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STUDIES IN BACTERIAL NUTRITION WITH SPECIAL REFERENCE TO CLOSTRIDIUM SORDELLII

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DOCTORAL DISSERTATION SERIES
title
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AUTHOR
UNIVERSITY
DEGREE
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I
PUBLICATION NO.
11 1 21
3
I UNIVERSITY MICROFILMS
ANN ARBOR ■ MICHIGAN
PURDUE UNIVERSITY
T H IS IS T O CERTIFY THAT T H E T H E S IS PREPA RED UNDER MY SUPERVISION
BY
Henry Dirk Plersaa
e n title d
TTUDILA In HACTHa I A UTRITI01'. WiTTI A?J:C L
R 7 I H T O
CLOCPHIDllLi ; OADCLLII
COMPLIES WITH T H E UNIVERSITY REGULATIONS O N GRADUATION T H E S E S AND IS APPROVED
BY ME AS FULFILLING T H IS PART O F T H E REQUIREM ENTS FO R T H E D EG REE OF
D o c t o r o f ■. P h i l o s o
.Thy
C »<>L
Professor ia Charge of Thesis
Head of School or Department
TO T H E LIBRARIAN:—
IS
T H IS T H E S IS IS NOT TO BE REG A RD ED AS CON FIDENTIAL.
Professor in Cha|(e~
Registrar Form 10—2-39—IM
STUDIES IN BACTERIAL NUTRITION
WITH SPECIAL REFERENCE TO CLOSTRIDIUM SOROELLII
A Thesis
Submitted to the Faculty
of
Purdue University
by
Henry Dirk Piersnia
in partial fulfillment of the
requirements for the Degree
of
Doctor of Philosophy
June, 19A1*
Abstract.
An investigation of the nutritional requirements of one of the
members of the gas gangrene group of bacteria, Clostridium sordellii,
was undertaken in an effort to produce the toxin elaborated by this
organism in a syn-thetio medium.
At the time this work was initiated,
no attempt had been previously made to grow this anaerobe in a medium
of known composition.
The results obtained in this study represent
the first report of the successful cultivation of Clostridium sordellii
in a chemically defined medium.
A basal medium was constructed containing gelatin hydrolysate,
potassium dihydrogen phosphate, certain amino acids, glucose, minerals,
and a reducing agent.
In this medium growth failed to take place
without the presence of certain growth factors, which were identified
as Vitamin B^, Vitamin Bg, pantothenic acid, and nicotinamide.
The
optimal concentration of these substances for growth in the described
basal medium was found to be 1.0 microgram per milliliter for Vitamin B^,
Vitamin Bg, and pantothenic acid, and 2.5 micrograms for nicotinamide.
The essential nature of these growth factors for Clostridium sordellii
was confirmed by an examination of three other strains of this organism.
It was found that these also required the same complex chemical substances
found essential to that strain employed in the original work.
It is
suggested, therefore, that possibly all strains of this pathogen require
Vitamin B^, Vitamin Bg, pantothenic acid, and nicotinamide for normal
growth.
The effect of the presence of biotin in the basal medium was
investigated, and it was observed that certain biotin-containing prepara­
tions were able to stimulate further development of this species.
How-
©ver, the methyl ester of biotin was unable to improve growth under the
described experimental conditions.
Several other compounds, some of
which are known to be important in the nutrition of other bacteria, were
found to be non-essential for Clostridium sordellii.
These substances
included uracil, para-amino benzoic acid, Vitamin B2, choline, glutamine,
pimelic acid, creatine, oadaverine, and betaine.
An alcohol soluble
fraction of liver extract was found to stimulate the growth of this
organism, which demonstrates that additional factors are required for
development in the synthetic medium.
In an attempt to construct an amino acid mixture to substitute for
the gelatin hydrolysate, certain amino acids were found to be more
essential than others.
Among those exerting the most pronounced
influence were d-alanine. d-glutamic acid, and I-cystine.
An experiment was carried out to determine the extent of toxin
production in the synthetic broth, and it was noted that practically no
toxin was elaborated in the medium of known composition.
It is apparent
that additional factors are important for the formation of toxin in this
medium.
Acknowle dgment
The author wishes to acknowledge the
inspiration, and aid received from Dr. C.A.
Behrens and Dr. Y. Subbarow during the course
of the work reported in this thesis.
