
losin
663
A . fumigatus
M. tuberculosis
Active filtrate
1015
and i4. albus
Chaetomium sp.
Various gram-positive
bacteria
Chaetomin
934
Gliocladium and
Various gram-positive and
Gliotoxin, highly
948
Trichoierma
gram-negative bacteria
bacteriostatic
P. citrinum
Various bacteria
Citrinin
714
P. claviforme
Gram-positive and gram-
negative bacteria
Claviformin
114, 115
P. notatum and
Mostly gram-positive and
Penicillin, active
3> 7, 79> i35>
P. chrysogenu7n
also certain gram-nega-
in vivo, low tox-
266,424,737,
tive (Neisseria, Gono-
icity
934
coccus) bacteria
P. notatum
All bacteria tested, in
Notatin, penatin,
59, i5i,493>
presence of glucose
penicillin B,
E. coli factor
494> 751.934
P. fuberulum
Various bacteria
Penicillic acid
56, 57,661,
and P. cyclofium
664
P. resticulosum
Various bacteria
Crude metabolic
product
58
Pe7iicilliu7Ti sp.
Gram-negative as well as
gram-positive bacteria
Penicidin
26
126
FUNGI AS ANTAGONISTS
Penlcillium luteum-furfurogenum
Pemc'ilUumnotatum-chrysogenimi
Tr-ichoderma-Gliocladium
Fusarium-Cefhalosforiufn
Chaetormum and other Ascomycetes
Miscellaneous other fun2:i
A comparative study of a number of fungi taken from a culture col-
lection brought out (986) the fact that about 40 per cent of the Asper-
gilli {Aspergillus fumarkuSy A. jum4gatusj Aspergillus schiemannii,
Aspergillus terreus) and 25 per cent of the Penicillia (Penicillium
chrysogenum, Penicillium daviforme, Penicillium funiculosum, Peni-
cillium exfansum) possessed antagonistic properties. Out of many
Phycomycetes tested, only Phythophthora erythroseftica showed some
activity. A few Ascomycetes were also found to be active. A summary
of the antibacterial properties of various fungi and of the antibiotic
substances produced by them is given in Table 25. In addition to the
specific strain of the organism, the composition of the medium and the
conditions of growth, especially aeration, are most important in con-
trolling the amount and nature of the antibiotic substance produced by
the organism, as shown in Table 26. The fact that different strains of
TABLE 26. EFFECT OF AERATION, AS ILLUSTRATED BY DEPTH OF MEDIUM,
ON ANTIBACTERIAL ACTIVITY OF SEVERAL FUNGI
VOLUME OF
MEDIUM PER
. ACTIVITY IN UNITS
ONE-LITER
FLASK,
E.
B.
B.
CULTURE
m MILLILITERS
coli
mycoides
subtilis
S. lutea
C haetomium sp.
100
0
20
20
600
A. fumigatus 20
100
0
300
150
800
A. fumigatus 20
300
0
300
60
800
A. fumigatus 84
100
0
600
300
>i
,000
A. fumigatus 84
300
0
300
100
>i
,000
P. luteum 1 08a
100
0
0
0
0
P. luteum 1 08a
300
0
0
20
10
P. notatum F
100
0
3
15
P. notatum F
700
10
10
>IOO
P. notatum W
100
0
45
70
70
P. notatum W
700
100
80
450
150
From Waksman and Horning (934).
Note. Cultures were incubated 5 to 6 days at 28° C.
ANTIBACTERIAL EFFECTS
127
the same organism when grown under identical conditions vary greatly
in the production of the antibiotic substance is brought out in Table 27.
PenicilUum notatum-chrysogenum Grouf
Because of the production by these organisms of penicillin, which has
already found a wide practical application, this group of fungi deserves
first consideration. Fleming (265) first observed that a fungus culture
growing on a staphylococcus plate brought about destruction of the bac-
teria, as shown by the fact that the colonies became transparent and
were undergoing lysis. The fungus was isolated in pure culture and was
later identified as P. notatum. It was found to possess marked bacterio-
static and bactericidal properties for some of the common pathogenic
bacteria, largely the gram-positive cocci and the staphylococci, the strep-
tococci, the diphtheria organism, and the gonococci and meningococci j
TABLE 27, PRODUCTION OF CLAVACIN BY FIFTEEN STRAINS OF
ASPERGILLUS CLAVATUS
ACTIVITY OF
5 -DAY-OLD
STRAIN
CULTURE IN
NUMBER
fYl OF MEDIUM
E. COLI UNITS
CLAVACIN
ISOLATED
5
14
Yield in
Activity, E. colt
days
days
grams per liter
units per gram
120
6.5
8.4
0
0.016
25,000
121
4.2
6.7
75
1.442
1,000,000
122
4-5
8.0
0
0.035
4,000
123
4.6
4-5
20
0.467
120,000
124
6.2
8.4
0
0.016
8,000
125
3.2
3-9
0
0.248
600
126
6.3
8.2
0
0.039
20,000
127
7.4
8.1
0
0.007
8,000
128
6.7
8.0
0
0.017
7,000
129
3-6
6.8
100
0.950
400,000
I29T
6.6
5-9
20
0.512
80,000
130
4.8
4-7
10
0.323
500,000
I30T
6.9
7-9
10
0.050
4,000
131
6.9
7.8
10
0.035
5,000
164
4.3
4.6
30
0.430
1,000,000
From Waksman and Schatz (942).
Note. Eight-day-old culture was used for extraction of the clavacin.
128 FUNGI AS ANTAGONISTS
bacteria belonging to the colon-typhoid-dysentery group were not af-
fected. The culture filtrate of the fungus was found to contain an active
substance, which was designated as penicillin.
A glucose-nitrate solution was used as the basic medium for the pro-
duction of penicillin. It was supplemented with yeast-extract or corn
steep liquor, or brown sugar was employed in place of glucose j the
growth of the organism and the production of the antibacterial sub-
stances were thus greatly facilitated (7, 282, 804). The reaction of the
medium was found to change from slight acidity initially (^H 6 to 7)
to distinct acidity (^H 3.0), followed later by alkalinity, finally reach-
ing a /)H of 8.0 or even 8.8. A faint to deep yellow color is produced in
the medium. Penicillin production is usually at its maximum at about
-pH. 7 and may remain constant for several days or may fall again rap-
idly. Aerobic conditions are essential for the formation of penicillin.
Once a fungus pellicle has been produced, the medium can be replaced
several times, giving fresh lots of penicillin in about half the time re-
quired during the initial growth period. Crude penicillin cultures are
capable of inhibiting the growth of staphylococci in dilutions of i : 800 j
recently, even more active preparations were obtained.
There is considerable variation in sensitivity to penicillin among bac-
teria belonging to the same group : 27 strains of enterococci and 6 strains
of S. lactis were shown to be resistant to the action of this agent, whereas
13 strains of S. viridans were susceptible (79). The ability of a strain
to resist the action of penicillin can be greatly increased by successive
transfers of the culture to media containing this substance (564, S^^)-'
Chain et al. (113) were the first to succeed in isolating from the cul-
ture medium of P. notatum a water-soluble, stable, brown powder
which had marked antibacterial activity. This preparation inhibited, in
dilutions of i to several hundred thousand, the growth of many aerobic
and anaerobic bacteria. The active material was relatively nontoxic to
laboratory animals. Intravenous and subcutaneous injections of 10 mg.
or more to mice had little or no effect. The material was active m vivo,
subcutaneous injections saving the lives of mice injected intraperitone-
ally with S. pyogenes or S. aureus. Intramuscular infections of mice
with CI. sefticum were also successfully treated by repeated subcutane-
ous injections of penicillin.
■i)3innnikM asd siNvaomuM ni avons ~ivnais3a
^ O (D (M
(\j rvj - - CO ^ o
1 1 1 1 1 1
Hd
1
o
en TO r- vo tf^ ^ n
'
1 1 1 1 1 1 r
fM
f X o o o 1
i/ ' !
/ /
/ \ <-/
/ < ,?/
/ -ij Eh i-
<n
/ \ */ <'■
/ \ >; w/
CO
5-
( v\ ^1 1/'
^
2
8
5S
a
O
1-
X,^X-^^^0 O^O
<
\ ^^ /
D
a
z
\ /\ ^\^
V-' A ^
X > \ ^-
■n
1- \
/■ ' \
/ '' \
/ / \
1
1 1 ./ I \ I \ 1 \
5;
nj o <D (o ^j fU o 1
2i3xnmiiN a3d siiwn A3aoij ni NniioiN3d
1 1 1 1 1
•o •^ (t) rvj - c
J
SlAlvaO NI 3-10ni3d JO XH0I3M K&Q
130 FUNGI AS ANTAGONISTS
An extensive literature soon began to accumulate on the production
(5, 117, 118, 164, 282, 332, 422), isolation (113, 469), and identifica-
tion (6, 8) of penicillin. The course of its formation in the culture of
the organism is illustrated in Figure 13. Conditions of nutrition were
found to be particularly important. Preparations having an activity of
2,000 Oxford units or 100,000,000 dilution units have been obtained.
The importance of the dual nature of P. notatu?n (the culture being
composed of two distinct cell constituents) must be recognized for maxi-
mum penicillin production (34, 376). The low toxicity of penicillin, its
solubility in water, and its in vivo activity make it an ideal agent for
combating disease caused by gram-positive bacteria (p. 232).
In addition to true penicillin, P. notatum was found to produce an-
other substance, which in glucose-containing media is active against not
only gram-positive but also gram-negative bacteria. It was designated
as the E. colt factor, penatin, notatin, and penicillin B (p. 179).
P. notatum represents an extremely variable group of organisms,
some of the strains producing considerable penicillin, others producing
little penicillin but large amounts of notatin. Some strains of a closely
related fungus, P. chrysogenum, are also capable of producing peni-
cillin that is apparently the same as the penicillin of P. notatum. The
P. notatum-chryso genum group of fungi is widely distributed in nature,
having been isolated from different soils (919) and from various moldy
food products J however, only a few strains produce enough penicillin
to justify their use for the commercial production of this substance
(732). Members of the A. flavus group of fungi, as well as strains of
A. niger, Aspergillus nldulans, A. oryzae, Penicillium citreo-roseum
(282), i4. gtganteus (688), yl. parasiticus (142), and others, are also
capable of producing penicillin or closely related compounds.
Among the other fungi that produce antibiotic substances largely ac-
tive against gram-positive bacteria may be listed Aspergillus flavifes
(976), Chaetomium cochliodeSy and others.
Certain species of Penicillium are also capable of producing other
antibacterial substances, namely, citrinin, penicillic acid, and claviformin
(p. 181), the first of which is also produced by certain species of Asper-
gillus belonging to the candidus group ( 883) .
ANTIBACTERIAL EFFECTS 131
Atkinson (26) tested 68 cultures of Penicillium and found that 18
possessed antibacterial properties. These cultures were divided into
two groups : first, those largely active against gram-positive bacteria and
producing substances like penicillin and citrininj second, those active
also against gram-negative bacteria and producing substances of the
penicillic acid and penicidin types.
Asfergillus jlavus-oryzae Group
The A . oryzae members of this group possess only limited antagonis-
tic properties. Many of the A . flavus strains, however, have apparently
the property of producing at least two antibacterial substances when
grown on suitable media and under suitable conditions.
White and Hill (978) isolated from cultures of a strain of this or-
ganism grown on tryptone media a crystalline substance, aspergillic
acid, that showed antibacterial activity against certain gram-negative as
well as gram-positive bacteria. The substance was produced when the
organism was grown on organic media, but not on synthetic. It was
soluble in ether, alcohol, acetone, or acetic acid, but not in petroleum
ether J it was soluble in dilute acid or alkaline aqueous solutions, and
was precipitated by phosphotungstic acid. Aspergillic acid proved to
have relatively high toxicity, and showed no protective action against
hemolytic streptococci or pneumococci infections in mice.
Glister, in England, isolated a strain of A. flavus (330) that also
produced an antibacterial agent with a wide range of activity, both
gram-positive and gram-negative bacteria being inhibited by the culture
filtrate. An extract was obtained that inhibited the growth of these bac-
teria in a dilution of approximately i : 200,000.
Jones, Rake, and Hamre (461 ) demonstrated that A . flavus of White
produces frequent variants 5 two strains were isolated and were found
to give consistently far higher yields of the antibiotic substance, asper-
gillic acid, than those reported by White. The substance was found to
have wide activity, being very active against gram-positive cocci and less
active against the anaerobes of gas gangrene and the gram-negative ba-
cilli. No significant differences were found in the spectrum of activity as
shown by filtrates or by solutions of purified aspergillic acid.
132 FUNGI AS ANTAGONISTS
Bush and Goth (lOO) isolated from A. flavus a second substance
designated as flavicin. They grew the organism for 6 to 8 days on a
nitrate-glucose medium containing 2 per cent corn steep. The filtrate
was acidified to fH 2.5 to 3.0 with 5 |j phosphoric acid and extracted
with purified isopropyl ether. The ether was treated with a slight ex-
cess of o.2A^ NaHC03 (5 to 10 cc. per liter of culture), giving a yield
of 75 to 100 per cent of active material obtained. Purification was ob-
tained by acidification of the NaHCOg extract with 5 [i H3PO4 to fH
2 to 3 and removal of the precipitate, the latter containing most of the
toxicity (due no doubt to aspergillic acid) and the filtrate most of the
activity. The filtrate was treated with ice-cold isopropyl ether, satu-
rated with CO2, washed with cold distilled water, and reextracted. The
combined extracts were distilled at 0° C. to dryness under CO2. A yel-
low-orange glassy residue was obtained. It had a low toxicity and was
active in vivo. In its properties it resembled penicillin.
McKee and MacPhillamy (56s, 56^) further established, by chemi-
cal isolation and composition, solubility and stability, biological behav-
ior, low toxicity to animals, and therapeutic activity, that the second anti-
biotic substance produced by A . flavus is similar to penicillin. A sodium
salt, assaying 240 O.U./mg. was obtained chromatographically and
gave the following composition : 45.36 per cent C, 4. 1 6 per cent H, 3.02
per cent N, and 13.36 per cent Na, [ajo = + 108° C. (in water).
Waksman and Bugie (929) have shown by means of the bacterio-
static spectrum that, under submerged conditions, diflFerent strains of
A. flavus produced two substances, one comparable to aspergillic acid
and the other to penicillin. Some strains produced little or no activity
in submerged cultures, and most strains produced very little activity in
stationary cultures. No activity was produced in synthetic media. The
production of gigantic acid by a species of A . giganteus and parasiticin
hy A. -parasiticus (142) appears to be comparable to that of flavicin.
Aspergillus jumigatus Group
Four antibacterial substances were isolated from strains of A . jumi-
gatus: the two pigments, spinulosin and fumigatin {66"^), which are
not selective in their action against bacteria, the colorless fumigacin
ANTIBACTERIAL EFFECTS
133
that Is active largely against gram-positive organisms (935) and glio-
toxin (331, 593). Helvolic acid, isolated from a strain of A. fumigatus
(116, 155) was found (593, 933) to be identical with purified fumi-
gacin.
Fumigacin is active against S. aureus in dilutions of i : 200,000 to
1:750,000 and is very stable. The pigment fumigatin, however, was
said to deteriorate on standing, inhibition of S. aureus being reduced
from 1 : 50,000 to 1 125,000 in 7 days. Fumigacin has a certain degree
of resistance to high temperatures. Boiling in aqueous solution for 5 to
10 minutes reduced but did not destroy completely its activity. Heat-
ing at 80° C. for 15 minutes reduced the activity only slightly. When
fumigacin was dissolved in alcohol and precipitated by addition of nine
volumes of water, the alcohol-water solution was found to contain 0.25
mg. per ml. A comparison of the antibacterial activity of fumigacin with
that of the other substances produced by A. fumigatus is given in
Table 28.
TABLE 28. CHEMICAL PROPERTIES AND BACTERIOSTATIC ACTIVITY OF FOUR
ANTIBIOTIC SUBSTANCES PRODUCED BY ASPERGILLUS FUMIGATUS
MELTING
CRYSTALLI- POINT
SUBSTANCE ZATION ° C. FORMULA
BACTERIOSTATIC ACTIVITY
IN DILUTION UNITS
B. sub-
E. coli S. aureus tills
Spinulosin Purplish-bronze
plates 201 CgHgOg
Fumigatin Maroon-colored
needles 116 CgHgO^
Fumigacin Very fine white
needles z 15-220 CgsH^^Og
Gliotoxin Elongated
plates 195 C13H14O4N2S2 6,000 1,500,000 750,000
1,200 200,000 40,000
1,200 2,000,000 100,000
Vaudremer (912) reported that a group of patients suffering from
tuberculosis were treated with extracts of A. jumigatuSy with varying
degrees of success. The disease-producing organism (M. tuberculosis)
was rendered nonpathogenic by such treatment.
134 FUNGI AS ANTAGONISTS
Asfergillus davatus Growp
This comprises a number of strains that produce highly active anti-
biotic substances. By treating the culture filtrate with charcoal and
eluting the active substance with ether, Wiesner ( 982 ) obtained a prepa-
ration having a bactericidal potency in dilutions of i : 100,000. This ac-
tivity was not inhibited by serum, pus, or urine j strains of bacteria that
proved to be resistant to sulfonamides or mandelic acid were inhibited
by this material.
The active substance was designated (935) as clavacin. A detailed
study was made of its production by a variety of strains of A. dava-
tus. The substance was found to be active against E. coli and other
gram-negative bacteria, as well as against gram-positive bacteria.
It is distinct, in this respect, from fumigacin. Whereas the latter acts
much more readily upon B. mycoldes than B. subtilisy clavacin shows
the opposite effect — greater activity against B. subtilis than against B.
mycoldes. Clavacin possesses a high bactericidal action, as compared
with other antibiotic substances. It has been suggested (942) that the
marked differences in the physiology of the different strains of A. da-
vatus explain the differences in the production of clavacin by different
strains. Those that change the reaction of the medium to alkaline, for
instance, tend to inactivate the clavacin (Table 27).
Since clavacin is produced by several fungi, this substance has re-
ceived a number of designations, including patulin produced by Pen'i-
cillium fatulum (713), claviformin by P. davijorme (114, 115), and
clavatin (47). It is also produced by strains of P. exfansuniy A. gigan-
teus, GymnoascuSy and other fungi (470).
Trichoderma and GUodadium Grouf
Certain strains of fungi of the genera Trichoderma and GUodadium
were found to exert a marked antagonistic action against various fungi
and bacteria. An antibiotic substance designated as gliotoxin was iso-
lated and found (962) to be highly bactericidal. In order to produce
this substance, the fungus is grown in a submerged condition in shake-
cultures. An abundant supply of oxygen and a high acidity {f¥L 5.0 or
lower) are essential. Ammonium salts as nitrogen sources give better
ACTION AGAINST FUNGI 135
results than peptone or nitrates. Glucose and sucrose were found to be
good carbon sources. It is of particular interest to note that whereas
penicillin and flavicin are produced in media containing complex or-
ganic materials as sources of nitrogen, fumigacin, clavacin, and glio-
toxin are produced in synthetic media, the presence of complex nitrogen
sources often being deleterious.
Gliotoxin was isolated from the culture filtrate by the use of lipoid
solvents, chloroform being most effective. Nonsterilized media ad-
justed to /)H 2.5 to 3.0 could be used for large-scale production, the high
acidity reducing the effect of contaminants (966). Gliotoxin is stable in
neutral and acid solutions at room temperature j at alkaline reactions,
it is very unstable, the rate of decomposition increasing with increasing
alkalinity and temperature. At -pH 2.4, heating to 122° C. for 30 min-
utes did not affect the active substance. With decreasing acidity, espe-
cially at -pH 5.0, it became less thermostable. As pointed out above,
gliotoxin is also produced by A. jumigatus (593).
Other Groups
Various other fungi, including A. albus, A. niger, and Monilia albi-
cans, were found (1015) to exert a marked antibacterial action against
human and bovine tubercle bacteria j active filtrates were obtained, but
the specific agents were not isolated. The nature of the antibiotic sub-
stances produced by the other groups listed above has not been suffi-
ciently studied.
ANTAGONISTIC ACTION OF FUNGI
AGAINST FUNGI
Numerous fungi were found to exert antagonistic effects either
against fungi belonging to the same species (64, 87, 152, 304, 547, 578,
801, 851) or against other fungi (Table 29). This phenomenon is par-
ticularly important in connection with the study of plant diseases ( 1 60,
225,644,695,738, 815, 851,852,959, loii). The effects are selective
in nature. The hyphae of Peziza will kill various Mucorales, whereas
different species of Aspergillus and Penicillium are able to kill Peziza
136
FUNGI AS ANTAGONISTS
(738). A single spore of P. luteum was found (955) capable of germi-
nating in cultures of CitromyceSy and of bringing about their destruc-
TABLE 29. ANTAGONISTIC INTERRELATIONSHIPS AMONG
DIFFERENT FUNGI
ANTAGONIST
Acrostalagmus sp.
A Itemaria tenuis
A. clavatus
A . flavus
A . niger
Botrytis allii
Botrytis cinerea
C efhalothecium roseum
Cunninghamella elegans
Fusarium lateritium
Fusarium sp.
Gliocladium sp.
H elmifUhosforium sp.
H. teres
H. sativum
Mucor sp.
Penicillium sp.
Penicillium sp.
Peziza sclerotiorum
Peziza trifoliorum
Sclerotium rolfsii
SterigVMtocystis sp.
Thamnidium elegans
Torula suganii
Torulofsis sp.
Trichoderma lignorum
T. lignorum
Verticillium sp.
ORGANISMS AFFECTED
Rhizoctonia
Ofhiobolus
Various fungi
Peziza
Peziza, Rhizoctonia
Monilia, Botrytis, etc.
Rhizoctonia
H elminthosforium
Monilia
Rhizoctonia
Deuterofhoma
H elmiiithosforiuTn, Mucor, etc.
CoUetotrichum, Fusariu?n,
Botrytis, etc.
Fusarium, Ustilago, Helmintho-
sforium, etc.
Ofhiobolus
Ofhiobolus, Mucor
Peziza, Rhizoctonia, etc.
Ofhiobolus, Fusarium., etc.
Mucor, Trichothecium, Dematiu?n,
etc.
Peziza
H elminthosforium
A Itemaria
Mucor
Asfergillus, Monascus, etc.
Blue-staining fungi
Rhizoctonia, ArTuillaria, Phy-
tofhthora, etc.
Rhizoctojiia, PythiuTn, etc.
Rhizoctonia
REFERENCES
964
88
928
738,911,964
911
964
353
911
964
790
695
695
695
88, 801
738
88,955
738
738
695
695
801
654
592
962-964
17, 60, 91,
911
964
From Novogrudskjr (644).
ACTION AGAINST FUNGI 137
tion. P. luteum-furpurogenum produces a thermostable substance, solu-
ble in ether and in chloroform, that is antagonistic to the growth and
acid production of A. niger (668). Coniofhora cerehella was inhibited
by a species of Penicillium, its mycelium being considerably modified j
however, in time the former organism adapted itself to the latter and
overgrew it, its rate of growth being eventually more rapid than that
of a pure culture (377). Certain fungi are able to parasitize other fungi:
Pi ftocef halts, for instance, attacks various species of Penicillium and As-
fergillus (176). The germination of the spores of one fungus may be
reduced by the presence of spores of another (519).
Different fungi produce different types of fungistatic and fungicidal
substances, which may be stable or unstable in nature. These are formed
particularly by the lower fungi or the molds, with the exception of the
Phycomycetes that have so far not been found to produce any antibiotic
substances. Their action consists in modifying or killing the mycelium
of the other fungus, or merely in preventing spore germination. Brom-
melhues (88), studying the effects of H. sativum and Penicillium sp.
against Ofhiobolus graminis, emphasized that the inhibitory action was
due to a toxic substance, thermostable in nature and diffusible in agar.
In some cases, no relation could be observed between the acidity pro-
duced by one organism and its ability to influence the growth of another
( lOi I ) j in other cases, as in the mutualistic effects of Sclerotium rolfsii
and Fusarium vasinjectum, the first overgrew completely the second at
^H 6.9, whereas in alkaline ranges the reverse took place (766).
Random isolations oi Penicillium cultur&s and of other soil-inhabiting
fungi were tested for their effects on the virulence of H. sativum on
wheat seedlings grown in steam-sterilized soil (785). Some forms ex-
erted a marked degree of suppression, some had no effect, and others
increased the virulence of the pathogeny marked variations in activity
were observed among the different species of Penicillium. Because
Hyphomycetes were found to be capable of parasitizing the oospores of
Pythium (184), Hyphomycetes were believed to serve as effective
agents in promoting soil sanitation. Various species of Torulopsis, in
addition to certain bacteria, are capable of inhibiting the growth of
Dematiaceae, fungi that cause the blue staining of wood pulp (592).
Certain fungi may affect the reproduction of others. Melanos-pora
13S FL*NGI AS ANTAGONISTS
pampeana^ for example, normally does not form any peritheda in cul-
ture but is able to do so in the presence of Basis porium gallarum or
Fiisjriuf77 momliforme. This effect was ascribed to a special substance
that resists heating at i io~ C. Different fungi have a special influence
on the germination of spores of various ascomycetes and of other fungi
(25, 742), these effects being characteristic of the antagonists.
The edible mushroom, PsalUota campestris^ exerts a deffnite antago-
nism against the parasitic fungus My co gone (124). This phenomenon
has been looked upon as a case of antibody formation. %>edes of Fusor-
r'lum are able to antagonize the mushroom fungus \ however, an actively
growin-g culture of the latter may become antagonistic to the former
(998). In the destruction of paper pulp by fungi, a marked antagonism
was shown {^I'l) ^^ take place between different organisms, especially
h\ Trichoderma Ugnorum against various species of Fusarium and other
fungi, as illustrated in Figures 14 and 15.
Certain spedes of Trichoderma and Gliodadium are able to inhibit
the growth of various plant pathogenic fungi, especially R. solani, as
well as of Blastomycoides derm-atitisy a causative agent of human skin
diseases (962—964). The active substance, gliotoxin, is liberated during
the early stages of growth. The mycelium of older cultures contains an-
other substance that is soluble in acetone j this has only an inhibiting ef-
fect and is not fungicidal as is gliotoxin. The fungicidal effect of glio-
toxin upon the germinating sp>ores of Sclerotwla americana and hv'phae
of R. solani was found to be greater than that of CUSO4 and less than
that of HgCU.
\'arious other fungi are able to exert antagonistic effects against plant
pathc^ns. T. lignorum and A . mger restricted the growth of the fungi
Macrophomina phaseoU and R. solani^ which produce cotton root rot,
and reduced the activity of the filtrates of the pathogens causing wilting
of the plants C911).
Satoh (788) has shown that Ophiobolus miyaheanus produces both
growth-promoting and growth-retarding substances, the first of which
is heat stable and passes through a Chamberland filter j the second is
inactivated at ICX)^ C. and does not pass through a filter. The formation
of two substances by Torula suganiiy both of which were thermostable,
however, was also demonstrated (654).
%..
FiGLTRE 14. Antagonistic etirect ot one tungus, P$. ■zmsatum (in center),
upon another, T. Ugnarttm. From Gofdaniich et al. C333).
FiGLTiE 15. Attack of an antagonstic i
fundus, F. sambiicinttm (in center). Frc
•w, upon anodier
(333)-
ACTION OF BACTERIA AGAINST FUNGI
139
ANTAGONISTIC EFFECTS OF BACTERIA AND
ACTINOMYCETES AGAINST FUNGI
Various bacteria and actinomycetes have marked selective fungistatic
and fungicidal effects (Table 30). Bacteria active against U. zeae were
isolated (37) from corn, these bacteria being capable of destroying the
colonies of the smut fungi. The widespread distribution of such bacteria
in the soil was believed to check the multiplication of the pathogenic
fungi. Four types of bacteria antagonistic to smuts and to certain other
fungi have been described (456). Some of these bacteria produce en-
zymes that are able to dissolve the chemical constituents of the cell
walls of the fungus sporidiaj they were also found to be active in the
TABLE 30. ANTAGONISTIC EFFECTS OF BACTERIA AGAINST FUNGI
ANTAGONIST
ORGANISMS AFFECTED
REFERENCES
Achromobacter sp.
Fusarium, Sclerotinia
130
Al. faecalis
H elminthosforium
695
Bacillus «Z)»
Ustilago, Penicillium
37
B. anthracis
S. cerevisiae
496
B. mesentericus
H elminthosforium
128,695
B. mycoides
H elminthosforium
695
B. simplex
Rhizoctonia
149
B. subtilis
Cefhalothecium, roseum
13
Bacterium sp.
Fusarium^ Sclerotinia, etc.
695
Bacterium sp.
Ustilago
456
Bacterium sp.
A Iternaria
231
Myxobacterium
Ustilago
248, 456
P. vulgaris
Basisforum, Phytofhthora, etc.
485,695
Ps. aeruginosa
Saccharomyces
496
Ps. juglandis
Dothiorella
248
Ps. fhaseoli
Fusarium
60, 248
Ps. translucens
Ofhiobolus
86
Ps. vulgaris
Ophiobolus
86
S. marcescens
Beauveria, etc.
12, 13, 14, 587
M. tuberculosis
Pythium
910
Spore-forming bacteria
Fungi
37,231,695,
734,738
From Novogrudsky (644).
140 FUNGI AS ANTAGONISTS
soil against the specific fungi. Brown (92) observed that H. sativum
and a certain bacterium produced thermostable mutually inhibiting
substances. The bacterium as well as its metabolic products inhibited the
growth not only of the particular fungus but also of other members of
the same genus, but not of Fusarium conglutinans. These bacteria pro-
duced a diffusible agent that inhibited the growth of H. sativum (108).
The active substance was not destroyed by autoclavingj it diffused into
fresh agar and water, producing "stale water" that was inhibitory to
the fungus.
Chudiakov (130) isolated from the soil two bacteria that were capable
of bringing about the lysis of different species of Fusarium as well as
other fungi. These bacteria were found to be widely distributed in most
soils j they were absent, however, in flax-sick soils, in spite of the abun-
dance of Fusarium. When this fungus was added to the soil containing
antagonistic bacteria, it did not develop, and the plants did not become
diseased. The antagonistic action of a variety of other bacteria against
plant pathogenic fungi has been definitely established, as in the case of
B. simflex against Rhizoctonia, P. vulgaris against Phytofkthora
(472), and B. mesentericus against H elminthosforium (128). B. sim-
flex was grown (475) for 7 days at 28° C. in potato-dextrose medium
containing i per cent peptone, and the active substance was removed by
charcoal and dissolved in alcohol. Different fungi differed in the de-
gree of tolerance to this substance. The majority were repressed by 10
per cent concentration of the stale medium added to fresh medium.
The ability to produce a thermostable substance toxic to the plant-
disease-producing fungus Rhizoctonia is widespread among spore-form-
ing bacteria. The toxic substance is insoluble in ether, chloroform, and
benzol, but is soluble in ethyl alcohol. It passes through collodion,
cellophane, and parchment membranes. It is readily destroyed on boil-
ing in alkaline media but is more resistant in acid media.
Nakhimovskaia (629) found that various bacteria are able to inhibit
the germination of rust spores. Nonspore-forming bacteria, such as Ps.
fuorescens and S. marcescenSy prevented the germination of the spores
of Ustilaga avenaey Ustilaga hordeiy Ustilaga nuda, and Ustilaga reae.
Spore-forming bacteria, including B. m-ycoides and B. mesentericus, as
well as sarcinae {S. ureae, S. lutea), exerted no antagonistic action on
ACTION OF BACTERIA AGAINST FUNGI
141
the rust spores. The presence of these bacteria, however, Influenced the
nature of the germination of the spores, which gave rise to mycelium-
like forms with great numbers of copulating filaments, whereas in the
control cultures yeast-like forms prevailed and copulating cells were
rarely encountered. The presence of a certain concentration of bacterial
cell substance was essential to this antagonistic effect. With a more lim-
ited amount of cell material, the bacteria ceased to inhibit the germina-
tion of the spores but influenced the germination process in the same
manner as do nonantagonistic bacteria, that is, they stimulated the sex-
ual process. An increase in concentration of cell substance, even of non-
antagonistic organisms, would inhibit spore germination.
The common occurrence of the fungus Pyronema confluens in freshly
burned-over soils, but not in natural soils, was explained ( 645 ) as due
to the destruction of the bacterial antagonists by heating of the soil. Ps.
fuorescens was particularly effective as an antagonizing agent. A com-
parative study of the fungistatic action of substances of bacterial origin
(855) has shown these to be more active than common disinfectants.
Tyrothricin inhibited the growth of animal pathogens in dilutions of
1:5,000 to 1:20,000, pyocyanin in 1:2,000 to 1:5,000, and hemi-
pyocyanin in i : 20,000 to i : 60,000.
Actinomycetes may also exert a marked depressive effect upon the
TABLE 31. FUNGISTATIC AND FUNGICIDAL ACTION OF ANTIBIOTIC
SUBSTANCES UPON CERATOSTOMELLA ULMI
MILLIGRAMS
1 OF SUBSTANCE
PER
6 CC. OF
SUBSTANCE
NUTRIENT BROTH
Complete fungi-
Partial fungi-
Fi
jngicidal action
static action
static action
in 48 hours
Penicillin
0
0
0
Actinomycin
O.I
0.03
O.I
Streptothricin
0
0
0
Clavacin
0.15
0.045
<o.i5
Fumigacin
0
5.0
0
Hemipyocyanin
0.5
O.I
0.1
Gliotoxin
0.5
O.I
^(^
From Waksman and Bugie (928).
142 FUNGI AS ANTAGONISTS
growth of fungi. The active substances produced by these organisms
show considerable selective action just as in the case of the bacteria.
Actinomycin was found (945) to inhibit the growth of Penicillium,
As-pergilliSy Ceratostom-ellay and yeasts in concentrations of i : 50,000 j
larger amounts (1:10,000) were required to inhibit other fungi, in-
cluding Rhizofus and Trichoderma. Streptothricin is less effective
against fungi, although it inhibits the growth of certain yeasts (1002).
A comparison of the fungistatic activity of several antibiotic substances
upon the causative agent of Dutch elm disease is brought out in
Table 31.
ACTIVITY OF FUNGI AGAINST INSECTS AND
OTHER ANIMAL FORMS
A number of fungi are capable of parasitizing insects and other ani-
mal forms. Comparatively little is known concerning the production
of antibiotic substances by these animal parasites.
CHAPTER 8
MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS
The microscopic animal world inhabiting the soil and water basins com-
prises protozoa, insects and insect larvae, nematodes and other worms.
Their relationships to the microbiological flora of soils and waters are
varied. Many, if not most, of these animals feed upon the bacteria and
fungi, as well as upon the smaller animal forms. Some carry a bacterial
population in their digestive tract and appear to depend upon these
bacteria for some of the digestion processes. Many of the animal forms
are parasitized by bacteria and fungi. Some of these forms are subject
to the action of specific substances produced by microbial antagonists.
No detailed discussion will be presented of these varied relationships,
but attention will be directed to a few specific phenomena which have a
bearing on the subject under consideration. The ability of higher ani-
mals to produce antibacterial substances has been amply established.
Some of these substances are well characterized, as in the case of ly-
sozyme found in mammalian tissues and secretions (262, 264) and in-
hibins found in fresh human urine (180).
INTERRELATIONSHIPS BETWEEN PROTOZOA
AND BACTERIA
The lower animal forms inhabiting the soil, manure piles, and water
basins often utilize bacteria in the synthesis of their foodstuffs. Al-
though many of the smallest organisms, namely the protozoa, are able
to obtain their nutrients from simple organic compounds and mineral
salts, they frequently depend upon the bacteria to concentrate the nu-
trients present in dilute forms in the natural substrate. It has been
shown (102), for example, that when carbohydrates are present in
water in very low concentration, the protozoa may not be able to use
them in that dilute formj however, the bacteria can assimilate these
carbohydrates and can build up extensive cell substance, and the pro-
144 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS
tozoa are then able to multiply by consuming the bacteria. Protozoa
are apparently also able to destroy pathogenic bacteria (747).
The fact that some of the protozoa feed upon bacteria served as the
basis for a theory designated as the "protozoan theory of soil fertility"
(776). According to this theory, the capacity of protozoa to consume
bacteria is responsible for the limited fertility of certain soils. The bac-
teria were viewed as the sole agents responsible for the liberation of
nutrients in the decomposition of soil organic matter and for the trans-
formation of these nutrients into forms available to higher plants. The
protozoa, because of their capacity to digest bacteria, were looked upon,
therefore, as the agents injurious to soil fertility. The increased fer-
tility which results from the treatment of soil with heat and with cer-
tain chemicals was believed to be due to the destruction of the protozoa,
considered as the "natural enemies of the bacteria."
Subsequent investigations did not support this theory. When proto-
zoa were added to cultures of bacteria responsible for certain specific
processes they did not exert any detrimental effect upon the particular
reactions brought about by the bacteria, despite the fact that they fed
upon and thereby considerably reduced the numbers of these bacteria.
In many cases, the effect of protozoa upon bacterial activities may actu-
ally be considered beneficial (156, 591, 630). This was found true for
such processes as the fixation of atmospheric nitrogen, the liberation of
ammonia from proteins, and the formation of carbon dioxide from car-
bohydrates. It has been suggested that the presence of protozoa in the
soil may keep the bacteria at a level of maximum efficiency (157).
Failure to confirm the protozoan theory of soil fertility was due pri-
marily to the fact that several assumptions were made that were not
fully justified, namely, (a) that bacteria are the only important soil or-
ganisms responsible for the decomposition of the soil organic matter j
(b) that protozoa, by consuming some of these bacteria, are capable of
restricting bacterial development and, if so facto, organic matter de-
composition. The fact was overlooked that the soil harbors, in addition
to the bacteria, many fungi and actinomycetes capable of bringing
about the decomposition of plant and animal residues, resulting in the
liberation of ammonia, and that this could take place even if all the bac-
teria were completely eliminated from the soil.
RELATIONS OF PROTOZOA AND BACTERIA 145
The favorable effect of partial sterilization of soil upon fertility still
remains to be explained. Various other theories have been proposed, the
most logical of which is one based upon a soil condition designated as
"microbiological equilibrium" (943). It has also been suggested (498)
that the phenomenon is due to the disappearance of the bacterial antago-
nists in the soil as a result of partial sterilization.
In many cases, however, protozoa are responsible for bringing about
extensive destruction of bacteria. This may find a practical application
in the purification of water and sewage. The action of the protozoa is
due in this case to the actual ingestion of the bacteria (440, 595, 743).
The idea (157) that protozoa may favor soil processes because of the
stimulation of bacterial development and hence the accelerated trans-
formation of soil materials is not always justified. The assumption is
usually made that these processes take place in the soil in a manner simi-
lar to those brought about in artificial culture media, a generalization
that may be justified only in very special cases. No consideration is given
to the fact that the presence of numerous other organisms in the soil
may modify considerably the activities of the protozoa. The use of arti-
ficial media gives only a one-sided conception of the significance of pro-
tozoa in soil processes. Although the more recent claim concerning the
function of protozoa in the soil (157) is based upon more direct experi-
mental evidence, it is still inadequate, because it gives insufficient con-
sideration to the numerous elements involved in the complex soil
population.
The protozoa make up only a small portion of the soil population,
both in numbers and in the actual amount of cell substance synthesized.
Their ability to reduce bacterial numbers in normal soil is not very sig-
nificant. The indirect method of studying protozoa in solution media,
whereby the types observed and the activities obtained are quite differ-
ent from those occurring in the natural soil, has been largely responsible
for the exaggerated importance attached to these organisms.
One may conclude that the protozoa, by consuming some of the bac-
teria, keep these organisms at a high state of efficiency, thus assisting in
the breakdown of the plant and animal residues in the soil. In other
words, the rate of energy transformation brought about by bacteria and
even the total amount of change produced in the substrate are increased
146 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS
by the presence of protozoa. Thus, an interrelationship among micro-
organisms which was at first thought to be antagonistic actually has
proved to be associative (943). The protozoan Oikomonas termo was
found capable of living at the expense of a large number of bacteria,
namely 83 per cent of those tested. The fact that Oikomonas causes
many species of bacteria to flocculate was suggested as explanation for
the ability of the protozoa to digest these bacteria (378).
The ability of protozoa to destroy bacteria was said (414) to be re-
sponsible for the protection of certain plants against attack by plant
pathogenic bacteria and fungi. This was said to hold true of attack of
potatoes by Bacterium aroideae and of other plants by Pseudomonas
hyacinthi and Pseudomonas citri, as well as by species of Fusarium and
Penicillium.
Various bacteria may exert a toxic action upon protozoa, thus limiting
the development or bringing about the destruction of the latter (122,
545, 687). Certain plant pathogenic bacteria inedible by amebae were
found to produce a toxin that was harmful to these amebae. The toxin,
however, appeared to be without effect on the flagellate Cercomonas,
which could eat all these bacteria partly or completely (826), In some
cases, the protozoa were capable of developing a certain resistance to
specific bacterial products (687).
Certain factors in the medium seem to affect the encystment of pro-
tozoa (874) 5 it remains to be determined to what extent these factors
can be classified with antibiotic substances.
RELATIONS OF PROTOZOA TO FUNGI
The presence of Colfoda and other infusoria in an active form was
found to repress the growth of Verticillium dahliae in culture media
and to prevent infection of tomato plants by this pathogeny Colfoda
was also active in soils and reduced the incidence of wilting (87).
Myxamoebae of the slime mold Dictyostileum discoideum also live
upon bacteria. They are able to utilize the gram-negative somewhat
better than the gram-positive types, with certain few exceptions. Bac-
terial spores are also ingested by these organisms, but they are not di-
gested. The ability of various fungi to destroy protozoa and nema-
todes has been studied in detail by Drechsler (183).
INSECT DISEASES AND MICROBIAL CONTROL 147
MALARIAL AND TRYPANOSOME PARASITES
In connection with the recent interest in antibiotic substances, con-
siderable work has also been done on the effect of these substances upon
different strains of Plasmodium causing malaria and upon different
trypanosomes causing various tropical diseases. Because of the war,
however, the results thus obtained have not yet been published. They
are highly interesting and offer promise of added application of these
substances.
Weinman found (967) that the general correlation between the
gram-stain of bacteria and their sensitivity to gramicidin also extends
to protozoa {Leishmaniay Tryfanosoma) and to the Leftosfira tested.
Tyrocidine had a marked effect, in concentration of 5 y per ml., upon
the flagellates 5 they remained active for many hours, gradually losing
their motility} a few escaped giving rise to delayed growth.
INSECT DISEASES AND MICROBIAL CONTROL
Insects are subject to attack by various groups of microorganisms,
including bacteria, fungi, protozoa, nematodes, and other insects. Many
attempts have been made to control insect pests by the use of pure or
mixed cultures of microorganisms. In this connection the following re-
lationships must be considered: the receptivity of the insect to microbial
attack during its various stages of development} the environmental
conditions favoring the attack on the insect by the disease-producing
organism} the influence of environment upon the virulence of the at-
tacking microbe} the manner in which the parasite attacks the host} the
coordination of the optimum activity of the disease-producing agent
with the abundance of the host and the proper stage of its develop-
ment (867).
The microbial agents that keep in check the spread of insects, some of
which are highly injurious to plants and animals, are far more impor-
tant than any other methods of control. These microbial agents can be
classified into three groups, depending upon the nature of the host: (a)
microbes that attack economically useful insects and that must be con-
trolled in order to avoid important losses from disease} (b) microbes
that attack injurious insects and that must therefore be favored and en-
148 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS
couragedj (c) microbial agents infectious to plants, animals, and man
that are spread by insects.
Various bacterial diseases that formerly caused considerable destruc-
tion of silkworms and bees have been controlled, once the nature of the
organisms concerned was established. One of Pasteur's important con-
tributions to microbiology was the control of Flacheria among silk-
worms. However, most of the problems of control of injurious insects
have been difficult to solve. A great number of bacterial, fungus, and
virus diseases of insects are now known, but the many attempts to em-
ploy these pathogens in combating the insect hosts have not always been
successful. The investigations so far carried out in this important field
may be considered as at a very primitive stage.
Metalnikoff (596) compared the bacterial treatment of caterpillars
of Pectinofhora gossyfiella with the action of arsenical poisoning. The
dry spores of Bacterium efhestiae. Bacterium gelechiae^ Bacterium^ 5,
and Bacterium cazaubon, in powder form, were mixed with water at the
rate of i to 4 ounces to 2>4 gallons of water, with the addition of 4 per
cent of molasses; this preparation was sprayed on the plants two to four
times, at regular intervals, at the rate of 196 gallons or less per acre.
The best results were obtained for plants treated with B. efhestiae^ the
infestation being reduced by about 50 per cent as compared with the
controls. A slightly smaller reduction occurred on plots sprayed with
B. cazaubofiy while B. gelechiae reduced the infestation by less than 40
per cent. Those plants that were treated with the arsenical spray showed
a reduction of only 1 8 per cent.
Recently microorganisms have been used for the control of the larvae
of Japanese and other beetles in the soil. A variety of bacteria, fungi,
and nematodes were found capable of destroying these larvae. Once the
attacking microorganisms have become established in the soil, the larvae
and the beetles themselves tend to disappear. Glaser (327) utilized for
this purpose Neoaflectana glaseri. This parasite possesses great repro-
ductive capacity and is capable of destroying large numbers of grubs.
Glaser demonstrated the presence of this nematode also in localities
where the grub was not present.
Fungi have also been utilized for the control of insects. Sweetman
(867) emphasized the importance of entomogenous fungi as destructive
INSECT DISEASES AND MICROBIAL CONTROL 149
enemies of insects. A limitation to their practical importance in the fight
against insects is that the fungi require special conditions for develop-
ment, especially high humidity and favorable temperature, which are
not always found under natural conditions.
Dutky (219) described two spore-forming bacteria {Bacillus fo-
filliae and Bacillus lentimorbus) which cause the milky disease of the
larvae of the Japanese beetle. These bacteria are grown in the larvae
and then inoculated into soil. They are capable of infecting the grub,
and are said to be responsible for the reduction in the beetle population.
Bacteria pathogenic to the citrus red scale have also been isolated from
the soil (840).
Glasgow (328) established that some of the caecal bacteria of Het-
eroptera show a marked antagonism toward other bacteria and proto-
zoan parasites that occur in the intestines of these insects. The caecal
system of the insects was removed and dropped into nutrient bouillon,
where it remained for a month or more without showing any bacterial
growth. This was believed to be proof of the fact that the caecal bac-
teria are antagonistic to ordinary saprophytic and parasitic bacteria and
prevent their development j also they apparently kill these bacteria
when they invade the alimentary canal of the insect.
According to Duncan (215), the bactericidal principle found in dif-
ferent insects and ticks shows differences in regard to the types of bac-
teria affected and the degree of their susceptibility. The gut-contents
of Argas and Stomoxys show the widest range of action j that of bugs,
the least. Spore-forming bacteria are especially affected by material
from Stomoxys J whereas staphylococci appear to be more susceptible to
the action of Argas material. The gut-contents of ticks was found to
have a v/eak activity upon P. festis, whereas the contents of certain in-
sects favored the growth of the latter. This phenomenon may have a
bearing upon the function of the plague flea. The action of the lethal
principle is greater and more rapid at 37° C. than at room temperature.
The lethal principle has been found to be active for at least six months
when kept in a dry state. It is thermostable, resisting temperatures as
high as 120° C, and is not destroyed by proteolytic enzymes. It appears
to be bound to proteins, since it is precipitated from solution by alcohol
and acetone, but it is not affected by these reagents. It is insoluble in the
150 MICROSCOPIC ANIMAL FORMS AS ANTAGONISTS
common fat solvents. It becomes inactivated when allowed to act upon
bacteria and appears to be adsorbed by killed bacteria, even by species
that are not destroyed by it. This substance does not have the properties
of either bacteriophage or lysozyme.
The presence in certain insects of a variety of other substances, such
as allantoin, which affect bacterial activities has also been established.
These observations give rise to the hope that man may in time succeed
in developing and utilizing microorganisms for the biological control of
injurious insects (849).
RELATION OF NEMATODES TO SOIL
MICROORGANISMS
Nematode worms are represented in the soil by a number of sapro-
phytes as well as by many plant and animal parasites. The latter vary
greatly in their relation to the host. The larvae of the cereal parasite
Tylenchus tritki penetrate the wheat seedlings between the leaf
sheaths, near the growing or apical points. When the head is formed,
the larvae enter the flowering parts and form galls. They become sexu-
ally mature, mate, and lay eggs which hatch in the galls, and then be-
come dormant. When the galls fall to the ground and decompose, the
larvae are liberated and proceed to find and attack new wheat plants
and cereal plants.
Some nematodes attack plants by feeding upon the roots. The meth-
ods of control require, therefore, a knowledge of their life history. Some
species produce resistant forms or cysts that may survive in the soil for
many years, even in the absence of the host plant. Soil sterilization by
steam or by chemicals is frequently employed as a measure of nematode
extermination.
Antagonistic relationships may be utilized for the control of nema-
todes. Linford et al. (528) found that the root-knot nematode of pine-
apple {Heterodera marioni) may be controlled by heavy applications of
organic material. The decomposition of this material results in a greatly
increased population of saprophytic nematodes in the soil. The decom-
posed organic residues also support large numbers of such other soil
microorganisms destructive to the parasitic nematodes, as the nema-
BACTERICIDAL ACTION OF MAGGOTS 151
capturing fungi (170, 184), the non-trapping fungal parasites, the
predacious nematodes, the predacious mites, and different bacteria ca-
pable of destroying nematodes.
BACTERICIDAL ACTION OF MAGGOTS
Surgical m.aggots are said to have a bactericidal effect in wounds, in
addition to removing necrotic debris. Simmons (825) demonstrated in
the maggot Lucilia sericata the presence of an active bactericidal sub-
stance which is thermostable and active against S. aureus, hemolytic
streptococci, and CI. welchii.
CHAPTER 9
ANTAGONISTIC RELATIONSHIPS BETWEEN
MICROORGANISMS, VIRUSES, AND OTHER
NONSPECIFIC PATHOGENIC FORMS
Antagonistic phenomena in relation to viruses have been but little in-
vestigated. It has been established, however, that certain microorgan-
isms are capable of destroying viruses, and particularly that some vi-
ruses possess the capacity of antagonizing other viruses. The rapid in-
activation of poliomyelitis virus in the process of aeration of sewage
sludge has also been indicated ( io6).
BACTERIA AND VIRUSES
B. subtilis was found (718) capable of inactivating the virus of vesicu-
lar stomatitis as well as staphylococcus phage, when in contact with
them for 15 to 18 hours at 35° C. This phenomenon has been ex-
plained as due to the process of adsorption. The facts that it is selective
in nature, that the phage cannot be reactivated, and that the virus is ren-
dered impotent by the action of the bacterium, all point to an antagonis-
tic eflFect rather than mere physical adsorption. The virus of rabies is
said to be influenced in certain ways by B. subtilis , the culture filtrate of
the organism suppressing the activity of the virus when a mixture of the
two is injected into rabbits (173).
However, different antibiotic substances, including penicillin, ty-
rothricin, and subtilin, when used either alone or in combination with
sulfonamides or acridine, have failed to prevent infection of mice with
influenza virus (508).
A "nontoxic" inactivator has been defined (306) as a substance that
inactivates plant viruses and is not detrimental to most forms of life.
Various microorganisms are capable of producing such inactivators.
Plant viruses differ in their sensitivity to "nontoxic" inactivators. Ac-
cording to Johnson (457, 458) various microorganisms are capable of
forming such inactivators against tobacco-mosaic virus j A. aero genes
INTERRELATIONSHIPS AMONG VIRUSES 153
was found to produce inactivators against a number of viruses. Taka-
hashi (868) isolated from yeast a substance which was capable of rap-
idly inactivating the tobacco-mosaic virus. A chemical reaction between
the inactivating principle and the virus was therefore suggested. The
inactivator in this instance was destroyed by heating with i N NaOH
solution, but not by 2 A^ HCl. It was not a protein and gave on analysis
39-7 per cent C and 5.85 per cent H. The substance was said to be a
polysaccharide. Fulton (306) demonstrated that A. niger forms in the
medium a substance capable of inactivating a number of different plant
viruses J the effect of the inactivator was found to be exerted upon the
virus itself and not upon the plant.
INTERRELATIONSHIPS AMONG VIRUSES
Andrews (20) reported that the cultivation of influenza virus in a
simple tissue-culture rendered the culture unable to support the growth
of a biologically distinct strain of the virus added 24 hours later. The
tissue-culture, however, was still capable of supporting multiplication
of a related virus such as that of lymphogranuloma venereum. When
two strains of the influenza virus were added to the tissue-culture simul-
taneously, the one added in larger concentration suppressed the growth
of the other.
Numerous reports have been made concerning the interference of one
virus by another, and even of inactivated bacteriophage with the active
agent of the same strain (1012, 1013). Henle and Henle (394) have
shown that even an inactivated virus, whether a homologous or a
heterologous strain, is capable of suppressing the development of the
influenza virus.
Jungeblut and Sanders (467) suggested that poliomyelitis in ani-
mals may be aborted by the injection of another virus. A strong antago-
nism was observed between a murine virus mutant (virus passed
through mice for many generations) and the parent strain of the virus.
The murine virus was capable of counteracting large paralytic doses of
poliomyelitis j the two viruses virtually counterbalanced each other.
Various other types of antagonism between viruses have been demon-
strated, as in the case of canine distemper or of lymphocytic chorio-
154 ANTAGONISMS BETWEEN NONSPECIFIC PATHOGENS
meningitis virus against experimental poliomyelitis (162). An intra-
muscular injection of a neurotropic strain of yellow fever virus was
found to protect animals against simultaneous infection with a highly
pathogenic viscerotropic strain (433). The antagonistic agent was be-
lieved to be a chemical substance produced by the murine virus, for
which the term "poliomyelitis inhibition" was proposed by Jungeblut.
The "interference phenomenon" of two viruses can be used to advan-
tage in bringing about immunity reactions.
A similar type of antagonism is frequently observed also among plant
viruses. Yellow mosaic virus will not grow in the tobacco tissue cells al-
ready infected with the agent causing common mosaic disease (569).
Other antagonistic phenomena between plant viruses have been ob-
served by McWhorter (573). Kunkel (510) demonstrated that the
peach-yellow virus prevented the invasion of the virus of little-peach
and that the latter prevented invasion of the former. McKinney (569)
concluded that virus domination in a plant may be looked upon as a
type of antagonism, quantitative in nature, the degree of domination
by a given virus being influenced by the host.
The ability of bacterial phages to interfere with the development of
other phages has been studied in detail by Delbriick and Luria (167,
546). They have shown that a certain phage, after inactivation by ultra-
violet radiation, retained its ability to interfere with the growth of an-
other phage acting upon the same host. The partly inactivated first
phage is adsorbed by the sensitive bacteria and inhibits their growth
without producing lysis. The partly inactivated phage interferes also
with the growth of the active phage. This interference between bac-
terial phages was explained as due to competition for a "key-enzyme"
present in limited amount in each bacterial cell. This enzyme was also
believed to be essential for bacterial growth.
BACTERIA AND TUMORS
The ability of certain bacteria to bring about hemorrhage in tumors
(446, 10 10) may also be classed among the antagonistic phenomena.
Laszlo and Leuchtenberger (515) described a rapid test for the detec-
tion of tumor-growth inhibitors. Inhibition was judged by comparing
BACTERIA AND TUMORS 155
tumor sizes and weights in treated and untreated groups of mice bear-
ing sarcoma, after a period of 48 hours of growth. The groups were
matched as to initial size of the tumors. The selective damage of sar-
coma cells, as compared with normal cells, said to be caused by penicillin
(150a) was later shown (525a) to be due not to the pure penicillin it-
self but to some impurity present in crude penicillin preparations.
CHAPTER 10
CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
CLASSIFICATION OF ANTIBIOTIC SUBSTANCES
Antimicrobial agents are of either chemical or biological origin. The
first comprise inorganic (heavy metals, halogens) and organic (phenols,
arsenicals, dyes, aromatic oils) compounds. The second include a variety
of products of higher plants (quinine, chaulmoogra oil, wheat flour pro-
tein), higher animals (lactenin, lysozyme), and microorganisms, to
which the term "antibiotic" is specifically applied.
The property possessed by culture filtrates of many bacteria of inhib-
iting the growth of bacterial cells has long been recognized (506). The
suggestion has even been made that all bacteria, when tested at the right
age and under proper conditions of culture, are able to produce anti-
bacterial substances (70). It is now definitely established, however,
that this property is characteristic of only certain strains of specific bac-
teria, fungi, and actinomycetes.
Antibiotic substances of microbial origin are primarily bacteriostatic
in nature. They are selective in their action. Some substances affect
largely gram-positive bacteria j their action upon gram-negative bacteria
is more limited as regards both the kinds affected and the concentration
required to bring about growth inhibition. Other substances may inhibit
alike the growth of certain members of both groups of bacteria. One is
fully justified, therefore, in speaking of a characteristic bacteriostatic
spectrum for each antibiotic substance. The production of antibiotic sub-
stances by specific microorganisms is influenced by the strain of the or-
ganism, the composition of the medium, the temperature of incubation,
the age of the culture, aeration, and certain other factors. Antibiotic sub-
stances also vary greatly in their mode of action upon the bacterial cells,
in their toxicity to animals, and in their practical utilization for the treat-
ment of human and animal diseases.
The more important antibiotic substances are described briefly in
Table 32. They may be classified on the basis of their origin from spe-
cific microorganisms, their chemical properties, or their biological ac-
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160 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
tion. Differences between various compounds may often be in degree
rather than in kind. Different organisms may produce the same kind of
compound. Many organisms are able to produce more than one anti-
biotic substance: B. hrev'ts produces tyrocidine and gramicidin j P. no-
tatum forms penicillin and notatinj 5. antibiot'icus produces actino-
mycin A and Bj ^4. fumigatus forms fumigatin, fumigacin, spinulosin,
and gliotoxinj A. flavus produces aspergillic acid and flavicin.
On the basis of their solubility, the antibiotic substances may be di-
vided into three groups :
Group A. Soluble in water at different reactions, and insoluble in ether.
These substances usually represent polypeptides, proteins, organic
bases, or adsorption compounds on protein molecules. Most of them
have not been isolated in a pure state. They comprise the bacterial
enzymes acting upon microbial polysaccharides, actinomycetin, mi-
crobial lysozyme, streptothricin, streptomycin, notatin, and pyo-
cyanin.
Group B. Soluble in ether and in water at proper reactions. Here belong
some of the most important antibiotic substances so far isolated and
described, namely, penicillin, flavicin, citrinin, clavacin, proactino-
mycin, penicillic acid, and aspergillic acid.
Group C. Insoluble in ether and in water. These include gramicidin, ty-
rocidine, subtilin, and the B. simflex factor.
Group D. Soluble in ether and insoluble in water. Here belong fumi-
gacin, fumigatin, gliotoxin, actinomycin, pyocyanase, and others.
Some of the antibiotic substances have been crystallized, and infor-
mation has been gained concerning the approximate chemical nature of
others j many others are still imperfectly known. On the basis of their
chemical nature, the antibiotic substances may be divided as follows:
Lipoids and lipoid-like bodies, including pyocyanase and certain little
known microbial extracts
Pigments, namely, pyocyanin, hemipyocyanin, prodigiosin, fumigatin,
chlororaphin, toxoflavin, and actinomycin
Polypeptides, comprising gramicidin, tyrocidine, subtilin, and actino-
mycetin
Sulfur-bearing compounds, such as gliotoxin and chaetomin
SUBSTANCES PRODUCED BY BACTERIA 161
Quinones and ketones, namely, fumi'gatin, citrinin, spinulosin, clavacin,
and penfcillic acid
Organic bases, including streptothricin, streptomycin, and proactinomycin
On the basis of their biological activity, the antibiotic substances also
vary considerably. They may be divided into three groups:
Primarily bacteriostatic agents, such as penicillin, actinomycin, and pro-
actinomycin
Substances which are bactericidal but not bacteriolytic, including pyocya-
nase, gliotoxin, fumigacin, clavacin, and pyocyanin
Bacteriolytic substances, namely, gramicidin, actinomycetin, and lysozyme
On the basis of their toxicity to animals, antibiotic substances may
also be divided into three groups:
Compounds that are nontoxic or but slightly toxic; here belong penicillin,
streptomycin, flavicin, pyocyanase, and actinomycetin
Compounds of limited toxicity, including gramicidin, tyrocidine, citrinin,
streptothricin, and fumigacin
Highly toxic compounds, such as actinomycin, gliotoxin, aspergillic acid,
and clavacin
Many of the antibiotic substances are thermostable, others are ther-
molabile ; some pass readily through Seitz and other filters, others are
adsorbed. The various methods of isolation of these substances are based
upon their chemical nature, solubility, and properties of adsorption.
SUBSTANCES PRODUCED BY BACTERIA
Lipoids and Pigments. Ps. aeruginosa, discovered by Gessard in
1882 (320) and formerly known under the names of Bacterium fyo-
cyaneuni and Bacillus fyocyaneus, was the first organism found to pro-
duce two antibiotic agents, the colorless pyocyanase and the pigment
pyocyanin. Pyocyanase, believed to be of the nature of an enzyme, is
now recognized as a lipoid containing unsaturated fatty acids {SS^ 4^9?
410). Recently this organism was shown to form (809) three com-
pounds that possess antibacterial properties, namely, pyocyanin,
162 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
a-hydroxyphenazine, and an oil that forms insoluble salts with calcium,
barium, and heavy metals. The last appears to be similar to what has
previously been described as pyocyanic acid, a substance strongly lytic
to V. comma. All three compounds were isolated by extraction with
chloroform.
Different strains of Ps. aeruginosa may produce either pyocyanase or
pyocyanin or both, the production of the two not proceeding in a paral-
lel manner. Among the amino acids, alanine and tyrosine were found to
be favorable to pyocyanin production (27, 320), although the effect
of tyrosine is not very significant (340, 341, 452, 529). It was suggested
(407) that the antagonistic action of young cultures of Ps. aeruginosa
is due primarily to the presence of pyocyanin, whereas in older cultures
pyocyanase is largely concerned.
The determination of the nature of the antibacterial substances of
Ps. aeruginosa can be carried out in the following manner (407) : the
organism is grown in bouillon for 14 daysj the cultures are heated for a
half hour at 75° C. to kill the living cells j they are then centrifuged,
the liquid is treated with chloroform which extracts the pigment, and
the chloroform solution is concentrated in vacuo at 50° C. j the aqueous
solution remaining after chloroform extraction is acidified with hydro-
chloric acid and again shaken five times with chloroform, thus extract-
ing the fatty acids. It was found that, on removing the pigment, the
antibacterial properties are very little diminished j however, when both
the pigment and the fatty acids are removed, no antibacterial action is
left in the culture. S. aureus is commonly used as the test bacterium.
In most cases the broth culture of the organism is first extracted with
ether, giving pyocyanase, and the residue treated with chloroform,
yielding pyocyanin. The solution left after the removal of the blue
chloroform extract may be again treated with ether, giving a yellow
pigment, which also has some activity (501 ). This pigment is a deriva-
tive of pyocyanin and is often designated ( 1006) as hemipyocyanin. It
may also be obtained by acidifying pyocyanin with acetic acid and heat-
ing. The fluorescin remaining in the culture after the ether and chloro-
form extraction was found to be inactive. In old cultures, pyocyanin is
changed into a brown pigment, pyoxanthose. A fourth pigment, which
is yellow in transmissible light and fluorescent-green in reflected light.
SUBSTANCES PRODUCED BY BACTERIA 163
may be produced under certain conditions. It was excreted into the me-
dium as a leuco base.
Pyocyanase is soluble in ether, benzol, benzene, and petrol ether. It
can be separated (370) into several lipoids, the action of which shows
slight variation. This preparation consists of a phosphatide, a neutral
fat, and a free fatty acid. The antibacterial properties have been attrib-
uted to the last constituent (410). A definite relation has been observed
between the number of double bonds and the activity of the substance
(SSy 409). According to Dressel (185), most fatty acids exert bacteri-
cidal and bacteriolytic effects upon gram-positive bacteria, whereas
gram-negative organisms are not lysed, Pyocyanase acts upon various
bacteria, including the colon-typhoid group, though the ability of the
substance to inhibit the growth of this group of bacteria has been denied
by some workers (370).
Many commercial pyocyanase preparations have been found to be of
little practical value. This is believed to be due largely to a lack of recog-
nition of the importance of strain specificity, conditions of cultivation
of organism, and methods of extraction of the active substance (501,
763).
Since Ps. aeruginosa is an extremely variable organism, the nature
and abundance of the pigment are also influenced by these conditions.
Keeping the organism for five minutes at 57° C. or cultivating it in
liquid egg-albumin has been found to result in destruction of some of
its pigment-producing properties (129, 321, 522).
Pyocyanin was first studied by Fordos in i860 (279). Since then
many contributions have appeared dealing with formation and nature
of this pigment. Several formulae have been suggested for pyocyanin
(452, 603, 913, 1006), one of which is shown in Figure 16. The struc-
ture of pyocyanin has considerable similarity to chlororaphin and io-
dinin, obtained from Chromobacterium {SSS^ SS^) ^^^ two synthetic
compounds, phenazine and acridine (919).
Besides Ps. aeruginosa, spore-forming bacteria, including B. mesen-
tericus, were also found to produce antibiotic agents of a lipoid nature.
The cell-free filtrate of this organism killed diphtheria bacteria in
4 minutes (1016), but when diluted to i per cent it required 24 hours
to effect a kill. The substance was not affected by heating for 30 seconds
164 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
at 100° C. but was weakened at 1 15° C. for 10 minutes. It is considered
similar in its bactericidal properties to pyocyanase.
Alcohol and acetone extracted from B. mesenterkus a weakly active
substance (408) that diffused through a cellophane membrane and
could be partly absorbed on a Berkfeld filter. When shaken directly
with ether, the culture lost its antibacterial properties. The ether extract
was concentrated and ammonia added, and the solution was treated with
50 per cent alcohol. The alcohol was then removed, and the residue was
1 ! !
c c c
CH,O.C C=CH2 H2C C=CH\ HOC CH H3CI
II I I I > II II ,
HC CH3 H2C CH.CO/ HC C.CH2OH li yOCHj
I V V „
O
COOH
PENICILLIC ACID
CLAVACIN
KOJIC ACID
H3C
PYOCYANIN
PHENAZINE
FUMIGATIN
CONH2
CHLORORAPHIN
Figure i6. Structural formulae of some antibiotic substances.
SUBSTANCES PRODUCED BY BACTERIA 165
acidified and treated with petrol ether, which brought the active sub-
stance into solution. The active substance was again dissolved in alcohol
and taken up in ether. The ether solution was washed with water, evapo-
rated, and dried. One liter of a 30-day-old culture of B. mesentericus
gave 1 62 mg. of petrol-ether-soluble fatty acids and an oily substance
of a brownish color. It was neutralized with NaOH solution and tested.
The extract diluted to 1:7,500 killed diphtheria j a 1:1,000 dilution
was required to kill staphylococci. Iso-valerianic acid and oleic acid,
isolated from this material, had a similar bactericidal action. Weaken-
ing of the substance by heating was demonstrated and was believed to
be due to a break in the double bond of the oleic acid.
E. colt exerts an antagonistic effect in vivo when injected subcutane-
ously or when used for feeding. It produces (365, 367) a thermolabile
substance that was considered to be a lipoid in character. According to
Hettche (408, 409), one is dealing, in the case of bactericidal constitu-
ents of the bacterial cell, with lipoids that contain unsaturated fatty
acids.
Chromobacterium iodinum produces {SSSi SS^) ^ purple-bronze pig-
ment designated as iodinin and found to be a di-N-oxide of dihy-
droxyphenzine. This substance inhibits the growth of streptococci (S.
hemolyticus) in concentrations of 1.2 to 2.0 x io"° M.
It may be added here that certain aromatic oils possess marked bac-
tericidal properties. Ordinary peptones have also been found to contain
a bacteriostatic substance that is active against various bacteria, especially
when small amounts of inoculum are used (191). The active substance
is thermostable and is associated with an acid-precipitated fraction that
is pigmented and changes color upon oxidation and reduction. The bac-
teriostatic effect of this material can be corrected by the addition of re-
ducing agents, such as thioglycollic acid. The bacteriostatic action of
dyes is well known and need hardly be discussed here. It is sufficient to
mention, for example, methylene blue and indophenols in oxidized
forms (197).
PoLYSACCHARiDASES. Among the antibiotic substances of microbial
origin may also be included the enzyme systems that have the capacity
of decomposing the capsular substance of certain bacteria, thereby ren-
dering them more readily subject to destruction in the blood stream or
166 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
in other substrates. The first enzyme of this type was isolated by Dubos
and Avery (195, 199, 202) from certain soil bacteria. These enzymes
are highly specific, some being able to act only upon one type of pneu-
mococci. As a result of their action, the pneumococcus cell is rendered
susceptible to destruction by phagocytosis (819,821,822). This enzyme
was produced by the soil bacteria under selective conditions of culture,
that is, when the capsular polysaccharide of the pneumococcus was pres-
ent in the medium j the only other substance that could be used for its
production was aldobionic acid, derived from the above polysaccharide.
Yields of the enzyme were increased by increasing the concentrations of
the specific substrate in the medium from o.Oi to o.i per cent. Above
0.1 per cent, the yields decreased, 0.3 to 0.4 per cent inhibiting the
growth of the bacterium. The addition of o. i per cent yeast extract fa-
vored the production of the enzyme j proper aeration was essential, the
bacterium making the best growth in shallow layers of medium. The
enzyme was concentrated by distillation in vacuo and by ultrafiltration.
Toxic substances accompanying the active preparation could be largely
removed by the use of an aluminum gel. The enzyme is associated with
a protein which passes through a collodion membrane with an average
pore size of 10.6 Mj but is held back by pores having a diameter of 8.2 m-
After filtration, the enzyme can be recovered in solution by immersing
the membrane in distilled water or in physiological salt solution (30,
195,293,337).
Dubos (188) believed that it is possible to develop "adaptive" bac-
terial enzymes against many organic substances. These enzymes exhibit
a great degree of specificity, as in the case of the enzyme that hydrolyzes
the capsular polysaccharide of the pneumococcus. The cell of this or-
ganism contains an enzyme that changes the cell from the gram-positive
to the gram-negative state, but is ineffective against streptococci or
staphylococci.
Active preparations of the enzyme protected mice against infection
with as many as i ,000,000 lethal doses of the specific pneumococcus. The
enzyme retained its activity for 24 to 48 hours after its injection into
normal mice ; it also exerted a favorable influence on the outcome of an
infection already established at the time of treatment. A definite rela-
SUBSTANCES PRODUCED BY BACTERIA 167
tionship was found to exist between the activity of the enzyme in vitro
and its protective power in the animal body.
Polypeptides. The credit for first isolating, in crystalline form from
spore-forming aerobic soil bacteria, specific chemical compounds of the
polypeptide type is due Dubos (190, 193, 203, 436, 530). The antago-
nistic organism {B. brevis) is grown in shallow layers of a medium
containing i per cent casein digest or tryptone and 0.5 per cent NaCl
in tap water, adjusted to /jH 7.0. After inoculation, the medium is
heated for 20 minutes at 70° C, in order to kill the vegetative cells of
the bacteria, leaving only the spores to develop. The culture is allowed
to grow for 72 hours. The reaction of the culture is then adjusted to /)H
4,5 by the use of about 3 or 4 cc. concentrated HCl per liter of culture.
A precipitate is formed which is removed by filtration through paper j
it is then suspended in 95 per cent alcohol (20 cc. of alcohol per liter of
culture) and allowed to stand 24 hours. The active substance is dis-
solved and is separated from the residue by filtration j when the alco-
holic solution is diluted with 10 volumes of i per cent NaCl, the sub-
stance is precipitated out. It carries all the activity and can be desiccated
in vacuo, over PoO-,, giving a yield of about 100 mg. of final dry sub-
stance per liter of culture medium. The protein-free, alcohol-soluble ac-
tive material was designated as tyrothricin. When an attempt was made
to produce tyrothricin in aerated submerged cultures, none was ob-
tained in complex nitrogenous media 5 however, simple amino com-
pounds, like asparagine, gave good growth and yielded the antibiotic
substance. The presence of cystine in the mixture of amino acids ap-
peared to inhibit growth ( 856) .
Tyrothricin can be separated into two crystalline preparations, grami-
cidin and tyrocidine. Gramicidin is obtained by treating tyrothricin with
a mixture of equal volumes of acetone and ether, evaporating, and dis-
solving in boiling acetone. On cooling, it crystallizes out as spear-
shaped colorless platelets, melting at 228° to 230° C, with a yield of
about 10 to 15 grams from 100 grams of the crude material. Gramicidin
is soluble in lower alcohols, acetic acid, and pyridine, and moderately
soluble in dry acetone and dioxanej it is almost insoluble in water,
ether, and hydrocarbons. When a solution containing 20 to 50 mg. per
168 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
milliliter alcohol is diluted to i mg. per milliliter, with distilled water
or with glucose solution, an opalescent solution is produced without
flocculation. On dilution with electrolyte solutions, an immediate floc-
culation occurs.
The specific rotation of gramicidin in 95 per cent alcohol solution is
approximately [a]^^ = -|- 5°. On analysis, it gives 62.7 per cent C,
7.5 per cent H, and 13.9 per cent N. The molecular weight, as
determined in camphor, is about 1,400. The empirical formula of
C74H106N14O14 has been suggested. On further study, the molecular
weight of gramicidin was found (885) to present an anomaly in that it
appeared to depend on the nature of the solvent and on the concentra-
tion of the solute, giving values from 600 to i,200j isothermal distil-
lation in methanol, however, indicated a molecular weight of 3,100.
Sulfur and carbon analyses of gramicidin flavianate gave a molecular
weight of 3,000. It gave neither free amino nor carboxyl groups (126,
434). Gramicidin is a polypeptide with 10 molecules of a-amino acids,
of which two or three are tryptophane residues. These and a saturated
aliphatic acid, with 14 to 16 carbons, account for about 85 to 90 per cent
of the weight of substance. Amino acids that have definitely been iden-
tified are /-tryptophane, <i-leucine, /-alanine, ^/-valine, and glycine
(339). A study of the configuration of the dipeptide valyvaline sepa-
rated from gramicidin brought out the fact that only valines of like
configuration have been joined together by the bacterium (125). About
45 per cent of the a-amino acids gave the d configuration (435, 436,
530). An unknown hydroxyamino compound has also been indicated
(339)-
Tyrocidine hydrochloride is moderately soluble in alcohol, acetic
acid, and pyridine ; it is sparingly soluble in water, acetone, and dioxane,
and is insoluble in ether and hydrocarbon solvents. An alcohol solution
can be diluted with water to give a clear solution containing 5 to 10 mg.
per milliliter j electrolytes produce an immediate precipitate. A solu-
tion in distilled water containing i mg. or even less per milliliter has a
low surface tension and behaves like a soap or detergent solution. Un-
like gramicidin, it precipitates a number of soluble proteins in a manner
similar to some of the cationic detergents.
Tyrocidine is dissolved in four times its weight of boiling absolute
SUBSTANCES PRODUCED BY BACTERIA
169
alcohol, to which is added alcoholic HCl (o.i mol. per liter). On cool-
ing, a precipitate is formed. This is recrystallized from absolute metha-
nol plus small amounts of HClj clusters of microscopic needles are ob-
tained, melting at 237-239° C, with decomposition j the specific rota-
tion is [a]'s = _ 102° ( I per cent in 95 per cent alcohol). Tyrocidine
BACILLUS BREVIS BG
PEPTONE CULTURES
ACID pH 4.8
PRECIPITATE
ALCOHOL EXTRACT
precipitate
(tyrothricin)
ACETONE -ETHER
SOLUBLE PART
CRYSTALLIZATION
FROM ACETONE
(GRAMICIDIN)
INSOLUBLE PART
CRYSTALLIZATION
FROM ALCOHOL + HCL
(TYROCIDINE HYDROCHLORIDE)
Figure 17. Preparation of tyrothricin, gramicidin, and tyrocidine. From
Dubos (192).
170 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
analyzes: 59.4 per cent C, 6.8 per cent H, 13.5 per cent N, 2.7 per cent
CI. The molecular weight Is about 1,260 or a multiple of this number.
Tyrocidine is a salt of a polypeptide having free basic amino groups.
The <^-amino acids make up 20 per cent of its a-amino groups. The most
probable molecule was shown to contain two amino groups, three amide
groups, and one weakly acidic carboxyl or phenolic group, with a molec-
ular weight of 2,534. Among the amino acids, tryptophane, tyrosine,
and dicarboxylic-amino acids, including aspartic acid, have been de-
tected (126, 434).
The tyrothricin type of antibiotic substance appears to be widely dis-
tributed among spore-forming aerobic soil bacteria (427, 428, 857).
The following method for its extraction has also been employed: A
seven-day-old bacterial culture was treated with 2 to 5 per cent of an
electrolyte and HCl added to give a ^H of 4.0. A precipitate was
formed which was centrifuged and extracted with 95 per cent alcohol,
until no more turbidity could be observed after dilution with an equal
volume of water. The alcoholic extracts were evaporated to dryness and
extracted with ether, petroleum ether, and benzol, in which the active
substances are insoluble. The residue was then dissolved in absolute
alcohol, and the concentrated solution dialyzed for 24 hours against
running tap-water and for 24 hours against distilled water. The active
substance was obtained partly in a precipitated form and partly in a
colloidal solution in the dialysis bag. Upon evaporation of the water, a
highly active, grayish-white powder was obtained. One hundred liters
of medium gave 15 grams of purified active substance. The activity
could be tested by inhibition of encapsulation of Friedlander's bac-
terium j this was brought about by the addition of 4 mg. to i ml. of cul-
ture medium. This preparation was later found to be identical with
gramicidin (427a, 885).
A thermostable substance was obtained (149) from B. simplex, an
organism capable of bringing about the destruction of various patho-
genic fungi. It was produced by the bacterium grown both on synthetic
and organic media. It can be adsorbed on activated charcoal and recov-
ered from the latter by the use of hot alcohol.
To what extent substances of bacterial origin that are toxic to brain
tissues, like toxoflavin (C6H6N4O2), are also effective against bacteria
.
v_^- ¥
/
^^^-V |\
<^
^^%^^
^
^w\
m
¥l // y
^jI
'm /
"7
Tyrocidine hydrochloride. From
Hotchkiss (435)
.-•^\
.*^\ #.->, v^^
Fumigacin. From Waksman and
Geiger (933)
Gramicidin. From Hotchkiss
(435)
^
\
Gliotoxin. From Waksman and
Geiger (933)
Citrinin. Prepared bv Timonin
Actinomycin. Prepared by Tischler
Figure 18. Crystalh'ne preparations of antibiotic substances.
SUBSTANCES PRODUCED BY ACTINOMYCETES 171
and other microorganisms still remains to be determined. Toxoflavin,
formed by Bacterium cocovenenanSy is extracted from the culture satu-
rated with salt by means of chloroform j from this it is recovered by an
aqueous solution and purified (908, 909). Other bacterial toxins, like
botulinus toxin, various amines and purine bases, and numerous toxins
produced by bacteria in living plant and animal systems, are beyond the
scope of this treatise.
SUBSTANCES PRODUCED BY ACTINOMYCETES
The antibacterial substances produced by actinomycetes can be di-
vided into three groups :
Water-soluble and alcohol-insoluble compounds of the protein type, in-
cluding actinomycetin (346, 347, 971-973), micromonosporin, and
the compounds of the lysozyme type (507)
Ether-soluble and alcohol-soluble pigmented compounds, including ac-
tinomycin
Basic substances, soluble or insoluble in ether and soluble in aqueous or al-
cohol acid solution, including streptothricin, streptomycin, and pro-
actinomycin
AcTiNOMYCiN is an ether-soluble and alcohol-soluble pigmented sub-
stance produced by only a few organisms, notably S. antibiotkus. The
culture medium is treated with ether, giving an orange-colored extract.
The residue is evaporated and separated (946) into two fractions:
A, soluble in ether and in alcohol but not in petrol ether, giving a clear
yellow-colored solution when diluted with water j B, soluble in ether
and petrol ether, soluble with difficulty in alcohol, and giving a turbid
suspension with water. Actinomycin A is bright redj it possesses ex-
tremely high bacteriostatic properties but is rather slowly bactericidal.
Actinomycin B is colorless j it has comparatively little bacteriostatic
action but possesses strong bactericidal properties. Despite the fact that
the organism produces a dark-brown pigment on organic media, actino-
mycin does not possess the enzymatic properties of a tyrosinase.
The purification of actinomycin A was effected by chromatographic
adsorption, followed by fractionation of eluate. The orange-brown resi-
due left after treatment with petroleum ether was dissolved in benzene,
172 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
filtered, and allowed to pass through a tower packed with aluminum
oxide. On washing the tower with large amounts of benzene, a chro-
matogram slowly developed. The column was then washed with a solu-
tion of 1 5 parts acetone to 85 parts benzene until the yellow-orange band
approached the bottom of the column. The elution of the pigment from
the column was accomplished finally by further washing with 30 per
cent acetone in benzene until the eluate was faintly yellow in color. The
later eluates were found by assays to contain all the active pigment,
whereas all previous eluates, as well as the fractions remaining on the
adsorbent, showed no bacteriostatic or bactericidal activity.
Pure actinomycin A was obtained by concentrating the 30 per cent
acetone-benzene eluates to dryness, then recrystallizing the red solid
residue from acetone-ether mixtures or from ethyl acetate. From these
solvents, the pigment separated as vermilion-red platelets which
melted at 250° C, with slow decomposition. The pigment is readily
soluble in chloroform, benzene, and ethanolj moderately in acetone
and hot ethyl acetate j and slightly in water and ether. The color of the
solid pigment depends on its state of subdivision; when ground very
fine, its color is orange-red.
Actinomycin A is optically active, a solution of 5 mg. in 2 cc. ethanol
in a I dm. tube having a rotation — 1.60°; [afj = —32.0° ± 5. Its
molecular weight was found to be around i ,000. Cryoscopic measure-
ments in cyclohexanol and in phenol gave molecular weights of 768 to
780 and 813, respectively. The approximate molecular formula was
found to be C41H56N8O11. Actinomycin A exhibits characteristic ab-
sorption in the visible and ultraviolet regions. In ethyl alcohol, it shows
strong absorption at 450 (E| ^ = 200) and between 230 and 250.
Actinomycin A is not soluble in dilute aqueous alkali or in dilute min-
eral acids. It is soluble in 10 per cent hydrochloric acid and appears to
be regenerated by diluting such solutions with water. With strong alco-
holic alkali a purple color is formed, which rapidly disappears. Actino-
mycin A is readily reduced by sodium hydrosulfite and by stannous
chloride, but is unaffected by sodium bisulfite. With sodium hydro-
sulfite the reduction is characterized by a change in color from red to
pale yellow. The color change is reversed by exposing the reduced pig-
ment to air. The same reversibility of color occurs when the pigment
SUBSTANCES PRODUCED BY ACTINOMYCETES 173
is subjected to catalytic hydrogenation in the presence of platinum
oxide. The pigment has one or more functional groups capable of re-
versible reduction-oxidation (probably quinone in nature) and several
others capable of acetylation (probably hydroxyls). The quinone-like
structure of the pigment is borne out by its sensitivity to alcoholic alkali,
and to hydrogen peroxide in the presence of sodium carbonate. In the
latter instance, the color rapidly disappears and a cleavage seems to
occur.
Actinomycin in alcohol-water solutions is resistant to the action of
heat, being able to withstand boiling for 30 minutes. When such solu-
tions are made acid, however, boiling has a destructive effect upon the
activity of the substance, the extent of destruction being directly pro-
portional to the concentration of acid. The effect of alkali, however, is
much greater. Dilute alkali changes the color of the substance to light
brown, accompanied by a reduction in activity, which can be largely re-
stored when the solution is made neutral again. At a higher alkalinity
(0.25 N), especially at boiling temperature, the activity and reversibil-
ity are destroyed. Exposure of solutions to light for i to 3 months re-
duces the activity of the pigment very little.
Streptothricin is produced by Stre-ptomyces lavendulae grown in a
medium containing glucose or starch ( i per cent) as a source of energy,
and tryptone, glycocoll, glutamic acid, or other organic nitrogenous
compound (0.3 to 0.5 per cent) as a source of nitrogen. Sodium nitrate
is a somewhat less favorable source of nitrogen. The organism is grown
in stationary, shallow cultures containing starch as a source of carbon
or glucose and a small amount of agar, or in submerged cultures. The
optimum temperature for the production of streptothricin is 23° to
25° C. (926). The relation between growth of the organism and pro-
duction of the antibiotic substance is brought out in Table 33.
Streptothricin is soluble in water and in dilute mineral acids, but is
destroyed by concentrated acids. It is insoluble in ether, petrol ether,
and chloroform. In the crude culture-filtrate and in the alcohol-precipi-
tated form, streptothricin is thermolabile, whereas in the purified state
it is thermostable, withstanding 100° C. for 15 minutes. Treatment
with proteolytic enzymes does not reduce its activity. On adjusting the
reaction of the medium, when growth is completed, to /)H ^.s with
174 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
acid, a precipitate is produced, the filtrate containing virtually all the
activity.
Streptothricin is completely adsorbed, at neutrality, on charcoal
(norit A), from which it can be removed by treatment for 8 to 12 hours
with dilute mineral acid or acid alcohol. The acid extract is neutralized
and concentrated in vacuo, at 50° C, just to dryness j the residue is ex-
tracted with absolute alcohol, filtered, evaporated, and taken up in
TABLE 33. GROWTH OF STREPTOMYCES LAVENDULAE AND PRODUCTION
OF STREPTOTHRICIN ON TRYPTONE-STARCH MEDIUM
INCU-
DRY WEIGHT
OF MYCE-
NITROGEN
IN MYCE-
ACTIVITY
IN UNITS
BATION
STARCH
LIUM IN
LIUM IN
E.
B. sub-
AERATION
IN DAYS
LEFT
MILLIGRAMS
MILLIGRAMS
coli
tilis
Shaken
2
+++
10
5
Shaken
3
+
225
18.2
10
50
Shaken
4
0
293
26.2
75
250
Shaken
6
0
231
17-3
100
300
Shaken
8
0
75
200
Shaken
12
0
142
9.6
30
50
Stationary
7
+++
50
200
Stationary
10
Tr
235
18.8
50
300
Stationary
14
Tr
60
250
FromWaksman (926).
water. It can also be precipitated from the neutralized solution with
ether. These preparations usually contain 40 to 50 per cent inorganic
matter and 2 to 3 per cent nitrogen, on an ash-free basis. Further con-
centration and reduction in ash content can be obtained by subsequent
treatments. On electrodialysis, the active substance moves to the cath-
ode at fH. 7.0. A highly active fraction has been isolated by chromato-
graphic adsorption on aluminum oxide.
Streptothricin acts as a base, with an optimum at /)H 8.0. It is re-
pressed by dextrose and by acid salts. Bacteria subject to the action of
streptothricin show greatly enlarged cells, due to incomplete fission
(287, 1002).
Another substance, designated as streptomycin (795), is similar in
SUBSTANCES PRODUCED BY FUNGI 175
many respects to streptothricin, although it differs somewhat in its anti-
bacterial spectrum and its lower toxicity for animals.
Proactinomycin is produced by N. gardneri grown in soft agar
media, from which it is extracted by organic solvents, such as ether, amyl
acetate, benzene, and carbon tetrachloride. It can be re-extracted in
water by adjusting the ^H to 4.0 with HCl or H2SO4. The aqueous
extract is concentrated in vacuo and evaporated to dryness from the
frozen state. A white powder, very easily soluble in water, is obtained.
The yield of the material is 60 mg. from i liter of culture. The sub-
stance is fairly stable, though boiling for 10 minutes at fVL 2.0 or fH
7.0 results in a small loss of activity. Boiling at />H lo.o destroys the
greater part of the antibacterial activity. Proactinomycin has basic prop-
erties and is precipitated from aqueous solution by such base precipitants
as picric acid, picrolonic acid, and flavianic acid.
SUBSTANCES PRODUCED BY FUNGI
The early studies of the phenomenon of staling accompanied by the
production of antibacterial and antifungal substances (83), some of
which could be removed from the acidified medium by ether or by col-
loidal clay (700), have recently been superseded by more exact and
detailed chemical studies. Only a few of the many antibiotic substances
produced by fungi have so far been identified, however. Some are pro-
duced in complex organic media, others in simple synthetic media. Only
the more important substances will be discussed here. Among these,
penicillin occupies a leading place because of its low toxicity and its ac-
tivity in vivo.
Penicillin is produced by various strains of P. notatum and P.
chrysogenum, and probably by a variety of other fungi (272a, 940a).
The penicillin-like nature of an antibiotic substance is usually estab-
lished by its biological and chemical properties: activity against S. au-
reus and not against E. colt; extraction in organic solvents at fH 2
and re-extraction in water at fH 7 ; inactivation by acid and alkali j par-
tial inactivation by heating at 100° C. and /)H 7 for 15 minutes j com-
plete inactivation by penicillinase and by copper ionsj inactivation by
methyl alcohol (272a).
176 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
The strain of the organism used, the composition of the medium, and
the conditions of growth greatly influence the yield of penicillin. Com-
plex organic media containing glucose or brown sugar as a source of
carbon are essential. Nitrate is used as a source of nitrogen j the medium
also must contain a phosphate and certain other minerals. The supple-
mentary addition of a stimulating substance in the form of yeast extract,
corn steep, or certain vegetable juices is essential for the maximum pro-
duction of penicillin. Since the organism produces an acid, probably glu-
conic, in the medium, some CaCOg must also be added. The metabolism
of P. notatum in relation to penicillin production is illustrated in Fig-
ure 13 (page 129).
Four methods have been proposed for the growth of the fungus and
production of penicillin. These are:
Surface growth in shallow liquid media; usually flasks, bottles, and other
containers are employed, the depth of the medium being 1.5 to
2.0 cm.
Submerged growth in liquid media; the vessels must be provided with
proper stirrers and aeration
Surface growth upon semi-solid media, including grain and bran (730)
Circulation of medium through a column, the supporting material being
made up of wood shavings or pebbles; the rate of flow of the me-
dium is very important
Since the various strains of penicillin-producing organisms vary
greatly in their optimum conditions for the production of this antibiotic
substance, different strains must be used for different conditions of cul-
tivation.
Penicillin is produced in the medium when active growth begins but
reaches a maximum soon after the growth maximum, which occurs in
7 to 14 days in stationary cultures and in 3 to 7 days in submerged cul-
tures, at 20° to 25° C.
Penicillin is soluble in ether, acetone, esters, and dioxanej it is mod-
erately soluble in chloroform, slightly soluble in benzene and in carbon
tetrachloride. It is soluble in water to the extent of 5 mg./ml.
It is inactivated by oxidation and by evaporation at 40° to 45° C. in
acid and in alkaline solutions, although it is fairly stable at /»H 5 to 6.
SUBSTANCES PRODUCED BY FUNGI 177
If the solutions are adjusted to /)H 6.8, it retains its potency for
3 months. The crude penicillin does not dialyze through a collodion
membrane and resists heating at 6o° to 90° C. for short periods j it
remains active when heated at 100° C. for 5 minutes but not for 10 min-
utes (737).
Fleming first reported that penicillin is insoluble in ether. This was
found (135) to be due to the alkaline reaction of the filtrate 5 for at fH
2.0 ether removes completely the antibacterial substance. The ether
extract is evaporated with some water in vacuo at 40° to 45° C, the
residual water containing the active substance, which is extremely labile.
For practical purposes, penicillin is extracted from the acidified cul-
ture by means of different organic solvents, such as ether or amyl ace-
tate (7, 8). It is then removed from the solvent by shaking with phos-
phate buffer or with water at /)H 6.7. Since penicillin is rapidly de-
stroyed at a high acidity, the first extraction must be carried out very
quickly and at a low temperature. In the presence of the solvents, peni-
cillin is stable for several days. The aqueous extract may be partly de-
colorized by shaking with charcoal and filtering. The solution is cooled,
acidified, and extracted several times with ether or amyl acetate j the
extracts are passed through an adsorption alumina column, or through
a 2.5 per cent precipitate of an alkaline earth carbonate on silica gel
(109). Water may often contain a pyrogenic or heat-producing sub-
stance that must be removed from the penicillin.
The following four main zones were recognized in the chromato-
grams, beginning from the top :
1 . A dark brownish-orange layer, the depth of which is inversely propor-
tional to the amount of charcoal used for the decolorization ; this zone
contains some penicillin
2. A light yellow layer containing most of the penicillin but none of the
pyrogen
3. An orange layer which contains some penicillin and some or all of the
pyrogen
4. A brownish or reddish-violet layer which contains almost no penicillin;
the pigment disappears on exposure to light
fThe fourth fraction is discarded, and the others are eluted with
M/15 phosphate buffer (-pH 7.2). The penicillin is again extracted
178 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
with ether, then with water, sodium hydroxide being used to adjust the
fH. Since penicillin is destroyed readily in alkaline solution, care must
be taken in adding the alkali. The "nonpyrogenic" or "therapeutic"
fraction, which contains about 8o per cent of the penicillin, is extracted
with pyrogen-free water. It is a deep reddish-orange liquid, yellow in
dilute solution, with a characteristic smell and bitter taste.
By means of adsorption, distribution between solvents, and reduc-
tion, a barium salt or penicillin was finally obtained (3, 7, 8) which was
homogeneous by chromatographic analysis and gave 450 to 500 Oxford
units per milligram of dry material. The active substance was found to
be a salt of a strong dibasic acid with ^H values approximately 2.3 and
2.5y having a formula of C24H;..OioNoBa. The molecule contained one
carboxyl, one latent carboxylic, two acetylatable, at least five C-Me
groups, and no easily reducible double bond. The penicillin thus pre-
pared was more sensitive to oxidizing agents than to reducing agents j it
was unstable toward dilute acids and alkalies, and to heat (loss of COo),
primary alcohols, and various heavy metal ions. Tentative suggestions
were made concerning its chemical nature as follows: (a) a polysubsti-
tuted hydroaromatic ring structure j (b) the acidic groups (carboxyl)
not conjugated with the chromophore responsible for the absorption j
(c) the possible presence of a trisubstituted a-unsaturated ketone
grouping.
Another method for obtaining penicillin has been suggested (598).
In this method, the culture medium was adjusted to -pH 3 to 4, satu-
rated with ammonium sulfate and extracted with chloroform. The con-
centrated chloroform extract was treated with phosphate buffer at fH
7.2 to remove the active substance. This process was repeated, the less
active substance being separated from the active fraction by extraction
with chloroform at different ranges. By precipitating the concentrated
extracts from petroleum ether, the free acid form of penicillin was ob-
tained. By saturating the chloroform-benzol solution with dry am-
monia gas, an ammonium salt was obtained which gave a dark yellow
microcrystalline powder. The salt was more stable than the acid form.
By acetylating or benzoylating the ammonium salt a further increase in
stability was obtained. The analysis of the penicillin prepared by this
method was found to fit best the formula Ci4Hic)NO,( or C]4Hi-N05 -f-
SUBSTANCES PRODUCED BY FUNGI 179
HoO. This penicillin was strongly dextrorotatory and had an adsorp-
tion maximum of 2,750 A°. The preparation had an activity of 32,000,-
000 dilution units against hemolytic streptococci, which corresponds to
about 240 Oxford units per milligram.
The most suitable form for general use is the barium salt. In this
form, it retains its antibacterial activity for an indefinite period. The Ba
salt of penicillin is soluble in absolute methyl alcohol, but is insoluble in
absolute ethyl alcohol. Penicillin forms water-soluble salts with most
heavy metals, except Fe^"^"^. It is inactivated by a number of heavy metal
ions, especially Cu, Pb, Zn, and Cd. Penicillin is stable toward atmos-
pheric oxygen, but is oxidized by H0O2 and KMn04, the antibacterial
activity being lost (2, 5, 1 1 1 ).
In assaying penicillin, tests are made for potency by one of several
procedures: sterility, moisture content, presence of pyrogenic sub-
stances, and toxicity (249).
Several derivatives of penicillin have been obtained:
1 . Penicillamine, a degradation product ( 6) , is produced by hydroly-
sis of the barium penicillin for one hour at 100° C. with N/io sulfuric
acid J the formula C«HiiN04.HCl has been suggested for itj the pres-
ence of a glyoxal nucleus has been indicated.
2. Penillic acid (210), a dextrorotatory substance having a pale blu-
ish fluorescence, is insoluble in ether and soluble in butyl alcohol; it is
produced by keeping penicillin in aqueous solution at ^H 2.0 ; it has
some of the properties of an amino acid.
3. Methyl, ethyl, «-butyl, and benzohydryl esters of penicillin have
been prepared (599, 600) ; these are insoluble in neutral or slightly al-
kaline buffers, but soluble in benzene j these esters are much less active
than true penicillin m vitro but are active in vivo.
The chemical nature and mode of action of the second antibacterial
substance produced by P. notatum- are given in Table 34. This sub-
stance is a protein and acts as a glucose oxidase, oxygen being required.
It is characterized by its action not only upon gram-positive but also
upon many gram-negative bacteria, and by the fact that the presence of
glucose is required for its activity. Its action is inhibited by the presence
of catalase (151, 416, 751).
Flavicin, a substance found (100, 461, s^s^ 5^7, 929) to be similar
180
CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
TABLE 34. NATURE OF SECOND ANTIBIOTIC FACTOR PRODUCED BY
PENICILLIUM NOTATUM
PHYSICAL AND
DESIGNATION
CHEMICAL
Penatin
Notatin
Penicillin B
PROPERTIES
(493)
(57>i5i)
(751,902)
Solubility
Soluble in water,
Soluble in water.
Soluble in water
insoluble in or-
insoluble in or-
ganic solvents
ganic solvents
Adsorption or pre-
Adsorbed on kaolin,
Precipitated by
Adsorbed on ben-
cipitation
at ;>H 4.0, eluted
acetone or
zoic acid, pre-
with sodium phos-
tannic acid
cipitated by
phate, precipitated
acetone
by dioxane and
phosphotungstic
acid
Chemical nature
Protein
Flavoproteln
Protein
Glucose required
Required
Required
Glucose and cer-
for activity
tain other car-
bohydrates re-
quired
Ox\-gen required
for activity
Activity against
gram-negative
bacteria in addi-
tion to gram-
positive bacteria
Sensitivity to alka-
lies
Biological nature
Apparently required Required
Required
Decidedlv active
Sensitive
Hydrogen peroxide
produced; not
verv toxic
Highly active Definitely active
Activity de-
stroyed at />H
above 8.0
Glucose-oxidase
Fairly toxic to
animals
Note. In view of the fact that the three preparations have been obtained by somewhat diflFerent
chemical procedures and in view of the variation of the strains of P. notatum producing this sub-
stance, there is a possibility that the different preparations may vary in chemical nature and pos-
sibly also in biological behavior.
SUBSTANCES PRODUCED BY FUNGI 181
in every respect to penicillin, is produced by A. flavus (929) j the same
is true of gigantic acid produced by A. giganteus (688). Preparations
of flavicin have also been designated as flavatin (716, 717), aspergillin
(100), flavacidin (461), and parasiticin.
AsPERGiLLic Acid is produced by A. -flavus (461, 978) grown on
tryptone-glucose media, as shown previously (page 131). The pure
acid has an m.p. of 93° C. (84° to 96'^) and has optical activity of
[ajo = +14°- The formula CioHoqNoOo has been proposed for this
substance. It possesses a hydroxyl group which gives it its acid nature
(^K S-S)- It is stable under acid and alkaline conditions and can be dis-
tilled with steam or in vacuo without loss of activity. When grown in
brown-sugar-containing media, a closely related substance is formed,
having the formula CioHooO.-jNo and an m.p, of 149°, with lower bio-
logical activity. Aspergillic acid is active against both gram-positive and
gram-negative bacteria.
CiTRiNiN was isolated from Penicillium ckrinum (714). It is pro-
duced by growing the organism on a synthetic medium, with inorganic
salts of nitrogen and with glucose as a source of carbon. The culture fil-
trate is acidified with HCl, and the substance crystallized from boiling
alcohol. Citrinin forms a monosodium salt which, at ^H 7.0 to 7.2, gives
virtually colorless solutions in water. Its bacteriostatic activity is much
lower than that of penicillin (33). It is a yellow crystalline solid, m.p.
170-171° (with decomposition). Its formula is C10H14O5.
Citrinin is a strong acid, changing in color from lemon-yellow at /)H
4.6 to orange-pink at fYl S-^ to 5.8 and to cherry-red at /)H 9.9 (352).
Addition of FeCla to the culture solution gives a heavy buff-colored
precipitate, which dissolves in excess of reagent to give an intense iodine-
brown solution (871 ). It has little if any activity against gram-negative
bacteria and about 50,000 dilution units against B. subtilis and S. aureus.
Penicillic Acid was first isolated in 1 9 1 3 by Alsberg and Black (19)
as a metabolic product of Penicillium fuberulum. A limited air supply
and an acid reaction of the medium favor the production of this acid, to
which the chemical formula C8H10O4 was given. This acid is a rather
weak antibiotic substance active largely against gram-positive bacteria j
however, it is more active against gram-negative bacteria than penicillin,
giving complete inhibition of E. coli m concentrations of 1:50,000,
182 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
whereas penicillin does not inhibit this organism even in concentrations
of 1 : 1,000. It was found to possess antibiotic properties also against
yeasts, and to be toxic to animals when injected subcutaneously in con-
centrations of 0.2 to 0.3 gm. per kilogram weight.
More recently, penicillic acid was isolated (661, 664) by evapora-
tion of the culture solution, the crude acid crystallizing on cooling. It
was purified by recrystallization from hot water. Yields greater than
2 gm. per liter of culture were obtained. The acid is a stable, colorless
compound which is appreciably soluble in cold water and gives a series
of colorless and readily soluble salts (s^j 470a).
Penicillic acid was shown to have the constitution y-keto-p-methoxy-
S-methylene-A-a-hexenoic acid, which exists in both the keto and lactone
forms :
CHo=CH(CH3)COC(OCH3)=CHCOOH -
CHo=CH-(CH3)-C(OH)-C(OCH,)=CH-CO
i o !
FuMiGATiN. Among the other quinones isolated from fungi, fumi-
gatin deserves consideration (21, 712). It is a 3-hydroxy, 4-methoxy,
2 : 5-toluquinone or C8H8O4 (Figure 16, page 164).
All quinones have been divided into three groups on the basis of their
action on Stafhylococcus: (a) those that have a markedly weaker anti-
bacterial action than fumigatin, including toluquinone and some of its
derivatives i (b) those that are somewhat more effective than fumi-
gatin, including 3:4 dimethoxytoluquinone 5 (c) those with activity
greater than that of fumigatin (methoxytoluquinones). The introduc-
tion of -OCH3 into the quinone nucleus results in an increase in anti-
bacterial activity. The introduction of an OH or the replacement of
-OCH3 by OH results in a decrease in activity. None of these quinones,
however, has any very striking action on gram-negative bacteria, such as
E. coU (660). Electrode potentials of quinones have been found to be
unrelated to their bacteriostatic action against E. coli, but for S. aureus,
the reduction potentials fall within certain limits (417, 6G6).
Clavacin is produced by A. clavatus (935), P. fatulum (patulin
[713]), P. clavijorme (114, 115),/*. exfansum (22), and probably a
number of other fungi (470). It can be isolated from the culture filtrate
SUBSTANCES PRODUCED BY FUNGI 183
either by preliminary adsorption on norit followed by removal with
ether or chloroform, or by the direct treatment of the culture with ether.
The extract is evaporated, leaving a brown substance j this is treated
with a small amount of water, and the aqueous solution again extracted
with ether, Clavacin crystallizes when the ether solution is concentrated,
or after preliminary purification over a silica gel column. Clavacin thus
isolated (429, 473) from A. davatus cultures showed the following
chemical properties: melting point, 109-110° C.j empirical formula,
C7H6O4J molecular weight (cryoscopic in benzophenone) 154^ semi-
carbazone, darkens at 200°, decomposes at 290° C. j 2,4-dinitrophenyl-
hydrazone, darkens above 190°, decomposes at about 300° C. j lactone
group indicated by slow reaction with alkali; saponification number 70
(evidently molecule cleaved); Zerewitinoff determination (in ;?-butyl
ether) shows slightly less than one active hydrogen per mol; esterifica-
tion by the acetic anhydride-pyridine method shows one hydroxyl per
mol. Clavacin, a neutral optically inactive compound, darkens and
loses activity in the presence of alkali, reduces Fehling's solution
strongly on heating, and readily decolorizes alkaline permanganate;
it does not react with aqueous ferric chloride or Schiff's reagent, and re-
duces ammoniacal silver nitrate.
Clavacin (patulin) is anhydro-3-hydroxymethylene-tetrahydro-Y-
pyrone-2-carboxylic acid, for which a formula has been suggested (713),
as shown in Figure 1 6.
Clavacin is soluble in water and in most of the more common organic
solvents except light petroleum. It is about equally active against gram-
positive and gram-negative bacteria, its growth inhibition being about
200,000 dilution units. Its lethal action upon mice is about 25 mg. per
kilogram body weight, when given intravenously or subcutaneously.
Claviformin isolated (114, 115) from P. clavijorme has recently
been shown to be identical with clavacin and patulin (47, 927).
FuMiGACiN is produced by different strains of A. jumigatus. It can
be extracted from the culture medium either by preliminary adsorption
on active charcoal followed by treatment with ether and alcohol, or by
direct extraction of culture in accordance with the following method
(593): The culture filtrate is acidified to /)H 2 with phosphoric acid
and extracted three times with ether, the combined extracts equalling
184 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
the volume of the filtrate. The ether is evaporated to one-tenth of its
volume and the concentrate is shaken repeatedly with saturated sodium
bicarbonate solution, which removes a dark-red pigment. The solution
is then exhaustively extracted with 6 per cent sodium carbonate solu-
tion. The ether phase, on evaporation, yields gliotoxin. The sodium
carbonate solution is acidified and distributed several times with ben-
zene j the partly crystalline residue from the benzene (7-12 mg. per
I L of culture filtrate), on repeated recrystallization from methanol,
yields pure fumigacin in the form of filamentous needles. Fumigacin
melts with some decomposition at 215-220° C, depending on the rate
of heating, [a]^^ =:= — 132 ± 2° (0.41 per cent in chloroform). The
ultraviolet absorption curve shows only strong end absorption below
260 m|_i with Ej^ =298 at 234 mp. Fumigacin is practically in-
soluble in water, sparingly soluble in cold methanol and ethanol, and
more readily soluble in acetone, ethyl acetate, benzene, and ether. It is
easily dissolved by chloroform, acetic acid, and dioxane.
The following reactions are negative: ferric chloride. Legal, fuchsin
sulfurous acid, Tollens, Molisch, Rosenheim, Hammersten (for cholic
acid), Jaffe-Tortelli, digitonin. The Zimmerman reaction with m-dini-
trobenzene for ketones is strongly positive. In the Chabrol-Charonnet
test for bile acid (phosphoric acid and vanillin) a strong red color is
obtained. Likewise, the Liebermann-Buchard test gives an intense
blood-red color. Fehling's solution is slowly but perceptibly reduced
at 100° C. The formula that has been suggested is Cof)H..8-4o07. Puri-
fied fumigacin has recently been shown to be identical with helvolic acid,
isolated from a strain of A . jumigatus.
Gliotoxin was isolated from cultures of Trichoderma, Gliocladium,
and A. jumigatus (459, 960, 961), as well as from other fungi (593,
933), the greatest activity being produced in 2 days. It was extracted
from the culture medium by the use of chloroform. The latter was dis-
tilled off, and the residue taken up in a small amount of hot benzene or
95 per cent alcohol, from which, on cooling, silky white needles crys-
tallized. It was recrystallized from benzene or alcohol. It was found
(965) to have a molecular weight of 347, an optical rotation of [a]^^ =
— 239°, and an m.p. of 121° to 122° C. It analyzed C14H16N0O4S0,
later shown (459) to be C13H14N2O4S0.
SUBSTANCES PRODUCED BY YEASTS 185
Gliotoxin is sparingly soluble in water and readily soluble in alcohol.
It is unstable, particularly in alkaline solutions, and is sensitive to
oxidation and to heating (961 ) j it is inactivated by heating for 10 min-
utes at 100° C. ( 17). Its potency was found to be destroyed by bubbling
oxygen for 5 minutes.
Gliotoxin is toxic to Rhizoctonia hyphae in a dilution of i : 300,000,
which is about two-thirds of the toxicity of HgCL. The crystals, as well
as the crude material, were found to be toxic also to Trichoderma, but
the minimum lethal dose was about 40 times greater than that required
for Rhi-zoctonia. Its antibacterial properties are brought out later.
Other Substances. Several other antibacterial substances have been
isolated from fungi, but have not been adequately studied either chemi-
cally or biologically. It is sufficient to mention the following:
Puberulic acid, CgHsOe, a colorless crystalline dibasic acid, with an
m.p. of 316-318°, and puberulonic acid, C8H4O6, a bright-yellow acid
with an m.p. of 298° C, produced (38, 58, 66$) by P. fuberulum. The
first is a quinol and the second is quinonoid. They have little activity
against gram-negative bacteria and some activity against gram-positive
types.
Penicidin was isolated (26) from a species of Penkillium. It is soluble
in ether, alcohol, chloroform, and dilute acids, but not in petrol ether.
It is destroyed by bases, and is adsorbed on active charcoal. It was found
to be active against E. tyfhosa.
Chaetomin is produced by a species of Chaetommm {Ch. cochliodes)
grown in complex organic media. It is active largely against gram-posi-
tive bacteria (930).
Kojic acid (Figure 16), produced by A. oryzae, apparently also pos-
sesses some antibacterial properties, more against gram-negative than
gram-positive bacteria (282).
SUBSTANCES PRODUCED BY YEASTS
According to Fernbach (253), certain yeasts produce volatile sub-
stances which are toxic not only to other yeasts but also to bacteria. Rose
yeasts {Torula suganii), either fresh or heated to 120 to 130° C, were
found (654) to contain a substance which has an antagonistic action
186 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
against fungi, especially in the young mycelial stage, but not against
yeasts ; the growth of A . niger was reduced by 60 to 70 per cent and
that of A. oryzae by 25 to 30 per cent. The substance was not found in
the ash of the organism and was not secreted in the filtrate, but re-
mained in the yeast cells. An alkaline reaction was unfavorable to its
formation and action. The active substance was soluble in acetone, alco-
hol, ether, and chloroform, and was adsorbed by kaolin, Seitz filter,
paper, and by the fungus mycelium. It could be removed from the
kaolin by treatment with ether or acetone. Acetone-treated yeast no
longer had an antagonistic effect, but only a stimulating one.
According to Schiller (798), yeasts produce a bacteriolytic substance
only in a state of "forced antagonism," that is, in the presence of staphy-
lococci and certain other bacteria. The substance is thermolabile, since
it is destroyed at 60° C. It is active also outside the cell. More recently
(144), the active substance of yeast was concentrated. In a crude state,
the active material was found to be nonvolatile and readily soluble in
water, in 95 per cent alcohol, and in acetone containing a trace of water.
It was stable at 100° C. at /)H 7.3. It contained nitrogen but no sulfur.
Although a positive biuret reaction was obtained, it appeared that the
protein was present as an impurity.
MICROBIAL LYSOZYME
The enzyme lysozyme or an antibiotic substance similar to it has
often been reported to be produced by microorganisms. Fleming (264)
found that the lysozyme of &gg white was soluble in water and in dilute
NaCl solution. It was precipitated by chloroform, acetone, ether, alco-
hol, and toluene. It was not acted upon by pepsin or trypsin. It was par-
ticularly active against micrococci, bringing about their lysis.
Lysozyme has been found in nearly all mammalian tissues and secre-
tions, in certain vegetables, and in bacteria (880). It was found to be a
polypeptide containing 1 6 per cent nitrogen and 2 to 3 per cent sulfur
and having a molecular weight of 1 8,000 to 25,000. It is soluble and
stable in acid solution, insoluble and inactivated in alkaline solutions,
and inactivated by oxidizing agents (601). It diffuses in agar and
through cellophane, and thus is markedly different from bacteriophage
MISCELLANEOUS ANTIBIOTIC SUBSTANCES 187
(323). It is fixed on the bacterial cells (264), It acts primarily upon
the cell membrane of bacteria, the highly viscous component of the bac-
terial cell (the mucoids), especially the sugar linkages of the complex
amino-carbohydrates (601), being disintegrated by the enzyme. The
degradation of the bacterial polysaccharide to water-soluble products
( N-acetylated amino-hexose and a keto-hexose) by lysozyme is accom-
panied by complete lysis of some of the bacteria. In the case of other
lysozyme-sensitive bacteria, such as B. subtilis, no lysis occurs 5 appar-
ently the morphological structure of these bacteria does not depend ex-
clusively on the unaltered state of the substrate for lysozyme (243),
The formation of a lysozyme-like material was demonstrated by
Fleming (262) for a coccus isolated from dust. A sarcina susceptible
to egg-white lysozyme also was found (601) to produce an autolytic
enzyme similar to it. It has been suggested that the antibacterial action
of saliva may be due to the presence in it of antagonistic bacteria (704).
The lysozyme of saliva is known to act primarily upon gram-positive
bacteria (880). Auerswald (28) tested a large number of bacteria for
their ability to antagonize diphtheria and pseudo-diphtheria organisms.
Only the spore-forming B. mesentericus and B. subtilis groups pro-
duced antagonistic substances, but these bacteria were not found in the
saliva. Cultures of bacteria isolated from the saliva had no antagonistic
effect, thus proving that the action of saliva need not be due to its bac-
terial content.
MISCELLANEOUS ANTIBIOTIC SUBSTANCES
In connection with the antibiotic substances of microbial origin, at-
tention may be directed to certain substances, of apparently similar na-
ture, of plant and animal origin. No attempt will be made to discuss
here the antibacterial blood reactions, including antibody formation,
precipitin reactions, agglutination, phagocytosis j lysin formation} ac-
tion of various body fluids j and other animal and plant reactions against
bacteria and in response to bacterial infection.
Unbleached wheat flour was shown (864) to contain a protein which
had bacteriostatic and bactericidal activity in vitro; although this activ-
ity was greatest against gram-positive organisms, it also had some ac-
188 CHEMICAL NATURE OF ANTIBIOTIC SUBSTANCES
tivity against gram-negative types. The antimicrobial action of this
protein can be neutralized by means of a phosphatide (1004), a reac-
tion which may be due to the formation of a lipoprotein that has no
longer any antibiotic activity. Sherman and Hodge (817) demon-
strated that the fresh juice of several plants has a marked bactericidal
effect. The active substance in the juice could be adsorbed on activated
carbon and by passage through fine Berkfeld filters. The substance was
thermolabile, being destroyed at 60° C. in 10 minutes. It has been re-
cently demonstrated (658) that antibacterial substances are widely dis-
tributed among plants.
Milk was found (657) to contain several thermolabile bactericidal
substances and two thermostable compounds which acted injuriously
upon lactic acid bacteria. Orla-Jensen emphasized that the growth of
bacteria in milk is influenced by a combination of activators or growth-
promoting substances and of inhibitors, the predominance of one or the
other being determined by various conditions. These substances influ-
ence the development of specific lactic acid bacteria during the spon-
taneous souring of milk.
CHAPTER I I
THE NATURE OF ANTIBIOTIC ACTION
Sulfanilamide^ fenicillin-j and gramicidin can be clearly set afart
from the classical antiseptics which are general frotoflasmric
foisons. All three substances are frimarily bacteriostatic rather
than bactericidal in their action. Since they do not destroy the res-
piration of bacteria y one may assume that the inhibition of growth
which they cause defends not upon interruption of the cellular
metabolism as a whole ^ but rather upon some subtle interference
with certain individual reactions. To interrupt the pathogenic
career of an infectious agenty therefore y it is not necessary to kill
the invading celly but only to block one step in its metabolic path
by some specific inhibitor. — Dubos (192).
ANTIBIOTIC SUBSTANCES AND CHEMICAL
DISINFECTANTS
Since antibiotic substances vary greatly in their origin and in their
chemical nature, they would be expected also to vary in their mode of
action upon the cells of bacteria and other microorganisms, and in the
effect upon the animal tissues when these agents are used for chemo-
therapeutic purposes. Comparatively little is known concerning these
mechanisms. It is known, however, that antibiotic substances act chiefly
by interfering with the growth of the bacterial cell, although in many
cases they are able to bring about the lysis of the cell as well. Because
of the first effect, it has been assumed that antibacterial agents are struc-
turally related to bacterial metabolites that usually function as co-
enzymes (560). In this connection, the following properties of anti-
biotic agents are of particular significance :
Most antibiotic substances are strongly bacteriostatic in nature and only
weakly bactericidal, though a few are also strongly bactericidal, and
some are even bacteriolytic.
Some substances act primarily in vitro and only to a limited extent in vivo
because of interference of the body tissues with their action; others,
however, act readily upon bacteria in vivo.
190 NATURE OF ANTIBIOTIC ACTION
A few antibiotic agents are fairly nontoxic to the animal body; others are
somewhat more toxic but can still be utilized; and some are so highly
toxic that they offer little promise as chemotherapeutic agents.
Antibiotic agents differ greatly in their solubility: some are water soluble;
others are alcohol soluble and only slightly soluble in water; and
some are acids and react with alkali solution to form soluble salts.
Some antibiotic agents are stable under a variety of conditions, whereas
others are unstable.
Some antibacterial substances are hemolytic; others have apparently no
injurious effect upon the blood cells. The latter can, therefore, be
used for general body treatment, whereas the former are suitable
only for local applications.
Since antibiotic substances are selective in their action upon microorgan-
isms, none can be expected to be utilized as general agents against all
bacteria. This also points to the remarkable physiological differences
in the morphology and physiology of the bacterial cells, and to the
differences in the mode of action of the different antibiotic substances
upon the various bacteria.
A comparison of the antibacterial action of the antibiotic substances
produced by two bacteria v^^ili serve to illustrate some of the foregoing
points. Pyocyanin, produced by Ps. aeruginosa, inhibits the growth of
many gram-positive and gram-negative bacteria in dilutions as high as
1 : 1 00,000 J pyocyanase and hemipyocyanin have less activity upon the
bacteria, but yeasts are more sensitive to them than to pyocyanin. Ty-
rothricin, produced by B. hrev'ts, is far more specific in its action, which
is limited largely to gram-positive bacteria. The sensitivity of patho-
genic fungi to these compounds also differs markedly (855). Other
striking differences are found on comparing two types of antibiotic sub-
stances produced by fungi, namely, penicillin and clavacin, and two
substances produced by actinomycetes, namely, streptothricin and ac-
tinomycin. The bacteriostatic spectra of these four substances are re-
corded in Table 35. The first of each pair has a limited toxicity to ani-
mals, and the second is highly toxic. Whereas penicillin acts largely
upon gram-positive bacteria and only upon a few gram-negative organ-
isms, streptothricin acts alike upon certain bacteria within each group.
Clavacin and actinomycin, both of which are highly toxic, differ simi-
larly in their action upon bacteria, the first being largely active against
ANTIBIOTIC SUBSTANCES AND DISINFECTANTS
191
gram-positive and the second active against members of both groups.
These four compounds show various other differences in the nature of
their antibacterial action. Differences in the bactericidal properties of
other antibiotic substances are brought out in Tables ^6 and 37.
Various attempts have been made to compare the antibacterial action
of antibiotic substances with that of organic antiseptics. According to
Suter (866), the bactericidal action of a compound depends upon cer-
tain physical and chemical characters j a property that determines the
bactericidal action of the compound upon E. typhosa may be relatively
unimportant in the case of another organism such as S. aureus. A sub-
stance may have the same activity, as expressed by the phenol coeffi-
cient, against two organisms and still differ markedly in its relative
TABLE 35. BACTERIOSTATIC SPECTRA OF FOUR ANTIBIOTIC SUBSTANCES
GRAM
PENI-
ACTINO-
STREPTO-
TEST ORGANISM
STAIN
CILLIN
MYCIN
THRICIN
CLAVACIN
5. aureus
+
9,500*
20,000
200
100
S. aureus
+
IjOOof
-
-
-
S. lutea
+
38,000*
60,000
100
500
B. subtilis
+
1 9,000*
60,000
750
200
B. megatherium
+
1,900*
40,000
200
100
B. mycoides
+
5*
40,000
<3
200
CI. welchii
+
i,50ot
1,000
-
-
Actinomyces sp.
+
i,ooot
10
10-50
-
"Neisseria sp.
-
2,OOOt
20
-
-
Br. abortus
-
2t
10
100
-
Sh. gallinarum
-
2t
20
300
-
Pasteur ell a sp.
-
it
<I0
100
-
Hemofhilus sp.
-
-
50
30
-
S. schottmulleri
-
<lt
<I0
200
60
S. aertrycke
-
10*
-
-
Ps. jiuorescens
-
<5*
10
<3
6
S. marcescens
-
<i*
<5
5
60
A . aerogenes
-
<5*
<5
30
50
E. coli
-
<it
-
-
-
E. coli
-
<5*
5
100
100
Note. Activity is indicated in thousands of dilution units per gram.
* Our own data, based on a sample having 470 Oxford units.
t Data reported by Abraham et al. (i), based on a less active preparation.
192
NATURE OF ANTIBIOTIC ACTION
TABLE 26. BACTERICIDAL EFFECTS OF PENICILLIN, GRAMICIDIN, AND
TYROCIDINE UPON S. HEMOLYTICUS
INHIBITING
AGENT*
At start
Penicillin
1,500
Gramicidin
1,500
Tyrocidine
1,500
NUMBER OF VIABLE ORGANISMsf
At At At
At I hour 3 hours 7 hours 24 hours
4,300 2,650 420 o
2,430 1,140 7 2.4
0.1 o 00
From Dawson, Hobby, Meyer, and Chaffee (164).
* 10 Y of each preparation was added to i milliliter ot culture.
t In thousands per milliliter.
TABLE 37. BACTERIOSTATIC AND BACTERICIDAL ACTION OF FUMIGACIN
AND CLAVACIN
CLAVACIN,
FUMIGACIN
CRUDE MATERIAL
Bacteriostatic actionf
Bacteri-
Bacterio-
Bacteri-
Crude
cidal
static
cidal
TEST ORGANISM*
Crystals^
material^
action^
actionf
action:]:
A . aerogenes
<40,ooo
<40,ooo
>200
50,000
20
E. coli
<40,ooo
40,000
>200
100,000
20
■S. schottmuUeri
<40,ooo
<40,ooo
>200
60,000
20
Salmo?tella sp.
(Breslau type)
<40,ooo
<40,ooo
>200
75,000
20
S. choleraesuis
<40,ooo
<40,ooo
>200
150,000
5
B. megatherium
1,250,000
1,000,000
20
100,000
5
B. cereus
500,000
500,000
200
125,000
5
B. mycoides
1,250,000
500,000
200
200,000
2
B. subtilis
750,000
500,000
200
200,000
2
S. aureus 3
750,000
750,000
200
100,000
2
S. aureus 2
500,000
250,000
>200
60,000
20
S. aureus H
750,000
500,000
>200
75,000
20
5. aureus W i
750,000
500,000
200II
200,000
5
5, aureus W2
500,000
500,000
200II
200,000
2
5. lutea
4,000,000
3,750,000
20
500,000
2
From Waksman, Horning, and Spencer (935).
* Staphylococcus cultures and gram-negative pathogens incubated at 37° C, others at 28° C.
t Units of activity ^ dilution in plate or tube inhibiting growth completely.
% Micrograms required to kill bacteria in i milliliter portions of 6-hour-old cultures.
§ Water saturated solution used.
If Crude mother liquor, from which 2 lots of crystals were removed on basis of dried material in
solution.
II Incomplete sterilization of culture even in 24 hours
ANTIBIOTIC SUBSTANCES AND DISINFECTANTS
193
lethal effects. The conclusion was reached that the mechanism of bac-
tericidal action must be considered as a separate problem for each type
of organism, and, one may add, for each type of compound.
Although the major difference in the action of antibiotic substances
and chemical antiseptics is based upon the selective antibacterial nature
of the former, still an attempt may be made to correlate the two types
of compounds. Marshall and Hrenoff (584) constructed a disinfectant
spectrum for antibacterial substances with a flexible blending of differ-
entiated degrees of activity. The first, or ineffective, band covers a
range of dilutions of an agent between zero concentration and the high-
est dilution which still exerts no action on bacteria. The second, or stimu-
lative, band comprises a range of relatively high dilutions in which
there is a slight stimulation of bacterial multiplication j this range is
ordinarily narrow, but it may become broad. The third, or inhibiting,
and the fourth, or germicidal, bands merge indistinguishably. The
fifth, or impractical, band covers a range of concentrations of the dis-
infectant that are too great for practical purposes (Figure 19).
By establishing the normal rate of multiplication of bacterial cells in
a given culture without the disinfectant, one can determine the retarda-
tion of that rate by the disinfectant. This rate approaches zero at com-
DISINFECTANT "SPECTRUM'
BACTERICIDAL
11111111111
HI
Space obout I/20 totol length
impractical
(insoluble,
too toxic,
OR )
100%
GENTIAN VIOLET
Space just perceptible (-OOlX)
Figure 19. Disinfectant spectrum. From Marshall and Hrenoff (584).
194
NATURE OF ANTIBIOTIC ACTION
plete inhibition with no multiplication and no deaths. A further increase
in the concentration of disinfectant results in the death of some organ-
isms per unit of time, and eventually a concentration is reached at which
all organisms die rapidly (Figure 20). Any rate of multiplication
LOGARITHM
OF
BACTERIA
PER
MILLILITER
^^^^^-^-"'''^ RATE = 0
I^nT--.
TIME
Figure 20. Disinfectant spectrum and rates of bacterial growth. From Mar-
shall and Hrenoff (584).
greater than zero but less than normal can be considered as the bacterio-
static zone, and the rate less than zero as the bactericidal zone. Accord-
ing to this concept of bacteriostasis, bacterial growth may be delayed
under the influence of a disinfectant for many days or for many hours j
or the bacteria may progressively die over a period of many days.
The following factors influence the selective action of an antibiotic
agent upon bacteria (189): the acidic and basic properties of the bac-
terial cell, the nature and property of its membrane, its permeability,
ANTIBIOTIC SUBSTANCES AND DISINFECTANTS 195
the relative importance for metabolism and viability of the specific bio-
chemical systems affected by the agent, the activity of autolytic enzymes
in the bacterial cell, as well as others.
Marked differences exist in the degree of sensitivity of various bac-
teria to different antibiotic substances and chemical agents. Gramicidin
is most specific in its action, being limited to the cocci and acting upon
actinomycetes to only a limited extent. Penicillin is next in its selective
action. Actinomycin, tyrocidine, and gliotoxin act primarily upon the
gram-positive organisms and actinomycetes, and much less upon gram-
negative bacteria. The selective action is in contrast to the generalized,
even if more limited, action of phenol and quinone, which act alike on
both gram-positive and gram-negative organisms. Pyocyanase, pyo-
cyanin, and the culture filtrate of P. notatum (due to the presence of
notatin) are similar in some respects but not in others to the chemical
compound in their action j they are found to be generally bacteriostatic
over a wide range of test organisms, no sharp division being obtained
upon the basis of the gram stain, Streptothricin is unique in its action j
the gram-positive spore-former B. suht'd'is is most sensitive, but the
other spore-former B. mycoides is not affected at all. The gram-negative
E. colt is more sensitive to streptothricin than either M. lysodeiktkus
or S. lutea. Sulfanilamide has a definite, even if limited, retarding effect
upon the growth of various organisms. The antibiotic substances of
microbial origin are generally found to be stronger bacteriostatic agents
than the chemicals tested. A high bacteriostatic effect is not necessarily
accompanied by a correspondingly high bactericidal action. Gliotoxin,
one of the most active bacteriostatic substances among those tested, pos-
sesses lower bactericidal properties than other preparations. Strepto-
thricin, on the other hand, is highly bacteriostatic and bactericidal
against certain gram-negative bacteria.
The specific morphological differences among the bacteria, based
upon the gram stain (205), as shown by their sensitivity toward anti-
biotic substances, are thus found to be relative rather than absolute.
Most of the gram-positive bacteria are more sensitive to the majority
of antibiotic substances than are the gram-negative bacteria. But other
an,tibiotic agents, such as streptothricin, streptomycin, and clavacin, act
quite differently and show marked variations within each group.
196 NATURE OF ANTIBIOTIC ACTION
MECHANISM OF ANTIBIOTIC ACTION
In an attempt to interpret the antibacterial activities of antibiotic
substances, one may benefit from a comparison of the action of these
substances and that of other antibacterial agents. Recent studies of the
mechanism of antibacterial action of chemotherapeutic agents led to
rather definite concepts concerning the nature of this action. The action
was believed to consist in depriving the bacteria of the use of enzymes
or metabolites by various types of interference (254). The nutritional
requirements of the organisms thus inhibited are more exacting than in
their normal state, E. coll and S. hemolyticus, when inhibited by acrifla-
vine components, were found to require for further growth two types
of material not normally added, one of which could best be replaced by
nucleotides, and the other by a concentrate of amino acids, especially
phenylalanine (557, 559).
On the basis of the information now available, the following mecha-
nisms may be tentatively presented here:
The antibiotic substance interferes with bacterial cell division, thus pre-
venting further growth of the organism. The cell, unable to divide,
gradually dies. It has been shown (359), by the use of the mano-
metric method, that certain bactericidal agents in bacteriostatic con-
centrations have no effect on the metabolic rates of bacteria, though
they do inhibit cell multiplication.
The antibiotic substance interferes with the metabolic processes of the mi-
crobial cells, by substituting for one of the essential nutrients. It has
been suggested (290a) that the antibiotic effect of certain polypep-
tides, such as gramicidin, may be due to the presence of a ^-amino
acid isomer of a natural amino acid, /-leucine, required for bacterial
growth.
The antibiotic substance may interfere with the vitamin utilization of the
organism. The staling effect of a medium, frequently spoken of in
connection with protozoa as "biological conditioning" of the organ-
ism, may serve as an illustration. Such effects have been overcome
by the addition of a mixture of thiamine, riboflavin, and nicotin-
amide (373).
The antibiotic agent brings about the oxidation of a metabolic substance
MECHANISM OF ANTIBIOTIC ACTION 197
which must be reduced in the process of bacterial nutrition, or other-
wise modifies the intermediary metabolism of the bacterial cell.
The agent combines with the substrate or with one of its constituents,
which is thereby rendered inactive for bacterial utilization.
The agent competes for an enzyme needed by the bacteria to carry out an
essential metabolic process.
The agent interferes with various enzymatic systems, such as the respira-
tory mechanism of the bacterial cell, especially the hydrogenase sys-
tem (435) and the phosphate uptake by the bacteria accompanying
glucose oxidation, as in the action of gramicidin. Penicillin, for ex-
ample, was shown (892) to be capable of inhibiting the activity of
urease. It was later proved (8ioa), however, that this was due not
to the penicillin itself but to certain impurities in crude penicillin
preparations.
The antibiotic substance may inhibit directly cellular oxidations, particu-
larly those involving nitrogenous compounds, an action similar to
that of propamidine (494).
The antibiotic substance acts as an enzyme system and produces, in the
medium, oxidation products, such as peroxides, injurious to the bac-
terial cell. The glucose oxidase produced by P. notatum (153, 492,
751, 902) catalyzes the following reaction:
Glucose -j- Oo — ^ Gluconic acid -f- H2O2.
Xanthine oxidase acts in a similar manner (531, 79l)-
The antibiotic substance favors certain lytic mechanisms in the cell,
whereby the latter is destroyed; this mechanism may be either sec-
ondary or primary in nature.
The antibiotic substance affects the surface tension of the bacteria, acting
as a detergent; tyrocidine lowers the surface tension of the bacterial
cell, thereby causing its death, possibly by forming a stable complex
with it (189).
The antibiotic substance may interfere with the sulfhydryl group which is
essential for cell multiplication. This was shown (254a) to hold true
for mercurials and other chemical antiseptics. The possible inter-
relationship between the sulfhydryl group and true antibiotics has re-
cently been indicated (109a).
On the other hand, bacteria subjected to the action of an antibiotic
substance may develop mechanisms that render them resistant to the
198 NATURE OF ANTIBIOTIC ACTION
action of the substance, and some bacteria and fungi even may produce
an enzyme, such as penicillinase, that brings about the destruction of the
antibiotic substance.
The antibacterial action of gramicidin was found (391, 401) to be
inhibited by a cationic detergent, phemerol, whereas penicillin was not
affected by either gramicidin or two cationic detergents, phemerol and
zephiran. When gramicidin and penicillin were used together, their
effect was only slightly additive (388) j however, penicillin and strepto-
thricin exerted a marked additive effect upon bacteria sensitive to both
of these substances (287).
The inhibition of the antibacterial action of sulfanilamide by
/(-amino-benzoic acid has been explained by the fact that the latter is a
growth factor in bacterial nutrition (538, 813, 1003). Competition for
this growth factor between the bacterial cell and the bacteriostatic agent
is responsible for the inhibition of the agent. In a similar manner pan-
toyltaurine, which is related to pantothenic acid as sulfanilamide is to
^-amino-benzoic acid, will inhibit the growth of hemolytic streptococci,
pneumococci, and C. difhtheriae, by preventing the utilization of panto-
thenic acid by these bacteria, for which it is an essential metabolite
(558). Fildes (254) emphasized that "chemotherapeutic research
might reasonably be directed to modification of the structure of known
essential metabolites to form products which can block the enzyme
without exhibiting the specific action of the metabolite." The antibac-
terial activity of iodinin is neutralized by quinones 5 this is probably due
to the destruction of the iodinin, since the N-oxide is reduced by the or-
ganism {SSS-) SS^)- Different anti-inhibitors are known for other anti-
biotic substances, as shown later.
The concentration of the active substance and the composition of the
medium are highly important in modifying the activity of the sub-
stance. Some antibiotic substances, like penicillic acid, lose considerable
bacteriostatic activity when incubated with sterile broth or with sterile
peptone water at /)H 7 and 37° C. for i to 3 days (662) j a similar ef-
fect was observed with certain simple amines and amino acids. The con-
centration of the substances reacting with penicillic acid is diminished
on autoclaving the peptone broth in the presence of 2 per cent glucose.
The neutralizing or anti-inhibiting agent interacts with the antibiotic
ANTIBACTERIAL ACTION 199
substance and neutralizes its antibacterial effect either in the absence or
in the presence of the organism.
Since only few antibiotic substances of microbial origin have been
isolated in a crystalline state, confusion often resulted from the use of
crude preparations. Welsch (971, 972) found that concentrated and
partly purified actinomycetin had no appreciable lytic action upon liv-
ing cells. However, the presence of a small amount of a highly bac-
tericidal substance, which was especially active against gram-positive
bacteria, resulted in the lysis of living bacteria by actinomycetin. This
action was thus a result of the activity of at least two different agents
present in one preparation.
ANTIBACTERIAL ACTION
Two antibiotic agents have recently received special consideration,
tyrothricin and penicillin. They will be considered here in further
detail.
Tyrothricin
The phenomenon of antibiotic action by a specific substance can best
be illustrated by the action of tyrothricin upon bacterial cells. Five dis-
tinct stages have been described (190) :
1. Inhibition of growth. Certain gram-positive bacteria are inhibited by
as little as I microgram or less of the substance per 10 milliliters of
nutrient broth or agar, thus giving an activity of i : 10,000,000 or
more.
2. Bactericidal action consists in the killing of the bacterial cells, either in
a washed state and suspended in saline, or in a growing state in broth
culture.
3. Lytic activity comprises the rate of lysis of a suspension of bacterial
cells. Streptococci, for example, are readily lysed by gramicidin,
whereas staphylococci are acted upon more slowly and less com-
pletely.
4. Inhibition of enzyme activity includes dehydrogenases or enzymes or
respiration. Gram-positive cocci, incubated at 37° C, lose their abil-
' ity to reduce methylene blue in the presence of glucose, upon addi-
tion of gramicidin. Since inactivation of the dehydrogenase takes
200 NATURE OF ANTIBIOTIC ACTION
place before any morphological changes are observed in the cells,
lysis was believed to be a secondary process, following cell injury;
hydrolytic enzymes, however, remained unaffected.
5. Protection of animals by the antibiotic substance against infection.
Tyrothricin is made up of two compounds, gramicidin and tyroci-
dine, that differ in their chemical properties and in their biological ac-
tivity (205). Gramicidin acts only against gram-positive bacteria, in-
cluding pneumococci, streptococci, staphylococci, diphtheria bacteria,
and aerobic spore-forming bacilli j meningococci and gonococci are not
readily acted upon. Tyrocidine affects both gram-positive and gram-
negative organisms. Gramicidin causes hemolysis of washed red cells,
this hemolytic action being destroyed on heating. Tyrocidine causes
lysis of many bacterial species. This action, however, is secondary,
autolysis following the death of the cells. Peptones and serum inhibit
the action of tyrocidine, but gramicidin is affected only to a limited ex-
tent by these agents (391, 579).
Tyrocidine behaves as a general protoplasmic poison. The effect of
gramicidin, on the other hand, is reversible. Staphylococci "killed"
with gramicidin and no longer able to grow on organic media can be
made to grow in the presence of certain tissue components. Gramicidin
is, therefore, not considered as a gross protoplasmic poison, but retains
a good deal of its activity in animal tissues. When applied locally at the
site of the infected area, gramicidin exhibits definite action against in-
fection with pneumococci and streptococci. When injected intrave-
nously, however, it is almost completely inactive against systemic in-
fection.
It was demonstrated by tissue culture technique (401) that the he-
molytic effect of tyrothricin was due to the presence of gramicidin.
When tyrothricin or gramicidin was heated in an aqueous suspension
there was a loss of hemolytic and bactericidal activity. Tyrocidine,
which is not very hemolytic, showed no marked toxic effect upon the
leucocytic elements of the human blood in amounts up to 100 mg. per
milliliter for 8 hours.
Other investigators (728) have reported that the hemolytic activity
of tyrothricin is inherent rather in the tyrocidine fraction, although
gramicidin also exhibits a definite hemolytic action. The addition of
ANTIBACTERIAL ACTION 201
glucose caused only slight inhibition of the hemolytic effect. Gramicidin
was found to be effective, in amounts as low as i mg., upon a billion
gram-positive organisms, whereas tyrocidine acted in 25 to 50 times that
concentration in the absence of inhibitors (435, 436). Tyrocidine ap-
peared to block all the oxidative systems of the bacteria studied, whereas
gramicidin seemed to affect only certain individual reactions. Both sub-
stances were found to exert a protective antibacterial action in mice in-
fected intraperitoneally with susceptible bacteria 5 gramicidin protected
the animals at a level one-fiftieth as high as that required for tyrocidine.
Both substances are toxic to animals when injected into the blood
stream J both are leucocytolyticj they show little toxicity when applied
locally by the subcutaneous, the intramuscular, or the intrapleural
route J oral administration is not accompanied by toxic effects, but such
treatment is ineffective (729).
Gramicidin remains active in the blood stream, but it has only weak
bacteriostatic properties and no bactericidal action. Tyrocidine is
strongly bactericidal but it is inactivated by blood serum, hence it is
limited to local applications. No specific effect was exerted by these sub-
stances on respiratory or circulatory systems (756).
According to Dubos (189, 201), the retention of the stain by gram-
positive bacteria indicates a peculiar property of the cell wall of these
organisms. The addition of 0.00 1 mg. of gramicidin to a billion pneu-
mococci, streptococci, staphylococci, and others is considered sufficient
to inhibit the growth of these organisms on subsequent transfers. This
effect was said to be due not to an alteration of the protoplasm but to
some specific interference with an essential metabolic function. Bacterial
cells which have become inhibited under the action of gramicidin be-
come viable again when cephalin is added to the medium. It was sug-
gested that the ineffectiveness of gramicidin on gram-negative bacteria
may be due to the presence of a phospholipid in these organisms.
Tyrothricin did not exert any effect upon staphylococcus bacterio-
phage (633). It did, however, inhibit the fibrinolytic activity of heta-
hemolytic streptococci as well as of the supernatant liquids of these bac-
teria but not of partially purified fibrinolysin. Although it prevented
the neutralization of hemotoxin by antitoxin, it did not inhibit the pro-
tective action of antitoxin against the toxin in mice (64). This substance,
202 NATURE OF ANTIBIOTIC ACTION
as well as actinomycin and clavacin, inhibited the coagulation of rabbit
plasma by staphylococci but did not prevent coagulation by sterile cul-
ture filtrates of these organisms j none of these three substances de-
stroyed the toxin, nor did they enhance its hemolytic or lethal action
(64).
Different strains of S. aureus differ in their susceptibility to the ac-
tion of tyrothricin. There is apparent adaptation of the organism to in-
creasing concentrations of the substance. A marked increase in resist-
ance of the infecting organism, after several weeks of therapy, was ob-
served in one patient (720). Various other observations have been made
(686) that staphylococci grown in the presence of increasing concentra-
tions of gramicidin become resistant to inhibition by this substance
(84,689).
Both gramicidin and tyrocidine are said (196) to be surface-active
compounds, their antibacterial action being inhibited by phospholipids.
Tyrocidine behaves like a cationic detergent j it is bactericidal in buffer
solutions for all bacterial species so far tested, with the exception of the
tubercle bacillus j it destroys immediately and irreversibly their meta-
bolic activity, such as oxygen uptake and acid production. For most
tissue cells, with the exception of spermatozoa, gramicidin is much
less toxic than tyrocidine. It behaves like a specific inhibitor of certain
metabolic reactions. It retains much of its activity in vivo.
It remains to be determined to what extent the action of tyrocidine,
as well as of other antibiotic substances, can be reversed by detoxication
with high molecular anions in a manner similar to their action upon sur-
face active cations. The bacteria were said (901) to function as cationic
exchanges, both the surface and the adsorbability depending on the
structure of the cation. The bacterial action of surface-active cations and
of toxic metallic ions and dye cations was considered as a phenomenon
of ionic exchange by bacteria. Harmless cations could thus exert a pro-
tective action on bacteria against the toxic cations.
Penicillin
Although penicillin is active primarily on gram-positive bacteria, it
also has an effect on certain gram-negative bacteria, but not on the colon
organism, Hemofhilus, or Brucella. The gram-negative cocci can be
ANTIBACTERIAL ACTION 203
divided into two groups, on the basis of the action of penicillin: first,
Neisseria gonorrhoea. Neisseria intracellular y and Neisseria catarrhalisy
which are sensitive j and second, A^. flavus and other nonpathogenic
Neisseriae, which are not sensitive (261, 266, 267).
Different strains of S. aureus vary little in their susceptibility to the
action of penicillin j however, by growing the organism in increasing
concentrations of this substance, it is possible to obtain more resistant
cultures. Strains of staphylococci possessing increasing resistance to peni-
cillin were also isolated from infections treated with the substance
(727)-
The oxygen uptake of suspensions of staphylococci was not inhibited
to any extent by the action of penicillin for 3 hours. In a concentration
of 1 : 1,000, after incubation for 24 hours at 37° C, the bacteria gave
larger numbers of colonies on plating (7). Although 0.0 1 to o.i mg. of
penicillin per milliliter was found (418-424) to be sufficient to inhibit
the growth of 2,500,000 hemolytic streptococci (Group A), no conclu-
sion could be reached as to whether its action is truly bactericidal or
bacteriostatic.
A comparison was made of the amounts of penicillin and gramicidin
required to bring about total inhibition of growth of bacteria, on the
basis of micrograms per milliliter of culture medium (rabbit's plasma
and a serum extract of chick embryo). The results were as follows:
Penicillin
Gramicidin
D. pneumoniae
2.5-5.0
0.5-1.0
S. fyogenes
2.5
5.0
S. salivarius
20-40
2.5-60
S. fae calls
200*
40-60
S. aureus
2.5-10
300*
* Inhibition not complete at these figures.
The two substances appeared to be as effective against bacteria in cul-
tures containing growth tissue as in cultures in which no tissue was
present (388, 389).
Inhibition of growth of 2 to 4 million hemolytic streptococci was ob-
tained by the use of 0.03 y penicillin with an activity of 240 to 250 Ox-
ford units per milligram (418-424). Peptone, ^-amino-benzoic acid,
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ANTIBACTERIAL ACTION
205
blood, or serum exerted no inhibiting effect, A marked difference was
found in the action of penicillin and sulfonamides, the latter merely de-
creasing the rate of multiplication and the former actually bringing
about a decrease in the number of organisms present. This is brought
out in Figure 21. The rates of activity of penicillin, gramicidin, and
tyrocidine are compared in Table 36 (page 192). The bactericidal ac-
tion of penicillin is not accompanied by lysis. No penicillin is absorbed
or destroyed by the bacteria.
Penicillin is not very stable j it is sensitive to reaction and temperature
changes. The effect of reaction upon the stability of penicillin is shown
in Figure 22.
Para-amino-benzoic acid and sulfapyridine were found to have a
synergistic effect on penicillin. A solution of sodium penicillin with
1,200 units per milliliter gave 100 B. suhtilu units in a synthetic casein
57 HOURS
2 4 6^
REACTION (pHJ
2 4 6 8 10
REACTION (pH)
Figure 22. Inactivating effect of reaction upon penicillin. From Foster and
Wilker (283).
206 NATURE OF ANTIBIOTIC ACTION
hydrolyzate medium j the activity was increased to 6,000 by addition
of /)-amino-benzoic acid in dilution of 1 12,500 to i : 10,000 j this was
also true in presence of glucose in test medium. A similar, although
somewhat lower, increase took place in case of S. aureus; no effect was
obtained on S. hemolytkus. The addition of a dilute solution of sulfa-
pyridine, which in itself had little inhibiting effect, exerted an even
greater synergistic action upon penicillin. This effect was exerted not
only in vitro but also in vivo (896).
Attention was directed previously to the production by P. notatum
of an oxidative enzyme. It is interesting to find that P. chrysogenum
also forms an oxytropic glucose-dehydrogenase that is not susceptible to
CO and cyanide (523) ; the glucose is oxidized to gluconic acid. The
active substances produced by both groups are thus similar (832).
Other Antibiotic Agents
Of the other agents, actinomycin, streptothricin, clavacin, gliotoxin,
and several other mold products deserve particular attention.
Actinomycin is a bacteriostatic agent, active primarily against gram-
positive bacteria. It is extremely toxic to animals, a factor which limits
its practical utilization. One milligram of actinomycin given to mice,
rats, or rabbits intravenously, intraperitoneally, subcutaneously, or
orally proved (757) to be lethal for i kilogram weight of the animals.
Doses as small as 50 y per kilogram injected intraperitoneally daily for
6 days caused death accompanied by severe gross pathological changes,
notably a marked shrinkage of the spleen. Actinomycin is rapidly re-
moved from the blood and excreted. It has no effect upon bacteriophage
or staphylococci, although o.i milligram per cent inhibits growth as
well as blood coagulation by these organisms {6'^';})).
A comparison of the effect of actinomycin with that of tyrothricin
and its constituents, tyrocidine and gramicidin, upon the growth of
rhizobia (890) showed that, whereas gramicidin inhibited all strains
alike, the other three substances inhibited the slow-growing rhizobia
much more than the fast-growing ones. Effective and ineffective strains
behaved alike. Of the four antibiotic substances, tyrocidine was usually
bactericidal, actinomycin was bacteriostatic, and the other two pos-
BACTERIOSTATIC AND BACTERICIDAL AGENTS 207
sessed both properties. Some strains of rhizobia were stimulated by lim-
ited concentrations of actinomycin.
Streptothricin is far less toxic than these four substances. It acts
largely upon gram-negative bacteria, and thus is quite distinct from the
other four antibiotic agents. In this respect, it is similar to clavacin,
which, however, is far more toxic.
Clavacin not only is bacteriostatic on gram-negative bacteria but pos-
sesses marked bactericidal properties, as is brought out in Table 37
(page 192) and in Figure 23. Fumigacin, on the other hand, is active
only upon gram-positive bacteria and has far more limited bactericidal
action.
Gliotoxin is active against both gram-positive and gram-negative bac-
teria (Table 38). The substance is rather toxic to animals, the minimum
lethal dose being 45 to 6$ mg. per kilogram body weight j hematusia
is caused by even lower concentrations (459).
TABLE 38. BACTERIOSTATIC ACTION OF GLIOTOXIN
TEST ORGANISM ACTIVITY
S. aureus 4,000,000
5. -pyogenes 1,000,000
Pneumococcl 4,000,000
5. enteritidis 250,000
A. aerogenes 200,000
K. fneumoniae 250,000
E. coli 80,000
From Johnson, Bruce, and Dutcher (459)-
Note. Units of activity by dilution method.
On comparing the action of citrinin with penicillic acid, the first was
found (661) to act largely upon gram-positive bacteria j the second,
like quinones, had a more widespread action, especially against the
colon-typhoid group,
BACTERIOSTATIC AND BACTERICIDAL AGENTS
Fleming (268) divided all selective bacteriostatic agents, exclusive
of the action of oxygen on anaerobic bacteria, into three groups: (a)
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BACTERIOSTATIC AND BACTERICIDAL AGENTS
209
physiological agents, including bile, serum, proteolytic enzymes, and
lysozymej (b) microbiological products, comprising the antibiotic sub-
stances} (c) chemicals of known composition, including dyes, salts (po-
tassium, tellurite, mercuric salts), and other agents.
Concentrations of antibiotic substance smaller than those needed to
cause inhibition often stimulate growth of the organisms or of their
metabolic processes (705). In this respect these agents are similar in
their action to synthetic detergents (35) and to other chemical disin-
fectants, as discussed previously. It may also be of interest to note here
that the antibacterial action of straight-chain mono-amines and di-
amines, amidines, guanidines, and quaternary bases increases with the
length of the chain up to a maximum and then decreases, the gram-
positive bacteria being more sensitive than the gram-negative organ-
isms. Serum increases the activity of the shorter-chain compounds and
decreases that of the longer-chain compounds, depending to a consider-
able extent upon the test bacteria (303).
The bactericidal action of antibiotic agents, as influenced by their
concentration, can be illustrated by the action of pyocyanase (Table 39).
In a study of the bactericidal action of actinomycin (946) it was
found that the addition of 0.5 mg. of actinomycin to a 10 ml. sus-
pension of E. coli reduced the number of viable cells from 6,400,000
to 493,000, the methylene blue reduction test remaining positive j
I mg. actinomycin reduced the number of cells to 4,800, the reduction
test becoming negative j 2 mg. of the agent brought about complete de-
TABLE 39. BACTERICIDAL ACTION OF PYOCYANASE UPON THREE BACTERIA
B.
ANTHRACIS
E.
TYPHOSA
M. TUBERCULOSIS
Bacteria per
Bacteria per
Bacteria per
Hours
milliliter
Hours
milliliter
Hours
milliliter
Start
11,060,000
Start
13,125,000
Start
2,105,000
24
6,890,000
3
1,242,000
3
980,000
72
1,360,000
9
105,000
8
71,500
96
654,000
24
0
120
329,000
H4
0
From Emmerich, Low, and Korschun (237).
210
NATURE OF ANTIBIOTIC ACTION
struction of all the cells. The bactericidal action of actinomycin seems
to be a result of a chemical interaction, similar to that of other anti-
septics. On adding o.i mg. actinomycin to a suspension of E. coli cells
in a 10 ml. buffer solution, the value of the constant K was found to
vary from 0.02 1 to 0.026 for different periods of incubation. Figure 24
illustrates graphically the effect of different concentrations of actino-
mycin on the death rate of E. coli in buffer solution.
9
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Mi.
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INCUBATION PERIOD IN HOURS
Figure 24. Action of actinomycin on E. colt; death rate in buffer solution.
Amounts are given in milligrams of actinomycin per 10 milliliters of solu-
tion. From Waksman and Woodruff (948).
BACTERIOSTATIC AND BACTERICIDAL AGENTS 211
Quinones have a high bactericidaJ power (145, 147, 615, 948), due
not to their chemical interaction with the cell proteins but to their re-
activity with the simpler cell constituents such as some of the amino
acids (146, 148). Only a slight difference was found in the apparent
activity of quinones toward yeasts, bacteria, proteins, peptones, pep-
tides, and certain amino acids. Alcohol increases the germicidal power
of the quinones. Actinomycin contains a quinone group ; however, it acts
differently toward gram-positive and gram-negative bacteria j alcohol
has no effect upon its action, thus pointing to marked differences in
chemical and biological nature of this antibiotic agent and of quinones.
On the other hand, many of the antibiotic substances produced by fungi
are typical quinones and act as such.
By varying the concentrations of disinfectants, the types of curves of
destruction of bacterial cells were found (694) to range from linear to
an abrupt drop to zero at critical concentrations. This is brought out in a
study of the spirocheticidal action of penicillin (Figure 25).
In general, chemical disinfectants act upon bacteria in four different
ways: (a) some affect the lag phase of the growth period, (b) some in-
fluence the mechanism of cell division, (c) some influence the metabolic
processes, and (d) some affect the death rate of the microbes. Similar, if
not greater, variations are found in the nature of the action of antibiotic
substances of microbial origin upon the bacterial cell.
Gramicidin inhibited dehydrogenase activity, since the antagonized
bacteria rapidly lost their capacity to reduce methylene blue in the pres-
ence of glucose. E. colt suspension treated with actinomycin lost its
capacity to reduce methylene blue before the cells were completely
killed. The oxidation of succinic acid by tissue preparations, which re-
quire the cooperation of succinic dehydrogenase and a cytochrome sys-
tem, was strongly inhibited by pyocyanin. This inhibition exhibited cer-
tain interesting peculiarities : in low concentrations, pyocyanin strongly
inhibited the activity of the complete succinic cytochrome system but
had no effect on the oxidation of succinic acid through methylene
blue J in the presence of KCN, pyocyanin acted as an autoxidizable hy-
drogen acceptor similar to methylene bluej glutaminic acid did not af-
fect the inhibitory action of pyocyanin. This inhibitory action was found
to be due not to the formation of oxalacetic acid but to a direct effect on
212
NATURE OF ANTIBIOTIC ACTION
Figure 25. Spirocheticidal action of various amounts of penicillin In vitro.
Inoculum, 4 x 10* organisms per milliliter. From Eagle and Musselman
(unpublished).
succinic dehydrogenase. The influence of pyocyanin on bacterial respira-
tion, as well as its ability to function as an accessory respiratory enzyme,
has aroused much interest (227, 301, 854).
A strong lytic action of some of the antibiotic substances, similar in
some cases to the action of enzymes, has also been indicated. This lytic
mechanism may be a product of the antagonized cell itself. It is to be re-
called that autolysis has usually been defined (865) as "the breaking
down and solution of some of the essential chemical constituents of the
cell by agencies (enzymes) originating within the cell," This does not
hold true, however, for most of the antibiotic substances.
EFFECT ON MORPHOLOGY OF MICROORGANISMS 213
The relation between antibiotics and bacteriophage has attracted con-
siderable attention. Gratia (345) observed a definite relation between
the action of lysozyme and the liberation of bacteriophage. The action
of antibiotic agents, however, usually exhibits a marked distinction from
that of bacteriophage (218, 344, 634). Filtrates of cultures of homolo-
gous bacteria are able to inactivate the anti-coli phage j at 27° C, the
inactivation is proportional to the phage and filtrate concentration j at
0° C, to the square root of the latter (232). Based upon the formation
of iso-antagonistic substances, a method has been suggested (121) for
the differentiation of bacteria belonging to the typhoid group.
EFFECT OF ANTIBIOTIC SUBSTANCES UPON THE
MORPHOLOGY OF MICROORGANISMS
Emmerich and Saida (238) were the first to report that anthrax bac-
teria undergo morphological changes as a result of the action of pyocya-
nase. Since that early work, the effect of bacterial filtrates upon cell
multiplication and cell growth has been made the subject of many in-
vestigations. It was reported (378), for example, that no complete ces-
sation of the fission process of bacteria results from the action of the
substance, but that growth itself is checked, the action being nonspecific
as far as bacterial species are concerned. The conclusion was reached that
this phenomenon is due to the production and accumulation of metabolic
products injurious to growth. Nonspecific antibiotic substances were
demonstrated (6s6) in filtrates of bacteria. They not only injured
growth of other bacteria but prevented the production of the ectoplas-
mic antigen. These substances could be partly removed by the use of
adsorbents.
The morphology of bacteria is greatly influenced by the presence of
other organisms or their antibiotic substances. In the case of diphtheria
bacteria this is accompanied by a reduction in virulence (406). The spe-
cific effect of the antagonistic B. mesentericus upon the morphology of
antagonized bacteria has been established by Pringsheim (705). The
antibiotic substances produced by actinomycetes were shown (80) to
affect the growth of B. mycoides as follows: cell division is delayed} the
cells become elongated, reaching enormous size and assuming most pe-
214 NATURE OF ANTIBIOTIC ACTION
culiar forms j spore formation or, with lower concentrations of agent,
the active substance is repressed j delayed nonspore-forming variants
are produced with a modified type of growth on nutrient media (Table
40).
TABLE 40. INFLUENCE OF CULTURE FILTRATE OF STREPTOMYCES SP. ON
MORPHOLOGY OF BACILLUS MYCOIDES
MORPHOLOGY OF
MACROSCOPIC
DAYS OF
ANTAGONIZED
GROWTH IN
SPORE
ROD
INCUBATION
BACTERIUM
BROTH
FORMATION
FORMATION
Medium plus
10 PER CENT CULTURE FILTRATE
2
Long filaments
X
-
+
4
Filaments have divided
into elongated cells
X
-
+
17
Cells altered
X
-
+
45
Cell fragments of vari-
ous shape and length x — -
Medium plus 5 per cent culture filtrate
2 Elongated cells x - +
4 Elongated cells x - +
17 Greatly deformed cells + - +
45 Greatly deformed cells + — +
Control medium
2 ++ - +
4 ++ + +
17 ++ + +
45 Deformed cells rare ++ + —
From Borodulina (80).
X indicates growth of B. mycoides in the shape of fluffy small balls inside liquid.
Gardner (308) reported that concentrations of penicillin lower than
those required for full inhibition caused a change in the type of growth
of CI. welchii in liquid media. The majority of the cells became greatly
elongated, giving rise to unsegmented filaments ten to twenty times
longer than the average normal cells. The same was found to hold true
for a number of other bacteria (Figure 26). Even gram-negative bac-
teria, which are relatively resistant to penicillin, showed the same ef-
fect. Many bacteria produced giant forms as a result of the autolytic
S. atirriis, normal cells. Preparctl by
Foster and Woodrutl"
S. tiiirrns, pcmcillin-inhihitccl cclU.
Prepared by Foster and Woodruff
^
'I /
/
/
B. subtilisy normal cells. Prepared
by Foster and Woodruff
"A%. mnlandu^ normal cells.
Prepared by Starkey
B. subtilis, penicillin-inhibited cells.
Prepared by Foster and Woodruff
A-z. v'lnlamiii^ actinomycin-inhibited
cells. Prepared by Starkey
Figure 26. Iniluence of antibiotic substances upon the morphology of bacteria.
Figure 27. Mechanism of antibacterial action as illustrated by the gradual
diffusion of an antibiotic substance in a bacterial agar plate. EflFect of strepto-
mycin on B. subtilis.
EFFECT ON PHYSIOLOGY OF THE BACTERIAL CELL 215
swelling and bursting of the elongated cells. It was recognized that
these changes were due to a failure of fission. Cell growth not accom-
panied by cell division underwent autolysis, Br. abortus and Br. meli-
tensis, which were not inhibited by penicillin even at i : i,000 dilution,
gave no enlargement of the cells but showed vacuolation even in lower
dilutions. CI. "xelchiiy which was inhibited by i : 6o,000 penicillin,
showed filament formation in a dilution of i : 1,500,000. The phenom-
ena of swelling and lysis were said ( 833 ) to be associated with the active
growth of the bacterial cell. Suspensions of fully grown bacterial cells
showed neither of these effects when added to concentrations of peni-
cillin many times higher. It was suggested that penicillin either has
some action on the cellular wall of S. aureus or that it interferes with
the assimilation of one or more growth factors necessary for the fission
of the growing cell.
A growth-depressing substance, which altered the type of growth of
both fungi and bacteria, was also isolated (144) from yeast. Fungi
treated with this substance produced thick gnarled mycelia and formed
no conidia or pigment. Increasing the concentrations of the depressing
agent changed the nature of the colony of E. colt from smooth to rough
and finally to grainy j this was associated with an increase in the length
of the cell and the formation of filaments. When the cultures thus modi-
fied were placed in media free of the agent, normal, highly motile cells
were again produced.
The mechanism of disintegration of the hyphae of a plant pathogenic
fungus Rhvzoctoma by an antagonistic fungus Trichoderma as well as
by the antibiotic product of the latter has been described by Weindling
(962). The hyphae are usually killed in less than 10 hours, as shown by
loss of the homogeneous appearance of the protoplasm and of the
vacuolate structure of the hyphae, which either become empty or as if
filled with granular material.
ANTIBIOTIC SUBSTANCES AND THE PHYSIOLOGY
OF THE BACTERIAL CELL
Half a century ago Smith (838) emphasized that bacteria growing
in mixed cultures undergo temporary and even permanent physiologi-
216 NATURE OF ANTIBIOTIC ACTION
cal modifications. Aside from cell proliferation, the important meta-
bolic processes commonly considered to be affected by antibiotic agents
were oxygen uptake, acid production, and dehydrogenase activity. Some
agents apparently can inhibit cell growth without destroying the viabil-
ity of the cells and their capacity for taking up oxygen.
Gramicidin and tyrocidine were believed to affect bacteria (390, 391,
579) by depressing the surface tension of aqueous solutions. This effect
was favored by the addition of organic solvents such as glycerin, which
increases the solubility of gramicidin. The addition of serum resulted
in a decrease in activity of tyrocidine, to a less extent, however, than of
gramicidin. Heat destroyed the bacterial and hemolytic effects of
gramicidin, but the property of altering surface tension was heat-stable.
It has further been shown (395) that gramicidin, after an initial stimu-
lation, inhibited oxygen consumption of bovine spermatozoa and ren-
dered them immobile J aerobic as well as anaerobic glycolysis was de-
pressed by about 40 per cent and motility of the spermatozoa impaired.
Tyrocidine, however, caused a small reduction in the oxygen consump-
tion and in glycolysis. The action of gramicidin upon the metabolic ac-
tivities of S. aureus and S. hemolyticus was shown (206) to be influ-
enced by the composition of the medium, the presence of potassium and
phosphate ions giving a prolonged stimulation of metabolism, whereas
ammonium ions favored a depression in oxygen uptake.
The specific effects of basic proteins, such as protamine and histone,
upon the activity of selective inhibitors offered a possible explanation
for the difference in the action of tyrothricin upon gram-positive and
gram-negative bacteria (606). These basic proteins also possess antibac-
terial properties. They have the capacity of sensitizing gram-negative
bacteria by means of substances which otherwise act only on gram-
positive forms. This is brought out in Figure 28.
Pneumococci grown in media containing the specific enzymes which
hydrolyze their capsular material are deprived of these capsules and
fail to agglutinate in the specific antiserum. The enzymes do not inter-
fere with the metabolic functions of the cells, but their action is directed
essentially against the capsule (193). These enzymes were found not
only to exhibit great selectivity but to be highly specific against the
particular polysaccharides.
EFFECT ON PHYSIOLOGY OF THE BACTERIAL CELL 217
150
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/ / MIXTURE OF TYteOTHRICIN AND PROTAMINE
^
jy^^-^ ^ ^ .
) 15 30 45 60
TIME IN MINUTES
Figure 28. Effect of tyrothricin and protamine on the respiration of E. coli
at 38° C. and />H 5.3. Number of cells per vessel: 5x10^. Concentrations:
tyrothricin 1:15,000; protamine 1:3,000; mixture, tyrothricin 1:15,000,
protamine 1:3,000. From Miller, Abrams, Dorfman, and Klein (606).
218 NATURE OF ANTIBIOTIC ACTION
INHIBITION OF BACTERIOSTATIC AND
BACTERICIDAL ACTION
The formation of specific chemical compounds capable of inhibiting,
inactivating, or even destroying bacteriostatic and bactericidal sub-
stances of microbial origin has been established for a number of anti-
biotic agents.
Yeasts were found to contain a substance which inhibits the action of
sulfanilamide against 5. hemolytkus as well as other streptococci and
pneumococci. This substance has been identified (771) with the
^-amino-benzoic acid referred to above. No relationship could be estab-
lished, however, between the growth-promoting properties and anti-
sulfanilamide activity of the yeast extract (544).
Br. abortus and certain other bacteria also contain (354, 355) a fac-
tor, designated as "p," which specifically inhibits the bacteriostatic ac-
tion of sulfanilamide. This factor stimulates markedly the growth of
many bacteria, and is not specific. The sensitivity of sulfanilamide de-
pends on the rate of release of the factor from the bacterial cell and not
on the total amount produced. This factor was believed to stimulate
some enzyme reaction concerned with bacterial reproduction, whereas
sulfanilamide inhibits this reaction. Similar factors have been isolated
from yeast (870) and from hemolytic streptococci (538). It is also
known that certain substances, like methionine, inhibit the growth-
stimulating effect of biotin (259).
To what extent antibiotic substances can be inhibited in their action
against bacteria still remains to be determined. Certain few facts have
so far been established.
Bacteria not inhibited by penicillin were found (4, lOOO) to be ca-
pable of producing a substance which destroys the growth-inhibiting
property of the antibiotic agent. The penicillin-destroying substance is
believed to be an enzyme, since it is destroyed by heating at 90° C. for
5 minutes. It was designated as "penicillinase." The optimum fYi of its
action was found to be 8 to 9. The presence or absence of this enzyme
in bacteria is independent of the sensitivity of the organism to peni-
cillin. Bacterial extracts, pus fluids, peptone, and ^-amino-benzoic acid,
which interfere with the action of sulfonamides, do not affect penicillin.
Cephalin and extracts of gram-negative bacteria, of milk, and of
DIFFERENTIATION OF BACTERIA 219
blood serum were found to inhibit the action of gramicidin. Because of
this, cephalin is capable of reviving bacterial cells killed with grami-
cidin. This phenomenon is similar to the inhibition by phospholipins of
the action of synthetic detergents upon bacterial metabolism (35).
Mucin inhibits the action of tyrothricin, especially on meningococci and
pneumococci (182).
Tannic acid is able to neutralize the antibiotic action of actinomycin.
Humus compounds of the soil have a similar capacity. This effect was
believed to be responsible for rendering harmless, to living plant and
animal forms, the actinomycin produced in the soil (947). Ascorbic acid
also has an effect in reducing the activity of this substance. Jungeblut
(466) demonstrated that vitamin C, interacting with atmospheric oxy-
gen, leads to the production of destructive peroxides in a medium. Since
vitamin C is a strong reducing agent and actinomycin is a reversible
oxidation-reduction system, it is conceivable that actinomycin may be
reduced through the action of the vitamin. Such an effect should be
greatly increased under anaerobic conditions, where no reoxidation due
to atmospheric oxygen could occur. Twenty-five to 50 times as great a
neutralizing effect of ascorbic acid upon actinomycin was obtained un-
der anaerobic conditions with CI. butyricum as a test organism as under
aerobic conditions with B. niycoides. Reduced actinomycin was inactive.
It was concluded, therefore, that the neutralizing action of ascorbic
acid upon actinomycin was due merely to its reducing properties. As far
as the common growth factors are concerned, the action of actinomycin,
like that of penicillin, differs from the mechanism proposed by Woods
(1003) and others for sulfanilamide inhibition.
DIFFERENTIATION OF BACTERIA BY MEANS
OF ANTIBIOTIC SUBSTANCES
Because of their selective action upon different bacteria, antibiotic
substances can be utilized for separating bacteria from one another.
Fleming (269) utilized penicillin to isolate Pfeiffer's bacillus and the
pertussis organism of various cocci, diphtheria, and neisseria organisms j
the substance was found to behave as the mirror image of tellurite in
inhibiting specific bacteria. Penicillin was also utilized for the separation
220 NATURE OF ANTIBIOTIC ACTION
of acne bacilli from accompanying staphylococci (153) and for the sepa-
ration of streptococci from staphylococci (266).
Actinomycin was used to separate gram-negative from gram-positive
bacteria (945).
Tyrothricin has been utilized (858) for the isolation of A'', gonor-
rhoeae from contamination with gram-positive bacteria. Usually a
1 : 15,000 dilution of the substance in "chocolate" agar was found quite
satisfactory for this purpose. By the use of tyrothricin, gram-negative
microorganisms have been isolated from the nasopharynx even in the
presence of overgrowth by gram-positive cocci. Hemophilus injiuenzae
is resistant to the action of tyrothricin, and its isolation is facilitated by
adding to the media on which it is cultured tyrothricin in a dilution
which inhibits the cocci (808).
Streptothricin was found to help in distinguishing B. mycoides from
B. subtilis (950).
SUMMARY
Comparatively little is yet known of the mode of action of antibiotic
substances. This field offers great opportunities for research and utiliza-
tion of bacterial activities. The solution to such important problems as
the morphology of the bacterial cell, taxonomic relations of bacteria,
various physiological reactions of microorganisms, the mechanism of
causation of disease, and the very control of disease-producing micro-
organisms— all fundamental problems in microbiology — will be fur-
thered by knowledge of the action of specific antibiotic substances upon
bacteria and other microorganisms.
a
CHAPTER 12 v'^^^^il*^
UTILIZATION OF ANTAGONISTIC MICROORGAN-
ISMS AND ANTIBIOTIC SUBSTANCES
FOR DISEASE CONTROL
Whether gra?mcidm or any other product of mtcrob'ic origin will
eventually be found to fulfill certain furfoses better than either
sulfhonamides or any other class of antiseftic remains to be seen.
That several classes of reagent should be competing for suprem-
acy in different aspects of a task which not long ago was consid-
ered impossible of any real fulfilment is a truly remarkable posi-
tion (343a)-
Microorganisms and products of their metabolism have been utilized
for the control of disease in man, animals, and plants with varying de-
grees of success. As early as 1 877, Pasteur (675) noted that the produc-
tion of anthrax in susceptible animals can be repressed by the simultane-
ous inoculation with B. anthrads and various other bacteria. This led
him to make the following significant suggestion: ". . . on peut intro-
duire a profusion dans un animal la bacteridie charbonneuse sans que
celui-ci contracte le charbon : il suffit qu'au liquide qui tient en suspension
la bacteridie on ait associe en meme temps des bacteries communes."
Pasteur may thus be looked upon as the first to advance the subject
of bacteriotherapy. Emmerich (233) reported that anthrax can be con-
trolled by the use of streptococci such as the erysipelas organism j these
bacteria were, therefore, looked upon as agents useful in bringing about
immunity against all bacterial infections. Pawlowsky (676) obtained
immunity against anthrax by inoculation with Friedlander's bacillus.
Bouchard (81) was successful in the control of anthrax by means of
Ps. aeruginosa. This organism, however, did not impart any immunity
to the animals, but by the use of a sterilized ten-day-old culture of the
antagonist, healing action was obtained against anthrax infection, or at
least a delay in the course of its development. Rabbits infected with an-
thrax were also cured by means of a pyocyanase preparation (872)}
many other cases of successful treatment of anthrax with pyocyanase
222 DISEASE CONTROL
have been reported (280). The pressed extract of Ps. aeruginosa had a
similar effect when injected in the animal simultaneously with the
pathogen (505).
Various methods of treating severe infections, like anthrax (97) or
malignant tumors (138), with mild infective agents have been sug-
gested. The reduction in pathogenicity of one organism by the presence
of others has thus been well recognized (62, 120, 239). Nonpatho-
genic organisms apparently have specific effects upon the pathogens, the
development of which was prevented or even suppressed. The very oc-
currence of specific types of pneumococci in healthy individuals and the
causation of specific forms of pneumonia were found to be controlled
by the antagonistic effects of other microorganisms (369).
It was thus definitely established that the growth of B. anthrads
could be inhibited by antagonists (48). Guinea pigs survived large in-
jections of washings from soil previously contaminated by B. anthrads
through the slaughtering of a diseased cow. When cultures of this or-
ganism were isolated from the soil and injected, however, characteristic
disease symptoms resulted. It was suggested that the anthrax spores are
digested by the leukocytes which have been attracted to the site of in-
jection by the accompanying bacteria (31).
Seitz (812), in discussing the problem of mixed infections, cited
many cases not only of decreased but also of increased virulence of the
pathogen as a result of accompanying bacteria. He warned, therefore,
against too sweeping generalizations concerning the healing effect of
antagonistic bacteria. He believed that in many cases of artificial infec-
tion, the favorable action of the antagonist may have been due entirely
to increased body resistance. Nevertheless, he accepted the possibility
of utilizing the antagonistic effects of microorganisms, provided it did
not concern tissue or blood infection, but only skin surfaces, including
those of the intestinal canal and the vagina.
Until very recent years, attempts to utilize the activities of antagonis-
tic microorganisms for the control of disease did not always meet with
success. This failure may have been due to an insufficient understanding
of the nature of the chemical agent produced by the antagonizing or-
ganism, to a lack of knowledge concerning the mechanism of its action.
MICROBIAL ANTAGONISTS 223
or to the variability of the antagonizing agent as regards strain specific-
ity and the production of the active antibacterial substance.
MICROBIAL ANTAGONISTS AND DISEASE CONTROL
In 1 885, Cantani treated a tubercular patient with a culture of a sapro-
phytic organism, designated as Bacterium termo; the results were
highly favorable (104). He expressed the hope that other infectious
diseases readily accessible and of a local nature could be effectively
treated with saprophytic bacteria which are antagonistic to the patho-
gens. Following this work of Cantani, Zageri ( 1009) inoculated S. "pyo-
genes into animals suffering from anthrax j the rise in temperature
caused by the streptococcus reduced the viability of the anthrax. The
growth of an antagonistic organism was found to change the environ-
mental conditions favorable to the pathogen, thus causing its attenua-
tion. These results received the immediate attention of other investi-
gators (226, 844).
Gate and Papacostas (318) observed that mixed infections were usu-
ally mild, a phenomenon later confirmed. Mixed cultures of the Fried-
lander bacillus and of C. difhtheriae gradually gave a predominance of
the former on repeated transfer} the morphology of the diphtheria
organism changed toward a more homogeneous state on staining. The
use of culture filtrates gave no evidence that the diphtheria toxin was
neutralized by the antagonist, either in vivo or in vitro; however, when
the two organisms were grown together no toxin was formed, nor was
toxin produced when the filtrate of the culture of the antagonist was
used to grow C. difhtheriae. The therapeutic use of filtrates was, there-
fore, suggested. Lactic acid bacteria were also employed successfully
i^'T^G) in the treatment of diphtheria.
By allowing an antagonist to act upon a disease-producing organism
that has previously been heated to 56° C, a hydrolyzate was obtained
which could be employed as a vaccine (347). Bezangon (53) treated
typhoid sufferers with a culture of E. typhosa lysed by means of Ps.
aeruginosa. Gratia (347) said, however, that this type of hydrolyzate
will bring about heat production, but the use of a preparation obtained
224 DISEASE CONTROL
by means of an Actinomyces, designated as a mycolysate, will not. The
use of living proteolytic bacteria (neocolysin) for treatment of chronic
purulent conditions, such as osteomyelitis, gave favorable results j the
bacteria were believed to continue growing as long as there was dead
tissue available ( 99 ) .
Besredka (51) used culture filtrates of bacteria for the treatment of
various diseases in man. A filtrate of the anthrax organism was em-
ployed for dressings or for intracutaneous injections j the results were
at least as good as those obtained with the bacterial vaccine. Staphy-
lococci and streptococci were also utilized for similar purposes. Besredka
believed that a substance, designated as antivirus, was secreted by the
bacteria into the filtrate. This was said to check further growth of the
bacteria. The mode of action of the antivirus was considered to be dif-
ferent from that of antibodies : the first affects the cells locally by stimu-
lating their resistance} the second acts upon the organism as a whole
and, through it, against the infecting agents. Antivirus was prepared by
allowing bacteria to grow in ordinary bouillon for a long time, until the
medium became unfavorable for further development of the bacteria.
Staphylococcus antivirus prevented the growth of the staphylococcus or-
ganism in a medium in which it had grown previously. In the presence
of the homologous antivirus, the organisms underwent active phagocy-
tosis, this action being specific. The antivirus was nontoxic and could
withstand a temperature of 100° C. It imparted to certain tissues a local
immunity against the specific bacteria.
The favorable therapeutic results obtained from the use of antivirus
have been confirmed, largely in France, Austria, and Germany. The
antivirus apparently acts not upon the bacterium but upon the tissue of
the host in such a way as to produce local immunization, thus prevent-
ing infection. Although unspecific filtrates may cause an occasional in-
crease of resistance, the protection produced by specific filtrates is said to
be more intense and more dependable {Gs'},-, 741). Antivirus therapy
was believed to offer some promise, although it was said not to give con-
sistent results (381). Further studies of antivirus led to suggestions that
its favorable effects were due entirely to the culture medium (6). The
whole question thus appears to be still debatable, with proponents and
opponents of the specific nature of the antivirus effect ( 1 10, 589).
MICROBIAL ANTAGONISTS 225
The application of bacteriotherapy for the treatment of chronic infec-
tions of the middle ear (706) and actinomycosis in man has also been
suggested. Filtrates of E. tyfhosa and of E. coli were found (810) to
check the growth of the typhoid organism, whereas E. coli grew readily
in such filtrates; the more sensitive typhoid bacterium was checked
earlier in its growth than the colon organism. In general, E. tyfhosa
was found to be readily inhibited by the growth of antagonistic bacteria.
Because of this, it was believed that pasteurized milk contaminated with
a pathogenic organism presents a particular danger, since no antago-
nists are present to inhibit the rapid multiplication of the pathogen.
Metchnikov (596a) suggested utilization of the antagonistic relations
between lactic acid bacteria and proteolytic bacteria for repressing the
growth of the latter. Thus, pure cultures of the former are introduced
into the food system of man, in order to repress in the intestinal canal
the proteolytic organisms that are supposed to bring about intoxication
in the system. In recent years, L. acidofhilusy an inhabitant of the hu-
man intestine possessing antagonistic properties against pathogenic in-
testinal bacteria, has come into general use (744). The problem of com-
bating pathogenic intestinal bacteria by means of nonpathogenic forms
(703) has thus been given wide consideration. The utilization of yeasts
for combating streptococci and staphylococci may also be classified
among the phenomena of antagonism (893). On the basis of the rapid
destruction of pathogenic bacteria added to natural water, the storage
of drinking water in large reservoirs was recommended as an important
safeguard against the water's becoming a carrier of bacterial diseases
(295-297).
Clinical methods have been proposed for evaluating the results ob-
tained by treating tooth gangrene by means of antagonists (325). Don-
aldson (181) found that CI. sforogenes or a closely related form had a
marked effect in suppressing the growth of pathogenic organisms in
septic wounds. He believed the antagonistic anaerobe is present in the
majority of gunshot wounds, but that its activities are held in abeyance
by the method of wound-dressing. This antagonist acts by virtue of its
proteolytic enzymes which hydrolyze the dead protein, from which the
pathogenic organisms operate, as well as the toxic degradation products
of other organisms.
226 DISEASE CONTROL
Dack (159) reported that CI. sforo genes formed in the soil was re-
sponsible for destroying the toxin of CI. botuUnum.
ANTIBIOTIC SUBSTANCES AND DISEASE CONTROL
In discussing the subject of antiseptics in war-time surgery, Fleming
emphasized that the treatment of war wounds has become far more
satisfactory during the second world war than it was during the first.
It is now known, for instance, that carbolic acid lacks value inside the
human body, as demonstrated by a diminution in efficiency with in-
creasing concentrations, due to its destructive effect upon the blood
leukocytes and body tissues. Dyes have been found also to be of little
value, since they are absorbed by the cotton used in dressing the
wounds. Fleming (260) warned against placing too much faith in anti-
septics belonging to the sulfonamide group, since they are not general
antiseptics but have specific effects upon certain bacteria, and their ac-
tion is neutralized by chemicals, by pus, and by dead bacterial cells.
They are, therefore, of little value in the treatment of seriously septic
wounds, in which pus and bacteria are inevitably present. Their major
importance is due chiefly to their great solubility, since they dissolve to
form high concentrations in the wound.
In view of these limitations in the use of chemical antiseptics, bacterio-
static and bactericidal (antibiotic) agents produced by microorganisms
may find particular application. Among the substances formerly utilized
for this purpose pyocyanase has received special consideration (239).
Unfortunately, the variation in the nature of the preparation of this
material and the difficulty of keeping it in an active condition for very
long periods of time have prevented its wider usefulness. Among the
more recent preparations, penicillin occupies a leading place.
Penicillin
Nature of Action. The action of penicillin upon bacteria is chiefly
bacteriostatic and not bactericidal (260). Penicillin shows in vitro a high
degree of specificity j pyogenic cocci, anaerobic Clostridia, and certain
pathogenic gram-negative cocci {GonococcuSy Meningococcus y and Mi-
crococcus catarrhalis) are sensitive, whereas the colon-typhoid, hemo-
ANTIBIOTIC SUBSTANCES 227
philic chromogenic bacilli and certain micrococci {Micrococcus flavus)
are resistant to its action (263) ; however, it has no effect upon M. tu-
berculosis (834), Trypanosoma equiferdum^ and the influenza virus
(753). The purest preparation of penicillin so far available completely
inhibited (276) the growth of S. aureus in a dilution of between
1 : 24,000,000 and 1 130,000,000. Partial inhibition was obtained up to
1 : 1 60,000,000. Salmonella organisms were also sensitive. The antibac-
terial activity of penicillin is not interfered with by substances that in-
hibit sulfonamides, namely, bacterial extracts, pus fluids (7), tissue
autolysates, peptones, and ^-amino-benzoic acid. It is nontoxic in con-
centrations far greater than those required for therapeutic purposes
(163, 164). However, it is rapidly excreted through the kidneys and
frequent administration is essential in order to maintain a proper blood
concentration.
In its biological properties, penicillin has been found, in general, to
resemble sulfonamide drugs, with certain significant differences (7)
which may be summarized as follows :
The bacteriostatic power of penicillin against streptococci and staphylo-
cocci is greater than that of sulfonamides, even when the tests are
made under conditions optimum for the action of the latter. Satu-
rated solutions of sulfapyridine and sulfathiazole showed no com-
plete inhibition of bacteria on the assay plate, whereas peniciUin, even
in a dilution of 1 1500,000, gave considerable inhibition.
The action of penicillin on streptococci and staphylococci, unlike that of
the sulfonamides, is influenced very little by the number of bacteria
to be inhibited. Bacterial multiplication could be completely pre-
vented by as low a concentration of penicillin as 1 : 1,000,000, even
if the inoculum contained several million bacterial cells. In the case
of smaller inocula, inhibition occurred in even higher dilutions. This
property of penicillin is believed to be of great importance in the
treatment of heavily infected wounds, on which the sulfonamide
drugs seem to have little beneficial action.
The bacteriostatic power of penicillin against streptococci and staphylo-
cocci is not inhibited- to any extent by protein breakdown products or
by pus, which neutralize the bacteriostatic action of sulfonamide
drugs. The leukocytes remain active in any concentration of peni-
cillin usually employed in intravenous injection.
228 DISEASE CONTROL
Penicillin is active against strains of bacteria that are resistant to the ac-
tion of sulfonamides (273, 566, 881). It is effective in the treat-
ment of hemolytic streptococcus, pneumococcus, and gonococcus in-
fections, which are resistant to sulfonamides. It has not been found
effective, however, in the treatment of subacute bacterial endocar-
ditis (748).
On repeated passage through broth containing penicillin, pneumo-
coccus cultures as well as Stafhylo coccus sp. and 5. -pyogenes (564) in-
creased in resistance to penicillin. This was accompanied by a propor-
tional loss of virulence. Small colony variants (G forms) of S. albus
showed a specially high resistance to penicillin (806). Two strains of
pneumococcus developed resistance to penicillin as a result of serial
passage through mice treated with it. The degrees of resistance devel-
oped and acquired varied significantly with the strains. In the case of one
strain, resistance was not impaired by 30 serial passages through nor-
mal mice. The development of resistance in vivo was accompanied by
an increase in resistance to penicillin in vitro. The response of the pneu-
mococci to sulfonamides was not altered by the development of resist-
ance to penicillin. The mechanisms whereby staphylococci become re-
sistant to sulfonamides and to penicillin appear to be distinctly differ-
ent (846, 847).
Toxicity. As to the toxicity of penicillin, it was found (7) that mice
were little affected by the intravenous injection of 10 mg. of penicillin j
they became ill from the use of 20 mg. but recovered shortly. One hun-
dred milligrams of crude penicillin given intravenously to man caused
a shivering attack with a rise of temperature in about an hour. The lat-
ter was due to the presence of a pyrogenic substance in the preparation.
Certain isolated fractions of penicillin had no such pyrogenic effect.
Penicillin was toxic to mice when given intravenously in single doses
of 0.5, i.O, 1.5, and 2.0 gm. per kilogram. More highly purified prepa-
rations were less toxic. Higher concentrations were required for lethal
effect from subcutaneous administration. The toxic dose is 64 times
greater than the effective dose (753).
The relative toxicity of various salts of penicillin was found (967) to
be, in increasing order, Na, NH4, Sr, Ca, Mg, and K. Based on milli-
grams of the cation at the LD-,o dose of salts of penicillin, the relative
ANTIBIOTIC SUBSTANCES 229
toxicity was Na, Sr, NH4, Ca, K, and Mg. It was concluded that the
toxicity of the salts of penicillin is primarily due to the cations used in
their preparation.
Penicillin is not inactivated by saliva, bile, or succus entericus, but is
destroyed rapidly by gastric juice, due not to the pepsin but to the HCl
in the juice (724, 725).
Penicillin is slowly absorbed and excreted, usually within one hour,
in the urine (725). The degree of its antibacterial action is proportional
to its concentration in the serum, maximum effects against hemolytic
streptococci being produced by concentrations of 0.019-0. 156 Oxford
units in i ml. of serum. The LD50 for an 18-gram mouse was 32 mg.
of the sodium salt (422, 424). The cardinal symptoms of toxicity were
choking, gasping, and rapid respiration. However, it is relatively non-
toxic in doses used for therapeutic purposes (163, 164, S^^, 753).
Penicillin was thus found to combine the two most desirable quali-
ties of a chemotherapeutic agent, namely, a low toxicity to tissue cells
and a highly bacteriostatic action against some of the most common and
destructive bacteria with which man may become infected. It was pos-
sible to maintain a bacteriostatic concentration of penicillin in the blood
without causing any toxic symptoms, and to recover a large proportion
of the substance from the urine j this recovered penicillin could then
be used again.
Animal Experiments. In animal experiments (699) it was estab-
lished that penicillin is an effective chemotherapeutic agent against
pneumococci, including sulfonamide-resistant types. In experiments
with S. aureus, a survival ratio of 2 : i was obtained in favor of penicillin
as compared with sulfathiazole, correction being made for the survival
of control mice. Penicillin, when administered subcutaneously, intra-
venously, or intraperitoneally, was also found to be effective against
hemolytic streptococci (418). Generalized staphylococcal infections
were cured by penicillin and local lesions healed during parenteral ad-
ministration. The best method for administering penicillin was by the
intramuscular route at 3-hour intervals j the blood should contain
enough penicillin (15,000 Oxford unit dose) to inhibit the growth of
the infecting agent (276, 399, 404). Intraocular infection caused by
D. fneumoniae was checked by local treatment with penicillin in solu-
230 DISEASE CONTROL
tions of 0.25 and o.i per cent j the application was continued for 2 to 4
days (780).
Since penicillin readily loses its activity in an acid solution, it is used
in the form of the sodium salt. Rabbits excreted in the urine as much as
50 per cent of the penicillin after intravenous injection, but less than
20 per cent after administration into the intestine j some excretion took
place in the bile. The penicillin could not be detected in the blood
within one-half hour after administration. Cats differed in this respect
from rabbits, since they maintained an antibacterial concentration of
penicillin in the blood for at least 1.5 hours after subcutaneous or intra-
venous injection, and for at least 3 hours after intestinal administration.
They differed also in excreting about 50 per cent of the penicillin in the
urine, even when the substance was injected into the intestine. In this
respect man appeared to resemble cats more closely than rabbits. The
excretion of penicillin could be blocked by simultaneous administration
of diodrast (723-725).
A comparison of antibiotic agents against the anaerobes causing gas
gangrene placed tyrothricin in first place, followed successively by peni-
cillin, the sulfa drugs, and other antibiotic agents j however, in vivo
treatment of mice infected intramuscularly with CI. ferfringens placed
penicillin first, with tyrothricin and aspergillic acid at the bottom of the
list (562)- Penicillin also proved superior to sulfonamides and amino
acridines in experimental infection with CI. welchii and CI. aedematiens
The in vivo activity of penicillin against CI. se-pticum and other
anaerobes, as well as many other bacterial pathogens ( 1 1 3 ) , is brought
out in Table 41. A single subcutaneous treatment of mice with 50
Florey units of penicillin at the time of intramuscular inoculation with
CI. welchii protected 98 per cent of the infected animals, and repeated
small doses gave as good protection as a single large dose. Delay in the
institution of therapy lowered the survival rate, but not appreciably un-
less the delay was over 3 hours. Local lesions were completely healed
within 3 weeks if penicillin was injected repeatedly into the site of in-
fection (371).
An intravenous injection of 20 mg. of the sodium salt of penicillin
was without apparent effect on a mouse, and human leukocytes survived
o >^
O CO O "
O
s N e
c^ iri tn
►J S "
<: w "
H S ^
H 5 fe
N u-1 cl
s i
^ ^
ffi Oh
6 -
o o o o
CIS rt
O O
6 6 e 6
C i> C (U
Or'-' Or'-'
o h u h
6 s e 6
^ n-j ^ n-i
O <u O (u
c S c S
c3h oh
S 6 6
r-l -d TJ
u u H h
232 DISEASE CONTROL
for an hour in a i per cent solution (273). The efficacy of penicillin
in protecting mice against streptococcal infections is brought out in
Table 42.
TABLE 42. IN VIVO EFFICACY OF PENICILLIN COMPARED WITH SULFANILAMIDE
IN STREPTOCOCCUS HEMOLYTICUS INFECTIONS IN MICE
DAILY
daily dosk
NUMBER OF MICE OF ORIG
INAL 20 SURVIVING AFTER
DOSE IN
1
[N OXFORD
I
2
3
4
5
6
7
8
9
10
MILLIGRAMS
UNITS
day
days
days
days
days
days
days
days
days
days
Penicillin
0.0625
3-75
0
0.125
7-5
16
0
0.250
15.0
18
4
I
I
I
I
1
I
0
0.500
30.0
20
4
0
i.o
60.0
20
20
18
15
15
15
15
13
9
9
2.0
120.0
20
20
20
20
20
20
20
20
20
20
4.0
240.0
20
20
20
20
20
20
20
20
20
20
Sulfanilamide
0.5
20
0
1.0
20
20
20
10
8
8
5
4
I
I
2.0
20
20
20
20
20
20
20
20
14
3
4.0
20
20
20
20
20
20
20
19
19
16
8.0
20
20
20
20
20
20
20
20
20
18
16.0
20
20
20
20
20
20
20
20
20
20
Controls
Culture dilut:
Ion
10-*
0
Culture diluti
ion
io-«
0
From Robinson (752).
Notes. Infection: 0.5 cc. of lO"* 6-hoiir-old culture dilution in broth. Treatment: Penicillin given sub-
cutaneously and sulfanilamide given orally immediately after the inoculation of bacteria, then every
3 hours day and night for 5 days.
The effectiveness of penicillin has also been tested against various
other infections in experimental animals, with varying degrees of suc-
cess. It was found, for example, that the administration of penicillin in
relatively large doses to mice after injection with murine typhus rick-
ettsiae resulted in marked reduction in mortality, particularly when the
initial dosage of the rickettsiae was relatively small (614).
Chemotherapeutic Action. Penicillin has also found an important
ANTIBIOTIC SUBSTANCES 233
place in the treatment of local and generalized infections in man. Flem-
ing was the first (265, 266) to recommend that it be employed for
dressing septic wounds. It appeared to be superior to dressings contain-
ing purely chemical agents. Isolated penicillin in a dry state was many
times more powerful than the most potent of the sulfonamide com-
pounds (267, 271 ). Local applications include those to lesions of the eye
produced by S. aureus, in which early treatment resulted in the elimina-
tion of the organism from the flora of the conjunctival sac (758).
Weight for weight, penicillin was found to be four times as potent as
sulfathiazole and 100 times as potent as sulfanilamide for the treat-
ment of wound infections (7, 272).
Penicillin proved to be an especially effective agent for the treatment
of staphylococcal and hemolytic streptococcal infections in man (725),
including streptococcal meningitis (270). Many cases of infected war
wounds treated with penicillin gave, in 24 hours, a uniform drop in the
number of gram-positive organisms, including Clostridia, staphylococci,
streptococci, and corynebacteriaj the gram-negative bacteria were not
affected. Excellent therapeutic effects were obtained. Even crude cul-
ture filtrates of P. nolatum, applied locally, gave good results (242,
707).
In order to lessen the frequency of effective invasion of the nose by
bacteria and the subsequent infection of the nasopharynx, the use of
penicillin as an antiseptic snuff was suggested (166). The material acts
as a prophylactic against bacterial infections of the upper respiratory
tract } the course of a cold could thus be checked by preventing second-
ary bacterial infection. The curing of nasal carriers of staphylococci or
even the reduction of the number of vegetative organisms was consid-
ered important in order to reduce the danger of the carrier as a source of
infection to others. Penicillin can also find application in certain chronic
cases, as in the treatment of chronic dermatitis and in preparing infected
surfaces of hands for skin-grafting and infected stumps for amputation
(277)-
Penicillin is an effective agent in the treatment of clinical infections
due to sulfonamide-resistant bacteria (39, 136, 137, 143,252,400,576).
Se\'eral strains of A^. gonorrheae, isolated from patients in whom the
infection was resistant to treatment with sulfonamide preparations.
234 DISEASE CONTROL
were found to be inhibited completely by penicillin. The number of
organisms decreased greatly at the end of i or 2 hours' contact with the
substance, and no viable organisms were found after 3 to 4 hours' con-
tact. The complete absence of toxicity following the intravenous admin-
istration of penicillin, the lack of discomfort to the patient, and the rapid
disappearance of clinical symptoms were observed in cases of sulfonam-
ide-resistant gonorrheal infections. In all the cases reported, in addi-
tion to the clinical response noted, negative bacterial cultures were ob-
tained some time between 1 7 and 48 hours after the institution of peni-
cillin therapy. Sulfonamide-resistant gonorrhea cases responded to in-
jections of 100,000 to 160,000 Oxford units (136, 479, 549, 863). Fa-
vorable responses have also been obtained in the treatment of sulfon-
amide-resistant strains causing staphylococcal pneumonia and empyema
(44, 6s) and other diseases. The susceptibility of various bacteria to
penicillin can be determined by means of a very simple technique (879).
A favorable therapeutic response was obtained by administering peni-
cillin intravenously to patients with staphylococcal infections and by
mouth to a baby with a persistent staphylococcal urinary infection. In
patients suffering from meningitis, penicillin was found to be absorbed
more rapidly than in normal persons, and a larger part of the dose was
excreted in the urine (723, 725).
Penicillin has not been found to be effective in trypanosome infec-
tions, but has been used successfully in the treatment of relapsing fever
(29, 393), although excessive doses were required (220, 221).
Treatment of early syphilis cases with penicillin (575) indicated that
the therapy was responsible for the rapid and complete disappearance of
the infecting agent from the blood stream, as determined by various
tests (112, 271, 275, 523a, 805). Penicillin was found to be actively
spirocheticidal (222). A comparative study has been made of the action
of penicillin and of other antibiotic agents upon Treponema -pallida
(2iy). The administration, at 3-hour intervals for a period of 15 hours,
of 20,000 units of penicillin intramuscularly was found satisfactory in
the control of gonorrhea in men (879a, 907). Penicillin was also found
to have an effect upon experimental typhus rickettsiae (357, 614).
As a result of treatment of 300 patients with penicillin, it has been
concluded (748) that this material is far superior to any of the sulfonam-
ANTIBIOTIC SUBSTANCES 23 5
ides in the treatment of S. aureus infections with and without bacteri-
emia, including acute and chronic osteomyelitis, cellulitis, carbuncles of
the lip and face, pneumonia and empyema, infected wounds and burns.
A study of 500 cases of infections treated with penicillin led to the
following conclusions (479, 549) : Penicillin can be administered intra-
venously, intramuscularly, or topically, but is ineffective when given by
mouth. As it is excreted rapidly in the urine, it must be injected continu-
ously or at intervals of 3 to 4 hours. Penicillin was found to be particu-
larly effective in the treatment of staphylococcic, gonococcic, pneumo-
coccic, and hemolytic streptococcus infections, especially sulfonamide-
resistant gonococcic infections, but not bacterial endocarditis. The usual
patient requires a total of 500,000 to i, 000,000 Oxford units, the best
results being obtained when treatment is continued for 10 to 14 days,
10,000 units to be given every 2 to 3 hours at the beginning of treat-
ment, either by continuous intravenous injection or by interrupted in-
travenous or intramuscular injections. Good results were obtained by
injections of 100,000 to 160,000 units over a period of 2 to 3 days. In
the treatment of empyema or meningitis it was found advisable to use
penicillin topically by injecting it directly into the pleural cavity or the
subarachnoid space. Toxic effects were extremely rare. Occasional chills
with fever or headache and flushing of the face were noted.
A summary of the response of different bacteria in septic gunshot
fractures is given in Table 43. Staphylococci and streptococci are rapidly
responsive to penicillin therapy. Anaerobic cellulitis due to the proteo-
lytic bacteria of putrid wound infection responds to penicillin, but the
bacteria may persist in the presence of devitalized tissue or wound
exudates. Pyocyaneus is not susceptible to penicillin and is considered to
be relatively unimportant as a single pathogen in the surgical manage-
ment of the wound (272, 548).
In view of the inefficacy of the sulfa drugs for the treatment of
burns, a detailed study has been made (133) of the utilization of peni-
cillin applied to the wound in the form of a cream. In 54 wounds thus
treated, penicillin had a lOO per cent effect upon the hemolytic strepto-
coccal flora, in 7 cases the strains being insensitive to sulfonamide.
The staphylococci also disappeared, although somewhat more slowly.
Healing was usually rapid and no toxic effects were observed. Gram-
236 DISEASE CONTROL
negative bacteria {E. coli, P. vulgaris, Ps. aeruginosa) y when present,
were not affected, as further shown by Bodenham (67).
A summary of the results of extensive use of penicillin in the North
African campaign of the present war led to the conclusion that in the
treatment of recent soft-tissue wounds penicillin brought about the vir-
tual elimination of infection and saved much hospitalization time.
Treatment of fractures also gave good results, though some penicillin-
resistant cocci appeared. Favorable results were also obtained in various
other infections. It is suggested that an average of 750,000 units of
sodium penicillin be allowed for systemic treatment and 50,000 units
of the calcium salt for local treatment (276, 316).
Penicillin is thus found to form a valuable addition to the growing
list of chemotherapeutic agents, to help man combat disease-producing
bacteria. It is commonly used not as a pure acid but as either a calcium
or a sodium salt, the former for local applications and the latter for
intramuscular or intravenous treatments (276, 479). Since penicillin
solutions are quite unstable, especially in the form of salts, the dry
preparations are stored and are dissolved either in water or in saline
just before required for use. Although penicillin has so far proved in-
effective when administered orally, certain of its esters (e-butyl) that
TABLE 43. RESPONSE OF DIFFERENT BACTERIA FOUND IN WOUNDS
TO PENICILLIN TREATMENT
PENICILLIN
RESPONSE
TYPE OF INFECTION
Systemic
Local
Putrid:
Proteolytic Clostridia
+ (large dosage)
+
Proteus vulgaris
0
0
Nonhemolytic streptococci:
Mesophilic
+
+
Thermophilic (S. faecalis)
0
0 (or slight)
Staphylococci
+ (3-5 days)
+ (often necessary)
Hemolytic streptococci
+ (1-3 days)
+ (not essential)
Pseudomonas aeruginosa
0
0
From Lyons (548).
ANTIBIOTIC SUBSTANCES 237
are inactive in vitro can, when given by the oral route, become highly
active against hemolytic streptococci (599, 600).
Production. Because of the limited amounts of penicillin available
at the present time, many attempts have been made by physicians and
hospitals to grow P. notatum on a suitable medium and use the crude
culture filtrate for the treatment of wounds and infections. Since such
cultures cannot be standardized and their activity cannot always be de-
termined and since unforeseen toxic substances may be produced by cer-
tain contaminants in the culture, this practice should not be encouraged,
unless carefully supervised by properly qualified bacteriologists.
The production of penicillin-destroying enzymes by bacteria and
fungi (4, 379, 516) can be utilized for the purpose of testing the steril-
ity of penicillin preparations. The penicillin, which would otherwise
cause inhibition of growth of the contaminating organism in the test
medium, is destroyed by the enzyme previous to the test. No apparent
relation was said ( lOOO) to exist between the resistance of an organism
to penicillin and its ability to produce penicillinase, a fact not generally
accepted (73-75). The mode of action of the enzyme is still not clearly
understood, although there is apparently an increase in the number of
carboxyl groups, as measured by ^H change ( lOOo).
Clavacin {Patulin)
The treatment of common colds that were prevalent in an English
naval establishment by the use of clavacin in the form of nasal sprays or
snuffed up by hand gave 57 per cent complete recovery in 48 hours, as
compared with 9.4 per cent for the controls} no ill effects were observed
(713). These results were not confirmed, however, the conclusion hav-
ing been reached that, compared with the natural evolution of the dis-
ease, patulin has no demonstrable effect on the course of a cold (847a).
This substance also proved to be unsatisfactory for the treatmnt of bo-
vine mastitis by udder infusion (681 a).
Tyrothricin
Dubos (193) reported that 0.002 mg. of gramicidin, one of the two
chemical constituents of tyrothricin, when injected intraperitoneally
into white mice, exerted a therapeutic action against experimental peri-
238 DISEASE CONTROL
tonitis caused by pneumococci and streptococci (Table 44). This sub-
stance was found to be effective against five different types of pneumo-
cocci, eleven types of group A streptococci, and three strains of group C
streptococci. It was, however, almost completely ineffective when ad-
ministered into animal tissues by the intravenous, intramuscular, or
subcutaneous route, because of its lack of activity under these conditions.
TABLE 44. BACTERICIDAL EFFECT OF TYROTHRICIN UPON
DIFFERENT BACTERIA
TYROTHRICIN IN MILLIGRAMS PER MILLILITER OF CULTURE
0.040
0.020
0.0 10
0.004
0.002
o.ooi
0.0
DiPLOCOCCUS PNEUMONIAE,
Type I
Viability*
-
-
-
-
+
1 1 1 1
Reductasef NR
NR
NR
NR
NR
NR
CR
Lysis§ C
C
C
C
C
P
N
Streptococcus hemolyticus, Group A, Tyi
pe6
Viability*
-
-
-
-
+++
1 1 1 1
Reductasef NR
NR
NR
NR
NR
PR
CR
Lysis§ N
N
N
N
N
N
N
Staphylococcus aureus
Viability*
-
-
^K+
+H-f
++-++
-H-+4-
Reductasef NR
NR
NR
CR
CR
CR
CR
Lysis§ C
C
N
N
N
N
N
From Dubos and Cattaneo (203).
*— no growth on blood agar, + reduced growth, MM abundant growth.
t NR no reduction of methylene blue, PR partial reduction, CR complete reduction.
§ N no lysis, P partial lysis, C complete lysis.
Tyrothricin exerted a lethal action m vitro on 1 8-hour broth cultures
of S. hemolyticus^ S. aureus, and C. difhtheriae, in a final dilution of
1 : 1,000,000; freshly isolated strains of meningococcus were affected in
a dilution of i: 100,000 (807). Two monkeys which carried in the
nasopharynx and throat gram-positive hemolytic streptococci and gram-
negative hemolytic bacilli showed disappearance of these bacteria within
1 hours following the administration of tyrothricin. Five days after a
single treatment no hemolytic organisms were found in one monkey,
and, in the other, only throat cultures were positive. A second applica-
tion of the material gave completely negative cultures within 3 hours.
ANTIBIOTIC SUBSTANCES 239
No local or general reactions to these treatments were observed. This
material was also administered to 5 human carriers of hemolytic strepto-
cocci, 2 of whom were persistent nasal carriers for two months following
scarlet fever and the other three convalescent in the third week of this
disease. In only one case was an immediate reduction in the number of
streptococci obtained ; a striking reduction or complete disappearance of
the organisms occurred in the others on the fifth day, after 3 to 4 spray-
ings. These observations were said to be sufficiently encouraging to jus-
tify the use of the material against carriers harboring streptococci, diph-
theria organisms, meningococci, and pneumococci. Injection of 3 to 40
mg. tyrothricin into the pleural cavity of rabbits with hemolytic strepto-
coccal empyema brought about the sterilization of the pleural cavity and
enabled the animal to survive. The injection of 10 mg. of tyrothricin
into the pleural cavity of normal rabbits produced certain local tissue re-
actions. In excess of 10 mg., adhesions, thickening of the pleura, sterile
abscesses, and other disturbances were produced (721).
The susceptibility of fecal streptococci to tyrothricin varies from
strain to strain. Oral administration of the substance may produce in-
hibition of the growth of streptococci in the intestines of mice. This
inhibition was most readily demonstrated when sulfasuxidine was ad-
ministered together with the tyrothricin (761 ). Application of tyrothri-
cin to ulcers brought about sterilization and healing of local infections.
Application to the mastoid cavity following mastoidectomy also gave
favorable results. In staphylococcic infections, resistant strains may de-
velop during therapy (722). Certain sulfonamide-resistant strains of
iS". fyogenes were eradicated by application of gramicidin (292).
Tyrothricin and tyrocidine exert a bactericidal effect, and gramicidin
is largely bacteriostatic (Figure 29) j the first two are affected by blood
and serum, but not the last. In order to be effective against bacteria,
the organisms must be in contact with the material (754). Gramicidin
is more toxic than tyrocidine, the toxic dose being larger, however, than
the dose necessary to kill most gram-positive cocci. Penicillin is one-
tenth as toxic as gramicidin (402, 403).
Both tyrothricin and tyrocidine cause hemolysis of erythrocytes, and
both are leukocytolytic, gramicidin being less so. Both tyrothricin and
gramicidin cause local and general toxic effects when injected into closed
vO
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ANTIBIOTIC SUBSTANCES 241
cavities of the body. Small amounts may bring about the sterilization of
local infections without producing general toxic effects, giving only
minimal local reactions. When injected into the skin, tyrothricin and
gramicidin produce local reaction, the latter to a lesser degree. Oral ad-
ministration is ineffective in reducing or destroying organisms which are
susceptible m vitro. Local application of these substances has not been
attended by toxic reactions even when large amounts were applied
(729). Tyrothricin in high concentrations caused cytoplasmic and nu-
clear disintegration of the exudative rabbit polymorphonuclear leuko-
cytes j in lower concentrations, it brought about altered staining reac-
tions. When there was no apparent microscopic injury to the cells,
phagocytosis of pneumococci took place. The presence of serum brought
about some protection of the cells from the effects of these substances
(132). Tyrothricin does not inhibit mitosis or migration of fibroblasts
or activities of leukocytes, following direct applications to tissue culture
(693).
The filtrate of B. mesentericus was found (956) to have a specific bac-
tericidal action on C. difhtheriae in a dilution of i: 1,250. When in-
jected parenterally into guinea pigs, it inhibited the toxic effect of the
diphtheria organism. The filtrate of the antagonist was found useful in
the treatment of diphtheria carriers.
Particularly favorable results were obtained from the use of grami-
cidin in the treatment of chronic mastitis (535). Several cows received
treatment with increasing amounts of gramicidin diluted with distilled
water. Following the morning milking, the residual milk in the cistern
and in the teat was flushed out with 100 to 200 ml. of the gramicidin so-
lution, containing 60 to 240 mg. in i ,000 ml. water j 800 to 900 ml. were
then injected under pressure into the quarter and allowed to remain
until the next milking. Within one hour after the injection, the treated
quarter became distended and rectal temperature began to increase,
reaching 41° C. at the fifth or sixth hour. The temperature returned
to nearly normal in about 3 hours thereafter, the acute swelling having
subsided at the next milking. The streptococci disappeared from most
of the quarters treated, without an appreciable decrease in milk pro-
duction. The fact that streptococci had been eliminated was established
242 DISEASE CONTROL
by daily bacteriological examination of the milk over periods ranging
from 1 5 to 8 1 days.
Sterile mineral oil was later found (536, 537) to be a suitable, non-
irritating medium for the administration of the gramicidin, though
some of the cows thus treated gave severe reactions. An alcoholic solu-
tion (2 to 3 ml.) of 80 to 120 mg. gramicidin was emulsified in 15 ml.
sterile distilled water, and the emulsion mixed with 25 ml. of heavy
mineral oil. The mixture was injected into the cistern shortly after the
morning milking and allowed to remain until the evening milking. The
treatment was repeated for several days in succession. Of 3 1 quarters
naturally infected with Streptococcus agalactiae and treated by the
gramicidin-oil mixture, 26 seemed to have responded by a complete dis-
appearance of the streptococci. The infection in some of the cases was of
severe chronic nature.
Less satisfactory results were obtained in the treatment of bovine
mastitis caused by Streftococcus uberis; of 4 cases treated, only one re-
sponded satisfactorily. The final recommendations consisted in using
20 to 40 mg. gramicidin in oil, daily, for four consecutive days. These
results were confirmed by various investigators (16, 888), who re-
ported 60 to 90 per cent cure for two to three treatments (585).
A comparison of tyrothricin, trypaflavin, and novoxil for eradicating
S. agalactiae from infected udders showed best results for the first. In-
fections with S. uberis and Streptococcus dysgalactiae also responded
well, but not staphylococcic infections (793). The efficacy of the treat-
ment is influenced by several factors, namely, (a) site of chronic infec-
tion, (b) selection of suitable cases, and (c) stage of lactation. Too ex-
tensive administration may damage the secretory tissues (622). In
some experiments, as many as 90 per cent of the cases were cleared up
with tyrothricin (95).
Gramicidin-like preparations were also used successfully in the treat-
ment of local infections in man, such as osteomyelitis (553), and for
various local administrations, such as conjunctivitis, as well as for in-
fected diabetic and ulcerating lesions of cancer ( 1007). The active ma-
terial must be used locally on infected cavities which do not communi-
cate with the blood stream (3 1 8a, 401 ). Tyrothricin has also been used
(78, 154) successfully in the treatment of acute otitis media, acute and
ANTIBIOTIC SUBSTANCES 243
chronic mastoiditis, and acute and chronic sinusitis. The substance does
not damage the tissue or interfere with wound healing. The growth of
most staphylococci, streptococci, and pneumococci is inhibited or the or-
ganisms are killed. When applied locally, tyrothricin does not reach
the blood stream. It has also been found that tyrothricin has an excellent
therapeutic effect when used for urethral irrigations in the male (187).
Tyrothricin possesses several limitations, from the point of view of
practical utilization: (a) development of bacterial, notably staphylococ-
cus, variants, which become resistant to this agent 5 (b) inhibition of its
action by phospholipinsj (c) hemolytic action, which prevents its intra-
venous use.
Tyrothricin was found to produce no lesions in the gastro-intestinal
tract (728, 968), but it is not very active when administered by mouth,
since it is destroyed by the proteolytic enzymes of the digestive system.
Streftothrkin
The fourth agent that was found to offer practical possibilities is
streptothricin. This agent gave favorable results for the treatment of
Br. abortus grown on chicken egg embryos. Its toxicity is low enough
to make possible the administration of doses sufficient to destroy this
pathogen in the living tissues (Table 45). Both the in vitro and the in
vivo activities of streptothricin against gram-negative bacteria sug-
gested the probability that it will prove useful in the local treatment of
infected wounds and burns, bacillary dysentery, typhoid fever, and food
poisoning produced by Salmonella organisms (755, 756a).
Other Agents
The protective action of the specific enzyme (polysaccharidase) of a
soil bacterium against type III pneumococcus infection has also been
established (30, 293). The specific protection induced in experimental
animals is determined by the nature of the polysaccharide of the pneu-
mococcus type. The polysaccharidase destroys the protective capsular
substance of the pneumococcus, thus rendering it susceptible to phagocy-
tosis.
The possibility of utilizing antagonistic bacteria for the control of
fungi causing skin infections has also been suggested (119).
244
DISEASE CONTROL
Virulent strains of M. tuberculosis were found to lose their virulence
in the presence of certain other organisms or their products. According
to Vaudremer (912), this phenomenon occurs when the tubercle or-
ganism is kept for 24 hours at 39° C. in contact with a filtered extract
oi A. jumigatusy and a similar effect can be exerted by certain bacteria
(708). On the other hand, extracts of A. jumigatus were used for the
treatment of 200 tubercular patients with rather inconclusive results
(912).
TABLE 45. IN VIVO EFFICACY OF STREPTOTHRICIN COMPARED WITH
SULFADIAZINE IN SALMONELLA AERTRYCKE INFECTION IN MICE
NUMBER OF
daily treat-
original
m:
ICE SURVIVING AFTER
ment
IN
number
I
2
3
4
5
INFECTION*
milligrams!
OF mice
day
days
days
days
days
Streptothricin
IO-*
I
10
10
5
5
4
2
I0-*
2
10
10
10
10
10
10
I0-*
4
10
10
10
10
10
10
10*
8
10
10
10
10
10
10
Sulfadiazine
10-*
16
10
10
10
10
4
4
Control
10-*
10
0
io-«
10
0
io-«
10
0
10-^
10
0
From Robinson (752).
* 0.5 cc. of a 6-hour-olcl culture dilution in broth.
t Streptothricin given subcutaneously immediately after inoculation of bacteria.
Treatments of intestinal disturbances by the use of antagonistic micro-
organisms, although highly promising, have not been sufficiently in-
vestigated as yet. It may be of interest to note, in this connection, that
the presence in human intestines of E. coU with a high antagonistic in-
dex is considered as important evidence of immunity of certain indi-
viduals to intestinal disturbances (383, 580, 619, 643).
TOXICITY OF ANTIBIOTIC SUBSTANCES
245
TOXICITY OF ANTIBIOTIC SUBSTANCES
The various antibiotic substances obtained from microorganisms vary
greatly in their toxicity to animals. The therapeutic use of many of these
agents, like actinomycin or clavacin, which are highly bacteriostatic,
may be considered as either entirely excluded for the present or limited
only to local applications. Some substances, as pyocyanase, penicillin,
and streptothricin, are relatively nontoxic (754) j others, like tyrothri-
cin (756) and fumigacin (752), are slightly toxicj and still others, like
actinomycin and clavacin, are highly toxic (757, 941). Some, like ty-
rothricin, are hemolytic (390, 579) j others, like penicillin, actinomycin,
and streptothricin, are not (Table 46).
TABLE 46. ACUTE TOXICITY TO MICE OF VARIOUS ANTIBIOTIC AGENTS
SUBCU-
INTRA-
ORAL
TANEOUS
PERITONEAL
INTRAVENOUS
ANTIBIOTIC
ADMIN-
ADMIN-
ADMIN-
ADMIN-
AGENT
ISTRATION
ISTRATION
ISTRATION
ISTRATION
Streptothricin
>2,000
> 1 ,000
3,000
1,000
Penicillin
> 2,000
1,600
2,000
500
Fumigacin (he
Ivolic
acid)
800
Citrlnin
100
100
Pyocyanin
100
80
Tyrocidine
> 1,000
> 1 ,000
20
1.25
Tyrothricin
> 1,000
> 1,000
10
1.2
Gramicidin
> 1 ,000
> 1,000
10
1.2
Gliotoxin
5
Clavacin (crude)
3-5
Actinomycin
5
0.15
0.15
0.15
From Robinson (752).
Note: Figures represent maximal tolerated dose in mgm./kgm.
CHAPTER I 3
MICROBIOLOGICAL CONTROL OF SOIL-BORNE
PLANT DISEASES
The possibility of controlling microorganisms, especially fungi, in the
soil by favoring the development of antagonistic microorganisms is sig-
nificant for several reasons: fungi are causative agents of some of the
most important diseases of plants and are added constantly and often
quite extensively to the soil, in plant residues and in diseased plant
products J fungi capable of causing certain diseases of animals and of
man also find their way sooner or later into the soil ; many soil-inhabit-
ing fungi have a marked antagonistic effect against fungus and bacterial
plant pathogens.
Some fungi that produce plant diseases are able to survive in the soil
for only short periods of timcj others become established in the soil
saprophytically and remain capable of attacking living plants when
proper conditions arise. Some of these fungi are specific, their ability to
attack different plants being limited, whereas others can cause diseases
of a great variety of plants and many survive in infected soil for long
periods. Some plant diseases, as in the case of virus infections, are trans-
mitted by specialized means, as by insect carriers. This complicates fur-
ther the interrelationship among the different organisms, in relation to
plant and animal diseases.
Microorganisms causing diseases of plants may either reduce the
vigor and productivity of the plants or destroy them completely.
Plants appear to develop at times a certain degree of resistance to mi-
crobial infection. Whether this is in the nature of a phenomenon of im-
munization, similar to that of animals, is still a matter of speculation.
Whatever the nature of the reaction, the degree of resistance depends
to a certain extent upon the imperviousness of the outer layers of the
plant tissues to penetration by the parasites, as well as upon the chemi-
cal composition of the plants. It is believed that an acid plant reaction,
combined with the presence of tannins and lignins, retards the growth of
many disease-producing agents. The survival of the pathogens outside
CONTROL OF SOIL-BORNE PLANT DISEASES 247
the host plant is due to the formation of resistant spores which remain
viable in the soil for long periods. Because of this, the growth of many
plants requires a long rotation if this system is to be used as a means of
controlling the specific diseases.
Many fungi and bacteria causing plant diseases were at first thought
capable of surviving in the soil for an indefinite time, even in the ab-
sence of the hosts. It has since been established, however, that, although
the majority of these pathogens are facultative saprophytes, some are
obligate parasites. The first can be grown easily on sterile soil and on
artificial culture media, whereas the second, such as Plasm-odiofhora
hrasskae and Synchytrmm endoh'iotkumy have not been cultivated so far
upon any artificial media and are known to die out in the soil in the ab-
sence of host plants.
Certain soil-borne plant diseases may be caused by more than one or-
ganism. In the pink-root of onions (375), Phoma terrestrls is followed
by Fusarium malli; in the take-all of wheat (314), O. graminis is fol-
lowed by Fusarium culmorum. This type of sequence occurs with other
diseases, where the primary parasite first attacks the root and is followed
by a succession of other fungi, both parasites and saprophytes. By means
of the direct microscopic technique, the sequence of microorganisms can
be demonstrated in the infected roots of the plants. Certain less special-
ized parasites are able to live saprophytically on the dead tissues,
whereas the saprophytes are found only in the later stages of decom-
position.
It has been suggested (313, 739) that the root-infecting fungi be
classified ecologically as soil inhabitants and soil invaders. The first may
be looked upon as primitive or unspecialized parasites with a wide host
range, their parasitism being considered incidental to their saprophytic
existence in the soil. The second group comprises a majority of root-
infecting fungi, the more highly specialized parasites. The presence of
these in the soil is closely associated with the occurrence of the host
plants: in the absence of a host, these fungi die out in the soil, because
of their inability to compete with the soil saprophytes. The close associa-
tion between this group of organisms and their host plants is believed to
be enforced by competition with the microbiological population of the
soil (312a).
248 CONTROL OF SOIL-BORNE PLANT DISEASES
ANTAGONISM OF SOI L- I N H AB ITI NG MICRO-
ORGANISMS TO PLANT PATHOGENS
The antagonistic interrelationships among the members of the micro-
biological population of the soil have received particular attention from
the point of view of modifying the virulence of those plant pathogens,
especially the fungi, that find temporary or permanent habitat in the
soil (23,310,554,696).
In the infection of wheat seedlings by O. graminis, a number of
fungi and bacteria are able to exert a marked antagonistic action against
the pathogen (784). Not only the living cultures of the antagonists,
but, in many cases, the culture filtrates are also effective (511, 992).
The growth of H. sativum and F. graminearum upon sterilized soil
was completely suppressed (397) by the addition of small amounts of
unsterilized soil or by the simultaneous inoculation with harmless fungi
and bacteria, with the result that no infection occurred when wheat seeds
were inoculated with this soil. Although H. sativum is able to sporulate
readily in sterilized soil, this does not take place in nonsterilized soil,
sporulation being inhibited by the soil microorganisms. Virulence of
H. sativum on wheat seedlings was reduced by 11 to 57 per cent by cer-
tain cultures of Penicillium; Trichoderma reduced virulence by 50 to
58 per cent, Absidia glauca by 39 per cent, and A. nidulans by 30 per
cent. Many fungi, however, had no effect on the virulence of the patho-
gen, and some even increased it (7B5). The fact that root-rot diseases
are less severe on wheat grown on summer-fallowed land than on land
cropped to wheat for several years was believed to be due to the soil
saprophytic microorganisms, which in bare fallow have an advantage
over the pathogenic organisms in competition for food (Figures 30
and 31).
The infection of wheat seedlings by O. graminis in sterile soil was
found to fall off rapidly with the reestablishment of the original soil
microflora (86). It was emphasized, however, that the effect of various
organisms upon the pathogen grown in artificial culture media is no
proof that the same organisms will be able to suppress the virulence of
the pathogen on wheat in soil. An inverse correlation was shown (617)
to exist between the degree of infection and the protective effect of the
SOIL MICROORGANISMS AND PLANT PATHOGENS 249
general soil microflora j this was determined by comparing infection in
an unsterilized soil with that obtained in a sterilized soil. An increase in
soil temperature was found (312, 398) to increase the antagonistic ac-
tion of the soil microflora against the parasitic fungi causing cereal root
rots.
Various actinomycetes were shown to be antagonistic (884) to species
of Pythiuniy a root parasite of sugar cane. The phenomenon of antago-
nism was independent of the f¥l changes j it has been ascribed to the
formation of a toxic, partly thermostable, principle. A marked influ-
ence of the soil microflora on grass diseases caused by Pythium (691 ) is
illustrated in Figures 32 and 33 (906). Clavacin (patulin) was found
capable of inhibiting the growth of various species of Pythium (cause of
damping-off disease of seedlings) in dilutions of about 1 1400,000 (22),
and of exerting a strong fungicidal action upon Ceratostomella ulmiy
the causative agent of the Dutch elm disease j the last effect could
partly be overcome by certain nutrients in the medium, especially
peptone (928).
Numerous soil microorganisms are moderately or strongly antago-
nistic to such pathogens as Hyfochnus centrifugus, Hyfochnus sasakii,
and Sderotium oryzae sativae (241 ) j culture filtrates from some of the
antagonistic fungi were also able to reduce the damage caused by the
pathogens.
Phytofhthora cactorum was found (974) to be inhibited in the rotted
tissues by the antagonistic effects of secondary organisms. In many
cases, the rotting of fruits was suppressed by mixtures of organisms as
compared with the pathogens j the type of rot was also modified, de-
pending on the temperature and the specific nature of the antagonists
(790).
The stimulating effect of mycorrhizal fungi on the host plant has
been explained (298) by the capacity of the fungi to inactivate, destroy,
or absorb certain plant-retarding principles found among the organic
constituents of peat and other humus materials, or produced by fungi.
Certain fungi are also known (332) to be antagonistic to ants and
their fungal symbionts. These antagonists are distributed by the insects,
thus spreading agents that are destructive to themselves and to their
fungus gardens.
250 CONTROL OF SOIL-BORNE PLANT DISEASES
GENEP.AL METHODS OF CONTROL OF
SOIL-BORNE DISEASES
Soil sterilization by heat and chemicals has long been practiced as a
method of control of soil-borne fungus diseases. This phenomenon is
usually designated as partial sterilization of soil, since not all micro-
organisms are killed by these treatments. However, once a soil thus
treated becomes reinfected with a disease-producing organism, the in-
fection may become much more severe. It has, therefore, been sug-
gested (380, 682) that partially sterilized soil be reinoculated with a
mixture of saprophytic microorganisms before it is used as a seedbed,
so as to counterbalance the injurious effect of the parasites (Table 47).
TABLE 47. EFFECT OF A BACTERIAL ANTAGONIST ON DAMPING-OFF
OF PLANTS IN THE SOIL
PERCENTAGE OF SEEDS PLANTED
PRODUCING NORMAL SEED-
TREATMENT OF SOIL LINGS IN 2 WEEKS
Cucumbers Peas
Control soil
35
52
Fresh medium added
65
55
Diluted medium added
61
77
Washed bacterial cells added
58
75
Culture of bacterial antagonist
added
55
80
Diluted culture added
87
90
Culture added continuously
81
90
From Cordon and Haenseler (149).
The importance of the soil microflora in modifying plant diseases
caused by soil-borne pathogens is being realized more and more clearly.
One of the earliest attempts to control a plant disease by microbiological
agents was made in 1908 by Potter (697). He found that Pseudomonas
destructansy the cause of rot of turnip, produces a potent, heat-resistant
toxin. The bacteria failed to grow in the presence of this toxin, and were
completely killed by the substance. By spraying turnips with this mate-
rial, the disease could be checked j the toxin was more or less specific for
the particular organism. Certain bacteria commonly found in soils were
shown (304) to have a deleterious effect on the growth, in artificial
-^T
Figure 30. An antagonistic fungus, Trichodermay attacking
a plant pathogenic fungus, S. rolfsiiy showing one break of a
septum. From Weindling (963).
Figure 31. Influence of antagonists upon the growth of Helminthosforiuni.
Distortion of mycelium by Bacterium sp. (A) and B. rnmosus (B). C is a
normal mycelium. From Porter (695).
m^ m~-tr-
\\
^ M^ ^ uM M M
Figure 32. Antagonism of soil organisms against parasitism of P.
volutum on Jgrostis. From van Luijk (906).
r ^i» - ^,, ^ 1 i
Figure 33. Inhibiting eflFect of sterilized liquid medium of P. exfan-
sum versus Pyth'mm de Baryanum on lucerne. From van Luijk (906 ) .
GENERAL METHODS OF CONTROL 251
media, of Ps. citri, which causes citrus canker. This effect was brought
about by inhibiting the growth of and by killing the pathogen. By the
use of an antagonistic bacterium, wheat seedlings were protected from
infection by H elminthosforiuni sp. (695). In a similar manner, flax
seedlings were protected from Fusarium sp.
A watermelon disease, caused by P hymatotrichwm omnlvoruniy was
considerably reduced when certain specific fungi and bacteria were pres-
ent in the soil together with the pathogen (91, lOi) j T. Ugnorum was
observed to attack and kill the hyphae of Phymatotrichum in culture.
The severity of the seedling blight of flax, caused by Fusarium Uni, was
diminished when the pathogen was accompanied in the soil by various
other fungi (875). The pathogenicity of H. sativum on wheat seed-
lings was suppressed by the antagonistic action of Trichothecium
roseum, this effect being due to a toxic substance produced by the latter
(353). T. Ugnorum prevented infection of wheat (60) by H. sativum
and Fusarium culmorum (Table 48). Novogrudsky (646) obtained
protection against infection of wheat with Fusarium by inoculating the
TABLE 48. EFFECT OF TRICHODERMA LIGNORUM ON GERMINATION AND
GROWTH OF BARLEY INFECTED WITH HELMINTHOSPORIUM
SATIVUM IN STERILIZED SOIL
STRAIN OF
H, SATIVUM
Emerg
ed
PERCENTAGE OF PLANTS
Stunted
Contorted leaves
H
H + T
H + SI
H
H + T
H + Sl
H
H + T H + SI
21
84
94
94
46
12
6
52
32 15
22
88
94
98
33
8
6
57
27 14
23
86
88
96
25
17
8
78
31 21
24
88
98
94
10
4
3
17
15 10
From Christensen (127).
Notes. Results are based on randomized duplicate pots, each sown with 50 seeds. H, seeds inoculated with
a spore suspension of H. sativum; H + T, seed inoculated with H. sativum plus T. Ugnorum; H + SI,
seed inoculated with H. sativum and soil with T. Ugnorum.
soil with the bacteria isolated by Chudiakov (130), provided the bac-
teria were introduced simultaneously with the fungus qr preceded it.
The role of microbiological antagonism in the natural control of soil-
borne fungus diseases of plants has thus been well emphasized (85,
252 CONTROL OF SOIL-BORNE PLANT DISEASES
398, 783, 784). Methods for combating plant pathogenic fungi by the
use of bacteria and other antagonists have been suggested by various in-
vestigators (46, 503, 841).
The principles underlying the biological control of soil-borne plant
diseases were outlined by Garrett (312) in terms of the soil population
in a state of dynamic equilibrium. When a given crop is grown continu-
ously in the sam.e soil, the parasitic organisms capable of attacking the
roots of that crop multiply (72). Organic manures stimulate the de-
velopment of saprophytic organisms in the soil, and are thus able to
check the activity of the pathogens, which are destroyed by the sapro-
phytes. Either the metabolic processes of the saprophytes check the
growth of the pathogens, or the saprophytes actually attack and destroy
the mycelium of the pathogens. The microbiological control of plant
diseases was said to be most effective against those organisms which have
become highly adapted to a parasitic form of life. The pathogenic
Ofhiobolus, when present in the form of mycelium inside the infected
wheat stubble buried in the soil, is able to tolerate adverse physical soil
conditions. Those soil treatments which favor increased activities of the
microbiological population, such as addition of organic matter, partial
sterilization followed by reinoculation with fresh soil, and improvement
in soil aeration, favored loss of viability of the pathogen.
Van Luijk (906) recommended the control of plant parasites by
inoculating the soil with specific microorganisms selected for their an-
tagonistic capacity, or by the addition of the growth products of these
microorganisms. Living soil fungi, including Trichoderma viridis and
Absidia sfinosa, exerted an adverse influence upon Rhizoctonia {Cor-
ticium) solanl and reduced its pathogenicity to cabbage seedlings (449).
Broadfoot (86) and others (248), however, emphasized that the an-
tagonism of a saprophyte to a plant pathogen, determined on artificial
culture media, is not a reliable measure of the actual control of the para-
site in the soil. A lack of specific microorganisms in the soil is not a suffi-
cient factor limiting biological control under natural conditions. There-
fore, no inoculation of soil with an antagonistic organism, such as T .
llgnoruniy can have more than a temporary effect in changing the micro-
biological balance of the soil population. Similar results have been ob-
GENERAL METHODS OF CONTROL 25 3
tained (966) in efforts to control R. solani, or the damping-off of citrus
seedlings (Figure 34), by the use of T. lignorum, and in the action of
B. sifnflex upon Rhizoctonia in the soil (149),
A number of antagonistic bacteria were found (48 1 ) to be able to pre-
vent scab formation by S. scabies on potatoes. Daines (161) found that
T. lignorum produces a diffusible substance which is toxic to S. scabies
in an artificial liquid medium. However, the toxic principle added to
potato soils is rapidly destroyed there by aeration j it can be removed
from solution by charcoal and by soil, where it is destroyed. It was sug-
gested, therefore, that it is highly doubtful whether antagonists will
be found to be of much assistance in combating potato scab in soil. The
physical and biological environments encountered in many cultivated
soils offer an important barrier against the establishment of the antago-
nist. When the latter was added to a 5-day-old culture of S. scabies, it
was greatly inhibited by the scab organism. Soil bacteria are also able to
produce substances toxic to both Trichoderma and Streftomyces alike.
In such a complex physical, chemical, and biological environment as the
natural soil, these antagonistic relationships may thus be modified or
even entirely destroyed.
The application to the soil of organic materials which favor the de-
velopment of antagonists has given much more favorable results than
the use of pure cultures. Fellows (251) obtained field control of the
take-all disease of wheat in Kansas by the application of chicken and
horse manure, alfalfa stems and leaves, boiled oats and barley, as well
as potato flour. Garrett believed (313, 314) that the factor chiefly con-
trolling the spread of pathogenic fungus along the roots of the wheat
plant was the accumulation of carbon dioxide, with a corresponding
lowering of oxygen tension in the microclimate of the root zone. A high
rate of soil respiration was, therefore, said to check the growth of O.
graminis. This can best be maintained, of course, by periodic additions
of organic manures. Materials low in nitrogen were found to be more
effective than those high in nitrogen. Garrett, therefore, postulated the
hypothesis that the soil microflora used the mycelium of the pathogen as
a source of nitrogen, in the process of decomposition of the nitrogen-
poor materials. The addition of nitrogenous substances, in either an or-
254 CONTROL OF SOIL-BORNE PLANT DISEASES
ganic or an inorganic form, was believed to protect the pathogenic or-
ganism against attack by the soil microflora, by offering a more readily
available source of nitrogen. Tyner (895) suggested that the differ-
ences in the microflora associated with the decomposition of different
plant residues are largely responsible for differences in persistence and
virulence of pathogens causing root rot of cereals.
Against some plant pathogens, however, high nitrogenous materials
were found to be very effective. Considerable reduction in the slime-
disease of tomato plants resulted from the addition of green manures
to the soil before planting (904) j organic materials high in nitrogen,
as well as the supplementary addition of nitrogenous materials suffi-
cient for complete decomposition of the organic matter, brought about
greater reduction of the disease. Organic matter was found to be most
effective during the process of decomposition 5 after it has undergone
extensive decomposition and reached a stage of slow decomposition,
when it is usually designated as humus, it becomes comparatively inert
(878).
The antagonistic action of soil microorganisms has been utilized in
several areas of the United States for the control of P. omnivorumy the
root rot of cotton. It was shown (484-486) that this pathogen can be
inactivated when organic manures are added to the soil before the crop-
growing season. Eaton and King (223) demonstrated, by the use of
the contact slide technique, that microbiological antagonism represents,
in this case, the true mechanism of the control process; the develop-
ment of saprophytic organisms was most profuse in the slides buried in
the manured plots, whereas the mycelium of the pathogen was most
abundant on the slides kept in the unmanured plots. The conclusion was
reached (345) that manuring definitely controls cotton root rot, as a
result of the parasitism by bacteria of the fungal strands of the causative
agent of the disease. Continuous growth of cotton on certain neutral or
alkaline soils in southern United States was believed to bring about an
unbalanced soil population in which P. omnivorum became a dominant
organism J this was accompanied by the absence or only the sporadic
presence of Trichoder-ma and other molds (878). The application of
organic matter to such soils results in the destruction of most of the
sclerotia and mycelium of the pathogen (609). Microbial antagonists
Figure 34. Sweet-orange seedlings in nonsterilized soil. A, control;
B, Rhi%octonia inoculated into soil layer in bottom of jar; C, Rh'i-zoc-
tonta as in B, plus Tr'ichodcrma in top layer of peat. From Weindling
and Fawcett (966).
GENERAL METHODS OF CONTROL 25 5
rather than food exhaustion were, therefore, considered to be respon-
sible for the destruction of the pathogen.
The Sclerotium rot of sugar beets was found (518) to be controlled
by the application of nitrogenous fertilizers. This effect was believed to
be due largely to a change in the metabolism of the fungus or of the
host. It was also suggested, however, that the possibility exists that the
suppression is due to a change in the balance of the soil microbiological
population.
The possibility of suppressing the growth and eliminating the infec-
tivity of plant pathogens by utilizing the activities of the soil microflora
was demonstrated also for a number of other diseases. It is sufficient to
cite the suppression of Monilia jructigena on apples by various fungi
and bacteria (911), of F. culmorum and H. sativum on wheat (398,
695), and of species of Rhizoctonia on citrus seedlings. These patho-
gens are markedly influenced by T. lignorum, a common soil sapro-
phyte. A species of Trichoderma was also found to cause a reduction in
the amount of Texas root rot of watermelons caused by P. omnivorum
(lOi).
The damping-off of citrus seedlings, caused by a number of fungi,
could be suppressed by T. lignorum, which parasitizes the fungi (962,
966). The addition of Trichoderma spores to acid sterilized soils pre-
vented the damping-off of the seedlings. When T. lignorum was inocu-
lated into pots containing Helminthosforium sp. and Fusarium sp., the
pathogenic action of these organisms was checked and rendered harm-
less to plants (60). T. lignorum was also found (17) to be decidedly
antagonistic to Rhizoctonia and Pythium, organisms responsible for
seed decay and damping-off of cucumbers.
The presence of Gibherella on corn inhibited infection due to T .
viridis; seed grains inoculated with the former gave more vigorous
growth than uninoculated seed grains (226). On the other hand, T.
viridis was found able to attack and to destroy the sclerotia as well as the
mycelium of such pathogenic fungi as Corticium rolfsii, Corticium sa-
dakii, and Sclerotinia lihertiana (S. sclerotiorum) . The utilization of
this organism for the biological control of plant diseases has, therefore,
been suggested (415).
Henry (398) believed that the biological control by the soil micro-
256 CONTROL OF SOIL-BORNE PLANT DISEASES
flora could even be directed against internal seed infection, since appre-
ciable damage to surface-sterilized flax seed was found to occur in steri-
lized but not in unsterilized soil.
CONTROL OF PATHOGENIC FUNGI IN SOIL BY
INOCULATION WITH ANTAGONISTIC
MICROORGANISMS
Despite the favorable results obtained from the action, in artificial
culture, of antagonistic bacteria and fungi upon plant pathogens, the
field results have often been rather disappointing. The soil microflora
seems to have no marked effect on certain diseases, such as the seedling-
blight of barley j the antagonistic action of the soil population appears
(127) to be insufficient to suppress the injury caused by diseased seed.
The addition of T. Ugnorum and certain other fungi and bacteria to in-
fected seed or to sterilized soil inoculated with H. sativum often pre-
vented seedling injury, increased the stand, and decreased the number
of deformed seed. Chudiakov ( 130) suggested inoculation of seed with
bacteria, for the control of flax against infection with Fusarium. It has
been said that wheat seedlings were protected from attack by the simul-
taneous introduction of lytic bacteria with the pathogenic fungus, but
when the bacteria were introduced 24 hours after the fungus, they were
unable to protect the wheat sown 3 days later.
On the basis of extensive studies on the control of plant-disease-pro-
ducing fungi by means of antagonistic soil microorganisms, Novogrud-
sky (646) came to the following conclusions: The distribution and
vigor of parasitic fungi are a result, on the one hand, of resistance and
immunity of plants to infection, and, on the other, of the antagonism
between soil microorganisms and pathogenic fungi. Among the numer-
ous forms of antagonism existing between soil microorganisms and
pathogens, those bacteria which produce lysis of fungi deserve particu-
lar attention. The bacteria are widely distributed in nature j they are
able to destroy and to dissolve the mycelium and the spores of different
fungi, including species of Fusarium, Colletotrichum, and other phyto-
pathogenic fungi. The lytic effect takes place not only in artificial me-
dia, but also in the soil. The inoculation of sterilized soil with F. grami-
CONTROL OF PATHOGENIC FUNGI 257
nearum led to the inevitable death of wheat plants, but additional inocu-
lation of the soil with lysogenic bacteria protected the wheat from the
disease. The addition of bacteria to unsterilized soil which has been
made sick by continuous growth of flax markedly lowered the per-
centage of plants diseased by F . lint.
The term "bacterization" was suggested by the Russian investigators
(46, 130, 631, 647) to designate the process of treatment of seed with
lysogenic bacteria, whereby the plants are protected against pathogenic
fungi. The susceptibility of plant seedlings to infection by fungi could be
decreased not only by the specific antagonistic bacteria, but also by the
presence of other bacteria which are able, in one way or another, to re-
tard the development of the fungi. The conclusion was reached that the
effect of bacteria on germinating seeds is due to the metabolic products
liberated by the bacteria, which are capable of depressing the develop-
ment of parasitic fungi. By treating flax seeds with the culture filtrate of
the antagonistic bacteria, a similar or even greater decrease in the num-
ber of diseased seedlings was obtained. The nature of the active sub-
stance produced by the antagonists was not investigated further. It was
said to accumulate in 5-to-io-day-old cultures. Heating at 80° C. for
10 minutes had no effect upon the substance, but heating at 100° C.
brought about its inactivation.
Jensen (455) concluded that the beneficial result of bacterization is
due not to nitrogen-fixation by Azotobacter or to production of growth-
promoting substances by microorganisms, but to the protection of the
seedlings against plant pathogens by specific bacterial antagonists.
Although it has now been definitely established that certain organ-
isms can repress or even destroy disease-producing fungi, the utilization
of specific microbial products for the control of plant diseases has made
comparatively little progress so far. Leemann (521) tested the action
of various secretions and extracts of microorganisms upon H. sativum.
He concluded that microorganisms, pathogenic or nonpathogenic, can
supply substances useful as preventive measures against plant patho-
gens. No favorable effects upon the development of Rhizoctonia disease
in plants could be observed, however, from the application to soil of cer-
tain bacterial and fungus products.
The only important procedure which has thus far found a practical
258 CONTROL OF SOIL-BORNE PLANT DISEASES
application is the treatment of the soil in such a manner as to modify its
microbiological population, which in its turn can destroy or in other
ways control the activities of plant pathogens. Many such instances can
be cited for illustrative purposes. In the case of root rots of the straw-
berry, it was found that carbohydrate decomposition induces a favor-
able change in the soil microflora from pathogenic or potentially harm-
ful organisms to beneficial or rather innocuous types (413). Studies of
the eradication of cotton root rot led to recommendations of treatments
of soil with organic materials, especially during the period of increased
microbial activity (610). The survival of the fungus is considered
to be limited by microbial interrelationships rather than by food ex-
haustion. During the early stages of incubation, viable sclerotia are de-
stroyed more rapidly than dead sclerotia in soils treated with organic
matter. It was suggested, therefore, that the germination of the scle-
rotia is an important factor for their elimination from the soil. Field
application of organic materials, accompanied by early October plowing,
results in an increase in microbial activity. This brings about a reduc-
tion of the incidence of dead cotton in the succeeding crop and greater
difficulty of finding sclerotia.
It was reported recently (63) that R. solani is able to cause lOO per
cent damping-off of radish seedlings planted at a distance of 4 cm. from
the inoculum j when the seeds were planted at a distance of 9 cm. the
damage was 40 per cent. The addition of i per cent ground wheat
straw or dried grass to the soil had a marked depressing effect upon the
growth of the fungus. This was ascribed to the nitrogen starvation of
the mycelium, accompanied by the rapid utilization of the available soil
nitrogen by the cellulose-decomposing microorganisms multiplying at
the expense of the fresh organic material j carbon dioxide produced by
the cellulose-decomposers was believed to cause a marked fungistatic
action on the Rhizoctonia.
Treatment of the soil with organic materials, which results in the de-
struction of certain plant pathogens by stimulating the development of
saprophytic microorganisms, may be called "partial disinfection."
The possible injury caused to leguminous plants through the antago-
nistic action of soil bacteria upon the root nodule bacteria has been but
little investigated (495).
CHAPTER 14
THE OUTLOOK FOR THE FUTURE
The production of antibiotic substances by microorganisms under con-
trolled laboratory or factory conditions and the utilization of these sub-
stances for disease control are of very recent origin. It has been known
for more than half a century that certain microbes are capable of com-
bating others, and it has even been suggested that saprophytic micro-
organisms may in time be utilized for controlling the growth of patho-
genic forms. Until recently, however, these suggestions were largely
speculative. Such positive facts as were available were merely isolated
items, the full significance of which was not sufficiently well recognized.
This is true, for example, of the claims and counterclaims concerning
pyocyanase and pyocyanin, two antibiotic substances of bacterial origin,
the antibacterial properties of which have long been known. It is true
also of certain antibiotic substances produced by fungi, such as gliotoxin.
The existing confusion is due largely to the fact that the utilization of
these antibiotic substances for disease control gave results that v/ere
rather disappointing.
In 1929, Fleming observed that the growth of a mold, which was
later identified as P. notatum, on a plate seeded with staphylococci pre-
vented the growth of these bacteria. In liquid media, this mold pro-
duced a soluble substance, designated as penicillin, that inhibited pyo-
genic cocci and members of the diphtheria group but not gram-negative
rods. Fleming, however, did not go beyond the suggestion that this sub-
stance be utilized for disease control or beyond the statement that
it might prove useful because of these antibacterial properties. This
contribution received no further attention for more than a decade, with
the exception of very few experiments that served to prove Fleming's
original observations. The full realization of the fact that we are dealing
here with an entirely new field of biology and chemotherapy that is
bound to enrich the subject of control of disease came only about half a
decade ago.
In 1939, Dubos announced that new antibacterial agents can be iso-
260 THE OUTLOOK FOR THE FUTURE
lated from soil microorganisms that are active not only m vitro but also
in vivo. He proceeded with the isolation of such substances in a novel
and unique manner, which consisted in enriching the soil with patho-
genic bacteria, isolating from such enriched soil specific antagonistic
organisms capable of destroying the pathogens, and finally isolating the
antibiotic substances from the bacteria. This work pointed to a new
method of approach to the isolation of antibiotic substances and attracted
the immediate attention of bacteriologists, chemists, and medical inves-
tigators. One no longer had to depend for the isolation of antagonistic
organisms upon mere air contaminations of exposed bacterial plates, or
upon accidental observations of the destructive action of one microbe
upon another. One could now proceed systematically with the isolation
of microorganisms capable of destroying sfecific disease-producing bac-
teria. The mechanisms whereby such destruction was brought about
were found to be due to the production of characteristic substances,
known as antibiotic agents. The isolation of each required special meth-
ods, because of its specific chemical nature. Since most of the antibiotic
substances are selective in their action upon different bacteria, affecting
some and not others, the method proved to be of great importance in
pointing a way to the isolation of a variety of agents active upon gram-
positive bacteria as well as gram-negative forms.
It was not sufficient, however, merely to isolate these protoplasmic
poisons. It was essential to determine the effect of animal tissues upon
the action of these agents and to establish their selective activity upon
the bacteria in the animal body as compared to the test tube. However,
once it was recognized that some of these antibiotic substances act in
vivo against a variety of diseases, that branch of medical science known
as chemotherapy acquired a new group of tools. This work led to a re-
examination of the potentialities of penicillin, which in turn led to the
second important contribution, namely, the work of Chain and Florey
and their co-workers. These investigators succeeded in obtaining crude
active preparations of penicillin, which, they demonstrated, had a
marked effect upon various gram-positive bacteria In the animal body.
The importance of this discovery was accentuated by the great need of
the moment and the urgency for new methods of combating infections
resulting from World War II. These investigations immediately at-
THE OUTLOOK FOR THE FUTURE 261
tracted universal attention, and were soon followed by remarkable de-
velopment in the manufacture, isolation, and study of the chemical na-
ture of this highly important antibiotic agent. Penicillin fully deserves
the designation "Wonder Drug" given to it by the popular press.
These two epoch-making contributions were rapidly followed by a
series of investigations that resulted in the isolation of a number of
other antibiotic substances. It was soon recognized that one is not deal-
ing here with only two types of chemical compounds capable of destroy-
ing various pathogenic bacteria and fungi, but that a new field of science
bordering on microbiology, chemistry, and pathology was being opened
that was bound to result in many chemotherapeutic applications. The
fact that many of these agents, including penicillin, are produced by sev-
eral different organisms and, further, the fact that many of these com-
pounds are produced in different chemical modifications open to the
chemist new fields for the synthesis of types of compounds heretofore
unknown, and point out to the medical world new ways of combating
infections and epidemics.
The rapid progress made in the utilization of antibiotic substances in
so brief a period of time can best be illustrated by the following two
citations :
On May 4, 1 940, Garrod ( 3 1 5 ) , in discussing the use of antiseptics in
wounds, wrote : "Only a few years ago it was thought impossible to kill
bacteria within the body with chemicals and likely always to remain so.
This belief was shaken by the discovery of a urinary antiseptic which
really worked, and it was shattered by the introduction of Prontosil,
with all its manifold consequences. Are we still to deny the possibility of
killing bacteria which are merely lying on a body surface? "
Less than four years later, Florey (275) summarized the value of
penicillin: "i. As a preventive of infection in wounds, enabling a po-
tentially septic wound to be treated in much the same way as an aseptic
one, 2. in the promotion of healing in burns and for ensuring the suc-
cess of skin grafts, 3. in infections (due to sensitive organisms) either
(a) chronic, or (b) of such severity as to render the prospect of death
likely, which have not responded to other forms of treatment, 4. in
acutfe infections due to sensitive organisms, 5. in the rapid curing of
gonorrhoea including sulphonamide-resistant cases, 6. in pneumonia.
262 . THE OUTLOOK FOR THE FUTURE
7. probably in gas gangrene, but here numbers have been few and meth-
ods not fully tried out."*
It appears, therefore, that certain generalizations concerning possible
future developments in the field of antibiotic substances are justified.
A SEARCH FOR NEW ANTIBIOTIC AGENTS:
A PROBLEM FOR THE MICROBIOLOGIST
Although some fifty compounds or preparations possessing bacterio-
static and fungistatic properties have already been isolated from micro-
organisms, there is sufficient evidence that many more can be obtained
without too great difficulty, if enough organisms are studied in greater
detail. In this connection, three methods of approach have been fol-
lowed: (a) testing organisms found in culture collections for antibac-
terial activity in general, followed by a detailed study of one or more
substances produced by one or more organisms j (b) isolating specific
organisms, such as members of the P. notatumr-P. chrysogenum groups,
from different soils and from moldy food materials and testing them for
the production of penicillin, in the hope of finding more active organ-
isms than those now known to exist j (c) enriching the soil with specific
bacteria, followed by the isolation of organisms capable of inhibiting the
growth of or of destroying such bacteria.
Several surveys (26, 282, 504, 628, 644, 934, 936, 986) have already
been made concerning the distribution of organisms capable of produc-
ing antibiotic substances among certain groups of bacteria and fungi.
Only very few such organisms were selected for more detailed investi-
gation. The reasons for this are quite obvious and are based largely
upon the great amount of time and experimentation required for the
isolation of any one substance. The selection of a particular substance
was largely governed by its specific antibiotic spectrum, or its activity
upon particular bacteria, its toxicity to animals, and its activity in vivo.
The following illustrations will suffice:
Of all the aerobic spore-forming bacteria known to produce anti-
biotic substances, only B. brevis has been utilized for the isolation of
* Further information on this subject is found in the various reports listed in Chapter 12 of
this book and in a group of papers presented before a symposium on antibiotic agents (65, 163,
189, 399a).
THE OUTLOOK FOR THE FUTURE 263
tyrothricin. It is known, for example, that various strains of B. mesen-
tericuSy B. mycoideSy B. subtiUsy and B. simplex are capable of produc-
ing antibiotic substances, some of which are markedly different chemi-
cally, biologically, or in selective activity. A more detailed study of these
organisms and the substances produced by them is bound to enlarge
greatly our knowledge of this group of chemical compounds and their
therapeutic potentialities.
Of all the nonspore-forming bacteria possessing antagonistic prop-
erties found in soils and water basins, only two have been studied in de-
tail : Ps. aerugwosa has been utilized for the production of pyocyanase
and pyocyanin, and C. iodinum for the production of iodinin. It is
known, however, that a large number of other nonspore-forming bac-
teria are capable of producing a variety of antibiotic substances, the
chemical nature and biological activities of which are still but little
understood.
Only very few of the antibiotic substances produced by actinomycetes
have so far been investigated, isolated, or concentrated j namely, ac-
tinomycetin, actinomycin, streptothricin, and proactinomycin. Even
these few substances, however, differ markedly in chemical nature and
in biological activity. In view of the fact that as many as 20 to 40 per
cent of all the actinomycetes are known to be capable of producing
antibiotic substances, many of which undoubtedly differ from those that
have already been isolated, the wealth of material that is awaiting in-
vestigation can only be surmised. Some of these possibilities have been
definitely indicated. Here belong the lysozyme-like agents discussed by
Russian investigators (507), micromonosporin which is active largely
against gram-positive bacteria, and streptomycin (795). The latter was
found, on the one hand, to resemble streptothricin in its chemical prop-
erties and activity in vivo, and, on the other hand, to differ from it in its
antibacterial spectrum.
The production of antibiotic agents by fungi likewise has been but
insufficiently studied. The following pertinent facts may direct attention
to the many problems still awaiting investigation :
(a) Some antibiotic substances, like penicillin, clavacin, and gliotoxin, are
produced by a number of different organisms; the nature of the or-
264 THE OUTLOOK FOR THE FUTURE
ganism often influences not only the yield of the substance but its
chemical nature and its biological activity.
(b) The mode of nutrition and the manner of growth of a single organ-
ism have often been found to influence the concentration and the na-
ture of the antibiotic substance, which may be formed in one me-
dium and not at all or in much lower amounts in another medium.
Some organisms are greatly favored in the production of antibiotic
substances by the presence in the medium of certain vitamin-like
complexes. Moreover, the formation of a substance is usually asso-
ciated with a certain stage of growth of the organism, since the sub-
stance is produced at one time and then rapidly destroyed, the range
of its accumulation often being very narrow. These facts point to
certain fundamental aspects in the physiology of the organism pro-
ducing the antibiotic substance that are still little understood.
(c) Certain organisms, such as members of the Fusarium group, produce
bacteriostatic substances, the action of which, however, is rapidly
overcome by the bacteria. This points to problems on the stability of
the antibiotic substance and on the adaptation of bacteria to the sub-
stance.
(d) Although it is known that certain yeasts produce antibiotic substances,
either of an adaptive or of a nonadaptive kind, very little is known
concerning the nature and mode of action of such substances.
These and many other problems are awaiting solution. The micro-
biologist is faced with a new field of research second only to that of the
very discovery of the causation of disease by microorganisms.
THE OPPORTUNITY FOR THE CHEMIST
The chemist has been searching far and wide for new chemotherapeu-
tic agents. He has synthesized many thousands of compounds, only
very few of which have proved to be of practical chemotherapeutic
value. The chemist has started from a certain lead, such as the arsenical
group in the salvarsan type of compounds and the sulfa-radical in the
sulfanilamides. The discovery of new chemical agents possessing anti-
bacterial or antifungal properties offers the chemist many new models
to draw upon for varied types of syntheses.
Although only very few antibiotic agents have so far been isolated,
THE OUTLOOK FOR THE FUTURE 265
and even fewer crystallized, it is already well established that we are
dealing here with a great variety of chemical compounds. It is sufficient
to mention the polypeptides (tyrothricin), oxidation-reduction systems
(pyocyanin, actinomycin), sulfur compounds (gliotoxin), quinones
(citrinin), various other non-nitrogenous simple (clavacin) and more
complex (fumigacin) compounds, a variety of nitrogenous compounds
comprising both bases (streptothricin, proactinomycin) and acids (peni-
cillin). Compounds, like actinomycin, that are highly active against bac-
teria but also highly toxic to animals, may possibly be modified in such a
manner as to reduce their toxicity without impairing their activity. This
is also true of simpler compounds, such as the less toxic but also less ac-
tive clavacin and gliotoxin. Many a chemist is awaiting the solution of
the problem of the chemical nature of penicillin before beginning new
syntheses.
Doubtless most of the compounds that prove to be useful as chemo-
therapeutic agents will sooner or later be synthesized. The contribution
of the bacteriologist may be all but forgotten in the light of the forth-
coming chemical developments, but even the bacteriologist will be
grateful for new tools to help combat disease-producing agents.
THE FIELD OF CHEMOTHERAPY
The utilization of the activities of antagonistic microorganisms for
the control of human and animal diseases has only begun. The same
may be said of the control of plant diseases. Many practices in surgery
and many old-time remedies are based on the creation of conditions fa-
vorable to the development of antagonistic microbes. Consider, for ex-
ample, the method of cast surgery developed during the Spanish Civil
War. To what extent the application of pure cultures of antagonists
may improve these and similar practices still remains to be determined.
Plaster treatment of wounds, without the use of antiseptics, has often
given marvelous results. Such wounds have been found to contain
aerobic bacteria with no one group predominating, except that Ps. aeru-
ginosa tends to become more numerous when the healing process has
been established (889). It still remains to be determined whether this
organism exerts a favorable effect due to its antagonistic properties or
is only another wound-infecting agent.
266 THE OUTLOOK FOR THE FUTURE
Of particular importance is the development of the manufacture of
antibiotic substances. Largely because of the stimulus given by World
War II when the need for new antibacterial agents became very acute,
an intensive study was made of the practicability of utilizing some of
the agents already known, and search was made for new ones. Among
these, penicillin occupies a leading place. As these lines are written, a
large number of great concerns in this country, in Great Britain, and
elsewhere are engaged in the manufacture of this drug by utilizing
several strains of P. notatum and P. chrysogenum. An intensive search
is being made for new agents capable of inhibiting the growth of and
destroying other pathogens resistant to the action of penicillin.
The progress made in the isolation of antibiotic substances from many
microorganisms has not kept pace with their evaluation as chemothera-
peutic agents. In discussing antimicrobial agents of biological origin,
Dubos (189) emphasizes that students of infectious diseases are pri-
marily concerned with the action of these substances upon certain strains
and stages of the parasites, with the mechanism of their action upon the
susceptible cells, and with physiologic and pathologic effects on the host.
Mcllwain (560), on the other hand, believes that animal testing in
chemotherapy is not necessarily much nearer to the conditions under
which the drug will be finally used than are properly chosen in vitro
conditions j although in vitro testing does not reproduce all the condi-
tions of the normal environment of the parasite, it is less likely, under
present conditions of testing, to introduce new and unknown factors
than is testing in another host. The in vitro and in vivo studies of an
agent are considered as complementary.
The utilization of fungi and bacteria against plant diseases has also
been variously attempted (472). The main difficulty involved is to es-
tablish the antagonist in the soil. This can be done by modifying soil
conditions, as by the addition of stable manure or other plant and animal
residues, in order to favor the development of the antagonist.
The activities of antagonistic microorganisms are also utilized for
combating injurious insects and other lower animal forms destructive to
plants and to animals. Among the insects, the Japanese and other
Asiatic beetles have been treated rather successfully by the use of nema-
todes and certain specific bacteria. Extensive use has already been made
THE OUTLOOK FOR THE FUTURE 267
of these bacteria, by inoculating the soil with grubs heavily infected
with them.
Comparatively little is yet known of the ability to control, by means
of antibiotic agents, diseases caused by protozoa, such as malaria and
trypanosomes, virus infections, and certain bacterial diseases such as
tuberculosis.
These instances suffice to arouse hope that even greater progress can
be expected in the control of disease by utilization of the activities of
antagonistic microorganisms. So far, most energies have been directed
to the treatment of acute infections caused by bacteria. Less is known
of chronic infections. Whether or not man will ever be able to control
all diseases caused by the numerous microscopic and ultramicroscopic
forms of life through the utilization of the activities of antagonistic
microorganisms, he will have gained sufficient knowledge from the
mode of action of these organisms, and of the substances produced by
them, to justify further hope in the possibilities thus opened.
MODE OF ACTION OF ANTIBIOTIC SUBSTANCES:
A FIELD FOR THE PHYSIOLOGIST
Finally, there remains the fourth important group of problems in-
volved in the study of antibiotic substances, namely, the mode of action
of these substances upon bacteria. The fact that different agents vary
greatly in their bacteriostatic and bactericidal action upon different, bac-
teria is well established. A number of mechanisms have been pro-
pounded, some of which hold true for one substance and some for more
than one. Each of these mechanisms involves some extremely puzzling
physiological problems. To take only two illustrations:
(a) If a given substance interferes with the utilization by the bacteria
of a certain metabolite in the medium, as in the relation of sulfa-drugs to
^-amino-benzoic acid, one must assume that the sensitive bacteria re-
quire the metabolite in question and the resistant forms do not, or that
the resistant bacteria synthesize larger concentrations of the particular
metabolite than the sensitive forms. Since the sensitivity of the bacteria
to an antibiotic substance is often more of degree than of kind, as in the
case of actinomycin, the assumption would be that the metabolite is
268 THE OUTLOOK FOR THE FUTURE
either required in different concentrations by the various organisms or is
synthesized to a different extent.
(b) The adsorption of the antibiotic substance by the bacterial cell,
rendering the cell incapable of multiplying or dividing, points to an-
other type of mechanism that may be rather common. This may often
express itself in the abnormal enlargement of the cell. A clear under-
standing of this phenomenon will have to await a better knowledge of
the mechanism of cell fission. Should one assume that the resistant cells
and the sensitive cells divide by different mechanisms?
All these and many other problems point directly to the fact that a
better understanding of the physiology of the microbial cell will be
gained from a clearer appreciation of the mode of action of antibiotic
substances upon the bacterial cell.
It is thus to the smallest of living systems, the microbe, that we must
look for the solution of some of the most important problems that have
faced man as well as his domesticated and friendly animals and plants.
CLASSIFICATION OF ANTIBIOTIC SUBSTANCES
GLOSSARY
BIBLIOGRAPHY
INDEX OF MICROORGANIS?vlS
GENERAL INDEX
CLASSIFICATION OF ANTIBIOTIC SUBSTANCES
PRODUCED BY ACTINOMYCETES
Actinomyces lysozyme {Streftomyces
sp.)
Actinomycetin (5. albus)
Actinomycin (5. antibioticus)
Micromonosporin {Micromonospora
sp.)
Proactinomycin {N. gard?ieri)
Streptomycin (5. griseus)
Streptothricin (5. laz'endulae)
PRODUCED BY ALGAE
Chlorellin {Chlorella i'p.)
PRODUCED BY BACTERIA
B. sim-plex factor {B. simflex)
Diplococcin (Streptococci)
Gramicidin {B. brevis)
lodinin {Ch. iodinum)
Pyocyanase {Ps. aeruginosa)
Pyocyanin {Ps. aeruginosa)
Subtilin (5. subtilis)
Toxoflavin {B. cocoveftenans)
Tyrocidine {B. brevis)
Tyrothricin {B. brevis)
Violacein {B. z'iolaceuni)
PRODUCED BY FUNGI
Aspergillic acid {A. fa-jus)
■ Chaetomin {Ch. cochliodes)
Citrinin {P. citrinum, A. candidus)
*Clavacin {A. clavatus, etc.)
*C]avatin {A. clavatus)
*Claviformin {P. clavifornie)
'\E. coli factor {P. notatum)
JFIavicin {A. flavus)
§Fumigacin {A, fumigatus)
Fumigatin {A. fumigatus)
iGigantic acid {A. giganteus)
Gliotoxin {Trichoderma^ Gliocla-
dium, A . fumigatus)
§Helvolic acid {A. fumigatus)
Kojic acid {A. oryzae)
fNotatin {P. notatum)
^Parasiticin (^4. parasiticus)
*Patulin {P. fatulum)
fPenatin {P. notatum)
Penicidin {Penicillium sp.)
Penicillic acid {P. pdberulum,
P. cyclofium)
Penicillin {P. notatu7n, P. chrysoge-
ntim)
f Penicillin B (P. 7iotatum)
Puberulic acid {P. fuberulum)
Spinulosin {A. sfinulosum, A. fumi-
gatus)
Note. Terms marked with the same symbol are synonyms.
GLOSSARY
Antagonism. The phenomenon of a living organism inhibiting the
growth or interfering with the activities of another living organism
as a result of the creation of unfavorable conditions in the medium
or the production of a specific antimicrobial substance.
Antagonist. An organism having the capacity to inhibit the growth or
interfere with the activity of another organism.
Antagonistic substance. A term frequently used to designate a substance
that neutralizes the bacteriostatic action of an antibiotic substance.
Antibiosis. The inhibition of growth of one organism by another.
Antibiotic. Inhibiting the growth or the metabolic activities of bacteria
and other microorganisms by a chemical substance of microbial origin.
Antibiotic substance, antibiotic. A chemical substance, of microbial ori-
gin, that possesses antibiotic properties.
Anti-inhibitor, inhibitor, suppressor are terms of similar significance to
antagonistic substance.
Bactericidal. Causing the death of bacteria.
Bacterioantagonistic. Inhibiting the growth of bacteria.
Bacteriolytic. Causing not only the death of bacteria but also their lysis
or disintegration.
Bacteriostatic. Inhibiting the growth of bacteria.
Bacteriostatic or antibiotic spectrum. A range of inhibition of growth of
different bacteria by different concentrations of an antibiotic sub-
stance. It may be expressed graphically, the bands of the spectrum
representing the concentrations of the substance.
Biostatic complex. The sum total of factors that limit microbial develop-
ment in a medium. The absence of such factors may result in the for-
mation of toxic products.
Fungicidal. Causing the death of fungi.
Fungistatic. Inhibiting the growth of fungi.
Inactivator, nontoxic. A substance that inactivates plant viruses and is
not detrimental to most forms of life.
272 GLOSSARY
Inhibitor or inhibitive substance. A term variously applied, but usually
used to designate a substance that inhibits the growth of bacteria and
other microorganisms.
Lysogenesis. The production by an organism of substances that cause
the lysis of bacterial cells.
Lyso-zyme. A substance produced by living tissues (white of egg, tears,
and also certain microorganisms) that is capable of dissolving living
bacterial cells, especially certain micrococci.
BIBLIOGRAPHY
1. Abbott, A. C, and Gildersleeve, N. A study of the proteolytic enzymes
and of the so-called hemolysins of some of the common saprophytic bacteria.
J. Med. Research 10:42-62 (1903).
2. Abraham, E. P. Mode of action of chemotherapeutic agents. Lancet 2:
761-762 (1941).
3. Abraham, E. P., Baker, W., Chain, E., Florey, H.W., Holiday, E.R.,
and Robinson, R. Nitrogenous character of penicillin. Nature 149:356
(1942).
4. Abraham, E. P., and Chain, E. An enzyme from bacteria able to destroy
penicillin. Nature 146:837 (1940).
5. Abraham, E. P., and Chain, E. Purification of penicillin. Nature 149:
328 (1942).
6. Abraham, E. P., Chain, E., Baker, W., and Robinson, R. Penicilla-
mine, a characteristic degradation product of penicillin. Nature 151:107
(1943).
7. Abraham, E. P., Chain, E., Fletcher, C. M., Gardner, A. D., Heat-
ley, N. G., Jennings, M. A., and Florey, H. W. Further observations
on penicillin. Lancet 2: 177-188 (1941).
8. Abraham, E. P., Chain, E., and Holiday, E. R. Purification and some
physical and chemical properties of penicillin; with a note on the spectro-
graphic examination of penicillin preparations. Brit. J. Exper. Path. 23:
103-120 (1942).
9. Acs, L. Ueber echte mitogenetische Depressionen, Bakterienantagonismus
und mitogenetische Strahlung. Zentralbl. f. Bakteriol., I, Or. 127:342—
350(1933).
10. Aldershoff, H. Untersuchungen in vitro uber die Art des Besredkaschen
Antivirus. Zentralbl. f. Bakteriol., I, Or. 112:273-281 (1929).
11. Alexandre, A., and Cacchi, R. Recherches sur quelques facteurs pro-
bables determinant I'antagonisme entre le B. coll dans la phase "s" et le L.
bulgaricum. Soc. internaz. di microbiol., Boll. d. sez. ital. 10:291—298
(1938).
12. Alexopoulos, C. a. Studies in antibiosis between bacteria and fungi; spe-
cies of actinomyces inhibiting the growth of Colletotrichum gloeosporoides
Penz. in culture. Ohio J. Sc. 41 : 425-430 (1941).
13. Alexopoulos, C. A., Arnett, R., and McIntosh, A. V. Studies in anti-
biosis between bacteria and fungi. Ohio J. Sc. 38: 221-235 (i937)'
14. Alexopoulos, C. A., and Herrick, J. A. Studies in antibiosis between
bacteria and fungi ; inhibitory action of some actinomycetes on various spe-
cies of fungi in culture. Bull. Torrey Bot. Club 69:257-261 (1942).
15. Alivisatos, G. P. Ueber Antagonlsmus zwischen Pneumokokken und
Staphylokokken. Centralbl. f. Bakteriol., I, Or. 94:66-73 (1925).
16. Allcroft, R. Antibacterial agents derived from microorganisms, with spe-
cial reference to gramicidin. Vet. Bull. i2:Ri-R6 (1942).
274 BIBLIOGRAPHY
17. Allen, M. C, and Haenseler, C. M. Antagonistic action of Tricho-
derma on Rhizoctonia and other soil fungi. Phytopath. 25:244-252
(1935)-
18. Allen, W.E. Cutthroat competition in the sea. Scient. Monthly 49: 1 1 1 —
II9(l939)•
19. Alsberg, C. L., and Black, O. F. Contribution to the study of maize de-
terioration; biochemical and toxicological investigations of Penicillium
puberulum and Penicillium stoloniferum. U.S.D.A., Bur. of Plant Indus-
try, Bull. 270 (191 3).
20. Andrews, C. H. Interference by one virus with the growth of another in
tissue-culture. Brit. J. Exper. Path. 23:214-220 (1942).
21. Anslow, W. K., and Raistrick, H. Studies in the biochemistry of micro-
organisms; fumigatin (3-hydroxy-4-methoxy-2: 5-toluquinone) and spinu-
losin (3:6-dihydroxy-4-methoxy-2: 5-toIuquinone), metabolic products re-
spectively of Aspergillus fumigatus Fresenius and Penicillium spinulosum
Thorn. Biochem. J. 32:687-696 (1938).
22. Anslow, W. K., Raistrick, H., and Smith, G. Anti-fungal substances
from moulds; patulin (anhydro-3-hydroxymethylenetetrahydro-i :4-pyrone-
2-carboxylic acid), a metabolic product of Penicillium patulum Bainier and
Penicillium expansum (Link) Thom. J. Soc. Chem. Ind. 62:236—238
(1943)-
23. Ark, P. A., and Hunt, M. L. Saprophytes antagonistic to phytopathogenic
and other microorganisms. Science 93: 354-355 (1941).
24. Arnaudi, C, Kopazcewski, W., and Rosnowski, M. Les antagonismes
physico-chimiques des microbes. Compt. rend. Acad. d. sc. 185:153-156
(1927).
25. AsTHANA, R. P., and Hawker, L. E. The influence of certain fungi on the
sporulation of Melanospora destruens Shear and some other Ascomycetes.
Ann. Bot. 50:325-344 (1936).
26. Atkinson, N. Antibacterial substances produced by moulds. Australian J.
Exper. Biol. & M. Sc. 20:287-288 (1942) ; 21 : i 5-16, i 27-1 3 i, 249-257
(1943) ; M.J. Australia 1:359-362 (1943).
27. AuBEL, E. Recherches biochimiques sur la nutrition du bac. pyocyanique.
Compt. rend. Acad. d. sc. 173:179-181 (1921).
28. AuERSWALD, H. Welche Mikroorganismen wirken auf Diphtheric- und
Pseudodiphtheriebazillen antagonistisch? Zentralbl. f. BakterioL, I 142:
32-41 (1938).
29. Augustine, D. L., Weinman, D., and McAllister, J. Rapid and
sterilizing effect of penicillin sodium in experimental relapsing fever infec-
tions and its ineffectiveness in the treatment of Trypanosomiasis (Trypano-
soma lewisi) and Toxoplasmosis. Science 99: 19—20 (1943).
30. Avery, O. T., and Dubos, R. The protective action of a specific enzyme
against type III pneumococcus infection in mice. J. Exper. Med. 54: 73-89
(1930-
31. Bail, O. ^'crsuche an Bakterienpopulationen. Arch. f. Hyg. 95: i (1925) ;
Ztschr. f. d. ges. exper. Med. 50: 1 1 (1926) ; Deutsche med. Wchnschr. p.
1289 (1929); Ztschr. f. Immunitatsforsch, u. exper. Therap. 60:1-22
(1929).
BIBLIOGRAPHY 275
32. Bail, O., and Breinl, F. V'ersuche iiber das seitliche Vcrdringen von
Verunreinigungen im Boden. Arch. f. Hyg. 82:33-56 (1914).
33. Bailey, J. H., and Cavallito, C. J. Production of citrinin. J. Bact. 45:
30-31 (1943)-
34. Baker, G. E. Heterokaryosis in Penicillium notatum. Bull. Torrey Bot.
Club 71:367-373 (1944); Science 99:436 (i944)-
35. Baker, Z., Harrison, R. W., and Miller, B. F. Action of synthetic de-
tergents on the metabolism of bacteria. J. Exper. Med. 73:249—271
(1941); 74:621-637 (1941).
36. Ballantine, E. N. On certain factors influencing survival of bacteria in
water and saline solutions. J. Bact. 19:303-320 (1930).
37. Bamberg, R. H. Bacteria antibiotic to Ustilago zeae. Phytopath. 20: 140
(1930); 21:881-890 (1931).
38. Barger, G., and Dorrer, O. Chemical properties of puberulic acid,
CgHgOe, and a yellow acid, CgH^Og. Biochem. J. 28:11-15 (1933).
39. Barr, J. S. The use of penicillin in the navy. J. Bone & Joint Surg. 26:
380-386 (1944)-
40. Barrieu, a. R. Contribution a I'etude du role des associations micro-
biennes; les bacilles sporules aerobies; leur action pathogene probable dans les
plaies de guerre. Thesis, Paris (191 9); Presse med. 28:40 (191 9); ab-
stract in Abstr. Bact. 4: 1 1 40 ( 1 920) .
41 . Beard, P. J. Longevity of Eberthella typhosus in various soils. Am. J. Pub.
Health 30:1077-1082 (1940).
42. Behrens, J. Wechselwirkungcn zwischen verschiedenen Organismen
(Symbiose, Metabiose, Antagonismus). Lafars Handb. techn. Mykol. Jena
1:501-513 (1904).
43. Beijerinck, M. W. Ueber Chinonbildung durch Streptothrix chromo-
gena und Lebensweise dieser Microben. Centralbl. f. Bakteriol., Abt. II 6:
2-12 (1900).
44. Bennett, T. I., and Parkes, T. Penicillin in sulphonamide-resistant
pneumonias. Lancet 1:305-308 (1944).
45. Berdnikoff, A. Les milieux de culture dits "vaccines" et I'antagonisme
des microbes in vitro. Compt. rend. Soc. de biol. 91:859-861 (1924).
46. Berezova, E. F. Bacteriological method of combatting fungus diseases of
agricultural plants. Microbiologia (U.S.S.R.) 8:186-197, 695—699
(1939)-
47. Bergel, F., Morrison, A. L., Moss, A. R., Klein, R., Rinderknecht,
H., and Ward, J. L. An antibacterial substance from Aspergillus clavatus
and Penicillium claviforme and its probable identity with patulin. Nature
152:750 (1943)-
48. Bergonzini, C. Contributo sperimentale alio studio dei mezzi che I'orga-
nismo oppone all'infezione. Rassegna di scienze mediche 5:551-568
, (1890).
49. Bershova, O. I. The dependence of the development of ammonifying bac-
teria on the soil bacteriophage. Mikrobiol. Zhur. 7:97—119 (1940).
276 BIBLIOGRAPHY
50. Bertarelli, E. Untersuchungen und Beobachtungen uber die Biologic und
Pathogenitat des Bacillus prodigiosus. Centralbl. f. Bakteriol., I, Or. 34:
193-202, 312-322 (1903).
51. Besredka, A, Les immunites locales, Paris, Masson et Cie, 1925, 1937.
52. Besta, B., and Kuhn, H. Untersuchungen iiber Antagonismus zwischen
Diphtheriebacillen und anderen Bakterien. Ztschr. f. Hyg. u. Infcktionskr.
116:520-536 (1934).
53. Bezan^on, F., Duchon et DuRuy. Les difficultes du probleme de la vac-
cinotherapie de la fievre typhoide. Presse med. 41: 1941— 1942 (1933).
54. BiENSTocK, Dr. Untersuchungen iiber die Aetiologie des Eiweissfaulnis;
Milchfiiulnis, Verhinderung der Faulnis durch Milch, Darmfaulnis. Arch,
f. Hyg. 39:390-427 (1901).
54a. BiouRGE, Ph. Les moississures du groupe Penicillium Link. Etude mono-
graphique. La cellule 33: 5-33 I (1923).
55. BiRCH-HiRSCHFELD, L. Versuche zur Analyse der Pyocyanase. Ztschr. f.
Hyg. u. Infektionskr. 116:304-314 (1934).
56. BiRKiNSHAw, J. H., Oxford, A. E., and Raistrick, H. Studies in the
biochemistry of microorganisms; penicillic acid, a metabolic product of Peni-
cillium puberulum Bainier and P. cyclopium Westling. Biochem. J. 30:
394-411 (1936).
57. Birkinshaw, J. H., and Raistrick, H. Notatin; an antibacterial glucose
aerodehydrogenase from Penicillium notatum Westling. J. Biol. Chem.
148:459-460 (1943)-
58. Birkinshaw, J. H., and Raistrick, H. Studies in the biochemistry of
micro-organisms; puberulic acid CgHijOg and an acid CgH^Og, new prod-
ucts of the metabolism of glucose by Penicillium puberulum Bainier and
Penicillium aurantio-virens Biourge. Biochem. J. 26:441—453 (1932).
59. Birkinshaw, J. H., Raistrick, H., and Smith, G. Studies in the bio-
chemistry of micro-organisms; fumaryl-dl-alanine (fumaromono-dl-alan-
ide), a metabolic product of Penicillium resticulosum sp. nov. Biochem. J.
36:829-835 (1942).
60. BiSBY, G. R., James, N., and Timonin, M. Fungi isolated from Mani-
toba soils by the plate method. Canad. J. Research 8:253-275 (1933).
61. Bitter, C. R. Bacterial antibiosis. J. Colorado-Wyoming Acad. Sc. 3
(i):i6-i7 (1941).
62. Blagovestchensky, N. Sur I'antagonlsme entre les bacilles du charbon et
ceux du pus bleu. Ann. Inst. Pasteur 4:689-715 (1890).
63. Blair, I. D. Behaviour of the fungus Rhizoctonia solani Kiihn in the soil.
Ann. App. Biol. 30: 1 18-127 (1943).
64. Blair, J. E., and Hallman, F. A. The effect of actinomycin, clavacin,
and tyrothricin on staphylococcal toxin. J. Infect. Dis. 72:246-252
(1943)-
65. Bloomfield, a. L., Rantz, L. A., and Kirby, W. M. M. The clinical
use of penicillin. J.A.M.A. 124:627-633 (1944).
66. Bocchia, I. Die Pyocyanase. Centralbl. f. Bakteriol., I, Or. 50:220-225
(1909).
BIBLIOGRAPHY 277
67. BoDENHAM, D. C. Infected burns and surface wounds; the value of peni-
cillin. Lancet 2:725-728 (1943).
68. BoDiNE, E. W, Antagonism between strains of the peach-mosaic virus in
Western Colorado. Phytopath. 32: 1 (1942).
69. RoEHM, M. M., and Kopaczewski, W. Etudes sur les phenomenes elec-
trocapillaires; I'antagonisme microbien et la therapeutique du cancer. Proto-
plasma 6:302-320 (1929).
70. BoGENDORFER, L. Hemmungsstoffe aus Bakterien und ihren Kultursub-
straten. Ztschr. f. d. ges. exper. Med. 41:620 (1924); abstract in Cen-
tralbl. f. Bakteriol., I, Ref. 77:372 (1924).
71. BoHNHOFF, Dr. Ueber die Wirkung der Streptokokken auf Diphtheriekul-
turen. Hyg. Rundschau. 5:97-100 (1896); abstract in Centralbl. f.
Bakteriol. 19:914 (1896).
72. BoLLEY, H. L. Wheat; soil troubles and seed deterioration. North Dakota
Agr. Exper. Sta. Bull, 1 07 : 5-94 ( 1 9 1 3 ) .
73. BoNDi,A., and DiETZ, C. C. Destruction of penicillin by bacteria. J. Bact.
47:20(1944).
74. BoNDi, A., and Dietz, C. C. Production of penicillinase by bacteria.
Proc. Soc. Exper. Biol. & Med. 56: 132-134 (1944).
75. BoNDi, A., and Dietz, C. C. Relationship of penicillinase to the action of
penicillin. Proc. Soc. Exper. Biol. & Med. 56:135-137 (1944).
76. BoNSKA, F. Studien iiber den Antagonismus zwischen Milchsauerfermenten
und Bakterien der Gruppe des Bac. subtilis. Landw. Jahresb. Schweiz i 7
(6):249(i903).
77. BoRDET, J. Apparition spontanee du pouvoir lysogene dans les cultures
pures. Compt. rend. Soc. de biol. 90:96-98 (1924); 93:1054-1056
(1925).
78. BoRDLEY, J. E., Crowe, S. J., Dolowitz, A., and Pickrell, K. L. The
local use of the sulfonamides, gramicidin (tyrothricin) and penicillin in
otolaryngology. Ann. Otol. Rhin. & Laryng. 51:936 (1942).
79. BoRNSTEiN, S. Action of penicillin on enterococci and other streptococci.
J. Bact. 39:383-387(1940).
80. BoRODULiNA, J. A. Interrelations of soil actinomyces and B. mycoides.
Microbiologia (U.S.S.R.) 4:561-586 (1935).
81. Bouchard, C. Influence, qu'exerce sur la maladie charbonneuse I'inocula-
tion du bacille pyocyanique. Compt. rend. Acad. d. sc. 108:713-714
(1889).
82. Bowser, B. M. A study of the action of certain soil bacteria on Mycobac-
terium tuberculosis, varieties Hominis and Bovis. Thesis, Univ. Pittsburgh
(1942).
83. Boyle, C. Studies in the physiology of parasitism; the growth reactions of
certain fungi to their staling products. Ann. Bot. 38:113-135 (1924).
84. Brewer, C. M. Use and abuse of Staphylococcus aureus as a test organism.
Am. J. Pub. Health 32:401-405 (1942),
85. Briscoe, C. F. Fate of tubercle bacilli outside the animal body. Illinois
Agr. Exper. Sta. Bull. 161:279-375 (1912).
278 BIBLIOGRAPHY
86. Broadfoot, W. C. Studies on foot- and root-rot of wheat; effect of age of
the wheat plant upon the development of foot- and root-rot. Canad. J.
Research 8:483-491, 545-552 (i933)-
87. Brodski, A. L. Antagonism between soil infusoria and (plant) pathogenic
fungi. Compt. rend. Acad. d. sc. (U.S.S.R.), n.s, 33:81—83 (1941).
88. Brommelhues, M. Die wechselseitige Beeinflussung von Pilzen und die
Bedeutung der Pilzkonkurrenz fiir das Ausmass der Schadigung an Weizen
durch Ophiobolus graminis Sacc. Zentralbl. f. Bakteriol., II 92:81-116
(1935)-
89. Broom, J. C. The exhaustion of media in bacterial culture. Brit. J, Exper.
Path, 10:71-83 (1929).
90. Brown, A. J. The influences regulating the reproductive functions of
Saccharomyces cerevisiae. J. Chem. Soc, Trans. 87:1395—1412 (1905).
91. Brown, J. G. Watermelon susceptible to Texas root-rot. Science 78:509
(1933).
92. Brown, W. Experiments on the growth of fungi in culture media. Ann.
Bot. 37:105-129 (1923); Bot. Rev. 2:236-281 (1936).
93. Browning, C. H., et al. Chronic enteric carriers and their treatment.
Spec. Rept. Ser. 179, Med. Res. Council, London, 1933.
94. Bruner, D. W. Differentiation between gram-positive and gram-negative
microorganisms by the use of enzymes. J. Bact. 26:361-371 (1933).
95. Bryan, C. S., Weldy, M. L., and Greenberg, J. The results obtained
with tyrothricin in the treatment of 157 cows with streptococcic mastitis.
Vet. Med. 37:364-369 (1942).
96. Buchanan, R. E., and Fulmer, E. I. Physiology and biochemistry of bac-
teria. Baltimore, Williams and Wilkins, 1930. Vol. 3, pp. 3-28.
97. BucHNER, M. Ueber Hemmung der Milzbrandinfektionen und uber das
aseptische Fieber. Klin. Wchnschr., Berlin, No. 18 (1890).
98. BuLTHUis, G. Welche Bakteriengruppen wirken auf Typhusbazillen an-
tagonistisch? Zentralbl. f. Bakteriol., I, Or. 145:462-469 (1940).
99. BuMM, R. Behandlung chronlsch-eiteriger Prozesse mit toten Gewebsruck-
standen durch Dauerpraparate von proteolytischen Bakterien. Arch. f. klin.
Chir. 138:111 (1925); abstract iu CentTnlhl. f. Bakteriol., I, Ref. 81:417
(1926).
99a. Burkholder, P. R., Evans, A. W., McVeigh, I., and Thornton, H. K.
Antibiotic activity of lichens. Proc. Nat. Acad. Sc. 30:250-255 (1944).
100. Bush, M. T., and Goth, A. Flavicin; an antibacterial substance produced
by an Aspergillus flavus. J. Pharmacol. & Exper. Therap. 78:164-169
( 1 943 ) ; Fed. Proc, Am. Soc. Exper. Biol. 2:75 ( 1 943 ) .
101. Butler, K. D. The cotton root rot fungus Phymatotrichum omnivorum,
parasitic on the watermelon, Citrillus vulgaris. Phytopath. 25:559—577
(1935)-
102. Butterfield, C. T., and Purdy, W. C. Some interrelationships of plank-
ton and bacteria in natural purification of polluted water. Ind. & Eng.
Chem. 23:213-218 (1931).
103. Callaham, J. R. Large-scale production by deep fermentation. Chem. &
Met. Eng. 5 1 : 94-98 ( 1 944) .
BIBLIOGRAPHY 279
104. Cantani, A. Tentativl di bacterioterapia. Riforma Medica, Napoli, 147
(1885) ; abstract in Centralbl, medicin, WIssensch. 23:513-514 (1885).
105. Cantani, F. Sulle antibiose microbica. Ann. d'ig. 40: 257-271 (1930);
abstract in Jior. di Immunol., p. 665 (1930).
106. Carlson, H. J., Ridenour, G. M., and McKhann, C. F. Effect of the
activated sludge process of sewage treatment on poliomyelitis virus. Am. J.
Pub, Health 33:1083-1087 (1943)-
107. Carra, J. Die Aminosauren in ihrer Beziehung zur Pigmentbildung des
Bacillus pyocyaneus. Centralbl. f. BakterioL, I, Or. 91:154-159 (1924)-
108. Carter, J. C. Diffusible nature of the inhibitory agent produced by fungi.
Phytopath. 25:1031-1034 (1935).
109. Catch, J. R., Cook, A. H., and Heilbron, I. M. Purification and chem-
istry of penicillin. Nature 150:633-634 (1942).
109a. Cavallito, C. J., and Bailey, J. H. Preliminary note on the inactivation
of antibiotics. Science 100:390 (1944).
no. Chaillot, L. Etude in vitro sur les antivirus. Compt. rend. Soc. de biol.
103:206-207 (1930).
111. Chain, E. Mode of action of chemotherapeutic agents. Lancet 2: 761-762
(1941).
112. Chain, E., and Florey, H. W. Penicillin. Endeavour 3 : 3-14 (1944).
113. Chain, E., Florey, H. W., Gardner, A. D., Heatley, N. G., Jen-
nings, M. A., Orr-Ewing, J., and Sanders, A. G. Penicillin as a chemo-
therapeutic agent. Lancet 2:226-228 (1940).
114. Chain, E., Florey, H. W., and Jennings, M. A. An antibacterial sub-
stance produced by Penicillium claviforme. Brit. J. Exper. Path. 23:202-
205 (1942).
115. Chain, E., Florey, H. W., and Jennings, M. A. Identity of patulin
and claviformin. Lancet 1:112-114 (1944).
116. Chain, E., Florey, H. W., Jennings, M. A., and Williams, T. I. Hel-
volic acid, an antibiotic produced by Aspergillus fumigatus, mut. helvola
Yuill. Brit. J. Exper. Path. 24:108-119 (1943).
117. Challinor, S. W. Production of penicillin. Nature i 50:688 (1942).
118. Challinor, S. W,, and MacNaughtan, J. Production of penicillin.
J, Path. & Bact. 55:441-446 (1943).
119. Chambers, S. O., and Weidman, F. D. A fungistatic strain of Bacillus
subtilis isolated from normal toes. Arch. Dermat. & Syph. 18:568—572
(1928).
120. Charrin, M., and Guignard, L. Action du bacille pyocyanique sur la
bacteridie charbonneuse. Compt. rend. Acad. d. sc. 108:764-766 (1889).
121. Chatterjee, G. C. On a new test for differentiation' of the bacilli of the
typhoid group. Centralbl. f. BakterioL, I, Or. 48:246-249 (1909).
122. Chatton, E. and M. L'influence des facteurs bacteriens sur la nutrition,
t la multiplication et la sexualite des infusoires. Compt. rend. Acad. d. sc.
176:1262-1265 (1923); 188:1315-1317 (1929); Compt. rend. Soc. de
biol. 93:675-678 (1925).
280 BIBLIOGRAPHY
123. Chatton, E. and M. Sur le pouvoir cytolytique immediat des cultures de
quelques bacteries chromogenes. Compt. rend, Soc, de biol. 97:289—292
(1927).
I 24. Chaze, J., and Sarazin, A. Nouvelles donnees biologiques et experimen-
tales sur la mole maladie du champignon de couche. Ann. sc. nat., bot. 18
(io):i-86 (1936).
125. Christensen, H. N. The configuration of valylvaline in gramicidin. J.
Biol. Chem. 154:427-436 (1944).
126. Christensen, H. N., Edwards, R. R., and Piersma, H. D. The compo-
sition of gramicidin and tyrocidine. J. Biol. Chem. 141:187-195 (1941).
127. Christensen, J. J. Associations of microorganisms in relation to seedling
injury arising from infected seed. Phytopath. 26:1091-1105 (1936).
128. Christensen, J. J., and Davies, F, R. Variation in Helminthosporium
sativum induced by a toxic substance produced by Bacillus mesentericus.
Phytopath. 30:1017-1033 (1940).
129. Christomanos, A. A. Zur Farbstoffproduktion des Bacillus pyocyaneus.
Ztschr. f. Hyg. u. Infektionskr. 36:258 (1902); abstract in Centralbl. f.
Bakteriol., I, Ref. 31:149 (1902).
130. Chudiakov, J. P. The lytic action of soil bacteria on parasitic fungi.
Microbiologia (U.S.S.R.) 4:193-204 (1935).
131. Chuiton, F. Le, Bideau, J., Pennaneac'h, J., and Mollaret, J.
Etude du pouvoir antibiotique exerce vis-a-vis du bacille diphterique par un
streptocoque, isole de la gorge d'un malade. Compt. rend. Soc. de biol. 129:
927-928 (1938).
132. Clapp, M. p. In vitro effect of tyrothricin and tryocidine hydrochloride
on polymorphonuclear leucocytes. Proc. Soc. Exper. Biol. & Med. 51 :279—
281 (1942).
133. Clark, A. M., Colebrook, L., Gibson, T., and Thomson, M. L. Peni-
cillin and propamidine in burns; elimination of haemolytic streptococci and
staphylococci. Lancet 1:605—609 (1943).
134. Clifton, C. E. Large scale production of penicillin. Science 98:69—70
(1943)-
135. Clutt^rbuck, P. W., Lovell, R., and Raistrick, H. Studies on the
biochemistry of microorganisms; the formation from glucose by members of
the Penicillium chrysogenum series of a pigment, an alkali-soluble protein
and penicillin — the antibacterial substance of Fleming. Biochem. J. 26:
1907-1918 (1932).
136. CoHN, A., and Seijo, I. H. The in vitro effect of penicillin on sulfonam-
ide resistant and sulfonamide susceptible strains of gonococci. J.A.M.A. 1 24:
I 125-1 126 (1944).
137. CoHN, A., Studdiford, W. E., and Grunstein, I. Penicillin treatment
of sulfonamide resistant gonococcic infections in female patients; preliminary
report. J.A.M.A. 124:1124-1125 (1944).
138. Coley, W. B. The treatment of malignant tumors by repeated inocula-
tions of erysipelas; with a report of ten original cases. Am. J. M. Sc. 105:
487-511 (1893).
BIBLIOGRAPHY 281
139. Conn, H. J. Soil flora studies; non-spore-forming bacteria in soil. New
York State Agr. Exper. Sta. Tech. Bull. 59 (191 7).
140. Conn, H. J., and Bright, J. W. Ammonification of manure in soil. New
York State Agr. Exper, Sta. Tech. Bull. 67 (1919); J. Agr. Research 16:
313-350(1919).
141. CoNRADi, H., and Karpjuweit, O. Ueber die Bedeutung der bakteriellen
Hemmungsstoffe f iir die Physiologie und Pathologic des Darmes. Miinchen.
med. Wchnschr., pp. 522, 1761, 2164, 2228 (1905) ; abstract itt Centralbl.
f. Bakteriol., I, Ref. 38:69-70 (1906).
142. CooK, A, H., and Lacey, M. S. An antibiotic from Aspergillus parasiticus.
Nature 153:460 (1944).
143. Cook, E. N., Pool, T. L., and Herrell, W. E. Further observations on
penicillin in sulfonamide resistant gonorrhea. Proc. Staff Meet., Mayo
Clinic 18:433-437 (1943).
144. Cook, E. S., Kreke, C. W., Giersch, M. C, and Schroeder, M. P. A
growth-depressant substance from yeast. Science 93:616-617 (1941).
145. Cooper, E. A., and Forstner, G. E. Studies on selective bactericidal ac-
tion. Biochem. J. 18:941-947 (1924).
146. Cooper, E. A., and Haines, R. B. The chemical action of quinones on
proteins and amino-acids. Biochem. J. 22:317-325 (1928); 23:4-9
(1929).
147. Cooper, E. A., and Mason, J. Studies of selective bactericidal action.
J. Hyg. 26: 1 19-126 (1927).
148. Cooper, E. A., and Nicholas, S. D. The chemical action of p-quinones
on proteins and other substances. J. Soc. Chem. Ind. 46:59T-6oT (1927).
149. Cordon, T. C, and Haenseler, C. M. A bacterium antagonistic to
Rhizoctonia solani. Soil Sc. 47:207—215 (1939).
150. CoRNiL, A. v., and Babes, V. Concurrence vitale des bacteries; attenua-
tion de leurs proprietes dans des milieux nutritifs modifies par d'autres bac-
teries; tentatives de therapeutique bacteriologique. J. conn. med. prat.
Paris 7:321-323 (1885).
150a. CoRNMAN, I. Survival of normal cells in penicillin solutions lethal to
malignant cells. Science 99:247 (1944); J, Gen. Physiol. 28:113-118
(1944).
151. CouLTHARD, C. E., Short, W. F., Skrimshire, G. H. E., Birkinshaw,
J. H., MicHAELiA, R., Sykes, G., STANDFAST, A. F. B., and Raistrick, H.
Notatin; an anti-bacterial glucose-aerodehydrogenase from Penicillium no-
tatum Westling. Nature 150:634-645 (1942).
152. Crabill, C. H. Dimorphism in Coniothyrium pirinum Sheldon. Am. J.
Bot. 2:449-467 (1915)-
153. Craddock, S. Use of penicillin in cultivation of the acne bacillus. Lancet
1:558-559(1942).
I 54. Crowe, S. J., Fisher, A. M., Ward, A. T., Jr., and Foley, M. K. Peni-
cillin and tyrothricin in otolaryngology. Ann. Otol. Rhin. & Laryng. 52:
' 541-572 (1943)-
282 BIBLIOGRAPHY
155. Crowfoot, D. M., and Low, B. W. Note on crystallography of helvolic
acid and methyl ester of helvolic acid. Brit. J. Exper. Path. 24:120
(1943)-
156. Cutler, D. W., and Bal, D. V. Influence of protozoa on the process of
nitrogen fixation by Azotobacter chroococcum. Ann. App. Biol. 13: 516-534
(1926).
I 57. Cutler, D. W., and Crump, L. M. Problems in soil microbiology. Lon-
don, Longmans, 1935.
158. Dack, G. M. Food poisoning. Chicago, Univ. Chicago Press, 1943.
159. Dack, G, M, Influence of anaerobic species on toxin of CI. botulinum with
special reference to CI. sporogenes. J. Infect. Dis. 38: 165—173 (1926).
160. D'Aeth, H. R. X. A survey of interaction between fungi. Biol. Rev. 14:
105-131 (1939)-
161. Daines, R. H. Antagonistic action of Trichoderma on Actinomyces scabies
and Rhizoctonia solanl. Am. Potato J. 14:85-93 (1937).
162. Dalldorf, G., Douglass, M., and Robinson, H. E. The sparing eff"ect
of canine distemper on poliomyelitis in Macaca mulatta. J. Exper. Med. 67:
333-343 (1938).
163. Dawson, M. H., and Hobby, G. L. The clinical use of penicillin; obser-
vations in 100 cases. J.A.M.A. 124:611-622 (1944).
164. Dawson, M. H., Hobby, G. L., Meyer, K., and Chaffee, E. Penicil-
lin as a chemotherapeutic agent. Ann. Int. Med. 19:707—717 (1943).
165. DeBary, A. Die Erscheinungen der Symbiose. Strassburg, 1879.
166. Delafield, M. E., Straker, E., and Topley, W. W. C. Antiseptic
snuffs. Brit. M. J. 1:145-150 (1941).
167. Delbruck, M., and Luria, S. E. Interference between bacterial viruses;
Interference between two bacterial viruses acting upon the same host, and the
mechanism of virus growth. Archiv. Blochem. 1:111—143 (1942).
168. Demelenne-Jaminon, G. a propos d'un cas de variation microbienne.
Compt. rend. Soc. de blol. 133:440-442 (1940).
169. Demolon, a., and Dunez, A. Nouvelles observations sur le bacteriophage
et la fatigue des sols cultlves en luzerne, Ann. Agron. 6:434—454 (1936).
170. Deschiens, R. Considerations relatives a la destruction des larves de
Nematodes parasites par des Hyphomycetes predateurs. Bull. Soc. path,
exot. 32:459-464 (1939) ; Compt. rend. Acad. d. sc. 215: 1 48-1 51 (1941).
171. Dietrich, A. Sind alle EInwande die Natur und Wirkungswelse der so-
genannten Nukleasen widerlegt? Centralbl. f. Bakteriol. 31:165—170
(1901); Arb. path. Inst. Tubingen 3:H. 2 (1901).
172. DiMicK, K. P. A quantitative method for the determination of tyrothricln.
J. Biol. Chem. 149:387-393 (1943).
173. Dimitrijevic-Speth, V. Die Schwiirmkultur Insbesondere Schwiirmhem-
mung, Antagonismus und Farbindlkatoren. Zentralbl. f. Bakteriol., I, Or.
116:332-338 (1930).
174. Dmitrevskaya, N. A., and Tchebotarewitch, M. F. On the phenome-
non of antagonism among microorganisms. Arch. biol. nauk (U.S.S.R.) 43:
337-344(1936).
BIBLIOGRAPHY 283
175. Dmitrieff, S., and Souteeff, G. Sur les phenomenes de dissociation et
de lyse observes dans les cultures de I'Actinomyces bovis Bostroem; essais
d'application des filtrats de cultures lysees au traitement de I'actinomycose.
Ann. Inst. Pasteur 56:470-476 (1936).
176. DoBBS, C. G. A mucorine parasite on Penicillia. Nature i 50: 3 19 ( 1942).
177. DoEBELT, H. Beitrage zur Kenntnis eines pigmentbildenden Penicilliums.
Ann. Mvcol. 7:315-338 (1909).
178. DoEHLE, Dr. Beobachtungen uber einen Antagonisten des Milzbrandes.
Habilitationschr. Kiel, 1889.
179. DoLD, H. Der bakterienantagonistische Anteil an der keimschadigenden
Wirkung des Speichels (der Mundhohlenfliissigkeit). Ztschr. f. Hyg. u.
Infektionskr. 124:579-596 (1942).
180. DoLD, H., and Deck, F. W. The antibacterial inhibition substances (in-
hibins) in normal fresh human urine. Ztschr. Hyg. Immunitat. 123:383
(1941) ; Klin. Wchnschr. 21:823 (1942).
181. Donaldson, R. Character and properties of the "Reading" bacillus, on
which a new method of treatment of wounds has been based. J. Path. &
Bact. 22:129-151 (1918).
182. Downs, C. M. The effect of bactericidal agents on gram-negative cocci.
J. Bact. 45:137-142 (1943)-
183. Drechsler, C. Some hyphomycetes parasitic on free-living terricolous
nematodes. Phytopath. 31:773-802 (1941); Biol. Rev. 16:265-290
(1941).
1 84. Drechsler, C. Two hyphomycetes parasitic on oospores of root-rotting
oomycetes. Phytopath. 28:81-103 (1938).
185. Dresel, E. G. Bakteriolyse durch Fettsauren und deren Abkommlings.
Centralbl. f. Bakteriol., I, Or. 97:178-181 (1926).
186. Druckrey, H., Richter, R., and Vierthaler, R. Formation of can-
cer-forming substances in man. Naturwiss. 29:63-64 (1941); abstract in
Chem. Abstr. 35:7446 (1941).
187. Dub, L. Urethral tyrothricin irrigations in the male; an experimental and
clinical study. Am. J. Syph., Gonor. & Ven. Dis. 28:325-333 (1944).
188. DuBos, R. J. The adaptive production of enzymes by bacteria. Bact. Rev.
4: 1-16 (1940).
189. DuBOS, R. Antimicrobial agents of biological origin. J. A.M. A. 124:633-
636 (1944)-
190. DuBos, R.J. Bactericidal effect of an extract of a soil bacillus on gram-
positive cocci. Proc. Soc. Exper. Biol, & Med. 40:311-312 (1939); J.
Exper. Med. 70:1-10, 11-17 (1939).
191. DuBos, R. The bacteriostatic action of certain components of commercial
peptones as affected by conditions of oxidation and reduction, J. Exper.
Med. 52:331-345 (1930).
192. DuBos, R. J. Bacteriostatic and bactericidal agents obtained from sapro-
phytic microorganisms. J. Pediat. 19:588—595 (1941).
193. DuBos, R. J. The effect of specific agents extracted from soil microorgan-
isms upon experimental bacterial infections. Ann. Int. Med. i 3 : 202 5— 2037
(1940).
284 BIBLIOGRAPHY
194. DuBos, R. J. Enzymatic analysis of the antigenic structure of pneumococci.
Ergebn. d. Enzymforsch. 8:135-148 (1939).
195. DuBOS, R. Factors affecting the yield of specific enzyme in cultures of the
bacillus decomposing the capsular polysaccharide of type III pneumococcus.
J. Exper. Med. 55:377-391 (1932).
196. DuBOS, R. J. Microbiology. Ann. Rev. Biochem. 11:659-678 (1942).
197. DuBOS, R. The relation of the bacteriostatic action of certain dyes to oxida-
tion-reduction process. J. Exper. Med. 49:575-592 (1929).
198. DuBOS, R. J. The significance of the structure of the bacterial cell in the
problems of antisepsis and chemotherapy. Univ. Penn. Bicentennial Conf.
Chemotherapy (1941), pp. 29-42.
199. DuBos, R. J. Studies on the mechanism of production of a specific bac-
terial enzyme which decomposes the capsular polysaccharide of type III pneu-
mococcus. J. Exper. Med. 62:259-269 (1935).
200. DuBos, R. J. Utilization of selective microbial agents in the study of bio-
logical problems. Bull. New York Acad. Med. 17:405-422 (1941).
201. DuBos, R. J. \'ulnerable structures of the bacterial cell. Journal -Lancet
61:405-407 (1941)-
202. DuBos, R. J., and Avery, O. T. Decomposition of the capsular polysac-
charide of pneumococcus type III by a bacterial enzyme. J. Exper. Med. 54:
51-71 (1931).
203. DuBos, R. J., and Cattaneo, C. Studies on a bactericidal agent extracted
from a soil bacillus; preparation and activity of a protein-free fraction.
J. Exper. Med. 70:249-256 (1939).
204. DuBos, R. J., and Hotchkiss, R. D. Origin, nature and properties of
gramicidin and tyrocidine. Tr. & Stud., Coll. Physicians, Philadelphia 10:
11-19 (1942)-
205. DuBos, R. J., and Hotchkiss, R. D. The production of bactericidal sub-
stances by aerobic sporulating bacilli. J. Exper. Med. 73:629—640 (1941).
206. DuBos, R. J., Hotchkiss, R. D., and Coburn, A. F. The effect of grami-
cidin and tyrocidine on bacterial metabolism. J. Biol. Chem. 146:421—426
(1942).
207. Duchesne, E. Contribution a I'etude de la concurrence vitale ; antagonisme
entre les moisissures et les microbes. Thesis, Lyon (1897).
208. DucLAUX, E. Reaction sur le microbe des produits de sa vie cellulaire.
Traite de Microbiologic. Paris, Masson, 1898. Vol. 1, pp. 236-249.
209. DuENSCHMANN, H. Etude experlmentale sur le charbon symptomatique et
ses relations avec I'oedeme malin. Ann. Inst. Pasteur 8:403—434 (1894).
210. DuFFiN, W. M., and Smith, S. Penillic acid, an optically active acid from
penicillin. Nature 1 5 1 : 25 1 (1943).
211. Dujardin-Beaumetz, E. Action antibiotique excercee "in vitro" par cer-
tains streptocoques et en particulier par un microcoque saprophyte. Compt.
rend. Soc. de biol. 117:1 178-1 180 (1934).
212. Dujardin-Beaumetz, E. Propriete antibiotique du pneumocoque. Compt.
rend. Soc. de biol. 124:890-891 (1937).
BIBLIOGRAPHY 285
213. DuLiscouET, R. Action probiotique et antibiotique des staphylocoques chez
les porteurs des germes diphteriques. Compt. rend. Soc. de biol. 118:1 277—
1280 (1935); Arch. med. pharm. colon. 129:410-443 (1939).
214. DuLiscouET, R., and Ballet, B. Curleuses proprietes des staphylocoques
chez les porteurs de bacilles diphteriques; indications prognostiques et appli-
cations therapeutiques. Presse med. 43:1297—1300 (1935).
215. Duncan, J. T. On a bactericidal principle present in the alimentary canal
of insects and arachnids. Parasitology 18:238—252 (1926).
216. DuNGERN, F. VON. Ueber die Hemmung der Milzbrandinfektion durch
Friedlandersche Bakterien. Ztschr. Hyg. Immunitat. 18: 177-208 (1894).
217. Dunham, W. B., Hamre, D. M., McKee, C. M., and Rake, G. W. Ac-
tion of penicillin and other antibiotics on Treponema pallidum. Proc. Soc.
Exper. Biol. & Med. 55:15 8-1 60 ( 1 944) .
218. Duran-Reynals, F. Bacteriophage et microbes tues. Compt. rend. Soc. de
biol. 94:242-243 (1926).
219. DuTKY, S. R. Two new spore-forming bacteria causing milky diseases of
Japanese beetle larvae. J. Agr. Research 61:57—68 (1940).
220. Eagle, H., and Magnuson, H.J. The therapeutic efficacy of penicillin
in experimental relapsing fever. J. Bact. 47:21—22 (1944).
221. Eagle, H., Magnuson, H. J., and Musselman, A. D. The therapeutic
efficacy of penicillin in relapsing fever infections in mice and rats. Pub.
Health Rep. 59:583-588 (1944).
222. Eagle, H., and Musselman, A. D. The spirocheticidal action of peni-
cillin in vitro (Treponema pallidum — Reiter strain). J. Bact. 47:22
(1944); J. Exper. Med. 80:493-497 (1944)-
223. Eaton, E. D., and King, C. J. A study of the cotton root rot fungus
(Phymatotrichum omnivorum) in the soil by the Cholodny method. J. Agr.
Research 49: 1 1 09-1 1 13 (1934).
224. Ebius, R. On the so-called antagonism among bacteria. Diss., luriev
( Dorpat ) Univ. ( 1 906 ) , Cited by Nakhimovskaia {62-/).
225. Edgerton, C. W. Plus and minus strains in the genus Glomerella. Am.
J. Bot. 1:244-254 (191 4).
226. Edwards, E. T. The biological antagonism of Gibberella fujikuroi and
Gibberella fujikuroi var. subglutinans to Trichoderma viride, with notes on
the pathological effects of the latter fungus on maize. J. Australian Inst.
Agr. Sc. 6:91-100 (1940).
227. Ehrismann, O. Pyocyanin und Bakterienatmung. Zentralbl. f. Bakteriol.,
I, Ref. 112:285-286 (1934).
228. Eijkman, C. Ueber thermolabile Stoffwechselprodukte als Ursache der
naturlichen Wachstumshemmung der Mikroorganismen. Centralbl. f. Bak-
teriol., I, Or. 37:436-449 (1904); 41:367-369, 471-474 (1906).
229. Eisenberg, F. Selective bacteriostasis and bactericidal action. Proc. Sec-
ond Internat. Cong. Microbiol. (1936), pp. 34-35.
230. Eisler, M., and Jacobsohn, I. Uber die antagonistische Wirkung steriler
Bouillonextrakte aus Bacterium prodigiosus. Ztschr. Hyg. Immunitat. 117:
76-91 (1936).
286 BIBLIOGRAPHY
231. Elliott, J. A. Taxonomic characters of the genera Alternaria and Macro-
sporium. Am. J. Bot. 4:439-476 (1917).
232. Ellis, E. L., and Spizizen, J. The rate of bacteriophage inactivation by
filtrates of Escherichia coli cultures. J. Gen. Physiol. 24:437-445 (1941).
233. Emmerich, R. Die Heilung des Milzbrandes. Arch. f. Hyg. 6:442-501
(1886).
234. Emmerich, R. Sind alle Einwlinde gegen die Natur und Wirkungsweise
der sogenannten Nukleasen widerlegt? Centralbl. f. Bakteriol., I, Or. 31:
585-588(1902).
235. Emmerich, R., and Low, O. Bakteriologische Enzyme als Ursache der
erworbenen Immunitat und die Heilung von Infektionskrankheiten durch
dieselben. Ztschr. Hyg. Immunitat. 31:1—65 (1899).
236. Emmerich, R., and Low, O. Die kunstliche Darstellung der immuni-
sierenden Substanzen (Nukleasen-Immunproteide) und ihre Verwendung
zur Therapie der Infektionskrankheiten und zur Schutzimpfung an Stelle des
Heilserums. Ztschr. f. Hyg. u. Infektionskr. 36:9 (1901); abstract in
Centralbl. f. Bakteriol. 29:577-579 (1901).
237. Emmerich, R., Low, O., and Korschun, A. Die bakteriolytische Wir-
kung der Nucleasen und Nucleasen-Immunproteidine als Ursache der natur-
lichen und kiinstlichen Immunitat. Centralbl. f. Bakteriol., I, Or. 3 i : 1-25
(1902).
238. Emmerich, R., and Saida, Dr. Ueber die morphologischen Verander-
ungen der Milzbrandbacillen bei ihrer Auflosung durch Pyocyanase. Cen-
tralbl. f. Bakteriol., I, Or. 27:776-787 (1900).
239. Emmerich, R., and Tromsdorf, R. Ueber die erfolgreiche Behandlung
todlicher intraperitonaler Streptokokkeninfektion beim Kaninchen durch
praventive Pyocyanase-Immunproteiden-Injektionen. Centralbl. f. Bak-
teriol., I, Or. 33:627-633 (1903).
240. Endo, S. Influence of carbohydrates and organic nitrogen compounds on
the antagonistic action of microorganisms. Ann. Jap. Phytopath. Soc. 2:2
(1932)-
241. Endo, S. Studies on the antagonism of microorganisms; growth of Hy-
pochnus sasakii Shirai as influenced by the antagonistic action of other micro-
organisms. Bui. Miyazaki Coll. Agr. For. 3 : 95-119 (193 l) ; 4: 133-1 58,
159-185 (1932); 5:51-73 (1933)-
242. Enoch, H. E., and Wallersteiner, W. K. S. A standardized antibac-
terial pyrogen-free metabolite preparation containing living Penicillium no-
tatum. Nature 153:380-381 (1944).
243. Epstein, L. A., and Chain, E. Some observations on the preparation and
properties of the substrate of lysozyme. Brit. J. Exper. Path. 21:339-355
(1940).
244. Etinger-Tulczinska, R. Ueber Bakterienantagonismus, Ztschr. f. Hyg.
u. Infektionskr. 113:762-780 (1932); 116:72-80 (1934).
245. Fabry, P., and van Beneden, J. Serum anti-lytique et anti-serum anti-
antilytique. Compt. rend. Soc. de biol. 90:111—113 (1924).
BIBLIOGRAPHY 287
246. Fadeeva, T. D., and Tchernobaiev, V. S. Bacillus pestis in mixed cul-
tures; antagonism of various species of bacteria towards plague bacilli,
Viestnik mikr. epidemiol. i parasitol. 14:346-356 (1935). Cited by Na-
khimovskaia {62y).
247. Faltin, R. Studien uber Hetero- und Isantagonismus, mit besonderer Be-
riicksichtigung der Verhaltnisse bei infektiosen Erkrankungen der Harn-
wege. Centralbl.f.BakterioL, I, Or. 46:6-20, 109-128, 222-229 (1908).
248. Fawcett, H. S. The importance of investigations on the effects of known
mixtures of microorganisms. Phytopath. 2 1 : 545-550 ( 1 93 1 ) .
249. FDA reveals penicillin assay methods. Drug Trade News 18:3 3—42 ( 1 943) .
250. Fehrs, Dr. Die Beeinflussung der Lebensdauer von Krankheitskeimen im
Wasser durch Protozoen. Hyg. Rundschau. 16:113-121 (1906).
251. Fellows, H. Studies of certain soil phases of the wheat take-all problem.
Phytopath. 19:103 (1929).
252. Ferguson, C, and Buchholtz, M. Penicillin therapy of gonorrhea in
men. J.A.M.A. 125:22-23 (1944).
253. Fernbach, A. Sur un poison elabore par la levure. Compt. rend. Acad,
d.sc. 149:437-439 (1909).
254. FiLDES, P. A rational approach to research in chemotherapy. Lancet i:
955-957(1940).
254a. FiLDES, P. The mechanism of the anti-bacterial action of mercury. Brit.
J. Exper. Path. 21:67-73 (1940).
255. Findlay, G. M., Fleming, A., and others. The mode of action of chemo-
therapeutic agents. Biochem. J. 36: i— 17 (1942).
256. Firth, R. H., and Horrocks, W. H. An inquiry into the influence of
soils, fabrics, and flies in the dissemination of enteric infection. Brit. M. J.,
pp. 936-943 (1902).
257. Fischer, R. A., Thornton, H. G., and MacKenzie, W. A. The accu-
racy of the plating method of estimating the density of bacterial populations.
Ann. App. Biol. 9:325-359 (1922).
258. Fisher, A. M. The antibacterial properties of crude penicillin. Bull.
Johns Hopkins Hosp. 73:343-378 (1943).
259. Fisher, C. v., and Martin, G. P. Anti-biotin activity of methionine.
J. Bact. 45:33 (1943).
260. Fleming, A. Chemotherapy and wound infection. Lancet i : 278 (1941).
261. Fleming, A. In vitro tests of penicillin potency. Lancet 1:732-733
(1942).
262. Fleming, A. Lysozyme; president's address. Proc. Roy. Soc. Med. (Lon-
don) 26:71-84 (1932).
263. Fleming, A. Mode of action of chemotherapeutic agents. Lancet 2:761-
762 (1940-
264. Fleming, A. On a remarkable bacteriolytic element found in tissues and
secretions. Proc. Roy. Soc. (London), s.B. 93:306-317 (1922).
265. Fleming, A. On the antibacterial action of cultures of a Penicillium, with
special reference to their use in the isolation of B. influenzae. Brit. J. Exper.
Path. 10:226-236 (1929).
288 BIBLIOGRAPHY
266. Fleming, A. On the specific antibacterial properties of penicillin and po-
tassium tellurite; incorporating method of demonstrating some bacterial an-
tagonisms. J. Path. & Bact. 35:831-842 (1932).
267. Fleming, A. Penicillin. Brit. M. J., No, 4210, p. 386 (1941).
268. Fleming, A. Selective bacteriostasis. Proc. Second Internat. Cong. Micro-
biol. (1936), pp. 33-34-
269. Fleming, A, A simple method of using penicillin, tellurite, and gentian
violet for differential culture. Brit. M. J. 1:547-548 (1942).
270. Fleming, A. Streptococcal meningitis treated with penicillin; measurement
of bacteriostatic power of blood and cerebrospinal fluid. Lancet 2:434—438
(1943)-
271. Flore Y, H. W. Penicillin; its development for medical uses. Nature 153:
40-42 (1944)-
272. Florey, H. W., and Cairns, H. Penicillin in war wounds; a report from
the Mediterranean. Lancet 2: 742-745 (1943).
272a. Florey, H. W., Heatley, N. G., Jennings, M. A., and Williams,
T. I. Penicillin-like antibiotics from various species of moulds. Nature
154:268 (1944)-
273. Florey, H. W., and Jennings, M. A. Some biological properties of highly
purified penicillin. Brit. J. Exper. Path. 23:120-123 (1942).
274. Florey, H. W., Jennings, M. A., and Philpot, F. J. Claviformin from
Aspergillus giganteus Wehm. Nature 153:139 (1944).
275. Florey, M. E. Clinical uses of penicillin. Brit. Med. Bull. 2:9—13
(1944).
276. Florey, M. E., and Florey, H. W. General and local administration of
penicillin. Lancet 1:387-397 (1943); 2:638,639 (1943).
277. Florey, M. E., and Williams, R. E. O. Hand infections treated with
penicillin. Lancet 1 : 73— 8 1 (1944).
278. Flugge, C. Die Mikroorganismen, mit besonderer Beriicksichtigung der
Otiologie der Infektionskrankheiten. 2 vols. Leipzig, Vogel, 1896.
279. FoRDOS, M. Recherches sur la matiere colorante des suppurations bleues;
pyocyanine. Compt. rend. Acad. d. sc. 51:21 5-2 17(1 860) .
280. FoRTiNEAU, L. Note sur le traitement du charbon par la pyocyanase. Ann.
Inst. Pasteur 24:955-972 (1910).
281. Foster, J. W. Quantitative estimation of penicillin. |. Biol. Chem. 144:
285-286 (1942).
282. Foster, J. W., and Karow, E. O. Microbiological aspects of penicillin;
penicillin production by different fungi. J. Bact. {To be fublished in ig^^).
283. Foster, J. W., and Wilker, B. L. Microbiological aspects of penicillin;
turbidimetric studies on penicillin inhibition. J. Bact. 46:377-389
(1943).
284. Foster, J. W., and Woodruff, H. B. Improvements in the cup assay for
penicillin. J. Biol. Chem. 148:723 (1943).
285. Foster, J. W., and Woodruff, H. B. Microbiological aspects of peni-
cillin; methods of assay. J. Bact. 46:187-202 (1943).
BIBLIOGRAPHY 289
286. Foster, J. W., and Woodruff, H. B. Microbiological aspects of penicil-
lin; procedure for the cup assay for penicillin. J. Bact. 47:43-58 (1944).
287. Foster, J. W., and Woodruff, H. B. Microbiological aspects of strepto-
thricin; antibiotic activity of streptothricin. Arch. Biochem. 3:241—255
(1943).
288. Foster, J. W., and Woodruff, H. B. Quantitative estimation of strepto-
thricin. J. Bact. 45:408-409 (1943).
289. Foster, J. W., Woodruff, H. B., and McDaniel, L. E. Microbiologi-
cal aspects of penicillin; production of penicillin in surface cultures of
Penicillium notatum. J. Bact. 46:421—433 (1943).
290. Fowler, G. J. An introduction to the biochemistry of nitrogen conserva-
tion. London, Arnold, 1934.
290a. Fox, S. W., Fling, M., and Bollenback, G. N. Inhibition of bacterial
growth by ^/-leucine. J. Biol. Chem. 155:465-468 (1944).
291. Francillon, M. Einfluss der aeroben Mischinfektion auf Entwicklung
und Toxinbildung dcs Bacillus botulinus. Arch. f. Hyg. 95:121-139
(1925).
292. Francis, A. E. Sulphonamide-resistant streptococci in a plastic-surgery
ward. Lancet 1 : 408-409 ( 1 942) .
293. Francis, T., Jr., Terrell, E. E., Dubos, R. J., and Avery, O. T. Ex-
perimental type III pneumococcus pneumonia in monkeys; treatment with an
enzyme which decomposes the specific capsular polysaccharide of pneumo-
coccus type III. J. Exper. Med. 59:641-668 (1934).
294. Franke, H., and Ismet, A. Ueber Cytolyse. Centralbl. f. Baktcriol., I,
Or., 99:570-576 (1926).
295. Frankland, p. F. The bacteriology of water; its present position. J. Soc.
Chem. Ind. 30:319-334 (1911).
296. Frankland, P. F. Microorganisms in water. New York, Longmans,
1894.
297. Frankland, P. F., and Ward, H. M. The vitality and virulence of Ba-
cillus anthracis and its spores in potable waters. Proc. Roy. Soc. (London),
53:164-317 (1893).
298. Freisleben, R. Weitere Untersuchungen iiber die Mykotrophie der Erica-
ceen. Jahrb. wiss. Bot. 82:413-459 (1935).
299. Freudenreich, E. de. De I'antagonisme des bacteries et de I'immunite
qu'il confere aux milieux de culture. Ann. Inst. Pasteur 2:200—206
(1888) ; abstract in Jahresber. path. Mikroorg. 5:530 (1889).
300. Friedenwald, J., and Leitz, T. F. Experiments relating to the bacterial
content of the feces, with some researches on the value of certain intestinal
antiseptics. Am. J. M. Sc. 138:653-661 (1909).
301. Friedmann, E. Pyocyanine an accessory respiratory enzyme. J. Exper.
Med. 54:207-221 (1931).
302. Frost, W. D. The antagonism exhibited by certain saprophytic bacteria
against Bacillus typhosus Gaffky. J. Infect. Dis. 1:599-640 (1904).
303J Fuller, A. T. Antibacterial action and chemical constitution in long-chain
aliphatic bases. Biochem. J. 36:548—558 (1942).
290 BIBLIOGRAPHY
304. Fulton, H. R. Chemotropism of fungi. Hot. Gaz. 41:81-108 (1908).
305. Fulton, M. Antibiosis in the coli-typhoid group; growth curves of two
strains in a synthetic medium. J. Bact. 34:301—315 (1937).
306. Fulton, R. W. The sensitivity of plant viruses to certain inactivators.
Phytopath. 33:674-682 (1943).
307. Gabritschewsky, G., and Maljutin, E. Ueber die bakterienfeindlichen
Eigenschaften des Cholerabacillus. Centralbl. f. Balcteriol. 13:780—785
(1893).
308. Gardner, A. D. Morphological effects of penicillin on bacteria. Nature
146:837-838 (1940).
309. Gardner, A. D., and Chain, E. Proactinomycin; a "bacteriostatic" pro-
duced by a species of Proactinomyces. Brit. J. Exper, Path. 23:123—127
(1942).
310. Garrard, E. H., and Lochhead, A. G. Relationships between soil micro-
organisms and soil-borne plant pathogens. Sc. Agr. 18:719—737 (1938).
311. Garre, C. Uber Antagonisten unter den Bakterien. Centralbl. f. Bak-
teriol. 2:312-313 (1887).
312. Garrett, S. D. Factors affecting the severity of take-all. J. Agr. S. Aus-
tralia 37:664-674, 799-805, 976-983 (1934); Biol. Rev. 9:351-361
(1934)-
312a. Garrett, S. D. Root disease fungi. Waltham, Mass., Chronica Botanica
Co., 1944.
313. Garrett, S. D. Soil conditions and the root-infecting fungi. Biol. Rev.
13:159-185 (1938); Imp. Bur. Sc. Tech. Commun. No. 38 (1939), No.
41 (1942).
314. Garrett, S. D. Soil conditions and the take-all disease of wheat. Ann.
App. Biol. 23:667-699 (1936); 24:747-751 (1937); 25:742-766
(1938); 27:199-204 (1940).
315. Garrod, L. p. Action of antiseptics on wounds. Lancet i : 798-802, 845-
848 (1940).
316. Garrod, L. P. The treatment of war wounds with penicillin. Brit. M. J.,
No. 4327, PP- 755-756 (1943)-
317. Gasperini, G. Recherches morphologiques et biologiques sur un micro-
organisme de I'atmosphere, le Streptothrix Foersteri Cohn. Ann. Microgr.
10:449-474(1890).
318. Gate, J., and Papacostas, G. Antagonisme biologique entre le Bacille de
LofHer et le Pneumobacille de Friedlander. Compt. rend. Soc. de biol. 85:
859-861, 1038-1040 (1921); 86:929 (1922); Paris med. 61:205-210
(1926).
318a. Gause, G. F., and Brazhnikova, M. G. Gramicidin S and its use in the
treatment of infected wounds. War Med. 6: 180— 181 (1944).
319. Gershenfeld, L., and Witlin, B. Surface tension reducents in bacteri-
cidal solutions; their in vitro and in vivo efficiencies. Am. J. Pharm. 113:
215-236 (1940-
320. Gessard, C. De la pyocyanine et son microbe. Thesis, Paris (1882).
BIBLIOGRAPHY 29t
321. Gessard, C, Diagnose pigmcntaire du bacille pyoqyanique. Ann. Inst,
Pasteur 33:241-260 (1919).
322. Gessard, C. Nouvelles rechcrches sur le microbe pyocyanique. Ann. Inst.
Pasteur 4:88-102 (1890).
323. GiLDEMEiSTER, E. Untersuchungen uber das Lysozym. Centralbl. f. Bak-
teriol., I, 136:408-412 (1936).
324. GiLDEMEiSTER, E., and Neustat, M. Beitrag zur Bakterienvermehrung
und zur Bakteriensymbiose. Zentralbl. f. Bakteriol., I, Or., 133:101-106
(1934).
325. GiLERSON, C, and Levinson, A. Clinical evaluation of the treatment of
gangrene of teeth pulp on the basis of bacterial antagonism. Modern Prob-
lems of Somatology (U.S.S.R.), M.L. 533 (1933).
326. GiLMouR, R. T. Further notes on the isolation of the Micrococcus meliten-
sis from peripheral blood; and experiments on the duration of life of this
microbe in earth and in water. Reports of the Commission for the Investi-
gation of Mediterranean Fever, 1906. Part IV, pp. 3-7.
327. Glaser, R. W. Studies on Neoaplectana glaseri, a nematode parasite of the
Japanese beetle (Popillia Japonica). N.J. Dept. Agr., Cir. 211, pp. 1-34
(1932); J- N.Y.Ent.Soc. 43:345-371 (i935)-
328. Glasgow, H. The gastric caeca and the caecal bacteria of the Heteroptera.
Biol. Bull. 26:101-155 (1914).
329. Glathe, H. Uber die Rotte des Stalldiingers unter besonderer Beriick-
sichtigung der Anaeroben-Flora. Zentralbl. f. Bakteriol., II, 91:65—101
(1934).
330. Glister, G. A. A new antibacterial agent produced by a mould. Nature
148:470 (1940-
331. Glister, G. A., and Williams, T. I. Production of gliotoxin by Asper-
gillus fumigatus mut. helvola Yuill. Nature 153:651-652 (1944).
332. GoETSCH, W., and Gruger, R. Die Pilze der Blattschneider-Ameisen und
ihre Vernichtung. Naturwiss. 28: 764-765 (1940).
333. GoiDANicH, G., BoRZiNi, G., Mezzetti, A., and Vivani, W. Ricerche
sulle alterazioni e sulla conservazione della pasta di legno destlnata alia fab-
bricazione della carta. Rome, Ministre delle Corporazioni, Commissario
deir Ente Nazionale per la cellulosa e per la carta, 1938.
334. GoLDiE, H. Absorption et destruction des substances inhibitrices de filtrats
microbiens. Compt. rend. Soc. de biol. 108:762-764 (1931).
335. GoLDiE, H. Pathogenic bacteria in sewage; bacteriolysis. Rev. d'hyg. 55:
5-23 (1933); al>siract in Chem. AhstT. 27:1929 (1933).
336. Goldman, W. Untersuchungen uber den Coli-Milzbrandantagonismus.
Zentralbl. f. Bakteriol., I, Or., 136:345-352 (1936).
337. Goodner, K., and Dubos, R. Studies on quantitative action of specific
enzyme in type III pneumococcus dermal infection in rabbits. J. Exper.
Med. 56:521-530 (1932).
338. Goodner, K. G., Dubos, R. J., and Avery, O. T. The action of a spe-
( cific enzyme upon the dermal infection of rabbits with type III pneumococ-
cus. J. Exper. Med. 55:393-404 (1932).
292 BIBLIOGRAPHY
339. Gordon, A. H., Martin, A. J. P., and Synge, R. L. M. The amino-acid
composition of gramicidin. Biochem. J. 37:86—92, 313—318 (1943).
340. GoRis, A., and Liot, A. Importance des sels ammoniacaux organiques dans
la production de la pyocyanine par le bacille pyocyanique. Compt. rend.
Acad. d. sc. 176: 191-193 (1923).
341. GoRis, A., and Liot, A. Nouvelles observations sur la culture du bac. pyo-
cyanique sur milieux artificiels definis. Compt. rend. Acad. d. sc. 172:
1622-1624 (1921); 174:575-578 (1922).
342. Goss, R. W. The influence of various soil factors upon potato scab caused
by Actinomyces scabies. Res. Bull. Nebraska Agr. Exper. Sta. 93 (1937).
343. Goth, A., and Bush, M. T. Rapid method for estimation of penicillin.
Ind. & Eng. Chem. (Anal. Ed.) 16:451-452 (1944).
343a. Gramicidin (Editorial). Brit. Med. J. 2:17-18 (1942).
344. Gratia, A. Antagonisme microbien et "bacteriophagie." Ann. Inst. Pas-
teur 48:413-437 (1932).
345. Gratia, A. Des relations numeriques entre bacteries lysogenes et particules
de bacteriophage. Ann. Inst. Pasteur 57:652-676; 56:307-315 (1936).
346. Gratia, A. L'action lytique des staphylocoques vivants sur les staphylo-
coques tues. Compt, rend. Soc. de biol. 95:734—735 (1926).
347. Gratia, A. La dissolution des bacteries et ses applications therapeutiques.
Bull. Acad. roy. de med. de Belgique, May 19, 285-295 (1934).
348. Gratia, A. Sur un remarquable exemple d'antagonisme entre deux souches
de colibacille. Compt. rend. Soc. de biol. 93: 1040 (1925).
349. Gratia, A., and Alexander, J, Sur la "mycolyse" par Ic streptothrix.
Compt. rend. Soc. de biol. 106: 1288-1289 (1931).
350. Gracia, A., and Dath, S. De Taction bacteriolytiques des streptothrix.
Compt. rend. Soc. debiol. 91: 1442-1443 (1924) ; 92: 1125-1126 (1925) ;
93:451 (1925); 94:1267-1268 (1926).
351. Gratia, A., and Rhodes, B. De Taction lytique des staphylocoques vivants
sur les staphylocoques tues. Compt. rend. Soc. de biol. 90 : 640—642 ( 1 924) .
352. Graves, A. H. Chemotropism in Rhizopus nigricans. Bot. Gaz. 62:337-
369 (1916).
353. Greaney, F. J., and Machacek, J. E. Studies on the control of the root-
rot diseases of cereals caused by Fusarium culmorum (W.G.Sm.) Sacc. and
Helminthosporium sativum P.K. and B.; pathogenicity of Helminthosporium
sativum as influenced by Cephalothecium roseum Corda in greenhouse pot
tests. Sc. Agr. 15:377-386 (1935).
354. Green, H. N. Mode of action of sulphanilamide, with special reference to
bacterial growth-stimulating factor ("P" factor) obtained from Br. abortus
and other bacteria. Brit. J. Exper. Path, 21:38-64 (1940).
355. Green, H. N., and Bielschowsky, F. A factor ("P" factor) in bacterial
extracts stimulating bacterial growth and inhibiting the action of sulphan-
ilamide. Chem. & Ind. 59:135, 850 (1940); Brit. J. Exper. Path. 23:
1-12 (1942).
356. Greer, F. E,, and Nyhan, V. The sanitary significance of lactose-ferment-
ing bacteria not belonging to the B. coli group. J. Infect. Dis, 42: 527-536
(1928).
BIBLIOGRAPHY 293
357. Greiff, D., and PiNKERTON, H. Inhibition of growth of typhus rickettsiae
in the yolk sac by penicillin. Proc. Soc. Exper. Biol. & Med. 55:1 16-1 19
(1944).
358. Greig, E. D. W. The invasion of the tissues by the cholera vibrio and fur-
ther observations on pneumonia in cases of cholera. Indian J. M. Research
2:1-27 (1914-1915).
359. Greig, M. E,, and Hoogerheide, J. C. Evaluation of germicides by a
manometric method. J. Bact. 41:557-562 (1941).
360. Greig-Smith, R. Contributions to our knowledge of soil fertility; the ac-
tion of certain microorganisms upon the numbers of bacteria in the soil.
Proc. Linn. Soc. N.S. Wales, 42: 162-166 (191 7).
361. Greig-Smith, R. Contributions to our knowledge of soil fertility; the
agricere and bacteriotoxins of soil. Proc. Linn. Soc. N.S. Wales, 36:679—
699 (1912); 40:631-645 (1915).
362. Greig-Smith, R. Contributions to our knowledge of soil fertility; the
search for toxin-producers. Proc. Linn. Soc. N.S. Wales, 43:142—190
(1918).
363. Gromakowsky, D. Diplococcus im Sputum als Antagonist der pyogenen
Staphylo- und Streptokokken. Centralbl. f. Bakteriol., I, Or., 32:272-273
(1902).
364. Gruber, Th. Die Bakterienflora von Runkelruber, Steckriiben, Karotten,
von Milch wahrend der Stallfiitterung und des Weideganges einschlieszlich
der in Streu, Gras und Kot vorkommenden Mikroorganismen und deren
Mengenverhaltnisse in den 4 letzten Medien. Centralbl. f. Bakteriol., II,
22:401-416 (1909).
365. GuNDEL, M. Ueber den Antagonismus von Coli-Bakterien auf Milz-
brandbazillen. Centralbl. f. Bakteriol., I, Or., 104:463-473 (1927).
366. GuNDEL, M., and Himstedt, H. Ueber den Antagonismus zwischen Bak-
terien in kiinstlichen Niihrmedien. Miinchen. med. Wchnschr. 72:1674—
1676 (1925).
367. GuNDEL, M., and Kliewe, H. Experimentelle Untersuchungen iiber das
antagonistisch wirksame Prinzip der Coli- gegeniiber Milzbrandbazillen.
Zentralbl. f. Bakteriol., I, Or., 124:519-528 (1932).
368. Gundel, M., and Mayer, U. Ueber den Bakterienantagonismus innerhalb
einer Art bei den Pneumokokken. Zentralbl. f. Bakteriol., I, Or., 129: 305—
323 (1933)-
369. Gundel, M., and Okura, G. Untersuchungen iiber das gleichzeitige
Vorkommen mehrerer Pneumokokkentypen bei Gesunden und ihre Be-
deutung fiir die Epidemiologie. Ztschr. f. Hyg. u. Infektionskr. 1 14:678-
704(1933)-
370. Gundel, M., and Wagner, W. Weitere Studien iiber Bakterienlipoide.
Ztschr. Hyg. Immunitat. 69:63-76 (1930).
371. Hac, L. R., and Hubert, A. C. Penicillin in treatment of experimental
Clostridium welchii infection. Proc. Soc. Exper. Biol. & Med. 53:61—62
(1943)-
372. Hall, I. C, and Peterson, E. The effect of certain bacteria upon the
toxin production of Bacillus botulinus in vitro. J. Bact. 8: 3 19-341 (1923).
294 BIBLIOGRAPHY
373. Hall, R. P. Vitamin deficiency as one explanation for inhibition of pro-
tozoan growth by conditioned medium, Proc. Soc. Exper. Biol. & Med. 47:
306-308 (1941).
374. Hamre, D. M., Rake, G., McKee, C. M., and MacPhillamy, H. G.
The toxicity of penicillin as prepared for clinical use. Am. J. M. Sc. 206:
642-652 (1943)-
375. Hansen, H. N. Etiology of the pink-root disease of onions. Phytopath.
19:691-704 (1929).
376. Hansen, H. N., and Snyder, W. C. Relation of dual phenomenon in
Penicillium notatum to penicillin production. Science 99: 264-265 (1944).
377. Harder, R. Uber das Verhalten von Basidiomyceten und Ascomyceten in
Mischkulturen. Naturw. Ztschr. Forst. Landw. 9:129—160 (191 1 ).
378. Hardin, G. Physiological observations and their ecological significance; a
study of the protozoan, Oikomonas termo. Ecology 25:192—201 (1944).
379. Harper, G. J. Inhibition of penicillin in routine culture media. Lancet
2:569-571 (1943)-
380. Hartley, C. Damping-off in forest nurseries. U.S.D.A. Bull. 934
(l92l),pp. 1-99.
381. Hartoch, O., and JoFFE, V. Ueber die Wirkung von Bakterienfiltraten in
vitro und in vivo. Arb. a. d. Staats. Inst. f. exper. Therap., Hft. 21, pp.
83-97 (1928).
382. Harvey, D. The causation and prevention of enteric fever in military
service with special reference to the importance of the carrier. J. Roy. Army
M. Corps 24:491 (1915).
383. Hashimoto, K. Wachstumhemmende Wirkung von Coli-Bazillen patho-
genen Darmbakterien gegeniiber. Centralbl. f. Bakteriol., I, Or., 103:1-9
(1927).
384. Havens, L. C, and Dehler, S. A. The effect of Gambusia affinis on the
B. coli index of pollution of water. Am. J. Hyg. 3:296—299 (1923).
385. Heatley, N. G. A method for the assay of penicillin. Biochem. J. 38:
61-65 (1944)-
386. Hegemann, F. The antibacterial action of human saliva. Ztschr. f. Hyg.
u. Infektionskr. 124:202-210 (1942).
387. Heilman, D. H. A method for standardizing penicillin. Am. J. M. Sc.
207:477-483 (1944)-
388. Heilman, D. H., and Herrell, W. E. Comparative antibacterial activity
of penicillin and gramicidin; tissue culture studies. Proc. Staff Meet., Mayo
Clinic 17:321-327 (1942).
389. Heilman, D. H., and Herrell, W. E. Comparative bacteriostatic activ-
ity of penicillin and gramicidin. J. Bact. 43:12-13 (1942).
390. Heilman, D., and Herrell, W. E. Hemolytic effect of gramicidin.
Proc. Soc. Exper. Biol. & Med. 46: 1 82-1 84 (i 941).
391. Heilman, D., and Herrell, W. E. Mode of action of gramicidin.
Proc. Soc. Exper. Biol. & Med. 47:480-484 (1941).
392. Heilman, D. H., and Herrell, W. E. Penicillin in treatment of experi-
mental ornithosis. Proc. Staff Meet., Mayo Clinic 19:57-65 (1944).
BIBLIOGRAPHY 295
393. Heilman, F. R., and Herrell, W. E. Penicillin in the treatment of ex-
perimental relapsing fever. Proc. Staff Meet., Mayo Clinic 18:457—467
(1943)-
394. Henle, W., and Henle, G. Interference of inactive virus with the
propagation of virus of influenza. Science 98:87-89 (1943).
395. Henle, G., and Zittle, C. A. Effect of gramicidin on metabolism of bo-
vine spermatozoa. Proc. Soc. Exper. Biol. & Med. 47: 193-198 (1941).
396. Henrici, a. T. Characteristics of fungous diseases. J. Bact. 39: 1 13-138
(1940).
397. Henry, A. W. The influence of soil temperature and soil sterilization on
the reaction of wheat seedlings to Ophiobolus graminis. ' Canad. J. Research
7:198-203 (1932).
398. Henry, A. W, The natural microflora of the soil in relation to the root-rot
problem of wheat. Canad. J. Research 4:69—77 (193 1); 5:407-413
(1930-
399. Herrell, W. E. Further observations on the clinical use of penicillin.
Proc. Staff Meet., Mayo Clinic 18:65-76 (1943).
399a. Herrell, W. E. The clinical use of penicillin; an antibacterial agent of
biologic origin. J. A.M. A. 124:622-627 (1944).
400. Herrell, W. E., Cook, E. N., and Thompson, L. Use of penicillin in
sulfonamide resistant gonorrheal infections. J.A.M.A. 122:289—292
(1943)-
401. Herrell, W. E., and Heilman, D. Experimental and clinical studies on
gramicidin. J. Clin. Investigation 20:433, 583-591 (1941); J.A.M.A.
1 18: 1401-1402 (1942).
402. Herrell, W. E., and Heilman, D. Tissue culture studies on cytotoxicity
of bacterial agents; cytotoxic and antibacterial activity of gramicidin and
penicillin; comparison with other germicides. Am. J. M. Sc. 206: 221—226
(1943)-
403. Herrell, W, E., and Heilman, D. Tissue culture studies on cytotoxicity
of bacterial agents; effects of gramicidin, tyrocidine and penicillin on cul-
tures of mammalian lymph node. Am. J. M. Sc. 205 : i 57-162 (1943).
404. Herrell, W. E., Heilman, D. H., and Williams, H. L. The clinical
use of penicillin. Proc. Staff Meet., Mayo Clinic 17:609-616 (1942).
405. Herrell, W. E., and Nichols, D. R. The calcium salt of penicillin.
Proc. Staff Meet., Mayo Clinic 18:313—319 (1943).
406. Hettche, H. O. Der Einfluss der Umwelt auf die Form der Diphtherie-
bazillen. Zentralbl. f. Bakteriol., I, Or., 134:433-438 (1934).
407. Hettche, H. O., and Vogel, W. Vergleichende Untersuchungen iiber
die antagonistische Wirkung von Bacterium fluorescens und Bacterium pyo-
cyaneum. Arch. f. Hyg. 117:234-244 (1937).
408. Hettche, H. O., and Weber, B. Die Ursache der bakteriziden Wirkung
von Mesentericus-filtraten, Arch. f. Hyg. 123:69—80 (1939).
409. Hettche, O. Untersuchungen iiber die bakteriziden und anthrakoziden
Bestandteile von Bacillus pyocyaneus und Bacillus prodigiosus. Arch, f . Hyg.
107:337-353(1932)-
296 BIBLIOGRAPHY
410. Hettche, O. Untersuchungen uber die Natur der bakteriziden und
hamolitischen Bestandteile der Pyocyaneuslypoiden. Ztschr. f. Hyg. u. Im-
munitatsforsch u. exper. Therap. 83:499-505, 506— 511 (1934).
411. Heukelekian, H., and Schulhoff, H. B. Studies on the survival of
B. typhosus in surface vi^aters and sewage. New Jersey Agr. Exper. Sta.
Bull. 589(1935).
412. HiLDEBRAND, A. A., and KocH, L. W. A microscopical study of infection
of the roots of strawberry and tobacco seedlings by microorganisms of the
soil. Canad. J. Research 14: 1 1-26 (1936).
413. HiLDEBRAND, A. A., and West, p. M. Strawberry root rot in relation to
microbiological changes induced in root rot soil by the incorporation of cer-
tain cover crops. Canad. J. Research 19: 183-198, 199-210 (1941).
414. HiNO, I. Antagonistic action of soil microbes with special reference to plant
hygiene. Trans. Third Internat. Cong. Soil Sc. i: 173—174 (1935).
415. Hind, I., and Endo, S. Trichoderma parasitic on sclerotial fungi. Ann.
Phytopath. Soc. Japan 10:231-241 (1940).
416. HiRSCH, J. Die Sekretion eines Glukose-oxydierenden Enzyms mit bak-
teriostatischer Wirkung durch Penicillium notatum Fleming. Istanbul
Seririyati 25:3-20 (1943).
417. HiRSCHY, H. W., and Ruoff, P. M. Polarographic determination of cit-
rinin. J. Am. Chem. Soc. 64: 1490-1491 (1942).
418. Hobby, G. L., and Dawson, M. H. Bacteriostatic action of penicillin on
hemolytic streptococci in vitro. Proc. Soc. Exper. Biol. & Med. 56:178—
181 (1944)-
419. Hobby, G. L., and Dawson, M. H. Effect of rate of growth of bacteria
on action of penicillin. Proc. Soc. Exper. Biol. & Med. 56:181-184
(1944).
420. Hobby, G. L., and Dawson, M. H. Relationship of penicillin to sulfonam-
ide action. Proc. Soc. Exper. Biol. & Med. 56:1 84-1 87(1 944) .
421. Hobby, G. L., Meyer, K., and Chaffee, E. Activity of penicillin in
vitro. Proc. Soc. Exper. Biol. & Med. 50:277-280(1942).
422. Hobby, G. L., Meyer, K., and Chaffee, E. Chemotherapeutic activity
of penicillin. Proc. Soc. Exper. Biol. & Med. 50:285-288 (1942).
423. Hobby, G. L., Meyer, K., and Chaffee, E. Observations on the mecha-
nism of action of penicillin. Proc. Soc. Exper. Biol. & Med. 50:281-285
(1942).
424. Hobby, G. L., Meyer, K., Dawson, M. H., Chaffee, E., and Falk-
ner, D. The antibacterial action of penicillin. J. Bact. 43:11—12 ( 1 942) .
425. HoLMAN, W. L. Bacterial associations. In The newer knowledge of bac-
teriology and immunology, edited by E. O. Jordan and I. S. Falk. Chicago,
University of Chicago Press, 1928, pp. 102— 1 19.
426. HoLMAN, W. L., and Meekison, D. M. Gas production by bacterial syner-
gism. J. Infect. Dis. 39: 145-172 (1926).
427. Hoogerheide, J. C. An agent, isolated from a soil bacillus, which inhibits
encapsulation of Friedlander's bacterium and is highly bactericidal for gram-
positive microorganisms. J. Franklin Inst. 229:677-680 (1940).
BIBLIOGRAPHY 297
427a. HooGERHEiDE, J. C. Aiitibiotic substances produced by soil bacteria. Bot.
Rev. 10:599-638 (1944).
428. HooGERHEiDE, J. C. Studies on capsule formation; inhibition of capsule
formation of Klebsiella pneumoniae (Friedlander's bacterium) by an agent
produced by a soil bacillus. J. Bact. 40:41 5— 422 (1940).
429. Hooper, I. R., Anderson, H. W., Skell, P., and Carter, H. E. The
identity of clavacin with patulin. Science 99: 16 (1944).
430. Horrocks, W. H. Further studies on the saprophytic existence of Micro-
coccus melitensis. Reports of the Commission for the Investigation of Medi-
terranean Fever, 1905, Part I, pp. 14-20,
43 1 . Horrocks, W. H. On the duration of life of the Micrococcus melitensis in
unsterillsed soil. Reports of the Commission for the Investigation of Medi-
terranean Fever, 1906, Part IV, pp. 27—31.
432. Horrocks, W. H. On the duration of life of the Micrococcus melitensis
outside the human body. Reports of the Commission for the Investigation
of Mediterranean Fever, 1 90 5, Part I, pp. 5—13.
433. HosKiNS, M. A protective action of neurotropic against viscerotropic yel-
low fever virus in Macacus rhesus. Am. J. Trop. Med. 15:675-680 (1935).
434. HoTCHKiss, R. D. The chemical nature of gramicidin and tyrocidine.
J. Biol. Chem. 141:171-185 (1941).
435. HoTCHKiss, R. D. Gramicidin, tyrocidine, and tyrothricin. In Advances
in enzymology, vol. 4, pp. 153— 199. New York, Intersclence, 1944.
436. HoTCHKiss, R. D., and Dubos, R. J. Fractionation of the bactericidal
agent from cultures of a soil bacillus. J. Biol. Chem. 132:791-792, 793-
794 (1940); 136:803-804 (1940); 141:155-162 (1941).
437. Houston, A. C. Report on inoculation of soil with particular microbes,
pathogenic and other. Local Govt. Board, Rep. Med. Officer 28:413-438
( I 898-1 899).
438. Houston, A. C. Studies in water supply. New York, Macmillan, 1913.
439. Howard, A., and Wad, Y. D. The waste products of agriculture; their
utilization as humus. Bombay, Oxford, 1931.
440. Huntemuller, O. Vernichtung der Bakterien im Wasser durch Protozoen.
Arch. f. Hyg. 54:89-100 (1905).
441. HiJTTERMANN, W. Beitrage zur Kenntnis der Bakterienflora in normalen
Darmtraktus des Rindes. Diss., Bern (1905); abstract in Koch's Jahresb.
Giirungs. 16:402 (1905).
442. HuMFELD, H., and Feustel, I. C. Utilization of asparagus juice in mi-
crobiological culture media. Proc. Soc. Exper. Biol. & Med. 54:232—235
(1943)-
443. Hunter, A. C, and Randall, W. A. Standardization of assay of penicil-
lin. J. Assn. Off. Agr. Chemists 27:430-438 (1944).
444. Hutchinson, H. P., and Thaysen, A. D. The non-persistence of bac-
teriotoxins in the soil. J. Agr. Sc. 9:43-62 (1918).
445. Hutchison, D., Weaver, R. H., and Scherago, M. The incidence and
significance of microorganisms antagonistic to Escherichia coli in water.
' J. Bact. 45:29 (1943).
298 BIBLIOGRAPHY
446. HuTNER, S. H., and Zahl, P. A. Action of bacterial toxins on tumors;
distribution of tumor-hemorrhage agents among bacterial species. Proc. Soc.
Exper. Biol. & Med, 52:364-368 (1943).
447. Iaroslavski. On the question of microbes— antagonists. Viestnik Bakt,,
Agron. Sta. No. 20, p. 89 (1913). Cited by Nakhimovskaia {diy).
448. IsABOLiNSKi, M. P., and Sobolewa, R. M. Ueber den Antagonismus der
Bakterien. Zentralbl. f. Bakteriol., I, Or., 133:107-110 (1934).
449. Jaarsveld, a. Der Einfluss verschiedener Bodenpilze auf die Virulenz von
Rhizoctonia solani Kuhn. Phytopath. Ztschr. 14:1—75 (1942).
450. Jacobson, K. a. Untersuchungen iiber die Lebensfahigkeit der Cholera-
vibrionen im Meerwasser. Centralbl. f. Bakteriol., I, Or., 56:201—207
(1910).
451. Jansen, E. F., and Hirschmann, D. J. Subtilin; an antibacterial product
of Bacillus subtilis, culturing conditions and properties. Arch. Biochem. 4:
297-309 (1944).
452. Jelinek, B., and Hof, T. L'influence de la nature des aliments azotes
sur le pouvoir pigmentaire du bacille pyocyanique. Ann. Ferment. 4: 141-
160(1938).
453. Jeney, a. von. Experimentelle Untersuchungen iibcr antagonistische Wir-
kuhg innerhalt der Typhus-Coli Gruppe. Ztschr. f. Hyg. u. Infektionskr.
100:47-58 (1923).
454. Jenkins, S. H. Organic manures. Imp. Bur. Soil Sc. (England), Tech.
Commun. 33 (1935)-
455. Jensen, H. L. Bacterial treatment of non-leguminous seeds as an agricul-
tural practice. Australian J. Exper. Biol. & M. Sc. 4: 1 1 7-1 20 (1942).
456. Johnson, D. E. The antibiosis of certain bacteria to smuts and some other
fungi. Phytopath. 21:843-863 (1931); Sixth Internat, Bot. Cong. 2:
221-222 (1935).
457. Johnson, J. Plant virus inhibitors produced by microorganisms. Science
88:552-553 (1938); Phytopath. 31:679-701 (1941).
458. Johnson, J., and Hoggan, I. A. The inactivation of the ordinary tobacco-
mosaic virus by microorganisms. Phytopath. 27:1014-1027 (1937).
459. Johnson, J. R., Bruce, W. F., and Dutcher, J. D. Gliotoxin, the anti-
biotic principle of Gliocladium fimbriatum; production, physical and bio-
logical properties. J. Am. Chcm. Soc. 65:2005-2009 (1943); 66:501,
614-616,617-619,619-621 (1944),
460. Johnson, S. D. The prognosis and treatment of mastitis. Cornell Vet. 34:
99-131 (1944).
460a. Jones, D., Metzger, H. J., Schatz, A., and Waksman, S. A. Control
of gram-negative bacteria in experimental animals by streptomycin. Science
100:103-105 (1944)-
461. Jones, H., Rake, G., and Hamre, D. M. Studies on Aspergillus flavus;
biological properties of crude and purified aspergillic acid. J. Bact. 45:
461-469 (1943)-
462. Jordan, E. O. The production of fluorescent pigment by bacteria. Bot.
Gaz. 27: 19-36 (1899).
BIBLIOGRAPHY 299
463. Jordan, E. O., and Dack, G. M. The effect of CI. sporogcnes on CI. botu-
iinum. J. Infect. Dis. 35:576-580 (1924).
464. Jordan, E. O., Russeli., H. L., and Zeit, F. R. The longevity of the
typhoid bacillus in water. J. Infect. Dis. 1:641-689 (1904).
465. JosLYN, D. A. Penicillin assay; outline of four-hour turbidimetric method.
Science 99:21-22 (1943).
466. JuNGEBLUT, C. W. Studies on the inactivation of diphtheria toxin by vita-
min C (1-ascorbic acid). J. Infect. Dis. 69:70-80 (1941).
467. JuNGEBLUT, C. W., and Sanders, M. Studies of a murine strain of polio-
myelitis virus in cotton rats and white mice. J. Exper. Med. 72:407-436
(1940); 76:127-142 (1942).
468. Kahn, M. C. Anaerobic spore-bearing bacteria of the human intestine in
health and in certain diseases. J. Infect. Dis. 35:423-478 (1924).
469. Kamada, K. Antagonismus und Mutation in Mischkulturen. Zentralbl. f.
Bakteriol., I, Or., 118:316-330 (1930).
470. Karow, E. O., and Foster, J. W. An antibiotic substance from species of
Gymnoascus and Penicillium. Science 99:265—266 (1944).
470a. Karow, E. O., Woodruff, H. B., and Foster, J. W. Penicillic acid
from Aspergillus ochraceus, Penicillium Thomii, and Penicillium suavolens.
Arch. Biochem. 5:279-282 (1944).
471. Kasarnowsky, S., and KuPERMANN, T. Zur Frage des Bakterienantagonis-
mus. Arch. biol. nauk (U.S.S.R.) 35 (3) (1934) ; abstract hi Zentralbl. f.
Bakteriol., I, Ref ., 118:151 (1935).
472. Katser, a. Ein Beitrag zur Anwendung des Antagonismus als biologische
Bekampfungsmethode unter besonderer Berucksichtigung der Gattungen
Trichoderma und Phytophthora. Boll. Staz. Pat. veg. Roma, N.S. 18:
1-134 (1938); 19:75-86 (1939)-
473. Katzman, P. A., Hayes, E. E., Cain, C. K., Van Wyk, J, J., Reithel,
F. J., Thayer, S. A., Doisy, E. A., Gaby, W. L., Carroll, C. J., MuiR,
R. D., Jones, L. R., and Wade, N. J. Clavacin, an antibiotic substance
from Aspergillus clavatus. J. Biol. Chem. 154:475-486 (1944)-
474. Katznelson, H. Bacteriophage in relation to plant diseases. Bot. Rev. 3:
499-521 (1937).
475. Katznelson, H. Inhibition of microorganisms by a toxic substance pro-
duced by an aerobic spore-forming bacillus. Canad. J. Research 20:169—
173 (1942).
476. Katznelson, H. A staining technique for evaluating the toxicity of an
antibiotic substance of microbiological origin. Canad. J. Research 20:602—
608 (1942).
477. Katznelson, H. Survival of microorganisms Introduced into the soil. Soil
Sc. 49:21-35, 83-93, 211-217, 283-293 (1940).
478. Kayukova, N. I., and Kremer, T. A. Development and toxin formation
of Bacillus botulinus in mixed cultures. Microbiologia (U.S.S.R.), 9: 585-
593 (1940).
479. Keeper, C. S., Blake, F. G., Marshall, E. K., Jr., Lockwood, J, S.,
and Wood, W. B., Jr. Penicillin in the treatment of infections; a report
of 500 cases. J.A.M.A. 122: 1217-1224 (1943)-
300 BIBLIOGRAPHY
480. Kempner, W. Ueber den vermeintlichen Antagonismus zwischen dem
Choleravibrio und dem Bacterium coli commune. Centralbl. f, Bakteriol.
17:32-35 (1895).
481. KiESSLiNG, L. E. Biologische Masznahmen zur Unterdriickung des Kartof-
felschorfes. Kiihn Archiv 38: 184-201 (1933).
482. KiMMEi^TiEL, p. Ueber einige biologische Eigenschaft eines Wurzelbazil-
lus. Centralbl. f. Bakteriol,, I, Or., 89:113-115 (1923).
483. KiMMELSTiEL, P. Weitere Versuche uber die bakteriolytische Fahigkeiten
des Bacillus mycoides. Med. Klinik 20:419-421 (1924).
484. King, C. J. Further observations on the natural distribution of the cotton
root rot fungus. Phytopath. 24: 55 1-553 (i934)-
485. King, C. J., Hope, C, and Eaton, E. D. Some microbiological activities
effected in manurial control of cotton root-rot. J. Agr. Research 49: 1093—
1107 (1934); U.S.D.A. Circular 425 (1937).
486. King, C. J., and Loomis, H. F. Experiments on the control of cotton root-
rot in Arizona. J. Agr. Research 32:297-310 (1926).
487. KiRBY, W. M. M. Extraction of a highly potent penicillin inactivator from
penicillin resistant staphylococci. Science 99:452-453 (1944).
488. Kligler, I. J. Investigation on soil pollution and the relation of the vari-
ous types of privies to the spread of intestinal infections. Mon. 15, Rocke-
feller Inst. Medical Research (1921).
489. Klimoff, J. Zur frage der Immunstoffe des Organismus. Ztschr. f . Hyg.
u. Infektionskr. 37: 120 (1901) ; abstract m Centralbl. f. Bakteriol., I, Ref.,
31:319(1901).
490. Klinger, R. Zur Oetiologie der Aktinomykose. Centralbl. f. Bakteriol.,
I, Or., 85:357-359(1921).
491 . Koch, F. E., and Kraemer, E. Ueber den Antagonismus von Colibakterien
gegen Typhusbakterien, Staphylokokken und Streptokokken. Untersuchungen
in vitro. Zentralbl. f. Bakteriol., I, Or., 123:308-318 (1932).
492. KocHOLATY, W. Cultural characteristics of Penicillium notatum in relation
to the production of antibacterial substance; indication of the dual nature of
the antibacterial substance. J. Bact. 44:469-477 (1942).
493. KocHOLATY, W. Purification and properties of the second antibacterial sub-
stance produced by Penicillium notatum. Science 97:186—187 (1943);
Arch. Biochem. 2:73-86 (1943).
494. KoHN, H. L. The effect of propamidine on bacterial growth. Science 98:
224 (1943)-
495. KoNiSHi, K. Effect of soil bacteria on the growth of the root nodule bac-
teria. Mem. Col. Agr., Kyoto Imp. Univ. 16 (1931); J. Sc. Soil Man.
Japan 9:75-82 (1935).
496. KoPAczEWSKi, W. Conditions physico-chimiques de la vitalite micro-
bienne. Arch, microb. 2:187-244 (1931).
497. KoPELOFF, N. Lactobacillus acidophilus. Baltimore, Williams & Wilkins,
1926.
498. KoRiNEK, J. De I'influence des microbes banaux du sol sur les plantes.
Faculte Sci. Univ. Charles 60 (1926).
BIBLIOGRAPHY 301
499. KoROLEV, P. A. Symbiosis and antagonism of bacteria of the Brucella
group with yellow sarcinae and white staphylococci. Zhur. Microb. Epid.
Immun. 22 (4):35-39 (i939)-
500. KosAROFF, P. Beitrag zur Biologic von Pyronema confluens Tul. Arb. k.
biolog. Anstalt Land- u. Forstw. 5:126-138 (1906).
501. Kramer, H. Neue Untersuchungen uber antagonistische Wirkung des Ba-
cillus pyocyaneus. Ztschr. f. Immunitatsforsch. u. exp. Therap. 84:505—
534 (1935)-
502. Krassilnikov, N. a. The phenomenon of autolysis in Actinomycetales.
Microbiologia (U.S.S.R.) 7:708-720, 829-837 (1938).
503. Krassilnikov, N. A., and Garkina, N. P. Influence of bacteria on the
growth of isolated plant roots. Microbiologia (U.S.S.R.) 8:952-958
(1939)-
504. Krassilnikov, N. A., and Koreniako, A. I. The bactericidal substance of
the actinomycetes. Microbiologia (U.S.S.R.) 8:673-685 (1939).
505. Krause, p. Ueber durch Pressung gewonnenen Zellsaft des Bacillus pyo-
cyaneus. Centralbl. f. Bakteriol., I, Or., 31:673-678 (1902).
506. Krencker, E. p. Ueber Baktericidie von Bakterienfiltraten. Inaug. Diss.,
Strassburg (1903).
507. Kriss, a. E. The lysozyme in actinomycetes. Microbiologia (U.S.S.R.)
9:32-38 (1940).
508. Krueger, a. p., et al. Attempts to protect against influenza virus with vari-
ous sulfonamides, acridines and antibiotics. Science 98:348—349 (1943).
509. KusTER, E, Keimung und Entwicklung von Schimmelpilzen in gebrauchten
Nahrlosungen. Ber. Deut. Bot. Gesell. 26a:246-248 (1908).
510. Kunkel, L, O. Immunological studies on the three peach diseases, yel-
lows, rosette and little peach. Phytopath. 26:201-219 (1936).
511. Lal, a. Interaction of soil microorganisms with Ophiobolus graminis Sacc,
the fungus causing the take-all disease of wheat. Ann. App. Biol. 26:247—
261 (1939)-
512. Langer, M. Der antagonistische Index der Colibazillen. Deutsche med.
Wchnschr. 43:1317-1320 (191 7).
513. Lasseur, Ph., Dupaix-Lasseur, A., and Rehn, G. Attempt to explain
the mechanism of certain antagonisms. Trav. lab. microbiol. faculte pharm.
Nancy 7:91-104 (1934),
514. Lasseur, Ph., and Marchal, J. G. Associations bacteriennes. Antago-
nisme-Antibiose. Trav. lab. microbiol. faculte pharm. Nancy 7:75—89
(1934); 9:49-53 (1936).
515. Laszlo, D., and Leuchtenberger, C. A rapid test for tumor growth in-
hibitors. Cancer Research 3:401-410 (1943).
516. Lawrence, C. A. Sterility test for penicillin. Science 98:413-414
(1943); 99:15-16 (1944).
517. Laws, J. P., and Andrews, F. W. Report on the result of investigations of
, the microorganisms of sewage. Reports to the London County Council, Dec.
13, 1894.
302 BIBLIOGRAPHY
518. Leach, L. D., and Davey, A. E. Reducing southern Sclerotium rot of
sugar beets with nitrogenous fertilizers. J. Agr. Research 64: I — 18 (1942).
519. Ledingham, R. J. Antagonism in inoculation tests of wheat with Hel-
minthosporium sativum P.K. & B. and Fusarium culmorum. Sc. Agr. 22:
688-697 (1942).
520. Lee, S. W., Foley, E. J., Epstein, J. A., and Wallace, J. H., Jr. Im-
provements in the turbidimetric assay for penicillin. J. Biol. Chem. 152:
485-486 (1944)-
521. Leemann, A. C. The problem of active plant immunity. Zentralbl. f.
Bakteriol., II, 85:360-376 (193 1 ).
522. Legroux, R., and DjEMiL, K. Sur la lyse du bacille de la morve et du bac.
pyocyanique. Compt. rend. Acad. d. sc. 193:1117-1119 (1931); Ann.
Ferment. 1 : 193 (1935).
523. Lenti, C. The effect of Penicillium chrysogenum on poly-, oligo- and
mono-saccharide. Gior. di batteriol. e Immunol. 24:56—69 (1940); ab-
stracts in Chem. Zentralbl., II, 2759 (1940); Chem. Abstr. 36:5848
(1940).
523a. Lentt;, J. W., Ingraham, N. R., Beerman, H., and Stokes, J. H.
Penicillin in the prevention and treatment of congenital syphilis. J. A.M. A.
126:408-413 (1944).
524. Lewek, T. Ueber den Wachsthumseinfluss nichtpathogener Spaltpilze auf
pathogene. Beitr. path. Anat. 6:277—298 (1890) ; abstract in Centralbl. f.
Bakteriol. 7:107-109 (1890).
525. Lewis, J. M. Bacterial antagonism with special reference to the effect of
Pseudomonas fluorescens on spore-forming bacteria in soils. J. Bact. 17: 89—
103 (1929)-
525a. Lewis, M. R. The failure of purified penicillin to retard the growth of
grafts of sarcoma in mice. Science 100:314-315 (1944).
526. LiESEGANG, R. E. Gegenseitige Wachstumshemmung bei Pilzkulturen.
Centralbl. f. Bakteriol., II, 51:85-86 (1920).
527. LiESKE, R. Morphologic und Biologic der Strahlenpilze. Leipzig, Born-
traeger, 1921, pp. 138-143.
528. LiNFORD, M. B., Yap, F., and Oliveira, J. M. Reduction of soil popu-
lations of the root-knot nematode during decomposition of organic matter.
Soil Sc. 45:127-140 (1938).
529. LioT, A. Culture du bac. pyocyanique sur milieux chimiquement definis.
Ann. Inst. Pasteur 37:234-274 (1923).
530. LiPMANN, F., HoTCHKiss, R. D., and DuBos, R. J. The occurrence of
d-amino acids in gramicidin and tyrocidine. J. Biol. Chem. 141 : 163-169
(1941).
531. LiPMANN, F., and Owen, C. R. The antibacterial effect of enzymatic
xanthine oxidation. Science 98: 246-248 (1943).
532. Lisbonne, M., and Carrere, L. Antagonisme microbien et lyse transmis-
sible. Compt. rend. Soc. de biol. 86: 569-570 (1922) ; 87: 1 01 (1922).
533. LissAUER, M. Uber den Bakteriengehalt menschlicher und tierischer Faces.
Arch, f . Hyg. 58:1 36-1 49 ( 1 906) .
BIBLIOGRAPHY 303
534. L1TTI.E, R. B. The treatment of chronic streptococcic mastitis with various
bactericidal agents. Internat. Assn. Milk Dealers A. Bull. 34:345 (1942).
535. Little, R. B., Dubos, R. J., and Hotchkiss, R. D. Action of gramicidin
on Streptococci of bovine mastitis. Proc. Soc. Exper. Biol. & Med. 44:444-
445 (1940); 45:462-463 (1940); Vet. Digest. 3:111 (1941).
536. Little, R. B., Dubos, R. J., and Hotchkiss, R. D. Gramicidin, novoxil,
and acriflavine for the treatment of chronic form of streptococcic mastitis.
J. Am. Vet. M.A. 98 : i 89-1 99 ( 1 941 ) .
537. Little, R. B., Dubos, R. J., Hotchkiss, R. D., Bean, C. W., and Mil-
ler, W. T. The use of gramicidin and other agents for the elimination of
the chronic form of bovine mastitis. Am. J. Vet. Research 2:305—312
(1941).
538. LocKWooD, J. Progress toward an understanding of the mode of chemo-
therapeutic action of sulfonamide compounds. Univ. Penn. Bicentennial
Conf. Chemotherapy (1941), pp. 9-28.
539. Lodge, R. M., and Hinshelwood, C. N. Physicochemical aspects of bac-
terial growth; conditions determining stationary populations and growth
rates of Bact. lactis aerogenes in synthetic media. J. Chem. Soc. 2:1683—
1697 (1939)-
540. Lode, A. Bacillus pyocyaneus. In Kolle, Kraus and Ullenhut, Handbuch
der pathogenen Mikroorganismen. Vol. 6, p. 163, Jena, Fischer, 1927.
541. Lode, A. Experimentale Untersuchungen iiber Bakterienantagonismus.
Centralbl. f. Bakteriol., I, Or., 33:196—208 (1902); Verhandl. deut.
Naturf. Aerzte (1902).
542. LoHNis, F., and Smith, J. B. Die Veranderungen des Stalldiingers wah-
rend der Lagerung und seine Wirkung im Boden. Fiihlings landw. Ztg. 63:
153 (1914)-
543. LoHRiscH, H. Der Vorgang der Cellulose- und Hemicellulosenverdauung
beim Menschen und der Nahrwerth dieser Substanzen fiir den menschlichen
Organismus. Ztschr. exper. Path. u. Ther. 5:478-539 (1908).
544. LooMis, T. A., Hubbard, R. S., and Neter, E. Inhibition of bacterio-
static action of sulfanilamide by yeast extracts. Proc. Soc. Exper. Biol. &
Med. 47: 159-163 (1941).
545. Luck, M. J., Sheets, G., and Thomas, J. O. The role of bacteria in the
nutrition of protozoa. Quart. Rev. Biol. 6:46-58 (1931).
546. LuRiA, S. E., and Delbruck, M. Interference between inactivated bac-
terial virus and active virus of same strain and of different strains. Arch.
Biochem. 1:207-218 (1942).
547. LuTZ, O. Uber den Einflusz gebrauchter Nahrlosungen auf Keimung und
Entwicklung einiger Schimmelpilze. Ann. Mycol. 7:91—133 (1909).
548. Lyons, C. Penicillin therapy of surgical Infections in the U.S. Army.
J.A.M.A. 123:1007-1018 (1943).
549. Lyons, C. Symposium on management of Cocoanut Grove burns at Massa-
chusetts General Hospital; problems of infection and chemotherapy. Ann.
Surg. 117:894-902 (1943)-
550. McCallan, S. E. a., and Wellman, R. H. Fungicidal versus fungistatic.
Contrib. Boyce Thompson Inst. 12:451-463 (1942).
304 BIBLIOGRAPHY
551. McCallum, G. B., and Kirkpatrick, J. Tuberculosis in the Hebrides;
the rarity of tubercle bacilli in the milk of cows with a note on the viability of
tubercle bacilli preserved in frozen milk. J. Hyg. 34:141—143 (1934).
552. McCoRMACK, R. B. The associative action of some species of actinomyces.
Thesis, Cornell Univ. (1935).
553. McDonald, E. Notes from the Biochemical Research Foundation; further
studies on the bactericidal agents obtained from soil bacilli. J. Franklin Inst.
229:805-815 (1940).
554. Machacek, J. E. Studies on the association of certain phytopathogens.
Macdonald Col. McGill Univ. Tech. Bull. 7 (1928).
555. McIlwain, H. Antibacterial action of two bacterial products of known
structure. Nature 148:628, 758 (1941).
556. McIlwain, H. The anti-streptococcal action of iodinin; naphthaquinones
and anthraquinones as its main natural antagonists. Biochem. J. 37: 265—271
(1943)-
557. McIlwain, H. Bacterial inhibition by aminosulfonic analogs of some natu-
ral aminocarboxylic acids. Brit. J. Exper. Path. 22:148—155 (1941).
558. McIlwain, H. Bacterial inhibition by metabolite analogs; pantoyltaurine;
the antibacterial index of inhibitors. Brit. J. Exper. Path. 23:95—102
(1942).
559. McIlwain, H. Biochemical characterization of the actions of chemothera-
peutic agents; measurement of growth of streptococcal cultures through their
gaseous metabolism, and the effects of pantothenate and pantoyltaurine upon
the metabolism and growth. Biochem. J. 38:97—105 (1944).
560. McIlwain, H. Biochemistry and chemotherapy. Nature 151:270-272
(1943); 153:300-304 (1944); Trans. Faraday Soc. 39=359-367 (i943).
561. McIlwain, H. A nutritional investigation of the antibacterial action of
acriflavine. Biochem, J. 35:1311—1319 (1941).
562. McIntosh, J., and Selbie, F. R. Penicillin in America. Lancet 2:106
(1943)-
563. McKee, C. M., Hamre, D. M., and Rake, G. The action of antibiotics
on organisms producing gas gangrene. Proc. Soc. Exper. Biol. & Med. 54:
211-213 (1943)-
564. McKee, C. M., and Houck, L. Induced resistance to penicillin of cul-
tures of staphylococci, pneumococci and streptococci. Proc. Soc. Exper.
Biol. & Med. 53:33-34 (1943)-
565. McKee, C. M., and MacPhillany, H. B. An antibiotic substance pro-
duced by submerged cultivation of Aspergillus flavus. Proc. Soc. Exper.
Biol. & Med. 53:237-248 (1943).
566. McKee, C. M., and Rake, G. Activity of penicillin against strains of
pneumococci resistant to sulfonamide drugs. Proc. Soc. Exper. Biol. & Med.
51:275-278 (1942); J- Bact. 43:645 (1942)-
567. McKee, C. M., Rake, G., and Houck, C. L. Studies on Aspergillus
flavus; the production and properties of a penicillin-like substance — flava-
cidin. J. Bact. 47: 187-197 (1943)-
568. McKee, C. M., Rake, G., and Menzel, A. E. O. Studies on penicillin;
production and antibiotic activity. J. Immunol. 48: 259-270 (1944).
BIBLIOGRAPHY 305
569. McKiNNEY, H. H. Virus-antagonism tests and their limitations for estab-
lishing relationship between mutants, and nonrelationship between distinct
viruses. Am. J. Bot. 28:770-778 (i940-
570. MacLeod, C. M., Mirick, G. S., and Curnen, E. C. Toxicity for dogs
of a bactericidal substance derived from a soil bacillus. Proc. Soc. Exper.
Biol. & Med. 43:461-463 (1940).
571. McLeod, J. W., and Govenlock, P. The production of bactericidins by
microorganisms. Lancet 200:900-903 (1921).
572. McMahan, J. R. An improved short time turbidimetric assay for penicil-
lin. J. Biol. Chem. 153:249-258 (1944).
573. McWhorter, F. P. The antithetic virus theory of tulip-breaking. Ann.
App. Biol. 25:254-270 (1938).
574. Maddock, E. C. G, Studies on the survival time of the bovine tubercle ba-
cillus in soil, soil and dung, in dung and on grass, with experiments on the
preliminary treatment of infected organic matter and the cultivation of the
organism. J. Hyg. 33:1 03-1 17 ( 1 93 3 ) •
575. Mahoney, J. F., Arnold, R. C, and Harris, A. Penicillin treatment of
early syphilis. Am. J. Pub. Health 33:1387-1391 (1943).
576. Mahoney, J. F., Ferguson, C, Buchholtz, M., and Van Slyke, C. J.
The use of penicillin sodium in the treatment of sulfonamide-resistant gonor-
rhea in men; a preliminary report. Am. J. Syph., Gonor. & Ven. Dis. 27:
525-528 (1943)-
577. Mair, W. Experiments on the survival of B. typhosus in sterilized and un-
sterilized soil. J. Hyg. 8:37-47 (1908).
578. Manil, p. Phenomenes d'antagonisme microbien; actions inhibitrices exer-
cees par des filtrats de culture de quelques champignons. Acta Biol. Belg. i :
509, 512 (1941).
579. Mann, F. E., Heilman, D., and Herrell, W. E. Effect of serum on
hemolysis by gramicidin and tyrocidine. Proc. Soc. Exper. Biol. & Med. 52:
31-33 (1943)-
580. Manolov, D. Antagonism between B. coli and B. typhi in mixed cultures
in vitro. Zhur. Microb. Epid. Immun. 14:524 (1935). Cited by Na-
khimovskaia (62-/).
581. Manteufel. Untersuchungen iiber die "Autotoxine" (Conradi) und ihre
Bedeutung als Ursache der Wachstumshemmung in Bakterienkulturen. Ber-
lin klin. Wchnschr. 43:313-318 (1906).
582. Marie, A. C. Virus rabique et Bacillus subtilis. Compt. rend. Soc. de biol.
92:561-562 (1925).
583. Marmorek, A. La toxine streptococcique. Ann. Inst. Pasteur 16: 169-178
(1902).
584. Marshall, M. S., and Hrenoff, A. K. Bacteriostasis. J. Infect. Dis.
61:42-54 (1937)-
585. Martin, F. E. The eradication of streptococcic mastitis by treatment with
tyrothricin. J. Am. Vet. M. A. 101:23-25 (1942); 102:267-268
(1942); Vet. Med. 38:174-176 (1943).
586. Martin, S. Growth of the typhoid bacillus in soil. Local Govt. Board,
Rep. Med. Officer 27:308-317 (1897-1898); 28:382-412 (1898-
1899); 29:525-548 (1899-1900).
306 BIBLIOGRAPHY
587. Masera, E. Fenomeni di antagonlsmo e antibiosi fra "Bacillus prodigiosus
Fliigge" e "Beauveria Bassiana Vuill." Ann. speriment. agri. 15:117—150
(1934)-
588. Matzuschita, T. Untersuchungen iiber die Mikroorganismcn des mensch-
lichen Kotes. Arch. f. Hyg. 41 : 210-255 (1901).
589. Maurin, C. Konservierung des Antivirus in Verbindung mit verschiedenen
chemischen Substanzen. Zentralbl. f. Bakteriol., I, Or., 130:129—137
(1933)-
590. Mazzeo, M. Suir antagonismo batterico tra il B. bulgaricum e alcuni germi
intestinali. Rassegna internaz. di clin. e terap. 7:329-341 (1926).
591. Meiklejohn, J. The effect of Colpidium on ammonia production by soil
bacteria. Ann. App. Biol. 17:614-637 (1930); 19:584-608 (1932).
592. Melin, E. Zur Frage des Antagonisms zwischen freilebenden Mikro-
organismen. Untersuchungen an Holzschliff. Arch. f. Mikrobiol. 4: 509-
513 (1933); Svensk Skogsv. Tidskr. 3-4:397-616 (1934).
593. Menzel, A. E. O., WiNTERSTEiNER, O., and HooGERHEiDE, J. C. The
isolation of gliotoxin and fumigacin from culture filtrates of Aspergillus
fumigatus. J. Biol. Chem. 152:419-429 (1944).
594. Menzel, A. E. O., Wintersteiner, O., and Rake, G. Note on anti-
biotic substances elaborated by an Aspergillus flavus strain and by an un-
classified mold. J. Bact. 46: 1 09 (1943).
595. Meredith, C. H. The antagonism of actinomyces to Fusarium oxysporum
cubense. Phytopath. 33:403 (1943); 34:426-429 (1944).
596. Metalnikoff, M. S. Utilisation des methodes bacteriologiques dans la lutte
centre les insectes nuisibles. Compt. rend. Soc. de biol. 113:169—172
(1933)5 134:66-68 (1940); Acad. Agr. France Proc. Verb., Jan. 17, pp.
1-7 (1940).
596a. Metchnikoff, E. The prolongation of life. New York, Putnam, 1908.
597. Metzger, H. J., Waksman, S. A., and Pugh, L. H. In vivo activity of
streptothricin against Brucella abortus. Proc. Soc. Exper. Biol. & Med. 51:
251-252 (1942).
598. Meyer, K., Chaffee, E., Hobby, G. L., Dawson, M. H., Schwenk, E.,
and Fleischer, G. On penicillin. Science 96: 20— 21 (1942).
599. Meyer, K., Hobby, G. L., and Chaffee, E. On esters of penicillin.
Science 97:205-206 (1943).
600. Meyer, K., Hobby, G. L., and Dawson, M. H. The chemotherapeutic
effect of esters of penicillin. Proc. Soc. Exper. Biol. & Med. 53: 100-104
(1943)-
601. Meyer, K., Thompson, R., Palmer, J. W., and Khorazo, D. The
purification and properties of lysozyme. J. Biol. Chem. 113:303-309,
479-486 (1936).
602. Meyer, K. F., and Dubovsky, B. J. The distribution of the spores of B.
botulinus in California. J. Infect. Dis. 31:41-55, 56-58, 59-94, 95-99>
100-109 (1923).
603. Michaelis, L., Hill, E., and Schubert, M. P. Die reversible zweistu-
fige Red'uktion von Pyocyanin in Y-Oxyphenazin. Biochem. Ztschr, 255:
66-81 (1932).
BIBLIOGRAPHY 307
604. Millard, W. A. Common scab of potatoes. Ann. App. Biol. 10:70-88
(1923)-
605. Millard, W. A., and Taylor, C. B. Antagonisms of microorganisms as
the controlling factor in the inhibition of scab by green manuring. Ann.
App. Biol. 14:202-215 (1927).
606. Miller, B. F., Abrams, R., Dorfman, A., and Klein, M, Antibacterial
properties of protamine and histone. Science 96:428-430 (1942).
607. Miller, C. P. Action of certain germicides on meningococcus. Proc. Soc.
Exper. Biol. & Med. 49: 197-201 (1942)-
608. Miller, D. K., and Rekate, A. C. Inhibition of growth of Mycobac-
terium tuberculosis by a mold. Science 1 00: 172-173 (1944).
609. Mitchell, R. B., Adams, J. E., and Thom, C. Microbial responses to
organic amendments in Houston black clay. J. Agr. Research 63:527-534
(1941).
610. Mitchell, R. B., Hooton, D. R., and Clark, F. E. Soil bacteriologi-
cal studies on the control of the Phymatotrichum root rot of cotton. J. Agr.
Research 63:535-548 (1941).
611. MoHR, W. Untersuchungen iiber antagonistische Vorgange zwischen Vari-
anten desselben Stammes. Ztschr. f. Hyg. u. Infektionskr. 116:288—294
(1934); Arch. f. Hyg. 116:197 (1936).
612. Molliard, M. Role des bacteries dans la production des peritheces des
Ascobolus. Compt. rend. Acad. d. sc. 136:899-901 (1903).
613. Mom, C. P., and Schaeffer, C. O. Typhoid bacteria in sewage and in
sludge. Sewage Works J. 12:715-737 (1940).
614. MoRAGUES, v., PiNKERTON, H., and Greiff, D. Therapeutic effective-
ness of penicillin in experimental murine typhus infection in dba mice.
J. Exper. Med. 79:431-437 (1944); 80:561-574 (1944)-
615. Morgan, G. T., and Cooper, E. A. The bactericidal action of the qui-
nones and allied compounds. Biochem. J. 15:587-594 (1921)5 J- Soc.
Chem. Ind. 43:352T-354T (1924).
616. Morgan, J. C, and Harvey, D. An experimental research on the viability
of the Bacillus typhosus as excreted under natural conditions by the "chronic
carrier." J. Roy. Army M. Corps 12:587-598 (1909).
617. MoRiTZ, O. Weitere Studien iiber die Ophiobolose des Wiezens. Arb.
Biol. Reichsanst. Land. Forstw. 20:27-48 (1932).
618. MoRiYA, G. Ueber die Umwandlungshypothese und Lebensdauer des Tu-
berkelbacillus. Centralbl. f. Bakteriol., I, Or., 51:480-493 (1909).
619. MosTOVA, P. On the antagonistic index of B. coli in colitis. Zhur. Microb.
Epid. Immun. 44:533 (1935)- Cited by Nakhimovskaia {627).
620. Much, H. Ueber den Bac. cytoliticus. Miinchen. med. Wchnschr. 72:
374(1925)-
621. Much, H., and Sartorius, F. Uber die neuartige Lysine des Mycoides
"Much." Miinchen. med. Klinik 20:345-348 (1924).
622. MuLCHiNOCK, W. J. The role of gramicidin in bovine mastitis therapy.
' Canad. J. Comp. Med. 6:95-101 (1942).
623. Murray, T, J. A study of the bacteriology of fresh and decomposing ma-
nure. Virginia Agr. Exper. Sta. Tech. Bull. 15:93-117 (1917)-
308 BIBLIOGRAPHY
624. Nachimson, L. I. On the antagonistic influence of B. coli on cholera vibrio.
Zhur. Microb. Epid. Immun. 20 (i):70 (1937).
625. Nadson, A., and Adamovic, M. tJber die Beeinflussung der Entwicklung
des Bacillus mycoides Flugge durch seine Stoffwechselprodukte. Bull. Jard.
Bot., St. Petersbourg, 10:154-165 (1910); abstract in Centralbl. f. Bak-
teriol., II, 31:287-288 (1910).
626. Nadson, G. A., and Zolkiewicz, A. I. Spicaria purpurogenes n. sp. Con-
cerning the question of microbial antagonism. Bull. Jard. Bot. Repub. Russe,
Sup. I, 21:1-12, 13-18 (1922).
627. Nakhimovskaia, M. I. Antagonism among bacteria. Microbiologia
(U.S.S.R.) 7:238-264 (1938); 8:1014-1032 (1939).
628. Nakhimovskaia, M. I. The antagonism between actinomycetes and soil
bacteria. Microbiologia (U.S.S.R.) 6:131-157 (1937).
629. Nakhimovskaia, M. I. The influence of bacteria on the germination of rust
spores. Microbiologia (U.S.S.R.) 8:116-121 (1939).
630. Nasir, S. a. Some preliminary investigations on the relationship of protozoa
to soil fertility, with special reference to nitrogen fixation. Ann. App. Biol.
10:122-133 (1923).
631. Naumova, a. N. The influence of grain bacterization on the infection de-
gree of the seedlings of summer wheat by parasitic moulds and on the yield.
Microbiologia (U.S.S.R.) 8: 198-205 (1939).
632. Nencki, M. iJber Mischkulturen. Centralbl. f. Bakteriol. 11:225-228
(1892).
633. Neter, E. Effects of tyrothricin and actinomycin A upon bacteriophage
and bacterial toxins and toxin-like substances. J. Bact. 43:10—11 (1942);
Proc. Soc. Exper. Biol. & Med. 49:163-167 (1942); Science 96:209-210
(1942).
634. Neufeld, p., and Kuhn, H. Untersuchungen uber "direkten" Bakterien-
antagonismus. Ztschr. f. Hyg. u. Infektionskr. 116:95-110 (1934).
635. Newman, A. S., and Norman, A. G. The activity of subsurface soil popu-
lations. Soil Sc. 55:377-392 (1943).
636. Nicholson, S. T., and Hogan, J. F. Effect of lactic acid bacilli on diph-
theria. J.A.M.A. 62:510 (1914).
637. NicoL, H., and Thornton, H. G. Competition between related strains of
nodule bacteria and its influence on infection of the legume host. Proc. Roy.
Soc. 130:32-59 (1941).
638. Nicolaier, a. Beitriige zur Aetiologie des Wundstarrkrampfen. Inaug.
Diss., Gottingen (1885); abstract in Baumgart. Jahresber. 2:270-272
(1886).
639. Nicolaier, A. Ueber infectiosen Tetanus. Deutsche med. Wchnschr. 10:
842-844 (1884).
640. NicoLLE, M. Action du "Bacillus subtilis" sur diverses bacteries. Ann.
Inst. Pasteur 21 :6i 3-621 (1907).
641. NiKiTiNSKY, J. iJber die Beeinflussung der Entwicklung einiger Schim-
melpilze durch ihre Stoffwechselprodukte. Jahrb. Wiss. Bot. 40:1-93
(1904).
BIBLIOGRAPHY 309
642. NissLE, A. Die Heilung der chronischen Obstipationen mit Mutaflor, ihre
Grundlagen und ihre Bedeutung. Miinchen. med. Wchnschr. 76:1745-
1748 (1929).
643. NissLE, A. Ueber die Grundlagen einer neuen ursachlichen Bekampfung
der pathologischer Darmflora. Deutsche med. Wchnschr. 42:1181-1184
(1916); Med. Klinik 2:29 (1918).
644. NovoGRUDSKY, D. M. Antagonistic interrelations among microbes, and bio-
logical methods of combatting fungus diseases of cultivated plants. Adv.
Mod. Biol. (U.S.S.R.) 5:509-536 (1936).
645. NovoGRUDSKY, D. M. Pyronema confluens Tul. et ses rapports reciproques
avec les microorganismes du sol. Bull. Soc. Nat. Moscow, S. Biol. 45:384—
403 (1936).
646. NovoGRUDSKY, D. M. The use of microbes in the fight against fungous dis-
eases of cultivated plants. Bull. Acad. Sc. (U.S.S.R.) 1:277-293 (1936).
647. NovoGRUDSKY, D. M., Berezova, E., Nachimovskaja, M., and Pervia-
KOVA, M. The influence of bacterization of flax-seed on the susceptibility
of seedlings to infection with parasitic fungi. Compt. rend. Acad. d. sc.
(U.S.S.R.) 14:385-388(1937).
648. NovoGRUDSKY, D. M., KoRONENKO, E., and Rybalkina, a. The change
of bacteria after their introduction into the soil. Bull. Microb. Inst. Acad.
Sc. (U.S.S.R.), pp. 1089-1113 (1936).
649. NovY, F. G. Ein neuer anaerober Bacillus des malignen Oedems. Ztschr.
f. Hyg. u. Infektionskr. 17:209-232 (1894).
650. NovY, F. G., Jr. Microbic respiration; so-called aerobic growth of ana-
erobes; potato respiration. J. Infect. Dis. 36:343—382 (1925).
651. NovY, F. G., Jr., Roehm, H. R., and Soule, M. H. Microbic respira-
tion ; compensation manometer and other means for study of microbic respira-
tion. J. Infect. Dis. 36: 109-167 (1925).
652. NovY, F. G., Jr., and Soule, M. H. Microbic respiration; respiration of
tubercle bacillus. J. Infect. Dis. 36:168-232 (1925).
653. Oesterlin, E. J. Experimental studies with pyocyaneus filtrates. J. Im-
munol. 16:359-367 (1929).
654. Okunuki, K. Uber die Beeinflussung des Wachstums der Schimmelpilze
durch die von Rosa-hefen gebildeten Stofi"e. Jap. J. Bot. 5:401-455
(1930-
655. Olitski, L. Ueber die antagonistischen Wirkungen des Bacillus fluorescens
liquefaciens und seine hygienische Bedeutung. Inaug. Diss., Berne (1891).
656. Olitski, L. Ueber die das Ektoplasma schadigenden Substanzen und ihre
Beziehungen zu anderen hemmenden Substanzen in Filtraten alter Kulturen.
Zentralbl. f. Bakteriol., I, Or., 119:223-238 (1930).
657. Orla-Jensen, S., and Snog-Kjaer, A. Uber Faktoren, welche aktivierend
oder hemmend auf die Entwickelung der Milchsaurebakterien wirken. Det.
Kongelige Danske Videnskabernes Selskab. Biol. Skr. 1:5-19 (1940).
658..OSBORN, E. M. On the occurrence of antibacterial substances in green plants.
Brit. J. Exper. Path. 24:227-231 (1943).
310 BIBLIOGRAPHY
659. Osborne, T. B., and Mendel, L. B. The contribution of bacteria to the
feces after feeding diets free from indigestible components. J. Biol. Chem.
18:177-182(1914).
660. Oxford, A. E. Anti-bacterial substances from moulds; the bacteriostatic
powers of the methyl ethers of fumigatin and spinulosin and other hydroxy-,
methoxy- and hydroxymethoxy-derivatives of toluquinone and benzoquinone,
Chem. & Ind. 61: 189-192 (1942).
661. Oxford, A. E. Anti-bacterial substances from moulds; the bacteriostatic
powers of the mould products citrinin and penicillic acid. Chem. & Ind. 61 :
48-51 (1942).
66 1 a. Oxford, A. E. Diplococcin, an anti-bacterial protein elaborated by certain
milk streptococci. Biochem. J. 38:178-182 (1944).
662. Oxford, A. E. On the chemical reactions occurring between certain sub-
stances which inhibit bacterial growth and the constituents of bacteriological
media. Biochem. J. 36:438-444 (1942),
663. Oxford, A. E., and Raistrick, H. Anti-bacterial substances from moulds;
spinulosin and fumigatin, metabolic products of Penicillium spinulosum
Thom and Aspergillus fumigatus Fresenius. Chem. & Ind. 61:128-129
(1942).
664. Oxford, A. E., Raistrick, H., and Smith, G. Anti-bacterial substances
from moulds; penicillic acid, a metabolic product of Penicillium puberulum
Bainier and Penicillium cyclopium Westling. Chem. & Ind. 61:22—34
(1942).
665. Oxford, A. E., Raistrick, H., and Smith, G. Anti-bacterial substances
from moulds; puberulic acid, CgHgOg, and puberulonic acid, CgHj^Og,
metabolic products of a number of species of Penicillium. Chem. & Ind. 61 :
485-487 (1942).
666. Page, J. E., and Robinson, F. A. Polarographic studies; mold metabolites
and related quinones. J. Chem. Soc. 62: I 33— 135 (1943).
667. Palevici, M. S. Sui fattori che determinano I'esaurimento del terreno nella
coltivazione dei batteri. Gior. di batteriol. e Immunol. 2:721—725 (1927).
668. Paley, T., and Osicheva, P. Reciprocal action of Aspergillus niger and
Penicillium luteum purpurogenum Thom and Church. Trudi Nauchno-
Izslied. Inst. Pischtch. Promish. 3 (4): 146-156 (1936).
669. Pane, N. Ueber die bacteriziden, von einigen Milzbrandbacillen, Antago-
nisten-Mikroben ausziehbaren Substanzen. Centralbl. f. Bakteriol., I, Or.,
54:457-461 (1910).
670. Papacostas, G., and Gate, J. Les associations microbiennes, leurs applica-
tions therapeutiques. Paris, Doin, 1928.
671. Passini, F. Ueber die Lebensdauer der Tuberculbacillen in Kulturen anaero-
ber Faulnissbakterien. Wien. klin. Wchenschr. 38:1182-1184 (1925)5
abstract in Centralbl. f. Bakteriol., I, 81:447 (1926).
672. Pasteur, L. Experiences tendant a demontrer que les poules vaccinees pour
le cholera sont refractaires au charbon. Compt. rend. Acad. d. sc. 91:315
(1880).
BIBLIOGRAPHY 311
673. Pasteur, L, Nouvel exemple de fermentation determinee par des animal-
cules infusoires pouvant vivre sans gas oxygene libre, et en dehors de tout
contact avec I'air de I'atmosphere. Compt. rend. Acad. d. sc. 56:416—421,
1 189-1194 (1863).
674. Pasteur, L. Sur les decouvertes relatives a la maladie charbonneuse. Bull.
Soc. Centr. d'Agr. France 38:119-124 (1878).
675. Pasteur, L., and Joubert, J. Charbon et septicemic. Compt. rend. Acad,
d. sc. 85:101-105 (1877).
676. Pawlovvsky, a. Heilung des Milzbrandes durch Bakterien und das Ver-
halten der Milzbrandbacillen im Organismus. Archiv. pathol. Anat. Physiol.
108:494-521 (1887).
677. Pederson, C. S., and Fisher, P. Bacterial activities of vegetable juice.
J. Bact. 47:421-422 (1944)-
677a. Penau, M. H., Levaditi, C, and Hagemann, G. Essais d'extraction
d'une substance bactericide d'origine fungique. Bull. Soc. chim. biol. 25:
406-410 (1943)-
678. Penicillin, 1929-1943. Brit. Med. Bull. 2:1-28 (1944).
679. Peretz, L. H., and Mostova, P. On the interaction of microbes at a dis-
tance. Viestn. Rontgenol. Radiol. 14:352; 17:510. Cited by Nakhimov^
skaia {62 j).
680. Peretz, L. H., Newler, A. J., and Larionow, L. T. Antagonistic ac-
tion of B. coli on tuberculosis and diphtheria bacteria, B. proteus and B. para-
coli. Zhur. Microb. Epid. Immun. (U.S.S.R.) 17:78 (1936).
681. Peretz, L. H., and Slawsky, E. M. Die Bedeutung der normalen Mikro-
flora fur den Organismus. Bakterium coli als Immunitatsfaktor. Ztschr. f.
Immunitatsforsch. u. exper. Therap. 80:520—534 (1933).
68 1 a. Peterson, E. H., and Graham, R. Studies on bovine mastitis; a note on
clavacin therapy in bovine mastitis. Am. J. Vet. Research 5:316-317
(1944).
682. Pfaltzer, a, C. B. Cladosporium cucumerinum, Ell. et Arth. en Corynes-
pova melonis (Coora) Lindau. Diss., Utrecht (1927).
683. Pfalz, G. J. Ueber den Einfluss von Bacterium coli auf pathogene Darm-
keime. Ztschr. f. Hyg. u. Infektionskr. 106:504-514 (1926).
684. Pfeffer, W. The physiology of plants; a treatise upon the metabolism and
sources of energy in plants. 2nd ed.. New York, Oxford, 1900, Vol. I,
pp. 512-514.
685. Pfuhl, E. Vergleichende Untersuchungen iiber die Haltbarkeit der Ruhr-
bacillen und der Typhusbacillen ausserhalb des menschlichen Korpers.
Ztschr. f. Hyg. u. Infektionskr. 40:555-566 (1902).
686. Phillips, R. L., and Barnes, L. H. Development of resistance in staphy-
lococci to natural inhibitory substances (gramicidin). J. Franklin Inst. 233:
396-401 (1942).
687. Philpot, C. H. Growth of paramecium in pure cultures of pathogenic bac-
( teria and in the presence of soluble products of such bacteria. J. Morphol.
46:85-129 (1928).
312 BIBLIOGRAPHY
688. Philpot, F. J. A penicillin-like substance from Aspergillus giganteus
Wehm. Nature 152:725 (1943).
689. Pico, C. E. Accion de la gramicidina (Dubos) sobre el bacilo difterico.
Rev. Inst. Bacteriol. Dept. Nacion. HIg. (Buenos Aires) 10:166-171
(1941).
690. PiNOY, E. Role des bacteries dans le developpement de certaines Myxomy-
cetes. Ann. Inst. Pasteur 21:622-656, 686-700 (1907).
691. Plakidas, A. G. Pythium root rot of strawberries in Louisiana. Phytopath.
20: 121 (1930).
692. PoDWYSSOTZKi, W., and Adamoff, A. Ueber die verschiedene Wirkung
der Pyocyanase auf Mikroben in festen und flussigen Nahrboden. Centralbl,
f. Bakteriol., I, Or., 50:44-46 (1909).
693. PoMERAT, C. M. Effect of direct applications of tyrothricin and allantoin
to cells in vitro. Proc. Soc, Exper. Biol. & Med. 5 i : 345-348 ( 1 942) .
694. PooLE, E. A., and Hinshelwood, C. N. Physicochemical aspects of bac-
terial growth; the influence of toxic substances on growth rate, stationary
population, and fermentation reactions of Bact. lactis aerogenes. J. Chem.
Soc, Dec. I 565-1 572 (1940).
695. Porter, C. L. Concerning the characters of certain fungi as exhibited by
their growth in the presence of other fungi. Am. J. Bot. 11:168—188
(1924) ; Proc. Indiana Acad. Sc. 41 : 149-152 (1932).
696. Porter, C. L., and Carter, J. C. Competition among fungi. Bot. Rev.
4: 165-182 (1938).
697. Potter, M. C. On a method of checking parasitic diseases in plants. J.
Agr. Sc. 3: 102-107 (1908).
698. Powell, H. M., and Jamieson, W. A. Penicillin chemotherapy of mice
infected with Staphylococcus aureus. J. Indiana M.A, 35:361—362 (1942).
699. Powell, H. M., and Jamieson, W. A. Response of sulfonamide-fast pneu-
mococci to penicillin. Proc. Soc. Exper. Biol. & Med. 49:387-389 (1942).
700. Pratt, C. A. The staling of fungal cultures; general and chemical investi-
gation of staling by Fusarium. Ann. Bot. 38:563-594, 599-615 (1924).
701. Pratt, R., et al. Chlorellin, an antibacterial substance from Chlorella. Sci-
ence 99:351-352 (1944)-
702. Prausnik, W. Die Hygiene des Bodens. Handb. der Hygiene 1:520-
562(1911).
703. Prell, H. Zur Frage der biologischen Bekaempfung pathogener Darm-
bakterien durch apathogene. Centralbl. f. Bakteriol., I, Or., 80:225—242
(1918); Ztschr. Hyg. Immunitat. 88:507-528 (1919).
704. Prica, M. Uber die Frage der antibakteriellen Speichelwirkung auf die
kapseltragenden Bakterien. Ztschr. f. Hyg. u. Infektionskr. 119:306-321
(1937)-
705. Pringsheim, E. G. uber die gegenseitige Schadigung und Forderung von
Bakterien. Centralbl. f. Bakteriol., II, 51:72-85 (1920).
706. PsHENiTCHNiKov. On the question of bacteriotherapy of chronic putre-
factive infections of the middle ear. Ural Med. J, 6:61 (1921). Cited by
Nakhitnovskaia (62y).
BIBLIOGRAPHY 313
707. PuLVERTAFT, R. }. V. Local therapy of war wounds; with penicillin.
Lancet 2:341-346 (1943)-
708. PuNTONi, v., and Favia, N. La perte de la virulence du b. tuberculeux
comme suite de son association avec le b. tuberculophile. Soc, internaz. di
microbiol., Boll. d. sez. ital. 6: 158-160 (1934).
709. PuRDY, W. C, and Butterfield, C. T. The effect of plankton animals
upon bacterial death-rates. Am. J. Pub. Health 8:499-505 (191 8).
710. QuoRTRUP, E. R., and Holt, A. L. Detection of potential botulinus-
toxin-producing areas in western duck marshes with suggestions for control.
J. Bact. 41:363-372 (1941).
711. Rahn, O. Ueber den Einflusz der Stoffwechselprodukte auf das Wachstum
der Bakterien. Centralbl. f. Bakteriol., II, 16:417-429, 609-617 (1906).
712. Raistrick, H. The production of quinones by "moulds." J. Soc. Chem.
Ind. 16:293-294 (1938).
713. Raistrick, H., Birkinshaw, J. H., Michael, S. E., Bracken, A., Gye,
W. E., and Hopkins, W. A. Patulin in the common cold; collaborative re-
search on a derivative of Penicillium patulin Bainier. Lancet 2:625—635;
comment p. 684 (1943).
714. Raistrick, H., and Smith, G. Anti-bacterial substances from moulds;
citrinin, a metabolic product of Penicillium citrinum Thom. Chem. & Ind.
60:828-830 (1941).
715. Rake, G., and Jones, H. A rapid method for estimation of penicillin.
Proc. Soc. Exper. Biol. & Med. 54:189-190 (1943).
716. Rake, G., Jones, H., and McKee, C. M. Antiluminescent activity of
antibiotic substances. Proc. Soc. Exper. Biol. & Med. 52:136-138 (1943).
717. Rake, G., McKee, C. M., and Jones, H. A rapid test for the activity of
certain antibiotic substances. Proc. Soc. Exper. Biol. & Med. 51:273—274
(1942).
718. Rakieten, M. L., Rakieten, T. L., and Doff, S. Inhibition of staphy-
lococcus bacteriophage and the virus of vesicular stomatitis. J. Bact. 3 1 :
55-56 (1936).
719. Rammelkamp, C. H. a method for determining the concentration of peni-
cillin in body fluids and exudates. Proc. Soc. Exper, Biol. & Med. 5 1 : 95-97
(1942).
720. Rammelkamp, C. H. Observations on resistance of Staphylococcus aureus
to action of tyrothricin. Proc. Soc. Exper. Biol. & Med. 49:346—350
(1942).
721. Rammelkamp, C. H. Tyrothricin therapy of experimental hemolytic
streptococcal empyema. J. Infect. Dis. 71:40—46 (1942).
722. Rammelkamp, C. H. Use of tyrothricin in the treatment of infection.
War Med. 2:830-846 (1942).
723. Rammelkamp, C. H., and Bradley, S. W. Excretion of penicillin in
man. Proc. Soc. Exper. Biol. & Med. 53:30-32 (1943).
724. Rammelkamp, C. H., and Helm, J. D., Jr. Studies on the absorption of
• penicillin from the stomach. Proc. Soc. Exper. Biol. & Med. 54:324-327
(1943)-
314 BIBLIOGRAPHY
725. Rammelkamp, C. H., and Keeper, C. S. The absorption, excretion and
toxicity of penicillin administered by intrathecal injection. Am. J. Med. Sc.
205:342-350 (1943); J. Clin. Investigation 22:425-438, 649-657
(1943).
726. Rammelkamp, C. H., and Keeper, C. S. Observations on the use of
"gramicidin" (Dubos) in the treatment of streptococcal and staphylococcal
infections. J. Clin. Investigation 20:433—434 (1941).
727. Rammelkamp, C. H., and Maxon, T. Resistance of Staphylococcus au-
reus to the action of penicillin. Proc. Soc. Exper. Biol. & Med. 51:386—
389(1942).
728. Rammelkamp, C. H., and Weinstein, L. Hemolytic effect of tyrothri-
cin. Proc. Soc. Exper. Biol. & Med. 48:211-214 (1941).
729. Rammelkamp, C. H., and Weinstein, L. Toxic effects of tyrothricin,
gramicidin, and tyrocidine. J. Infect. Dis. 71:166-173 (1942).
730. Rao, S. S., and De, S. P. Production of penicillin. Current Sc. 12:209
(1943)-
731. Raper, K. B. Growth and development of Dictyostelium discoideum with
different bacterial associates. J. Agr. Research 55:289—316 (1937).
732. Raper, K. B., Alexander, D. F., and Coghill, R. D. Natural variation
and penicillin production by Penicillium notatum and allied species. J. Bact.
47:17 (1944)-
733. Raubitschek, H., and Russ, V. K. Zur Kenntnis der bakteriziden Eigen-
schaften der Pyocyanase. Centralbl. f. Bakteriol., I, Or., 48:114-122
(1909).
734. Ravn, F. K. Ucber einige Helminthosporium-Arten und die von derselben
hervorgerufenen Krankheiten bei Gerste und Hafer. Ztschr. Pflanzenkr. 1 1 :
1-64 (1901).
735. Reddish, G. F. Methods of testing antiseptics. J. Lab. & Clin. Med. 14:
649-658 (1930-
736. Regnier, J., and Lambin, S. Etude d'un cas d'antagonisme microbien.
Compt. rend. Acad. d. sc. 199: 1682-1686 (1934).
737. Reid, R. D. Some properties of a bacterial-inhibitory substance produced
by a mold. J. Bact. 29:215-220 (1935).
738. Reinhardt, M. O. Das Wachstum der Pilzhyphen. Jahrb. wiss. Bot. 23:
479-563 (1892).
739. Reinking, O. a., and Manns, M. M. Parasitic and other Fusaria counted
in tropical soils. Ztschr. Parasitenk. 6:23—75 (1933); Zentralbl. f. Bak-
teriol., II, 89:502-509 (1934).
740. Remlinger, p., and Nouri, O. Les geloses dites vaccinees. Compt. rend.
Soc. de biol. 65:361-363 (1908).
741. Renaux, E. Sur I'antivirus staphylococcique. Compt. rend. Soc. de biol.
104:129-130 (1930).
742. Rennerpelt, E. Beobachtungen iiber den gegenseitigen Einfluss einiger
Pilze aufeinander. Svensk Botan. Tidskr. 32:332-345 (1938).
743. Rettger, L. F. The antagonism of bacteria and their products to other bac-
teria. J. Infect. Dis. 2:562-568 (1905).
BIBLIOGRAPHY 315
744. Rettger, L. F., and Cheplin, H. A. Treatise on the transformation of
the intestinal flora, with special reference to the implantation of Bacillus
acidophilus. New Haven, Yale University Press, 192 1.
745. Rhines, C. The longevity of tubercle bacilli in sewage and stream-water.
Am-. Rev. Tuberc. 31:493-497 (1935).
746. Rhines, C. The persistence of avian tubercle bacilli in soil and in associa-
tion with soil microorganisms. J. Bact. 29:299—311 (1935).
747. Rhines, C. The relationship of soil protozoa to tubercle bacilli. J. Bact.
29:369-381 (1935)-
748. Richards, A. N. Penicillin; statement released by the Committee on Medi-
cal Research. J.A.M.A. 122:235-236 (1943).
749. Richards, E. H. Note on the effect of temperature on a mixed culture of
two organisms in symbiotic relation. J. Agr. Sc. 29:302-305 (1939).
750. Rizzi, I. Untersuchungen iiber die antagonistische Wirkung von Colibacil-
len verschiedener Herkunft. Ztschr. Hyg. Immunitat. 82:380—385
(1934).
751. RoBER-K, E. C, Cain,C. K., MuiR,R.D., Reithel, F. J., Gaby,W. L.,
VanBruggen, J. T., HoMAN, D. M., Katzman, p. a., Jones, L. R., and
DoiSY, E. A. Penicillin B, an anti-bacterial substance from Penicillium no-
tatum. J, Biol. Chem. 147:47-58 (1943).
752. Robinson, H. Some toxicological, bacteriological and pharmacological prop-
erties of antimicrobial agents produced by soil microorganisms. Thesis, Rut-
gers Univ. (1943).
753. Robinson, H. Toxicity and efficacy of penicillin. J. Pharmacol. & Exper.
Therap. 77:70-79 (i943)-
754. Robinson, H. J., and Graessle, O. E. In vitro and in vivo studies of
gramicidine, tyrothricin and tyrocidine. J. Pharmacol. & Exper. Therap.
76:316-325 (1942).
755. Robinson, H. J., Graessle, O. E., and Smith, D. G. Studies on the tox-
icity and activity of streptothricin. Science 99:540—542 (1944).
756. Robinson, H. J., and Molitor, H. Some toxicological and pharmacologi-
cal properties of gramicidin, tyrocidine and tyrothricin. J. Pharmacol. &
Exper. Therap. 74:75-82 (1942).
756a. Robinson, H. J., and Smith, D. G. Streptothricin as a chemotherapeutic
agent. J. Pharmacol. & Exper. Therap. 81:3 90-40 1 ( 1 944) .
757. Robinson, H. J., and Waksman, S. A. Studies on the toxicity of actino-
mycin. J. Pharmacol. & Exper. Therap. 74:25-32 (1942).
758. Robson, J. M., and Scott, G. I. Local chemotherapy in experimental le-
sions of the eye produced by Staph, aureus. Lancet 1:100—103 (1943);
Nature 149:581-582 (1942).
759. RocHAix, A., and Urtinette, M. Antagonisme du colibacille et des bac-
teries putrides dans les eaux. Compt. rend. Soc, de biol. 106:669—670
(1931).
760. RocHAix, A., and Vieux, G. Antagonisme du Bacille pyocyanique et du
Colibacille dan I'eau d'alimentation. Compt. rend. Soc. de biol. 1 24: 1 11 8—
1119(1937).
316 BIBLIOGRAPHY
761. RoDANiCHE, E. C, and Palmer, W. L. The action of tyrothricin on fecal
streptococci in vitro and in vivo. J. Infect. Dis. 72:154-156 (1943).
762. RoDioNovA, E. A. The influence of metabolism products of micro-organisms
on the development of others. Arch, biol, nauk 30:335-344 (1930).
763. RosLER, F. Die wachstumshemmende bezw. bakterizide Wirkung der Pyo-
cyanase auf verschiedene tierpathogene Bakterien. Prager Archiv. Tiermed.
vergl. Pathol. A 6:217-232 (1926).
764. Roger, G. H. Influence des produits solubles du B. prodigiosus sur I'infec-
tion charbonneuse. Compt. rend. Soc. de biol. 47:375 (1895).
765. Rogers, L. A. The inhibiting effect of Streptococcus lactis on Lactobacillus
bulgaricus. J. Bact. 16:321-325 (1928).
766. Rosen, H. R., and Shaw, L. Studies on Sclerotium rolfsii, with special ref-
erence to the metabolic interchange between soil inhabitants. J. Agr. Re-
search 39:41-61 (1929).
767. Rosenthal, L. La lyse des Bacilles diphteriques effectuee par un strepto-
thrix. Compt. rend. Soc. de biol. 93:77-78 (1925).
768. Rosenthal, L. Sur les lysobacteries thermophiles. Compt. rend. Soc. de
biol. 92:78-79, 472-473 (1925); 93:1569-1570 (1926); Proc. Soc.
Exper. Biol. & Med. 46:448-449 (1941).
769. Rosenthal, L., and Duran Reynals, F. Tyrothrix scaber et flore intesti-
nale. Compt. rend. Soc. de biol. 94:309-310, 1059-1060 (1926).
770. Rosenthal, L., and Ilitch, Z. Sur le pouvoir lytique des filtrats de Ty-
rothrix scaber. Compt. rend. Soc. de biol. 95:10-11 (1926).
771. RuBBo, S. D., and Gillespie, J. M. Para-amino benzoic acid as a bac-
terial growth factor. Nature 146:838-839 (1940).
772. Ruchhoft, C. C. Studies on the longevity of Bacillus typhosus (Eber-
thella typhi) in sewage sludge. Sewage Works J. 6:1054-1067 (1934).
773. Ruehle, G. a. a., and Brewer, C. M. United States food and drug ad-
ministration methods of testing antiseptics and disinfectants. U.S.D.A. Cir-
cular 198 (1931).
774. RuLLMANN, W. Ueber das Verhalten des in Erdboden eingesaten Typhus-
bacillus. Centralbl. f. Bakteriol., I, 30:321-335 (1901).
775. RuscHMANN, G. Natiirlicher und kiinstlicher Stalldiinger. Jauche und
Guile. Handb. Pflanzenern. Dungerl. (Honcamp), 2: 162-234 (193 1 ).
776. Russell, E. J., and Hutchinson, H. B. The effect of partial sterilization
of soil on the production of plant food. J. Agr. Sc. 3: 1 1 1-144 (1909) ;
5:152-221 (1913)-
777. Russell, H. L., and Fuller, C. A. The longevity of B. typhosus in natu-
ral water and sewage. J. Infect. Dis. Sup. 2:40-79 (1906).
778. Rybalkina, a. V. On the toxic substances in soils and their action upon soil
bacteria. Microbiologia (U.S.S.R.) 7:917-932 (1938).
779. Rybalkina, A. V. The vitality of cultures of Azotobacter chroococcum
Bei. in peat. Microbiologia (U.S.S.R.) 7:933-935 (1938).
780. Sallmann, L. von. Penicillin and sulfadiazine in treatment of experimental
intraocular infection with pneumococcus. Arch. Ophth. 30:426 (1943).
BIBLIOGRAPHY 317
781. Sanfelice, F. Der Antagonismus des Milzbrandbacillus gegeniibcr dem
"B. coli." Arch. f. Hyg. 110:348-354 (i933)-
782. Sanford, G. B. Some factors affecting the pathogenicity of Actinomyces
scabies. Phytopath. 16:525-547 (1926).
783. Sanford, G. B. Some soil microbiological aspects of plant pathology. Sc.
Agr. 13:638-641 (1933)-
784. Sanford, G. B., and Broadfoot, W. C. Studies of the effects of other
soil-inhabiting microorganisms on the virulence of Ophiobolus graminis
Sacch. Sc. Agr. 11:512-528 (1931).
785. Sanford, G. B., and Cormack, M. W. Variability in association effects of
other soil fungi on the virulence of Helminthosporium sativum on wheat
seedlings. Canad. J. Research i 8 : 562-566 ( 1 940) .
786. Sartorius. Neuartige Lysine bei Mycoidesbakterien. Centralbl. f. Bak-
teriol., I, Or., 93: 162-167 (1924).
787. Sartory, a. De I'influence d'une bacterie sur la production des peritheces
chez un Aspergillus. Compt. rend. Soc. de biol. 79: 174 (1916).
788. Satoh, S. Studien iiber die Wirkungen der durch Ophiobolus miyabeanus
gebrauchten Nahrlosungen auf die Keimung und Entwicklung eines andern
Pilzes. Mem. Col. Agr., Kyoto Imp. Univ. 13 (1931),
789. Savage, W. G. The self-purification of made-soil. J. Sanit. Inst. (Lon-
don) 24:442-459 ( 1 903-1 904).
790. Savastano, G., and Fawcett, H. S. A study of decay in citrus fruits pro-
duced by inoculations with known mixtures of fungi at different constant
temperatures. J. Agr. Research 39:163-198 (1929).
791. Schales, O. Mode of action of some antibacterial mould products. Arch.
Biochem. 2:487-489 (1943).
792. ScHALK, A. F, Results of some avian tuberculosis studies. J. Am. Vet.
M. A. 72:852-864 (1928).
793. ScHALM, O. W. Treatment of bovine mastitis. J. Am. Vet. M. A. 99: 196
(1941); 100:323-334 (1942).
794. ScHAPiRo, L. Ueber das bactericide Verhalten der Pyocyanase. Hyg.
Rundschau. 18:453 (1908).
795. ScHATZ, A., Bugie, E., and Waksman, S. A. Streptomycin, a substance
exhibiting antibiotic activity against gram-positive and gram-negative bac-
teria. Proc. Soc. Exper. Biol. & Med. 55:66-69 (1944).
796. Scheffler, W. Bakteriologisch-chemische Untersuchungen iiber den
Stalldiinger, speziell uber den Einfluss verschiedener Konservierungsmittel
auf die Bakterienflora und die Glirungsvorgange. Landw. Jahrb. 42:429-
547 (1912).
797. Schiller, I. Sur les produits des microbes en association. Centralbl. f.
Bakteriol., I, Or., 73:123-127 (1914).
798. Schiller, I. Uber erzwungene Antagonisten. Centralbl. f. Bakteriol., I,
Or., 91:68-72 (1924); 92:124-129 (1924); 94:64-66 (1925); 96:
54-56 (1925); 103:304-314 (1927); Compt. rend. Soc. de biol. 105:
< 423-425,550-552(1930).
318 BIBLIOGRAPHY
799. Schilling, C. Antagonismus bei Bacterien. Zentralbl. f, Bakteriol., I,
Or., 127:276-279 (1933)-
800. Schilling, C, and Califano, L. Antagonismus der Bakterienarten. Zen-
tralbl. f. Bakteriol., I, Or., 119:244-246 (1930).
801. Schmidt, B. Untersuchungen iiber das Myzelwachstum der Phycomyceten.
Jahrb. wiss. Bot. 64:509-586 (1925).
802. Schmidt, E. A., and Strassburger, J. Die Fazes des Menschen im nor-
malen und krankhaften Zustande mit besonderer Beriicksichtigung der
klinischen Untersuchungsmethoden. 4th ed. Berlin, Hirschwald, 191 5.
803. Schmidt, L. H., and Sesler, C. L. Development of resistance to penicillin
by pneumococci. Proc. Soc. Exper. Biol, & Med. 52:353-357 (1943).
804. Schmidt, W. H., and Mover, A. J. Penicillin; methods of assay. J. Bact.
47:199-209(1944).
805. Schmitt, G. F. Penicillin; a review of the literature through 1943. Am.
J. Med. Sc. 207:661-678 (1944).
806. ScHNiTZER, R. J., Camagni, L. J., and Buck, M, Resistance of small
colony variants (G-forms) of a Staphylococcus towards the bacteriostatic ac-
tivity of penicillin. Proc. Soc. Exper. Biol. & Med. 53:75-78 (1943).
807. Schoenbach, E. B., Enders, J. F., and Mueller, J. H. The apparent
effect of tyrothricin on Streptococcus hemolyticus in the rhino-pharynx of
carriers. Science 94: 217-21 8 (1941).
808. Schoenbach, E. B., and Seidman, L. R. A selective medium for isolation
of Hemophilus influenzae. Proc. Soc. Exper. Biol. & Med. 49:108-110
(1942).
809. ScHOENTAL, R. The nature of the antibacterial agents present in Pseudo-
monas pyocyanea cultures. Brit. J. Exper. Path. 22:137—147 (1941).
810. Schweinburg, F. Ueber Kulturversuche mit Antivirus. Wien. klin.
Wchnschr. 40:811-813 (1927).
8lOa. Scudi, J. v., and Jelinek, V. C. On the inhibition of urease by peni-
cillin. Science 100:312-313 (1944).
811. Sedgwick, W. T., and Winslow, C.-E. A. Experiments on the viability
of typhoid fever bacilli in earth at various temperatures. Mem. Am. Acad.
Arts & Sc. 12:508-515 (1902).
812. Seit^, A. Misch- und Sekundarinfektion. In Kolle, Kraus and UUenhut,
Handbuch der pathogenen Mikroorganismen. 3d ed., vol. I, pt. i, pp. 505—
522, Jena, Fischer, 1929.
813. Selbie, F. R. The inhibition of the action of sulphanilamide in mice by
p-aminobenzoic acid. Brit. J. Exper, Path. 21:90—93 (1940).
814. Sewertzowa, L. B. Zur Frage nach den mitogenetischen Strahlen. Biol.
Zentralbl. 49:212-225 (1929).
815. Shantz, H, L., and Piemeisel, R, L, Fungus fairy rings in eastern Colo-
rado and their eff"ect on vegetation, J, Agr, Research 11:191-245 (1917).
816. Shaughnessy, H, J., and Winslow, C.-E. A. The diffusion products of
bacterial cells as influenced by the presence of various electrolytes. J. Bact.
14:69-99 (1927).
BIBLIOGRAPHY 319
817. Sherman, J. M., and Hodge, H. M, The bactericidal properties of cer-
tain plant juices. J. Bact. 31 196 (1936).
818. Sherwood, M. B., Falco, E. A., and De Beer, E. J. A rapid, quantita-
tive method for the determination of penicillin. Science 99:247-248
(1944).
819. Sickles, G. M., and Shaw, M. Action of microorganisms from soil on
type-specific and nontype-specific pneumococcus type I carbohydrates. Proc.
Soc. Exper. Biol. & Med. 31:443-445 (1934).
820. Sickles, G. M., and Shaw, M. Activity of soil microorganisms and their
products. New York State Dept. Health, Div. Lab. Res., Annual Report
(1942), pp. 12-13.
821. Sickles, G. M., and Shaw, M. Micro-organisms which decompose the spe-
cific carbohydrate of pneumococcus types II and III. J. Infect. Dis. 53 '3^""
43 (1933)-
822. Sickles, G. M., and Shaw, M. A systematic study of microorganisms
which decompose the specific carbohydrates of the pneumococcus. J. Bact.
28:415-431 (1934)-
823. Silberschmidt, W., and Schoch, E. Contribution a. I'etude de microbes
antagonistes de la bactericide charbon (Bacillus anthracis). Ann. Inst.
Pasteur 34:669-683 (1920).
824. SiLVERTHORNE, N. Penicillin in the treatment of haemolytic staphylococcal
septicaemia. Canad. Med. Assn. J. 49:516-517 (1943).
825. Simmons, S. W. A bactericidal principle in excretions of surgical maggots
which destroys important etiological agents of pyogenic infections, J. Bact.
30:253-267 (1935)-
826. Singh, B. N. Selection of bacterial food by soil flagellates and amoebae.
Ann. App. Biol. 29:18-22 (1942); Nature 149:168 (1942).
827. SiROTiNiN, V. N. Ueber die entwicklungshemmenden Stofi"wechselproducte
der Bakterien und die sog. Retentionshypothese. Ztschr. f. Hyg. u. In-
fektionskr. 4:262 (1888) ; abstract m Centralbl. f. Bakteriol., I, Or., 4:636
(1888).
828. Skinner, C. E., and Murray, T. J. The viability of B. coli and B.
aerogenes in soil. J. Infect. Dis. 38:37-41 (1926).
829. Smith, E. F. Bacteria in relation to plant diseases. Carnegie Inst. Wash.
Pub, 27 (1905).
830. Smith, F. R., and Sherman, J. M. The hemolytic streptococci of human
feces. J. Infect. Dis. 62:186-189 (1938).
831. Smith, J. L. An investigation into the condition aflfecting the occurrence
of typhoid fever in Belfast. J. Hyg. 4:407-433 (1904).
832. Smith, L. D. The bacteriostatic agent of Penicillium chrysogenum. J.
Franklin Inst. 234:396-402 (1942).
833. Smith, L. D., and Hay, T. The effect of penicillin on the growth and
morphology of Staphylococcus aureus. J. Franklin Inst. 233:598-602
(1942).
834., Smith, M. I., and Emmart, E. W. The action of Penicillium extracts in
experimental tuberculosis. Pub. Health Rep. 59:417-423 (1944).
320 BIBLIOGRAPHY
835. Smith, O. The antagonistic action of Bacillus thermophilus, Bacillus sub-
tilis, Escherichia coli-communis and Alcaligenes fecalis on Sarcina lutea. J.
Bact. 36:659-660 (1938).
836. Smith, R., and Kilbourne, I. L. Investigations into the nature, causation
and prevention of Southern cattle fever. U.S.D.A., Bur. of Animal Indus-
try, Bull. I (1893).
837. Smith, R. P. The influence of B. coli on the growth of B. typhosus with
special reference to enrichment by brilliant green in typhoid carriers. J.
Path. & Bact. 26: 122-123 (1923)-
838. Smith, T. Modification, temporary and permanent of the physiological
characters of bacteria in mixed cultures. Trans. Am. Assn. Physicians 9:85—
109 (1894).
839. Snyder, M. L., and Lichstein, H. C. Sodium azide as an inhibiting sub-
stance for gram-negative bacteria. J. Infect. Dis. 67: 113— 115 (1940).
840. SoKOLOFF, V. P., and Klotz, L. J. A bacterial pathogen of the citrus red
scale. Science 94 : 40-4 1(1941).
841. SoLNT^EVA, L. I. On the lysis of phytopathogenic bacteria caused by Myxo-
bacteriales. Microbiologia (U.S.S.R.) 8:700-705 (1939).
842. SoPARKAR, M. B. The vitality of the tubercle bacillus outside the body.
Indian J. M. Research. 4:627—650 (1917).
843. Soule, M. H. Microbic respiration; respiration of Trypanosoma lewisi and
Leishmania tropica. J. Infect. Dis. 36:245—308 (1925).
844. SoYKA, J., and Bandler, A. Die Entwickelung von (pathogenen) Spalt-
pilzen unter dem wechselseitigen Einfluss ihrer Zersetzungsprodukte.
Fortschr. Med. 6:769-773 (1888).
845. Speakman, H. B., and Phillips, J. F. A study of a bacterial association;
the biochemistry of the production of lactic acid. J. Bact. 9:183—197
(1924).
846. Spink, W. W., Ferris, V., and Vivino, J. J. Antibacterial effect of whole
blood upon strains of staphylococci sensitive and resistant to penicillin.
Proc. Soc. Exper. Biol. & Med. 55:210-213 (1944).
847. Spink, W. W., Ferris, V., and Vivino, J. J. Comparative in vitro resist-
ance of staphylococci to penicillin and to sodium sulfathiazole. Proc. Soc.
Exper. Biol. & Med. 5 5 : 207-2 10(1 944) .
847a. Stansfeld, J. M., Francis, A. E., and Stuart-Harris, C. H. Labora-
tory and clinical trials of patulin. Lancet 2: 370-372, 372—375 (1944).
848. Starkey, R. L. Some influences of the development of higher plants upon
the microorganisms in the soil. Soil Sc. 27:319—334, 355—378, 433-444
(1929); 32:367-393 (193O; 45:207-249 (1938).
849. Steinhaus, E. A. The microbiology of insects; with special reference to
the biologic relationships between bacteria and insects. Bact. Rev. 4: 17—57
(1940).
850. Stevens, F. L., and Hall, J. G. A serious lettuce disease (sclerotiniose)
and a method of control. North Carolina Tech. Bull. 8 (191 1 ).
851. Stevens, F. L., and Hall, J. G. Variation of fungi due to environment.
Bot. Gaz. 48:1-30 (1909).
BIBLIOGRAPHY 321
852. Stevens, N. E, Pathological histology of strawberries affected by species of
Botrytis and Rhizopus. J. Agr. Research 6:361-366 (191 6).
853. Stewart, A. G., and Ghosal, S. C. The germicidal action of the activated
sludge process. Indian J. M, Research 16:989-992 (1929).
854. Stheeman, A. A. Die Rolle des Pyocyanins im Stoffwechsel von Pseudo-
monas pyocyanea. Biochem. Ztschr. 191:320—336 (1927).
855. Stokes, J. L., Peck, R. L., and Woodward, C. R., Jr. Antimicrobial ac-
tion of pyocyanine, hemipyocyanine, pyocyanase, and tyrothricin. Proc.
Soc. Exper. Biol. & Med. 5 i : 126-130 (1942).
856. Stokes, J. L., and Woodward, C. R., Jr. Formation of tyrothricin in sub-
merged cultures of Bacillus brevis. J. Bact. 45:29-30 (1943); 46:83-88
(1943)-
857. Stokes, J. L., and Woodward, C. R., Jr. The isolation of soil bacteria
that produce bactericidal substances. J. Bact. 43:253-263 (1942).
858. Stokinger, H. E., Ackerman, H., and Carpenter, C. M. The use of
tyrothricin in culture medium as an aid in the isolation of Neisseria gonor-
rhoeae. J. Bact. 45:31 (1943).
859. Stokvis, C. S. Protozoen und Selbstreinigung. Arch. f. Hyg. 71:46-59
(1909).
860. Stokvis, C. S., and Swellengrebel, N. H. Purification of water by in-
fusoria. J. Hyg. 11:481-486 ( 1 911).
861. Stovall, W. D., Scheid, E., and Nichols, M. S. The influence on the
morphology and staining of B. diphtheriae by growth in mixed cultures with
staphylococcus and streptococcus. Am. J. Pub. Health 13:748—753 (1923).
862. Strasburger, J. Untersuchungen iiber die Bakterienmenge in menschli-
chen Faces. Ztschr. f. klin. Med. 46:413—444 (1902); Miinchen. med.
Wchnschr. 50:2289—2291 (1903).
863. Strauss, H. Cure by penicillin following repeatedly unsuccessful sulfon-
amide therapy in a pregnant woman with gonorrhea. Am. J. Obst. & Gynec.
47:271-272 (1944).
863a. Strong, F. M. Isolation of violacein. Science 100:287 (1944).
864. Stuart, L. S., and Harris, T. H. Bactericidal and fungicidal properties
of a crystalline protein isolated from unbleached wheat flour. Cereal Chem.
19:288-300 (1942).
865. Sturges, W. S., and Rettger, L. F. Bacterial autolysis. J. Bact. 7: 55 i-
577 (1922).
866. Suter, C. M. Relationships between the structure and the bactericidal
properties of phenols. Chem. Rev. 28:269—300 (1941).
867. Sweetman, H. L. The biological control of insects. Ithaca, N. Y., Com-
stock Publishing Co., 1936.
868. Takahashi, W. N. A virus inactivator from yeast. Science 95:586—587
(1942).
869. Tanner, F. W., and Wilson, F. L. Germicidal action of aliphatic alco-
hols. Proc. Soc. Exper. Biol. & Med. 52:138-140 (1943).
870. Tatum, E. L., Peterson, W. H., and Fred, E. B. Effect of associated
growth on forms of lactic acid produced by certain bacteria. Biochem. J.
26:846-852 (1932).
322 BIBLIOGRAPHY
871. Tauber, H., Laufer, S., and Goll, M. A color test for citrinin and a
method for Its preparation. J. Am. Chem. Soc. 64:2228—2229 (1942).
872. Tavernari, L. Die Pyocyanase Emmerich's und Loew's bei dem experi-
mentellen Milzbrand. Centralbl. f. Bakteriol., 31:786—793 (1902).
873. Taylor, C. B. The ecology and significance of the different types of coli-
form bacteria found in water. J. Hyg. 42: 23-45 (1942).
874. Taylor, C. V., and Strickland, A. G. R. Reactions of Colpoda duode-
naria to environmental factors; factors influencing the formation of resting
cysts. Physiol. Zool. 12:219-230(1939).
875. Tervet, I. W. Effect of mixed inocula on the production of seedling
blight in flax. Phytopath. 28:21 (1938).
876. Tey, S. Untersuchungen ueber die Antibiose zwischen den Paratyphus
B-Bazillen und dem Bacillus coli. Ztschr. Hyg. Immunitat. 50:93 (1927);
abstract in Centralbl. f. Bakteriol., I, Ref., 87:454 (1927).
877. Thalhimer, W., and Palmer, B. The bactericidal action of quinone and
other phenol oxidation products as determined by the Rideal-Walker method.
J. Infect. Dis. 9: 172-180 (191 1).
878. Thom, C, and Morrow, M. B. Experiments with mold inoculation in
cotton root-rot areas. Proc. Soil Sc. Soc. Amer. 1:223 (1936).
879. Thomas, A. R., Jr., Levine, M., and Vitagliano, G. R. Simplified pro-
cedures for ascertaining concentration of and bacterial susceptibility to peni-
cillin. Proc. Soc. Exper. Biol. & Med. 55:264-267 (1944).
879a. Thompson, G. J. The clinical use of penicillin in genitourinary infec-
tions. J.A.M.A. 126:403-407 (1944).
880. Thompson, R. Lysozyme and its relation to the antibacterial properties of
various tissues and secretions. Arch. Path. 30:1096—1134 (1940).
881. TiLLETT, W. S., Cambier, M. J., and Harris, W. H., Jr. Sulfonamide-
fast pneumococci ; a clinical report of two cases of pneumonia together with
experimental studies on the effectiveness of penicillin and tyrothricin against
sulfonamide-resistant strains. J. Clin. Investigation 22:249-255 (1943).
882. TiLLETT, W. S., Cambier, M. J., and McCormack, H. E. The treat-
ment of lobar pneumonia and pneumococcal empyema with penicillin.
Bull. New York Acad. Med. 20:142-178 (1944).
883. TiMONiN, M. I. Another mould with anti-bacterial ability. Science 96:
494(1942).
884. Tims, E. C. An actinomycete antagonistic to a Pythium root parasite of
sugarcane. Phytopath. 22:27 (i932)-
885. Tishler, M., Stokes, J. L., Trenner, N. R., and Conn, J. B. Some
properties of gramicidin. J. Biol. Chem. 141:197-206 (1941).
886. Tissier, H., and Martelly. Recherches sur la putrefaction de la viande
de boucherie. Ann. Inst. Pasteur, 16:865-903 (1902).
887. Trawinski, a. Etudes sur la vitalite des bacilles pathogenes du groupe coli-
typhique dans I'eau de mer. Bull, de I'lnst. oceanograph. No, 542, pp. 1—3
(1929).
888. Tripp, L. H., and Lawrence, L. H. Clinical observations on use of grami-
cidin in the treatment of bovine mastitis. Cornell Vet. 32:90—95 (1942).
BIBLIOGRAPHY 323
889. Trueta, J. Treatment of war wounds and fractures. London, Hamish
Hamilton, 1939.
890. Trussell, p. C, and Sarles, W. B. Effect of antibiotic substances upon
Rhizobia. J. Bact. 45:29 (1942),
891. TsucHiYA, H. M., Drake, C.H., Halvorson, H. O., and Bieter, R. N.
An antibacterial substance from a plant. J. Bact. 47:422 (1944).
892. Turner, J. C, Heath, F. K., and Magasanik, B. Inhibition of urease
by penicillin. Nature 152:326 (1943).
893. TuRRO, R., Tarruella, J., and Presta, A. Die Bierhefe bei experi-
mentell erzeugter Streptokokken- und Staphylokokken-infektion. Centralbl.
f. Bakteriol., I, Or., 34:22-28 (1903).
894. TwiTCHELL, D. C. The vitality of tubercle bacilli in sputum. Trans. Nat.
Assn. Study Prev. Tuberc, N. Y. 1:221-230 (1906).
895. Tyner, L. E. The effect of crop debris on the pathogenicity of cereal root-
rotting fungi. Canad. J. Research 18:289-306 (1940).
896. Ungar, J. Synergistic effect of para-aminobenzoic acid and sulphapyridine
on penicillin. Nature 152:245-246 (1943),
897. Upton, M. F. The effect of filtrate of certain intestinal microbes upon bac-
terial growth. J. Bact. 17:315-327 (1929).
898. Urbain, a., and Kowarski, T. Sur I'antagonisme entre divers germes et la
bacteridie charbonneuse. Compt. rend. Soc. de biol. 115:1085-1087
(1934)-
899. Vacek, B. Examination of some conditions necessary for the survival of the
typhoid bacillus in water. Water Pollution Research 6 (8): 272-273
(1933)-
900. V'aerst, K. Immunisierung gegen Milzbrand mit Pyocyanase und Kombi-
nationen derselben. Centralbl. f. Bakteriol., I, Or., 31:293-317 (1902).
901. Valko, E. I., and DuBois, A. S. The antibacterial action of surface active
cations. J. Bact. 47:15-25 (1944).
902. Van Bruggen, J. T., and others. Penicillin B; preparation, purification,
and mode of action. J. Biol. Chem. 148:365-378 (1943).
903. VanCanneyt, J. Action du B. subtilis et de ses secretions sur le bacille de
la tuberculose. Compt. rend. Soc. de biol. 95:878-881 (1926).
904. Van der Poel, J. Overzicht van de thans verkregen resultaten bij het
onderzoek naar den invloed van verschillende meststoffen op de slijmziekte.
Mededel. Deli-Proefst. 2:99 (1938).
905. Van DER Reis. Der Antagonismus zwischen Coli- und Diphtheriebacillen
und der Versuch einer praktischen Nutzanwandung. Ztschr. f . d. ges. exper.
Med. 30:1 (1922); abstract in Centralbl. f. Bakteriol., I, Ref., 75:446-
447 (1922).
906. Van Luijk, A. Antagonism between various microorganisms and different
species of the genus Pythium, parasitizing upon grasses and lucerne. Mede-
del. Lab. Willie Com. Schol. Baarn. 14:43-83 (1938).
907. Van Slyke, C. J., Arnold, R. C, and Buchholtz, M. Penicillin ther-
' apy in sulfonamide-resistant gonorrhea in men. Am. J. Pub. Health 33:
1392-1394(1943).
324 BIBLIOGRAPHY
908. Van Veen, A. G., and Baars, J. K. The constitution of toxoflavin; provi-
sional communication. Rec. trav. chim. 57:248 (1938).
909. Van Veen, A. G., and Mertens, W. K. On the isolation of a toxic bac-
terial pigment. K. Akad. Wetensch. Amsterdam 36:666-670 (1933);
Rec. trav. chim. 53:257-266, 398-404 (1938).
910. Vanni, S. Recherche des variations sur des germes cultives en association
avec le bacille tuberculeux; observations sur une souche de Bact. coli. Soc.
internaz. di Microbiol., Boll, de sez. ital. 5:303-307 (1933).
911. Vasudeva, R. S. Studies in the physiology of parasitism; on the effect of
one organism in reducing the parasitic activity of another. Ann. Bot. 44:
557-564 (1930) ; Indian Jour. Agr. Sc. 6:904-916 (1936) ; 1 1 :422-43i
(1941).
912. Vaudremer, A. Action de I'extrait filtre d'Aspergillus fumigatus sur les
bacilles tuberculeux. Compt. rend. Soc. de bid. 74:278-280, 752-754
(1913)-
913. Vellinger, E. Sur les proprietes spectrales de la pyoflavine qui accom-
pagne normalement la pyocyanine dans les cultures du bacille de Gessard.
Compt. rend. Soc. de biol. 112:306-308 (1933).
914. Verner, A. R., and Altergot, V. F. On the phenomenon of mycophagy.
Compt. rend. Acad. d. sc. (U.S.S.R.) 15:219-224 (1937).
915. Vignati, J. Beitrag zur Kenntnis des Tvphus-Coli-Antagonismus und
seiner Ausniitzung zur Anreicherung der Typhusbazillen. Centralbl. f.
Bakteriol., I, Or., 107:54-69 (1928); Compt. rend. Soc. de biol. 94:212-
213 (1926); 96:212 (1926).
916. Vincent, H. Le bacille du tetanos se multiplie-t-il dans le tube digestif des
animaux.^ Compt. rend. Soc. de biol. 65:12-14 (1908).
917. Vincent, J. G., and Vincent, H. W. Filter paper disc modification of
the Oxford cup penicillin determination. Proc. Soc. Exper. Biol. & Med.
55:162-164 (1944).
918. Wagner, W. Untersuchung der bacteriziden Bestandteile des Bac. pyo-
cyaneus. Ztschr. Hyg. Immunitat. 63:483—491 (1929).
919. Wagner-Jauregg, Von Th. Die neueren biochemischen Erkenntnisse
und Probleme der Chemotherapie. Naturwiss. 31:335—344 (1943).
920. Waksman, S. A. Antagonistic relations of microorganisms. Bact. Rev. 5:
231-291 (1941).
921. Waksman, S. A. Associative and antagonistic effects of microorganisms; his-
torical review of antagonistic relationships. Soil Sc. 43:51-68 (1937).
922. Waksman, S. A. Humus; origin, chemical composition, and importance in
nature. 2d ed. rev. Baltimore, Williams & Wilkins, 1938.
923. Waksman, S. A. The microbe as a biological system. J. Bact. 45:1-10
(1943)-
924. Waksman, S. A. Microbes in a changing world. Scient. Monthly 5 i :422-
427 (1940).
925. Waksman, S. A. Principles of soil microbiology. 2d ed. Baltimore, Wil-
liams & Wilkins, 1932.
BIBLIOGRAPHY 325
926. Waksman, S. A. Production and activity of streptothricin. J. Bact. 46:
299-310 (1943)-
927. Waksman, S. A. Purification and antibacterial activity of fumigacin and
clavacin. Science 99: 220-221 (1944).
928. Waksman, S. A., and Bugie, E. Action of antibiotic substances upon
Ceratostomella ulmi. Proc. Soc. Exper. Biol. & Med. 54:79-82 (1943).
929. Waksman, S. A., and Bugie, E. Strain specificity and production of anti-
biotic substances; Aspergillus flavus-oryzae group. Proc. Nat. Acad. Sc. 29:
282-288 (1943)-
930. Waksman, S. A., Bugie, E., and Reilly, H. C. Bacteriostatic and bac-
tericidal properties of antibiotic substances, with special reference to plant
pathogenic bacteria. Bull. Torrey Bot. Club 71:1 07-2 21(1 944) .
931. Waksman, S. A., Cordon, T. C, and Hulpoi, N. Influence of tempera-
ture upon the microbiological population and decomposition processes in com-
posts of stable manure. Soil Sc. 47:83-113 (1939).
932. Waksman, S. A., and Foster, J. W. Associative and antagonistic effects of
microorganisms; antagonistic effects of microorganisms grown on artificial
substrates. Soil Sc. 43:69-76 (1937).
933. Waksman, S. A., and Geiger, W. B. The nature of the antibiotic sub-
stances produced by Aspergillus fumigatus. J. Bact. 47:391—397 (1944).
934. Waksman, S. A., and Horning, E. S. Distribution of antagonistic fungi
in nature and their antibiotic action. Mycologia 35:47—65 (1943).
935. Waksman, S. A., Horning, E. S., and Spencer, E. L. The production of
two antibacterial substances, fumigacin and clavacin. Science 96:202—203
(1942); J. Bact. 45:233-248 (1943)-
936. Waksman, S. A., Horning, E. S., Welsch, M., and Woodruff, H. B.
The distribution of antagonistic actinomycetes in nature. Soil Sc. 54:281-
296 (1941).
937. Waksman, S. A., and Hotchkiss, M. Viability of bacteria in sea water.
J. Bact. 33:389-400 (1937)-
938. Waksman, S. A., and Hutchings, I. J. Associative and antagonistic effects
of microorganisms; associative and antagonistic relationships in the decom-
position of plant residues. Soil Sc. 43:77-92 (1937).
939. Waksman, S. A., and Lomanitz, S. Contribution to the chemistry of de-
composition of proteins and amino acids by various groups of microorganisms.
J. Agr. Research 30:263-281 (1925).
940. Waksman, S. A., and Nissen, W. On the nutrition of the cultivated mush-
room, Agaricus campestris, and the chemical changes brought about by this
organism in the manure compost. Am. J. Bot. 19:514-537 (1932).
940a. Waksman, S. A., and Reilly, H. C. Strain specificity and production of
antibiotic substances; Penicillium notatum-chrysogenum group. Proc. Nat.
Acad. Sc. 30:99-105 (1944).
941. Waksman, S. A., Robinson, H., Metzger, H. J., and Woodruff, H. B.
Toxicity of actinomycin. Proc. Soc. Exper. Biol. & Med. 47:261-263
1(1941).
326 BIBLIOGRAPHY
942. Waksman, S, a., and Schatz, A, Strain specificity and production of anti-
biotic substances. Proc. Nat. Acad. Sc. 29:74-79 (1943).
943. Waksman, S. A., and Starkey, R. L. Partial sterilization of soil, micro-
biological activities and soil fertility. Soil Sc. 16: 137-157, 247-268, 343-
358 (1923)-
944. Waksman, S. A., and Tishler, M. The chemical nature of actinomycin,
an antimicrobial substance produced by Actinomyces antibioticus. J. Biol.
Chem. 142:519-528 (1942).
944a. Waksman, S. A., Umbreit, W. W., and Cordon, T. C. Thermophilic
actinomycetes and fungi in soils and in composts. Soil Sc. 47:37—61
(1939)-
945. Waksman, S. A., and Woodruff, H. B. Actinomyces antibioticus, a new
soil organism antagonistic to pathogenic and non-pathogenic bacteria. J.
Bact. 42:231-249 (1941).
946. Waksman, S. A., and Woodruff, H. B. Bacteriostatic and bactericidal
substances produced by a soil actinomyces. Proc. Soc. Exper. Biol. & Med.
45:609-614 (1940).
947. Waksman, S. A., and Woodruff, H. B. The occurrence of bacteriostatic
and bactericidal substances in the soil. Soil Sc. 53:233-239 (1942).
948. Waksman, S. A., and Woodruff, H. B. Selective bacteriostatic and bac-
tericidal action of various substances of microbial origin. J. Bact. 43:9—10
(1942); 44:373-384 (1942).
949. Waksman, S. A., and Woodruff, H. B. The soil as a source of micro-
organisms antagonistic to disease-producing bacteria. J. Bact. 40:581-600
(1940).
950. Waksman, S. A., and Woodruff, H. B. Streptothricin, a new selective bac-
teriostatic and bactericidal agent, particularly active against gram-negative
bacteria. Proc. Soc. Exper. Biol. & Med. 49:207-210 (1942).
95 I . Waksman, S. A., and Woodruff, H. B. Survival of bacteria added to soil
and the resultant modification of soil population. Soil Sc. 50:421—427
(1940).
952. Walker, E. L. Some new aspects of the etiology and endemiology of lep-
rosy. J. Prev. Med. 3:167-195 (1929).
953. Ward, H. M. Symbiosis. Ann. Bot. 13:549-562 (1899).
954. Wathelet, M. Recherches bacteriologiques sur les dejections dans la fievre
typhoide. Ann. Inst. Pasteur 9: 252-257 (1895).
955. Wehmer, C. Die Pilzgattung Aspergillus. Mem. Soc. Phys. d'Histoire
Nat. de Geneve. Vol. 33, pt. 2, no. 4 (1901).
956. Weiland, p. Bakterizide Wirkung von Mesentericusfiltraten auf Diph-
theriebazillen. Zentralbl. f. Bakteriol., I, Or., 136:451-456 (1936); 147:
321-334(1941).
957. Weinberg, M., Nativelle, R., and Prevot, A. R. Les microbes anaero-
bies. Paris, Masson, 1937.
958. Weinberg, M., and Otelesco, I. B. proteus des plaies de guerre. Compt.
rend. Soc. de biol. 84: 535-536 (1921).
BIBLIOGRAPHY 327
959. Weindling, R. Association effects of fungi. Bot. Rev. 4:475-496
(1938).
960. Weindling, R. Experimental consideration of the mold toxins of Gllo-
cladium and Trichoderma. Phytopath. 31 :99I-I003 (1941).
961. Weindling, R. Isolation of toxic substances from the culture filtrates of
Trichoderma and Gliocladium. Phytopath. 27:1175-1177 (1937).
962. Weindling, R. Studies on a lethal principle effective in the parasitic action
of Trichoderma lignorum on Rhizoctonia solani and other soil fungi. Phyto-
path. 24:1153-1179 (1934).
963. Weindling, R. Trichoderma lignorum as a parasite of other soil fungi.
Phytopath. 22:837-845 (1932).
964. Weindling, R. Various fungi recently found to be parasitic on Rhizoc-
tonia solani. Phytopath. 24: 1 141 (1934).
965. Weindling, R., and Emerson, O. H. The isolation of a toxic substance
from the culture filtrate of Trichoderma. Phytopath. 26:1068—1070
(1936).
966. Weindling, R., and Fawcett, H. S. Experiments in the control of
Rhizoctonia damping-off of citrus seedlings. Hilgardia 10:1-16 (1936).
967. Weinman, D. Effects of gramicidin and tyrocidine on pathogenic protozoa
and a spirochete. Proc. Soc. Exper. Biol. & Med. 54:38—40 (1943).
968. Weinstein, L., and Rammelkamp, C. H. Study of effect of gramicidin
administered by oral route. Proc. Soc. Exper. Biol. & Med. 48:147-149
(1941)-
969. Welch, H., Grove, D. C, Davis, R. P., and Hunter, A. C. The
relative toxicity of six salts of penicillin. Proc. Soc. Exper. Biol. & Med.
55:246-248 (1944).
970. Welsch, M. Bactericidal substances from sterile culture-media and bac-
terial cultures, with special reference to bacteriolytic properties of actino-
mycetes. J. Bact. 42:801-814 (1941).
971. Welsch, M. Bacterioly tic. properties of actinomyces. Proc. Third Inter-
nat. Cong, for Microbiol. (1939), pp. 260— 261 ; J. Bact. 43:10 (1942);
44:571-588(1942).
972. Welsch, M. La dissolution des germes vivants par les Streptothrix. Compt.
rend. Soc. de biol. 124:573-577 (1937).
973. Welsch, M. Proprietes bacteriolytiques du Streptothrix et sporulation.
Compt. rend. Soc. de biol. 123:1013-1017 (1936); 124:1240-1242
(1937); 125:1053 (1937); 126:244-246,247-249, 1254-1257 (i937h
127:347-349 (1938); 128:795-798, 799-801, 1170-1171, 1172-1174,
1175-1178 (1938); 130:104-107, 797-800, 800-804 (1939); 131:
1296(1939).
974. Welsh, M. F. Studies of crown rot of apple trees. Canad. J. Research 20:
457-490 (1942).
975. Whipple, G. C, and Mayer, A., Jr. On the relation between oxygen
in water and the longevity of the typhoid bacillus. J. Infect. Dis. Sup. 2:
' 76-79 (1906).
328 BIBLIOGRAPHY
976. White, E. C. Antibacterial filtrates from cultures of Aspergillus flavipes.
Proc. Soc. Exper. Biol. & Med. 54:258-259 (1943).
977. White, E. C. Bactericidal filtrates from a mold culture. Science 92: 127
(1940).
978. White, E. C, and Hill, J. J. Studies on antibacterial products formed
by molds; aspergillic acid, a product of a strain of Aspergillus flavus. J.
Bact. 45:433-442 (1943)-
979. Whitehead, H. R. A substance inhibiting bacterial growth, produced by
certain strains of lactic streptococci, Biochem. J. 27:1793—1800 (1933).
980. WiBAUT, N. L., and Moens, I. Het verdwijnen van typhus-bacillen met
water. K. Akad. Wetensch. Amsterdam, Natuurk. 36:129-139 (1927).
981. Wieringa, K. T., and Wiebols, G. L. W. De aardappelschurft en de
heterolyse der schurftparasiet. Tijdschr. Plantenziekten 42:235—240
(1936).
982. Wiesner, B. P. Bactericidal effect of Asperigillus clavatus. Nature 149:
356-357 (1942).
983. WiKULLiL, L. VON. Uber den quantitiven Dextroseabbau in Coli-Para-
typhus-B-Mischzuchten. Ztschr. f. Hyg. u. Infektionskr. 116:11-14
(1934).
984. WiKULLiL, L. VON. Wachstumsverhaltnisse in Bakterienmischpopulationen.
Zentralbl. f. BakterioL, I, Or., 126:488-508 (1932).
985. WiLKiNS, W. H., and Harris, G. C. M. Estimation of the antibacterial
activity of fungi that are difficult to grow on liquid media. Nature 153:
590-591 (1944).
986. WiLKiNS, W. H., and Harris, G. C. M. Investigation into the produc-
tion of bacteriostatic substances by fungi; preliminary examination of 1 00
fungal species. Brit. J. Exper. Path. 23:166-169 (1942); 24:141-143
(1943); Ann. App. Biol. 30:226-229 (1943).
987. Williams, R. S., and Hoy, W. A. The viability of B. tuberculosis (bo-
vlnus) on pasture land, in stored faeces and in liquid manure. J. Hyg. 30:
413-419 (1930).
988. Wilson, S. D., Winfield, G. F., Chen, S. C, and Chao, T. Y. Con-
trol of fecal-borne diseases in North China; chemical nature of Shantung
farm manure. Soil Sc. 49:379-392 (1940).
989. Wilson, U. A new rapid method for penicillin assay. Nature 152:475-
476 (1943)-
990. Winfield, G. F. Studies on the control of fecal-borne diseases in North
China. Chinese Med. J. 51:217-236, 502-518, 643-658, 919-926
(1937); Supplement, pp. 463-486 (1938); 54=233-254 (1938); 56:
265-286 (1939).
991. Winslow, C.-E. A., and Brooke, O. R. The viability of various species
of bacteria in aqueous suspensions. J. Bact. 13:235—243 (1927).
992. Winter, G. Untersuchungen iiber den Einfluss biotischer Faktoren auf die
Infektion des Weizens durch Ophiobolus graminis. Ztschr. Pflanzenk. 1 :
113-134 (1940).
BIBLIOGRAPHY 329
993. Wolf, J. E. Beitr-ige zur Biologic dcs Pfeiffcrschcn Influcnzabazillus.
Mischkulturen-Mischinfektion, Centralbl. f. Bakteriol., I, Or., 84:241-
255 (1920).
994. WoLLMAN, E. Action lytique des staphylocoques vivants sur les staphylo-
coques tues (A propos de la note de A. Gratia). Compt. rend. Soc. de biol.
95:679 (1926); Ann. Inst, Pasteur 56:316-324 (1936).
995. WoLLMAN, E. Recherchessur Tautolyse; lesautolysinesspecifiqucs. Compt.
rend. Acad. d. sc. 198: 1642-1644 (1934)-
996. WoLLMAN, E., and Reynals, F. D. Bacteriophage and autolysis. Compt.
rend. Soc. de biol. 94:1330-1331 (1926).
997. WoLMAN, A. Hygienic aspects of the use of sewage sludge as fertilizer.
Engr. News-Record 92: 198-202 (1924).
998. Wood, F. C. Studies on "damping off" of cultivated mushrooms and its as-
sociation with Fusarium species. Phytopath. 27:85-94 (1937); 29:728-
739 (1939)-
999. WooDHEAD, M. M., and Wood, C. De I'action antidotique exercee par les
liquides pyocyaniques sur le cours de la maladie charbonneuse. Compt.
rend. Acad. d. sc. 109:985-988 (1889).
1000. Woodruff, H. B., and Foster, J. W. Bacterial penicillinase. J. Bact.
47:19 (1944)-
1 00 1. Woodruff, H. B., and Foster, J. W. Cultivation of actinomycetes un-
der submerged conditions, with special reference to the formation of strep-
tothricin. J. Bact. 45: 30 (1943).
1002. Woodruff, H. B., and Foster, J. W, Microbiological aspects of strep-
tothricin; metabolism and streptothricin formation in stationary and sub-
merged cultures of Actinomyces lavendulae. Arch. Biochem. 2:301-315
(1943)-
1003. Woods, D. D. The relation of p-aminobenzoic acid to the mechanism of
the action of sulphanilamide. Brit. J. Exper. Path. 21:74-90 (1940);
Chem. & Ind. 59: 133-134 (1940).
1004. WooLLEY, D. W., and Krampitz, L. O. Reversal by phosphatides of the
antimicrobial action of a crystalline protein from wheat. J. Biol. Chem.
146:273-274 (1942)-
1005. Worpenberg, H. Sind die Grundlagen fuer eine Unterscheidung von
stark und schwach antagonistischen Colibacillen experimentell begruendet?
Tierarzt. Rund. 30:601-603 (1924).
1006. Wrede, F., and Strack, E. Uber das Pyocyanin, den blauen Farbstoflf des
Bacillus pyocyaneus, Ztschr. f. physiol. Chem. 140:1-15 (1924); 181:
58-76 (1929)-
1007. Wright, V. W. M. Treatment of infected wounds by H-i, a new germi-
cidal extract from soil bacilli cultures. J. Franklin Inst. 233:188-198
(1942).
1008. Young, C. C, and Greenfield, M. Observations on the viability of the
Bacterium coli group under natural and artificial conditions. Am. J. Pub.
Health 13:270-273 (1923)-
330 BIBLIOGRAPHY
1009. Zageri, G. Esperlenze sulk concorrenza vitale dei microorganismi e sopra
un nuovo mezzo di profilassi carbonchiosa. Gior. Intern. Scienz. Med.
Napoli 9:617-623 (1887).
1 010. Zahl, p. a., and Hutner, S. H. The occurrence among bacteria of
agents inducing hemorrhage in transplanted tumors. J. Bact. 45:81
(1943)-
lOil. Zeller, S. M., and Schmitz, H. Studies on the physiology of fungi;
mixed cultures. Ann. Missouri Bot. Garden 6:183—192 (1919).
1012. Ziegler, J. E., Jr., and Horsfall, F. L., Jr. Interference between the
influenza viruses; the effect of active virus upon the multiplication of influ-
enza viruses in the chick embryo. J. Exper. Med. 79:361-377 (1944).
1013. Ziegler, J. E., Jr., Lavin, G. I., and Horsfall, F. L., Jr. Interfer-
ence between the influenza viruses; the efi"ect of virus rendered non-infec-
tive by ultraviolet radiation upon the multiplication of influenza viruses in
the chick embryo. J. Exper. Med. 79:379-400 (1944).
1014. ZoBell, C. E. Bactericidal action of sea water. Proc. Soc. Exper. Biol.
& Med. 34: 1 1 3-1 16 (1936).
1015. ZoRZOLi, G. Influenza dei filtrati di alcuni miceti sul Bacillo tubercolare
umano e bovino. Ann. Inst. Carlo Forlanini 4: 208-220, 221-237 (i94o)'
1016. Zukerman, I., and Minkewitsch, I. Zur Frage des bakteriellen An-
tagonismus. Wratschebnoje Delo, No. 7 (1925) ; abstract in Centralbl. f.
BakterioL, I, Ref., 80:483-484 (1925).
INDEX OF MICROORGANISMS
Absidia glauca, 248
Ab. sfinosa, 252
Achromobacter, 139
A . lifolyticuni, qq
A . stutzeri, 1 1 5
Acrostalagmus, 60, 136
Actinomyces, 40, 70, 102, 191, 224
See also Strepomyces
A. a/ bus, 107
A. bovis, 72, 121
Actinomycetaceae, 102
Actinomycet-ales, 102
Actinomycetes. See General Index
A erobacter aero genes, 11, 13, 21, 34,
44, 70, 81, 90, 94, 95> 96, 115'
119, 152, 191, 192, 207
Agrostis, 251 (Fig. 32)
Algae, 6, 9, 54
Alkali genes fecalis, no, 139
Alternaria, 54, 60, 136, 139
^. tenuis, 136
Amebae, 9, 23, 146
Anthrax organism. See General Index
Argas, 149
Ar mill aria, 136
A scoria luTnbricoides, 27
Ascomycetes, 54, 126, 138
Aspergilli, 43, 126
Asfergillus, 9, 48, 124, 135, 137, 142
A. albus, 125, 135
A. candidus, 130, 157
A. clavatus, 124 (Fig. 12), 125, 126,
134, 136, 157, 182, 183
A. flavifes, 130
A. fiavus, 67, 124, 125, 130, 131,
132, 136, 157, 160, 181
A. fumaricus, 126
A. fumigatus, 67, 124 (Fig. 12),
125, 126, 132, 133, 135, 158,
159, 160, 183, 184, 244
Al giganteus, 130, 132, 134, 157,
181
Asfergillus (cont.)
A. nidulans, 1 30, 248
A.niger, 51,94, 130, 136, 137, 138,
153,186
A. oryzae, 124, 130, 131, 185, 186
A . -parasiticus, 130, 132, 157
A. schiemannii, 126
A. terreus, 126
Azotobacter, 16, 43, 44, 109, 257
Az. agile, 119
Az. chroococcum, 16, 106, 119
Az. indicum, 119
Az. vinelandii, 1 06, 115, 119, 214
(Fig. 26)
Bacillus, 22, 81, 139
B. anthracis, 52, 72, 80, 81, 83, 88,
90, 94, 96, 97, 99, I 20, I 39, 209,
221, 222
B. brevis, 49, 53, 67, 68, 82, 83, 87,
158, 159, 160, 167, 190, 262
B. cereus, 13, 22, 60, 81, 94, 115,
119, 192
B. lentimorbus, 149
B. macerans, 115, 119
B. megatherium, 81, 82, 94, 106,
115, 119, 120, 191, 192
B. mesentericus, 22, 49, 53, 82, 83,
84, 85, 86, 87, 106, 115, 139,
140, 163, 164, 165, 187, 213,
241, 263
B. m.esentericus-vulgatus, 83, 85
B. mucosus-cafsulatus, 80
B. mycoides, 53, 59 (Fig. 5), 60, 70,
82, 83, 84, 85, 94, 106, 108, 1 10,
115, 117, 119, 126, 134, 139,
140, 159, 191, 192, 195, 213,
214, 219, 263
B. mycoides-cytoliticus, 85, 86
B. fetasites, 22
332
INDEX OF MICROORGANISMS
Bacillus (cont.)
B. folymyxa, 115, 119
B. fofilliae, 149
B. futrificus verrucosus, 1 00
B. fyocyaneus. See Ps. aerugniosa
B. ramosus, 250 (Fig. 31)
B. simflex, 87, 139, 140, 157, 160,
170, 253, 263
B. subtilis, 22, 59, 60, 61, 69, 70,
7i>73,74>75,8i,82, 83,84, 85,
94, 106, 1 15, 1 16, 1 19, 126, 133,
134, 139. 152, i59> i74> 181,
187, 191, 192, 195, 205, 208,
2i4(Fig. 26),2i5 (Fig. 27), 263
B. suifestifer, 8 3
B. thermofhilus, 8 3
B. tumescens, 22, 106
B. vulgatus, 94
Bacterium, 139, 148, 250 (Fig. 31)
B. acidi lacticiy 99
B. aroideae, 146
B. cazaubon, 148
B. cocovenenans, 171
B. efhestiae, 148
B. gelechiae, 148
B. lactis aeroge?ies, 99
B. fyocyaneum. See Ps. aeruginosa
B. solanacearum, 121
B. termo, 223
B. tyrogenes, 82
B. violaceum, 5 i
Bacteroides, 21
Basidiomycetes, 54, 124
Basisporium, 139
B. gallarum, 1 3 8
Beauveria, 139
Blastomycoides der77ia(itidis, i 3 8
Boofhilus bovis, 1 7
Botrytis, 136
5. tf//«, 136
5. cinerea, 136
Botulinus organism, 29
Brucella, lOO, 202
5r. abortus, 59, 69, 72, lOO, 115,
119, 191, 215, 218, 243
Brucella (cont.)
Br. melitensis, 36, 72, 90, 98, 1 00,
215
Br. suis, 100
C ef halo s for ium, 9, i 26
CefhalotheciuTTi roseum, 136, 139
Ceratostomella, 142
C. «/w/, 78, 141, 249
Cercomonas, 146
Chaetomium, 125, 126, 185
C-^. cochliodes, 130, 157, 185
Chi or el la, 54, 157
Chroniobacterium, 1 6 3
CA. iodinum, 158, 165, 263
CA. violaceum, 81, 94, 159
Citromyces, 136
Cladosforium, 9
Clostridium acetohutylicum, 44
C/. botulinum, 100, 226
C/. butyricum, 100, 119, 219
C/. chauvoei, 29, 89, 93
C/. fall ax, 2 3
C/. granulobacter-fectinovorum, 44
C/. oedematiens, lOO, 230, 236
C/. oedematis, 23, 72
C/. ferfringens, lOO, 230
C/. sefticum, 23, 72, 128, 230, 231
C/. sforo genes, 90, lOO, 225, 226
C/. tetani, 29, 72
C/. welchii, 23, 72, lOO, 115, 151,
191, 214, 215, 230
Colletotrichum, 136, 256
C. gloeosfcn'oides, i i 3
Colfidium, 23
Colfoda, 146
Coniofhora cerebella, 137
Corticium rolfsii, 255
C sadakii, 255
Corynebacteria, 98, 233
CorynebacteriuTn, 106
C. difhtheriae, ']!, 83, 86, 89, 90,
92, 96, 198, 223, 238, 241
Cryftochilum nigricans, 23
INDEX OF MICROORGANISMS
333
Cunningha?nel la, 44, 45
C. elegans, 136
Dematiaceae, 137
Dematium, 136
Deuterofhoma, 136
Dictyostileum discoideufn, 146
D. mucoroides, 50
Diplococci, 89, 90
Diflococcus fneumojiiae, 120, 203,
229, 238
Dothiorella, 139
Dysentery bacteria, 29-30
Eberthella, 81
E. typhi, 94
E. typhosa, 32, 33, 34, 50, 61, 61
69, 80, 81, 83, 85, 86, 89, 9O;
95, 96, 98, 99, 107, 158, 185
209, 223, 225
Entamoeba histolytica, 27
Erzvinia carotovora, 1 1 5
Escherichia coli, 11, 21, 31, 32, 33, 34,
44,47^50, 59> 61, 69, 71,72, 80
81, 83, 84, 86, 89, 90, 92, 94, 95
96, 97, 99, 100, 106, 107, 108
115, 116, 119, 120, 125, 126
i27> 133, 134, i74> i75> 181
182, 191, 195, 196, 207, 208
210, 211, 215, 217, 225, 236
244
FlavobacteriuTTi, 21, 34
Fluorescent bacteria, 88-89, 93~95
Friedlander's bacillus, 87
Fungi Imperfecti, 54
Fusarium, 9, 40, 60, 63, 105, 114, I 26,
136, 138, 139, 146, 251, 255,
256, 264
Ff. conglutinans, 140
F. culmorum, 247, 251, 255
F. grarninearum, 248, 256
Fusarium (cont.)
F. lateritium, i 36
F. lini, 251, 257
F. main, 247
F. moniliforme, i 3 8
F. oxys forum cubense, 1 1 4
F. sambucinum, 138 (Fig. 15)
F. vasinfectum,, 137
Gajfkya tetragena, 1 1 5
Gambusia, 89
Gas-gangrene organisms, 28-29, 131,
230
Gibber ella, 255
Gleosforium, 60
Gliocladium, 125, 126, 134, 136, 138,
158, 184
Gonococcus, 125, 226
Gy?nnoascus, 134
Helminthosforium, 54, 63, 136, 139,
140, 250 (Fig. 31), 251, 255
H. sativum, 88, 136, 137, 140, 248,
251, 255, 256, 257
H. teres, 136
Hemophilus, 191, 202
H . influenzae, 119
H . pertussis, 1 1 5
H. suis, 1 1 9
Heterodera marioni, 150
Hyphomycetes, 137
Hypochnus centrifugus, 249
H . sasakii, 249
Klebsiella pneumoniae, 72, 90, 94, 99,
207
Lactobacillus acidophilus, 98, 1 00, 125
L. bulgaricus, 52, 90, 98, 99, 1 00
L. casei, 1 1 9
334
INDEX OF MICROORGANISMS
Leish-mania, ij[.j
Leftospira, 147
L. icterohaemorrhagiaey 72
Lucilia sericata^ i 5 i
Macrofhomina fhaseoli, 138
Melanosfora famfeana, 137
Meningococci, 91, 127, 219, 239
Metiingococcus, 226
Micrococci, 89, 90, 98, 108
Micrococcus, 33
M. antibioticus , 98
M. candicans, 21, 22, 106
M. catarrhalis, 226
M. ficwus, 94, 227
M. luteus, 22, 106
M. lysodeikticus, 61, 69, 70, 106,
119, 195
M. roseus, 106
M. ruber, 106
M. tetragenes, 98
Micromonosfora, 102, 104, 105, iii,
112, 158
M. vulgaris, 102 (Fig. 10)
Monascus, 136
Monilia, 136
M. albicans, 1 3 5
M. jnictigena, 255
Mucor, 9, 136
Mucorales, 135
Mycobacteriaceae, 102
Mycobacterium, 102
Af. citreum, 106
Af. /"/^/^Z, 1 06, 119
M. smegmae, 106
M. tuberculosis, 23, 34, 35, 72, 83,
85, 89, 90, 92, 96, 98, 99, 100,
106, 107, 115, 124, 125, 133,
139, 209, 227, 244
My CO gone, 138
Myxobacteria, 90
Myxobacteriales, 99
Myxobacterium, 60, 139
'Neisseria, 81, 125, 191
A'^. catarrhalis, 94, 115, 203
N . flavus, 203
N. gonorrhaeae, 72, 203, 233
iV. intracellular, 203
A'^. meningitiiis, 72, 120
Neoaflectana glaseri, 148
Nocardia, 102, 104, 108, i 1 1
A'', ^/i-^, 1 06
A'^, corallina, 1 06
A'^. gardneri, 105, 112, 114, 120,
I59> 175
A'^. rubra, 106
Oidium, 22
Oikomonas, 146
O. termo, 146
Ofhiobolus, 40, 136, 139, 252
O. graminis, 137, 247, 248, 253
O. miyabeanus, 138
Paratyphoid bacteria, 86, 90
Pasteur ell a, 191
F. avicida, 90
?. />^j/w, 72, 86, 90, 91, 97, 99, 149
P. fseudotuberculosis, 1 1 9
Pectinofhora gossyfiella, 148
Penicillium, 9,63, 124, 125, 130, 131,
i35> 136, 137, 139. 142, 146,
158, 185, 248
P. africanum, 5 I
P, chrysogenum, i 24 (Fig. 12), 125,
126, 127, 130, 158, 159, 175,
206, 262, 266
P. citreo-roseum, i 30
P. citrinum, 124 (Fig. 12), 125,
157, 181
P. claviforme, 124, 125, 126, 134,
157, 182, 183
P. cyclofium, 124, 125, 158
P. expansum, 126, 134, 157, 182,
251 (Fig. 33)
P. funiculosum, i 26
INDEX OF MICROORGANISMS
335
Pe/iicillium (cont.)
P. luteum, 51, 126, 136
P. luteum- fur fur ogenum, 126, 137
P. notatum, 56, 67, 124 (Fig. 12),
125, 126, 127, 128, 129, 130,
158, 159, 160, 175, 176, 179,
180, 195, 197, 206, 233, 237,
259, 262, 266
P. fatulum, 134, 157, 182
P. fuberulum, 125, 158, 159, 181,
185
P. resticulosum, i 2 5
P. sfinulosum, i 5 9
Pestalozzia, 63
Peziza, 1 3 5, 136
P. sclerotiorum, 1 3 6
P. trifoliorum, 136
Pfeijferella mallei, 89
Phoma terrestris, 247
Phycomycetes, 54, 124, 126, 137
Phymatotrichum, 2 5 i
Ph. omnivorum, 251, 254, 255
Pkytomonas, 81
Ph. bozvlesii, 94
Ph. tumefaciens, 90, 98
Phytofhthora, 136, 139, 140
Ph. cactorum, 249
Ph. erythroseftica, 1 26
Piftocefhalis, 137
Plasmodiofhora brassicae, 247
Plasmodium, 147
Pneumococci, 90, 91, 98, 99, 105, 198,
201, 204, 207, 216, 218, 219,
239, 241, 243
Pneumococcus, 72, 76, 86
Polytoma uvella, 23
Proteus vulgaris, 22, 72, 80, 85, 86, 92,
99, 100, 106, 139, 140,236
Psalliota camfestris, 138
Pseuioeurotium zonatum, 138 (Fig.
15)
P seudomonas , 22, 33, 80
Ps. aeruginosa, 33, 44, 49, 52, 67,
72, 81, 86, 88, 89, 91, 92, 94,
104, 105, 106, 107, 115, 117,
120, 139, 159, 161, 162, 163,
190, 221, 222, 223, 236, 263,
265
Ps. aviseftica, 99
Ps. citri, 146, 251
Ps. destructans, 250
Ps. fuorescens, 10, 13, 22, 44, 45,
53, 80, 81, 88, 89, 91, 94, 97,
106, 115, 119, 121, 140, 141,
191
Ps. hyacinthi, 146
Ps. juglcmdis, 139
Ps. fhaseoli, 139
Ps. futida, 22, 94
Ps. fyocyaneus. See Ps. aeruginosa
Ps. translucens, 139
Pyronema, 16
P. conjiuens, 141
Pythium, 105, 136, 137, 139, 249, 255
P. de Baryanum, 251 (Fig. 33)
P. volutum, 251 (Fig. 32)
Radiobacter, 106
Rhizobium, 46, 109
Rh. legU7ninosarum, 106
Rh'^zoctonia, 40, 136, 139, 140, 185,
215, 253, 254 (Fig. 34), 255,
257,258
R. solani, 87, 138, 252, 253, 258
Rhizofus, 9, 44, 45, 48, 142
Rhodococcus, 81
R. cinnebareus, 94
R. roseus, 94
Saccharomyces, 139
Sac. cereviseae, 91, 139
Sac. ellifsoideus, 94
Sac. marianus, 94
Sac. fastorianus, 94
Salmonella, 81, 192
5. ahortivoequina, 119
5. aertrycke, 191
S. cholerasuis, 119, 192
336
INDEX OF MICROORGANISMS
Sdmofiella (cont.)
S. enteritidis, 2 2, 94
S. gartnert, "jl
S. faratypki, 72, 90, 97, 120
S. fidlorum, 94
5. schottmulleri, 96, 119, 191, 192
5. suifestifer, 94
S.tyfhi, 72, 73, 120
5. tyfhimurium, 72, 119
Sarcina, 21, 33, 99
S. flava, 22
5. /i^/-^^, 50, 59 (Fig, 5), 61, 69, 70,
83, 94, 104, 106, 115, 119, 126,
140, 151, 191, 192, 195
S. ureae, 1 40
Schrottnia, 60, 139
5. arnericana, 138
5. libertiana, 138, 255
Sclerotium, 255
5. oryzae sativae, 249
S.rolfsii, 136, 137, 250 (Fig. 30)
Serratia, 36, 93, 95
S. marcescens, 53, 80, 81, 82, 88, 89,
91, 93, 94, 104, 106, 115, 119,
139, 140, 191
Shiga bacillus, 30, 97
Shigella, 86, 120
5h. dysenteriae, 72
Sh. gallinarum, 115, 119, 191
Sh. faradysenteriae, 94
Sficaria fur fur 0 genes, 5 1
Sforotrichum, 17
Staphylococci, 82, 85, 89, 90, 91, 98,
99, 105, 115, 200, 201, 202, 203,
220, 224, 225, 227, 233, 235,
236, 243
Stafhylococcus, 182, 228
5. albus, 22, 86, 92, 94, 96, 204, 228
S. aureus, 59, 60, 69, 71, 72, 73, 75,
76, 80, 86, 92, 94, 96, 100, 104,
106, III, 120, 128, 133, 162,
175, 181, 182, 191, 192, 202,
203, 206, 207, 214 (Fig. 26),
215, 216, 227, 229, 231, 233,
235, 238
Staphylococcus (cont.)
S. citreus, 86, 94
5. muscae, 119
5. viridis, 86
Sterigmatocystis, 136
Stomoxys, 149
Streptococci, 23, 72, 90, 91, 98, 105,
107, 115, 157, 198, 201, 218,
220, 224, 225, 227, 229, 233,
235, 236, 237, 238, 239, 243
Streftococcus agalactiae, 242
5. cremoris, 98, 157
S. dysgalactiae, 242
S. enteritidis, 207
5. faecal is, 203
S. hemolyticus, 71, 86, 92, 165, 192,
196, 204, 206, 216, 218, 232,
238
5. lactis, 90, 98, 128
S. mastidis, 98
5. viucosus, 86, 98
5. pyogenes, 21, 22, 72, 77, 97, 104,
120, 128, 203, 207, 228, 231,
239
5. salivarius, 203
5. sefticemiae, 2 1
5. therniophilus, lOO
5. uberis, l^l
S. viridans, 61, 72, 73, 128
Strepto?nyces, 44, 45, 102 (Fig. 10),
104, 105, 108, III, 112, 113,
121, 214, 253
5. albus, 105, 112, 113, 114, 119,
120, 157
S. albus, var. ochraleuceus, 1 1 2
S. annul atus, 1 1 2
5'. antibioticus, 59 (Fig. 5), 67, 1 02
(Fig. 10), 105, 112, 114, 116,
121, 157, 160, 171
5. aurantlacus, 106
5". aureus, i i 2
5. bovis, I I 2
5. californicus, 112, 115
S. candidus, i i 2
5. cellulosae, 112, 115
INDEX OF MICROORGANISMS
337
Streptomyces (cont.)
Tric/wdermay 9, 44, 45, 69, 125, 126,
5. coelicolor, 1 1 3
134, 138, 142, 184, 185, 215,
5. cretaceus, 1 1 2
248, 250 (Fig. 30), 253, 254
5". f radii, 1 1 2
(Fig. 34), 255
S. globisforusy 106
T. lignoruniy 136, 138 (Fig. 15),
5. griseus, 106, 112, 113,
114,
^ ' 7>
251, 252, 253, 255, 256
159
T. viridisy 252, 255
5. halstedii, 1 1 3
TrichomastriCy 23
S. hominisy 1 1 3
Trichomonas y 23
S. lavendulae, 102 (Fig.
10),
105,
Trie hot hecium roseum, 2 5 i
112, 114, 116, 117,
119,
159.
Tryfanosomdy 147
i73> 174
T. equiferduniy 227
5. lifmaniiy 1 1 3
Tylenchus Priticiy 150
5. microflavus, 1 1 3
Typhoid bacteria. See General Index
S. odorifer, 1 1 3
Tyrothrixy 82
S. fraecoxy 105, 1 13, 121,
, 122
5. reticuliy 1 1 2
5. roseusy 112
Us til a go, 139
5. rz^^r, 1 1 2
U. avenae, 140
5. rutgersensisy 1 1 3
U. hordei, 140
5. samfsoniiy 1 1 3
C/. ««</(7, I 40
5. safrophiticuSy 112
U. reae, 1 40
S. scabies y 105, 1 1 2, 113,
121,
122,
U. zeae, 60, 136
253
5. setoniiy 1 1 3
5. tetanusemuSy 1 1 3
Verticillium, 136
5. violaceusy 106, 1 10
F. dahliaCy 146
5. violaceus-ruber, 1 1 9
Fi^no commay 36, 50, 72, 80, 81, 82,
Streftomycetaecae-y 102
83, 84, 85, 89, 90, 91, 94, 97,
Streftothrix, 104
98, 162
Synchitrium endobioticuniy 7
^47
F. metchnikoviy 91, 92
Tetramitus rostratuSy 2 3
Thamnidium eleganSy 136
Torula sfhaericCy 94
r. suganiiy 136, 138, 185
TorulofsiSy 136, 137
Treponema palliday 234
Y-bacillus, 86, 92
Yeasts, ^^i? General Index
ZygorhynchuSy 60
Zygorsaccharomyces priorianuSy 94
GENERAL INDEX
See also Index of Microorganisms, page 331
Acridine, 164, 230
Acriflavine, 196
Actinomyces lysozyme, 270
Actinomycetes, 102-103
antagonistic properties of, 1 04-1 14
as antagonists, 102-123
against actinomycetes, I 20-1 2 1
against agents producing plant dis-
eases, I 21-122
against bacteria, 106, 108, 1 1 1-
113, 120
against fungi, 54, 11 3-1 14, 139,
141-142
in soil, 6, 8, 9, 109-1 12
substances produced by, 54, 11 4-1 21,
171-175
in vivo activity of, 122-123
Actinomycetin, 1 07-1 08, 114, 1 20,
263, 270
chemical and biological properties,
157, 160, 161, 171
effect on bacteria, 108
effect on cells, 120, 199
therapeutic value, 123
toxicity, 161
Actinomycin, 114-115, 270
antiluminescent activity, 77
chemical nature, 53, 157, 1 71-173
compared with chemical substances,
70
compared with penicillin, streptothri-
cin, and clavacin, 1 90-1 91
compared with proactinomycin, i 20
compared with tyrothricin, 206
crystals. Fig. 18 (p. 1 70)
differentiation of bacteria by, 220
effect on bacteria. Fig. 5 (p. 59), 77,
171, 206-207, 209-210, 211
by species, 70, 115, 191
effect on cells, 195
effect on fungi, 78, 141
pffect on soil bacteria, 21, 22
in vivo activity, 122
medium, 1 16
Actinomycin {cont.)
neutralizing agent, 219
toxicity, 190, 206, 245
Actinomycin A, 67, 160. See also Ac-
tinomycin
Actinomycin B, 67, 160, 171
Actinomycosis, 28, 225
Adaptive enzymes, 166
Aerobic bacteria as antagonists, 99— lOO
Agar diffusion method of measuring
antibiotic activity, 73-75
Agar method for testing antagonistic ac-
tion, 64
Agar plate-dilution method of measur-
ing antibiotic activity, 69, 71
Alfalfa, decomposition by microorgan-
isms, 44-45
Alfalfa-sick soils, 1 8
Algae, 6, 9, 54
Amebae, 9, 23, 146
Anaerobic bacteria, 22, lOO
Anaxogramic method of testing action
of antagonists, 62
Animal excreta. See Human and ani-
mal wastes
Animal pathogens, survival of, 28-32
Animals, microscopic
as antagonists, 145-146
in soil, 5, 6
Antagonism, 14, 54
defined, 271
types, 47, 49
Antagonist, defined, 271
Antagonistic action, 51-54, Fig. 5 (p.
59) . .
Antagonistic interrelationships among
microorganisms, 38-41, 46-51
Antagonistic microorganisms
isolation and cultivation, 55-64
production of antibiotic substances,
51-54,64-79
isolation and utilization of sub-
stances, 78-79
340
GENERAL INDEX
Antagonistic microorganisms (coni.)
production of antibiotic substances
(cont.)
measurement of activity of sub-
stances, 66- J J
measurement of bactericidal action
of substances, 77-78
measurement of in vivo activity of
substances, 78
methods of growing organisms,
64-66
utilization for disease control, 223-
226
See also Actlnomycetes; Animals, mi-
croscopic; Bacteria; Fungi
Antagonistic substance, defined, 271
Anthrax, 93, lOO
survival of organism in soil, 18, 28,
96
treatment, 221, 222, 223
use of organism in treating diseases
in man, 224
Antibacterial action, inhibition of, 197-
199
Antibiosis, 38-41, 271
Antibiotic, defined, 271
Antibiotic spectrum, defined, 271
Antibiotic substances
as means of differentiation of bac-
teria, 219-220
chemical nature. See Chemical na-
ture of antibiotic substances
classification, i 56-1 6 1
compared with chemical antiseptics,
189-195
defined, 271
Inhibition, 218-219
mode of action, 67, 189-199, 212-
217, 267, 268
produced by actlnomycetes, 54, 114-
121, 171-175
produced by bacteria, 161-171
produced by fungi, 175-185
produced by yeasts, 63, 185-186,
215
production, 64-66
properties, 1 89-1 90
structural formulae, 164
See also name of sfecific substance
Anti-inhibitors, 198, 271
Antiluminescent test for measuring anti-
biotic activity, 76, 77
Antiseptic snuff, penicillin in, 233
Antiseptics, 189-195, 261
Antivirus, 224
Ants, fungi antagonistic to, 249
Aromatic oils, as bacteriostatic agents,
165
Ascaris, 27
Ascorbic acid, effect on actinomycin,
219
Aspergillic acid, 270
antiluminescent activity, 77
chemical nature, 53, 157, 160, 181
effect on bacteria, 77, 125, 131
in experimental infection with CI.
ferfringens, 230
toxicity, 161
Aspergillin, 65, 157, 181
Assay value, 74
Associative interrelationships among mi-
croorganisms, 42-45
Autoantibiosis, 41
Autolysis, 2 1 2
Autophage, 96
Autotoxins, 53
Bacillus Tnesentericus filtrate, bacteri-
cidal action of, 241
Bacillus simplex izctor, 157, 160, 1 70,
270
Bacteria
aerobic and anaerobic, 9, 99-1 01
agents destructive to, 165, 207-213,
215-217
actlnomycetes, 106, 108, 1 1 1-
113, 120
insects, 1 49-1 50
fungi, 124-135
maggots, 151
protozoa, 23, 143-146
substance found in milk, 188
substance produced by yeast, 215
ticks, 149-150
as antagonists, 80-101
against agents producing plant dis-
eases, 138
against bacteria, 82-87, 88, 89-90
GENERAL INDEX
341
Bacteria {cont.)
as antagonists (coni.)
against fungi, 63, 87-88, 139-
142
against insects, 148-149
against protozoa, 143
against viruses, i 5 2
cocci, 97-99
colon-typhoid group, 32-34, 95-97
differentiation by means of anti-
biotic substances, 219—220
effect on tumors, 154-155
fluorescent bacteria, 88-89, 93~95
in fecal matter, 20-23, 84
in soil, 6, 8, 9, 1 1, 247
lysis of, 78, 82, 86, 200
nonspore-forming bacteria, 88-99
spore- forming bacteria, 82-88
substances produced by, 161-171
survival in soil, 28-36
Bacterial agar plate method of isolating
antagonistic microorganisms, 57-
58
Bacterial cell division, interference by
antibiotic substances, 196
Bactericidal, defined, 271
Bactericidal action, methods of measur-
ing, 77-7 8_
Bacterioantagonistic, defined, 271
Bacteriolytic, defined, 271
Bacteriophage, 96, 152, 153, 154, 201
different from lysozyme, i 86
relation to antibiotics, 2 1 3
Bacteriostatic, defined, 271
Bacteriostatic action, 66, 67
inhibition of, 218-219
Bacteriostatic and bactericidal agents.
See Bacteria, agents destructive to
Bacteriostatic spectrum, defined, 271
Bacteriotherapy, 221, 225
Bacterization, 257
Barley infection, 251
Biological conditioning, 196
Biostatic complex, defined, 271
Blackleg organism in soil, 18, 28, 29
Blood cells, hemolysis of
as test of antibiotic activity of tyroth-
ricin, 76
ty gramicidin, 239
by tyrocidine, 239, 240
Blood cells, hemolysis of {co?it.)
by tyrothricin, 200-201, 239, 240
Blood serum extract, inhibition of
gramicidin by, 218-219
Botulinus, 29, 226
Bovine mastitis
survival of organism, 30
treatment with clavacin, 237
treatment with gramicidin, 241-242
Brucella organism, survival of, 36
Bubonic plague organism, survival of,
30
Caecal bacteria, 149
Cancer, rectal, and E. coli, 97
Cationic detergent, 202
Cattle tick, relation to Texas fever, i 7
Cephalin, 218-219
Chaetomin, 125, 157, 160, 185, 270
Chemical composition of soils, 4-5
Chemical nature of antibiotic sub-
stances, 156-188
classification of substances, 1 56-161
microbial lysozyme, 186-187
substances produced by actinomycetes,
171-175
substances produced by bacteria, 161-
171
substances produced by fungi, 175-
185
substances produced by yeasts, 185-
186
Chemist, problems for, 264-265
Chemotherapy, 259, 261, 265-267
Chlorellin, 54, 157, 270
Chlororaphin, 160, 163, 164
Cholera bacteria, survival of, 30, 36
Citrinin, 130, 270
chemical nature, 53, 157, 160, 161,
181
crystals, Fig. 18 (p. 1 70)
effect on bacteria, 125, 131, 207
structural formula, 164
toxicity, 245
Citrus canker, 251
Clavacin, 182-183, 270
antiluminescent activity, 77
chemical nature, 157, 161
compared with fumigacin, 192
342
GENERAL INDEX
Clavacin (conf.)
compared with penicillin, strepto-
thricin, and actinomycin, 1 90-1 91
effect on bacteria, 77, 125, 134, 161,
195, 207, 208
by species, 190, 191, 192
effect on fungi, 141, 249
production, 127, 134, 263-264
structural formula, 164
therapeutic value, 237
toxicity, 245
Clavatin, 134, 157, 270
Claviformin, 125, 130, 134, 157, 183,
270
Clover-sick soils, i 8
Cocci as antagonists, 97-99
Coccidiosis organism, survival in soil, i 8
Colds
treatment with clavacin, 237
treatment with penicillin, 233
Collodion sac method of testing antago-
nistic action, 62
Colon-typhoid bacteria
as antagonists, 95-97
in manure, 21, 22
in soil, 1 1, 32-34
Competition among microorganisms,
45-46
Corylophilline, 158
Cotton root rot, control of, 254
Crowded plate method of isolating an-
tagonistic microorganisms, 58
Cultivation of antagonistic microorgan-
isms, methods, 64-66
Cup method of measuring antibiotic ac-
tivity, 73-75
Cytolytic bacteria, 86
Damping-off disease, 28, 249, 250,
255, 258
Definitions of terms, 271-272
Dehydrogenases, 76, 199, 211, 212,
216
Dermatitis, chronic, treatment with
penicillin, 233
Differentiation of bacteria by means
of antibiotic substances, 219-220
Dilution method of measuring anti-
biotic activity, 69—73
Diphtheria
neutralization of toxin, 223
survival of organism, 30
treatment of carriers with filtrate of
B. meseniericus, 241
Diplococcin, 157, 270
Direct antagonism, 47, 49
Direct microscopic method of determin-
ing abundance of microorganisms
in soil, 7
Direct soil inoculation method of iso-
lating antagonistic microorganisms,
58-59
Disease control, 221-245
of fecal-borne diseases in China, 27
of plant diseases. See under Plants
of soil-borne diseases, 14-15, 246-
toxicity of antibiotic substances, 245
use of antibiotic substances, 226-245
use of microbial antagonists, 223-
226
Disinfectant spectrum, 193-194
Disinfectants, chemical, compared with
antibiotic substances, 189-195
Double plate method of testing antago-
nistic action, 62, 64
Dutch elm disease, 141, 142, 249
Dyes as bacteriostatic agents, 165
Dysentery bacteria, survival of, 29-30
E. coli
and rectal cancer, 97
antagonists of, 33-34
influence of enrichment of soil with
organism, 31
survival of organism, 31, 32-34
E. coli factor, 125, 130, 158, 270
Enzyme action, inhibition by tyrothri-
cin, 199-200
Enzymes acting on polysaccharides, 1 60,
165-167, 197, 216
Equilibrium among soil microorgan-
isms, 40
Eye lesions, treatment with penicillin,
233
Fecal-borne diseases, control of, 27
GENERAL INDEX
343
Fecal residues. See Human and animal
wastes
Fertilizer
effect on microbial population, 8, 27
use for disease control, 255
Filter method of testing antagonistic
action, 61-62
Flavatin, 77, 157, i8l
Flavicidin, 157, 181
Flavicin, 67, 132, 135, 179, 181, 270
chemical nature, 157, 160
effect on bacteria, 125
toxicity, 161
Flax blight, 251
Flax-sick soils, 1 40
Fluorescent bacteria as antagonists, 88-
89> 93-95
Fluorescin, 162
"Forced antagonism" method of isolat-
ing antagonistic microorganisms,
56, 59, 186
Formulae, structural, of antibiotic sub-
stances, 164
Freudenreich's method of testing an-
tagonistic action, 61
Fumigacin, 67, 183-184, 270
antiluminescent activity, 77
chemical nature, 133, 158, 1 60
compared with clavacin, 192
crystals, 133, 158, Fig. 18 (p. 170)
effect on bacteria, 77, 125, 134, 161,
207
by species, 133, 192
effect on fungi, 141
inactivation, 158
medium, 135
toxicity, 245
Fumigatin, 67, 132-133, 182, 270
chemical nature, 53, 133, 158, 1 60,
161
effect on bacteria, 125, 133
structural formula, 164
Fungi
agents destructive to, 78, 137, 141,
142
actinomycetes, 54, 11 3-1 14, 139,
141-142
^ bacteria, 63, 87-88, 139-142
substance produced by yeast, 63,
215
Fungi {cont.)
as antagonists, 124-142
against agents producing plant dis-
eases, I 38
against bacteria, 124-135
against fungi, 63, 135-138
against insects and other animal
forms, 142, 148-150
in fecal matter, 21, 23
in soil, I, 6, 8, 9-10, 137, 247
lysis of, 140
pathogenic to plants, control of, 256-
258
relation to protozoa, 1 46
substances produced by, 175-185
Fungicidal, defined, 271
Fungistatic, defined, 271
Fungus infections
of animals, 17
of human skin, 243
Gangrene, 28-29, 131, 225, 230
Garbage disposal, 26
Gigantic acid, 132, 157, 181, 270
Gliotoxin, 69, 135, 184-185, 270
antiluminescent activity, 77
chemical nature, 53, 133, 158, 160
compared with chemical substances,
70
crystals, 133, Fig. 18 (p. i 70)
effect on bacteria, 77, 125, 195, 207
by species, 70, 133
effect on fungi, 138, 141
medium, i 35
toxicity, 245
Glucose-dehydrogenase, 206
Glucose-oxidase, 1 79, 197
Gonorrhea, treatment with penicillin,
233-234
Gramicidin, 1 67-1 70, 270
antiluminescent activity, 77
chemical nature, 53, 158, 160, 161,
216
compared with chemical substances,
70, 189
compared with penicillin, 192, 203
compared with tyrocidine, 192
crystals. Fig. 18 (p. 170)
344
GENERAL INDEX
Gramicidin (cont.)
eflfect on bacteria, 77, 192, 195, 199-
202, 206, 211, 216
by species, 70, 203
inhibition of, 198, 218-219
mode of action, 196, 216
therapeutic value, 239, 241-243
toxicity, 239, 241, 245
Gramidinic acid, 77
Gram stain and sensitivity to antibiotic
substances, 115, 157-159, 191,
195
Helvolic acid, 133, 158, 184, 245, 270
Hemipyocyanin, 53, 78, 141, 160, 162,
190
Hemolysin production method of meas-
uring antibiotic activity, 76
Hemolytic action. See Blood cells,
hemolysis of
Hetero-antagonism, 48
Heterotrophic bacteria in manure, 22
Human and animal wastes, 19-37
destruction of microorganisms, 27
garbage, 26
manure and fecal residues, 20-25
composition and decomposition,
23-25
microbial population, 20-23, 84
sewage, 25-26
survival of pathogens, 27-36
Humic acids, 4
Humus compounds, effect on actino-
mycin, 219
a-Hydroxyphenazine, 162
Implantation method of testing antago-
nistic action, 62
Inactivator, nontoxic, defined, 271
Inactivators of viruses, 152-153
Indirect antagonism, 47, 49
Infections, wound. See Wound infec-
tions
Influenza organism, 30, 81, 153
Inhibins, 143
Inhibition
of antibacterial action, 197-199
of antibiotic action, 218-219
Inhibitive substance, defined, 272
Inhibitor, defined, 271, 272
Inorganic constituents of soil, 5
Insects
activity of fungi against, 142
bactericidal action of, 149-150
control of, 147-150, 266
in soil, 6, 9
Interference phenomenon, 154
Intestinal disturbances, treatment of,
244
In vivo activities of antibiotic sub-
stances, methods of testing, 78
lodinin, 165, 263, 270
chemical nature, 53, 158
effect on bacteria, 165
inhibition of, 158, 198
structural formula, 164
Iso-antagonism, 48, 99, 213
Isolation of antagonistic microorgan-
isms, methods. See Methods of iso-
lating antagonistic microorganisms
Japanese beetle, control of, 1 48-1 50,
266
Ketones, 161
Key-enzyme, 154
Kojicacid, 164, 185, 270
Lactenin, 156
Lactic acid bacteria, lOO-lOl, 225
Lauryl sulfate, 70, 77
Leguminous plants, 258
Leprosy, 17, 30
Lipoids, 160, 161-165
Liquid media for testing antagonistic
action, 61-62
Lysin of bacteria, 85
Lysis
of bacteria, 78, 82, 86, 200
of fungi, 140
Lysobacteria, 84
Lysogenesis, defined, 272
Lysozyme, 143, 156, 186-187, 270
defined, 272
effect on bacteria, 70, 161
GENERAL INDEX
345
Lysozyme {cotit.)
relation to bacteriophage, 213
solubility, 160, 171
Lytic action
of actinomycetes, 104-109, 120-121
of antibiotic substances, 212
Maggots, bactericidal action of, 151
Malarial parasites, 147
Malignant tumors, 222
Manure. See Human and animal
wastes
Mastitis. See Bovine mastitis
Measurement of antibiotic activity,
methods. See Methods of measur-
ing antibiotic activity
Mechanical separation method of deter-
mining abundance of organisms in
soil, 7
Mechanism of antibiotic action. See un-
der Antibiotic substances
Medium, 61-64, 65
effect of aeration on antibacterial ac-
tivity of fungi, I 26
staling, 41, 52
Meningitis, treatment with penicillin,
233,234
Metabolic processes of cells, interfer-
ence by antibiotic substances, 1 96
Metabolic products, effect of, 61
Methods of determining abundance of
microorganisms in soil, 6-7
Methods of growing organisms for pro-
duction of antibiotic substances,
64-66
Methods of isolating antagonistic mi-
croorganisms, 56-60, 78-79
bacterial agar plate, 57-58
crowded plate, 58
direct soil inoculation, 58-59
"forced antagonism," 56, 59, 186
soil enrichment, 56-57
Methods of measuring antibiotic ac-
tivity, 66-77
agar diffusion or "agar cup," 73-75
agar plate-dilution, 69, 71
antiluminescent test, 76
interference with function, 76
lysis of red blood cells, 76
Methods of measuring antibiotic activity
{cont.)
serial dilution, 71, 73
turbidimetric, 75-76
Methods of measuring bactericidal ac-
tion, 77-78
Methods of testing antagonistic action
of microorganisms, 60-64
Methods of testing in vivo activity of
antibiotic substances, 78
Microbial cell, growth of, 13-14
Microbiologist, problems for, 94, 262-
264
Micromonosporin, 114, 158, 171, 263,
270
Microorganisms
in animal excreta, 20-23, 84
in soil, 1-2, 6-10, II, 36-37, 247
nutrition of, 12-13, 196
See also Actinomycetes; Animals, mi-
croscopic; Antagonistic microor-
ganisms; Bacteria; Fungi; Mixed
cultures
Microscopic methods of determining
abundance of microorganisms in
soil, 7
Milk
bactericidal action, 188
inhibition of gramicidin, 218-219
pasteurized, 225
Milky disease of larvae, 149
Mixed culture inoculation for testing
antagonistic action, 64
Mixed cultures
antagonistic interrelationships, 46-5 i
associative interrelationships, 42-45
competitive interrelationships, 45-46
growth of microbial cells, 13-14, 38,
81, 215-216
mutualistic relationships, 14, 38-41,
43
nature, 41-42
Mixed infections, 222, 223
Mixed population. See Mixed cultures
Mode of action of antibiotic substances.
See under Antibiotic substances
Morphology, 99, 2 1 3-2 1 5
Much-lysin, 85
Mucin, 219
Mushroom fungi, 9
346
GENERAL INDEX
Mutualistic relationships among micro-
organisms, 14, 38-41, 43
Mycoin, I 5 8
Mycolysate, 55, 107, 224
Mycophagy, 124
Mycorrhizal fungi, 249
Mytogenetic rays, 53
Nematodes
control of, 150-151
in control of insect pests, 1 48-1 50
Neocolysin, 224
Nitrite production method for measur-
ing activity of penicillin, 76
Nitrogenous bases, 53
Nitrogenous ring compounds, 53
Nonspore-forming bacteria, 88-99
Notatin, 67, 130, 270
chemical nature, 158, 160, I 80
effect on bacteria, 125
Nutrition of microorganisms, 12-13,
196
Organic bases, 161
Oxford unit, 74, 130
Oxygen supply of soil, 3
a-Oxyphenazine, 92, 93
Para-amino-benzoic acid, eif ect on peni-
cillin, 205-206
Parasiticin, 132, 157, 270
Parasitism, 1 4, 40
Paratyphoid, 86, 90
Partial disinfection of soil, 258
Partial sterilization of soil, 145, 250
Passive antagonism, 49
Pathogenic organisms
in fecal wastes, 23
In soil, 14-15, 16-18, 27-36
Patulin, 134, 157, 182-183, 237, 249,
270
Penatin, 125, I 30, 158, I 80, 270
Penicidin, 125, 131, 158, 185, 270
Penicillamine, 179
Penicillic acid, 1 30, 1 81-182, 198,
270
chemical nature, 158, 160, 161
Penicillic acid (cont.)
effect on bacteria, 125, 131, 207
structural formula, 164
Penicillin, 67, 175-179, 260-261, 270
animal experiments with, 128, 229-
232 ,
antiluminescent activity, 77
chemical nature, 53, 159, 177, 178-
179
compared with
actinomycin, streptothricin, and
clavacin, 1 90-1 91
chemical substances, 70, i 89
gramicidin, 192, 203
sulfanilamide, 229, 232, 233, 261
sulfathiazole, 204
sulfonamide, 205, 227-230, 234
crystals, frontispiece
differentiation of bacteria by, 219-
220
effect on bacteria, 75, 77, 125, 195,
198, 202-206, 211, 212, 234,
259
by species, 70, 72, 190, 191, 192,
203, 204
resistance of bacteria, 128, 228
effect on cell morphology, 2 1 4-2 1 5
effect on fungi, 141
effect of ^-amino-benzoic acid and
sulfapyridine, 205-206
effect of reaction, 205
effect on sulfanilamide, 205
esters of, 1 79
inactivation of, 175, 218, 229, 237
in vivo activity, 226-228
measurement, 71, 73, 76, 179
medium, 65, 135
mode of action, 202-206, 226-228
production, 128-130, 175-179, 237
stability, 205
therapeutic value, 226-237, 261
toxicity, 228-229, 245
Penicillinase, 77, 175, 198, 218, 237
Penicillin B, I 25, 1 30, I 58, 180, 270
Penicillin-like substances, 179, i8l
Penillic acid, 179
Peptones as bacteriostatic agents, 165
Phage in soil, 10
Phenazine, 164
Phenol, 66, 67, 70, 71, 77, 195
GENERAL INDEX
347
Physical properties of soil, 2-4
Physiologist, field for, 267-268
Physiology of bacteria, effect of anti-
biotic agents, 215-219
Pigment formation and antagonism, 50-
51
Pigments, 53, 102-103, 160, 161-165
Plants
agents pathogenic to, 18, 246-247
bactericidal action of juice, 188
diseases of, control, 246-258
by actinomycetes, i 21-122
by antibiotic substances, 141
by fungi, 138
by use of antagonistic microorgan-
isms, 1 21-122, 138, 248-249
methods, 250-256
of fungal diseases, 256-258
influence on microbial population of
soil, 7-8
Plate culture method of determining
abundance of microorganisms in
soil, 7
Pneumonia organism, survival, 30
Poliomyelitis, 153-154
Polypeptides, 53, 160, 1 67-1 71
Polysaccharidases, 165-167, 243
Potato scab, i 21-122, 253
Proactinomycin, 175, 270
chemical nature, 53, 159, 160, 161,
171
compared with actinomycin, i 20
effect on bacteria, 120, 161
toxicity, 158
Prodigiosin, 53, 160
Production of antibiotic substances,
methods of growing organisms for,
64-66
Protamine, 217
Protozoa
in manure and urine, 23
in soil, 6, 9
relation to bacteria, 23, 143-146
relation to fungi, 146
Protozoan theory of soil fertility, 144
Pseudomonas aeruginosa, antibacterial
substances of, 1 61-163
Puberulic acid, 159, 185, 270
Pure cultures, i 3
Pyocyanase, 67, 91-92, 162, 270
Pyocyanase {cont.)
antiluminescent activity, 77
chemical nature, 53, 159, 160, 163
effect on bacteria, 77, 190, 195
by species, 70, 209
practical value, 163, 221-222, 226
toxicity, 245
Pyocyaneus organism as antagonist, 89
Pyocyanic acid, 162
Pyocyanin, 67, 92-93, 270
antiluminescent activity, 77
chemical nature, 53, 159, 160, 163
effect on bacteria, 77, 190, 195
by species, 70
effect on fungi, 141
inhibitory action, 21 1
isolation, 162
structural formula, 164
toxicity, 245
Pyoxanthose, 162
Pyrogenic substances, 178, 179, 228
Quinones, 53, 102, 103, 161, 173
classification, 182
effect on bacteria, 195, 207, 211
neutralization of iodinin, 198
Radiations, 53
Rectal cancer and £■. coli, 97
Repressive antagonism, 49
Rhizosphere, 5
Rickettsiae, murine typhus, 232
Root parasites. Fig. 2 (p. 42), 247, 248,
249
Rust spores, inhibition of, 1 40-1 41
Saliva, antibacterial properties of, 27
Saprophytic organisms in soil, 15-18
Saprophytism, 40
Sarcoma cells, 155
Sea water, bactericidal action of, 34
Seed inoculation, 256
Selective culture method of determin-
ing abundance of microorganisms
in soil, 7
Semisolid media for testing antagonistic
action, 64
348
GENERAL INDEX
Serial dilution method of measuring
antibiotic action, 71,73
Sewage, 22, 25-26
Silkworms, destruction of, 148
Simultaneous inoculation method of
testing antagonistic action, 61, 62
Skin diseases, 138, 243
Smuts, bacteria antagonistic to, 139
Soil
actinomycetes in, 6, 8, 9, 109-1 1 2
as culture medium, lo-l i
as habitat of microorganisms, 1-2
bacteria in, 6, 8, 9, 1 1, 247
biological state, 5-6
chemical composition, 4-5
fungi in, I, 6, 8, 9-10, 137, 247
heating, effect of, 144, 250
microbial population. See Soil micro-
organisms
pathogens in, 14-15, 16-18, 27-36
physical properties, 2-4
saprophytes in, 15-18
sterilization. See Sterilization of soil
unsterilized, effect on plant growth.
Fig. 34 (p. 254)
Soil-borne diseases
effects of actinomycetes, i 21-122
effects of fungi, 138
methods of control, 14-15, 246-258
Soil enrichment method of isolating an-
tagonistic microorganisms, 56-57
Soil inoculation method of isolating an-
tagonistic microorganisms, 58-59
Soil microorganisms, 6-10, 36-37
and nematodes, 150-151
antagonism to plant pathogens, 248-
249
effect of bacteria, 3 i
equilibrium, 40
Solid media for testing antagonistic ac-
tion, 62-64
Space antagonism among microorgan-
isms, 49-50
Spinulosin, 67, 270
chemical nature, 133, 159, 160, 161
crystals, 133, 159
effect on bacteria, 125, 132
Spirocheticidal action of penicillin, 211,
212, 234
Spore-forming bacteria as antagonists,
82-88
Spore germination, 137, 141
Spores, preparation of, 65-66
Spot inoculation method of testing an-
tagonistic action, 64
Stable manures, 20-25
Staling of medium, 41, 52
Staphylococcus aureus infections, treat-
ment with penicillin, 234-235
Sterilization of soil
effect on coliform bacteria, 1 1
effect on germination of barley, 251
effect on potato scab, 122
partial, 145, 250, 258
Strawberry root rot, 258
Streptomycin, 174-175, 270
chemical nature, 53, 159, 160, 161,
171
effect on bacteria, 117, 195, Fig. 27
(p. 215)
practical value, 123
production, 117, 120
toxicity, 122
Streptothricin, 1 1 6-1 17, 118, 173-
174, 270
chemical nature, 53, 159, 160, 161,
171
compared with actinomycin, clavacin,
and penicillin, 190-191
differentiation of bacteria by, 220
effect on bacteria, 195, 198
by species, 70, 119, 191
effect on fungi, 141-142
therapeutic value, 123, 243, 244
toxicity, 245
Structural formulae of antibiotic sub-
stances, 164
Submerged growth, 176
Subtilin, 159, 160, 270
Successive inoculation method of test-
ing antagonistic action, 61, 62
Sulfanilamide
antiluminescent activity, 77
compared with penicillin, 229, 232,
233, 261
effect on bacteria, 70, 77, 189, 195,
229, 261
effect on penicillin, 205
GENERAL INDEX
349
Sulfanilamide (cout.)
inhibition of antibacterial action,
198, 218, 219
therapeutic value, 226
Sulfapyridine, 205-206, 227
Sulfathiazole, 204, 227, 229, 233
Sulfhydryl groups, 197
Sulfonamide
compared with clavacin, 134
compared with penicillin, 205, 227-
230, 234
Sulfur compounds, 53, 160
Suppressor, defined, 271
Surface tension, 197
Symbiosis, 14, 38-41, 43
Synergism, 40, 206
Syphilis, treatment with penicillin, 234
Tannic acid, effect on actinomycin, 219
Temperature for growth of antagonists,
Testing antagonistic action, methods.
See Methods of testing antagonis-
tic action
Testing in vivo activity, methods, 78
Tetanus organism, survival of, 29
Texas fever, i 7
Ticks, bactericidal action of, 1 49-1 50
Tissue culture, 200
Tolu-/>-quinone, 70, 77
Toxicity of antibiotic substances, 122,
i57-i59> 161, 245
Toxin destruction, 223
Toxin production, lOO
Toxoflavin, 160, 170, 171, 270
True antagonism, 47, 49
Trypanosome parasites, 1 47
Tubercle bacillus, 30, 34-36, 135
Tuberculosis, treatment with extracts of
A. fumigatus, 133, 244
Tumors, 154-155, 222
Turbidimetric method of measuring
antibiotic activity, 75-76
Typhoid organism, 90, 91, 213
as antagonist, 97
in soil, 29-30, 32-34, 80-81
Typhus rickettsiae, 234
Tyrbcidine, 67, 167-17 1, 270
Tyrocidine (cout.)
chemical nature, 53, 159, 160
compared with gramicidin, 192
crystals. Fig. 18 (p. 170)
eifect on bacteria, 70, 192, 195, 206
hemolytic effect of, 239, 240
mode of action, 197, 199-202, 216
therapeutic value, 230, 237-243
toxicity, 245
Tyrosinase, 171
Tyrothricin, 68, 76, 87, 167-171, 270
antiluminescent activity, 77
chemical nature, 159
compared with actinomycin, i 20
compared with protamine, 217
differentiation of bacteria by, 220
effect on bacteria, 70, 77, 190, 206,
217, 237-243
effect on fungi, 141
hemolytic effect of, 200-201, 239,
240
inhibition of, 219
mode of action, 199-202
therapeutic value, 230, 237-243
toxicity, 245
Udder infections. See Bovine mastitis
Urinary infections, 234
Urine, human, bacterial composition of,
21
Utilization of antibiotic substances. See
Disease control
Violacein, 159, 270
Viruses
antagonisms among, 153-154
inactivators of, 152-153
Vitamins, 12, 196, 219
War-time surgery, 226
Wastes, human and animal. See Human
and animal wastes
Water
as culture medium, lO-ii
as habitat of microorganisms, 1-2
Wheat, diseases of, 247, 248, 251
350
GENERAL INDEX
Wheat protein, antibiotic action of,
187-188
Worms, 6, 9
Wound infections, 225, 265
treatment, 232-233, 235, 236
Xanthin oxidase, 197
Yeasts
action against sulfanilamide, 218
in soil, 9
substances produced by, 63, 185-
186, 215
utilization of, 225
Yellow fever virus, 154
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