TABLE OF CONTENTS
Forward
1. The Nutritional Requirements of Bacteria
A. Introduction
B. Autotrophic Bacteria
C. The Heterotrophic Bacteria
D. Origin of the Growth Factor Concept
E. Chemical Compounds Recognized as Growth Factors
for Bacteria
Biotin
Choline
Codehydrogenases
Glutamine
Hematin
Nicotinic Acid
r&ntotnenic Acid
Para-amino Benzoic Acid
Phthiocol and 2-methy1-1:4 naphthoquinone
Phthioeol
2-Methyl-l:4 Naphthoquinone
Pimelic Acid
Uracil
Vitamin B^j Thiamin Chloride Hydrochloride
Vitamin B2; Riboflavin
Vitamin Bgj Pyridoxine
F. Bacteria Requiring Known Growth Factors
T54
Choline
Codehydrogena ses
Glutamine
Hematin
Nicotinic Acid
Pantothenic Acid
Para-amino Benzoic Acid
Phthiocol, 2-Methyl-l:4 Naphthoquinone, and
Vitamin K
Pimelic Acid
Uracil
Vitamin Bj; Thiamin Chloride Hydrochloride
Vitamin E^, Riboflavin
Vitamin Bg; Pyridoxine
U
X V
y~.
;
Summary of Bacteria Requiring Known Growth Factors
Page
II. Experimental Work
A. Experimental Methods
Sterilization
Methods of Assay of Growth Response
The Determination of Biotin in Biological
Materials
B. Experimental Study of the Nutrition of Clostridium
sordellii.
TJ ■?
Ql
Morphological Characteristics
Cultural Characteristics
Serological Reactions
Pathogenicity
Experimental Work
Preparation of a Basal Medium
Method of Amaerobiosis
Determination of Factor or Factors in the
"Shotgun" Mixture Stimulating the Growth
of Clostridium sordellii
Determination of the Optimal Concentration
of Vitamin B^, Vitamin Bg, Pantothenic Acid
and Nicotinamide
Growth Factor Requirements of Other Strains
of Clostridium sordellii
Effect of Eiotin upon Growth of Clostridium
sordellii
The Effect of Other Factors on the Gro*vth
of Clostridium sordellii
Some Amino Acid Requirements of Clostrid­
ium sordellii
Studies in Toxin Production of Clostridium
sordellii in a Synthetic Medium
O \
O
y
/jwviJ
ckAivx
P
Bibliography
A v 4 / % 1 i-i «■»■? A n
f
56
56
57
61
65
65
67
68
69
71
75
75
80
82
96
99
103
106
110
111
114
List of Tables.
Page
Table I
Table II
Table III
Table IV
Table V
Table VI
Table VII
Table VIII
Table IX
Table X
Table XI
Table XII
Table XIII
Table XIV
Growth of three Pathogenic Anaerobic Bacteria
on a Basal Medium
77-8
Effect of Various Methods of Anaerobiosis on
Growth of Clostridium Sordellii in a Synthetic
Medium
83-4
Effect of Omission of One Factor From the
Mixture of Growth Stimulants
86
Effect of Various Factors upon the Growth of
Clostridium Sordellii in a Basal Medium
88-9
Determination of the Optimal Concentration of
Each Growth Factor in the Presence of an
Excess of the Others .
91-2
Determination of the Optimal Concentration of
Each Growth Factor in the Presence of An
Optimal Concentration of Others
93-4-5
The Effect of Vitamin Bj, Vitamin Bg, Panto­
thenic Acid and Nicotinamide on the Growth
of Various Strains of Clostridium Sordellii
98
Determination of Biotin in Liver Protein
Hydrolysates
100
Effect of Liver Protein Hydrolysate on Growth
of Clostridium Sordellii
102
Effect of Biotin on Growth of Clostridium
Sordellii
104
Effect of Various Substances Upon Growth of
Clostridium Sordellii
105
The Amino Acids and Percentage Composition of
Gelatin
107
The Effect of Omission of Separate Amino Acids
on the Growth of Clostridium Sordellii
109
Toxin Production in a Synthetic and Beef Heart
Infusion Medium
112
FORff'vURD
It has been recognized since the early days of bacteriology that
the cultivation of any micro-organism upon an artificial medium depends
upon the successful duplication of all the important conditions of the
natural habitat of the organism.
Since bacteria are ubiquitous organisms,
it follows that a great variation exists in the culture media designed
for their growth.
The nutritional requirements of bacteria may be satisfied by simple
inorganic compounds, or by these and other substances so involved that
their chemical nature is obscure or unknown.
It can be understood,
therefore, that the cultivation of micro-organisms demanding complex
chemical compounds presents a particularly difficult problem.
Attempts
to grow these organisms upon artificial media have included the use of
various animal and plant extracts, frequently with irregular or unsatis­
factory results.
T/ithin recent years, considerable progress has been made in our
knowledge of the complex chemical compounds required for growth by cer­
tain bacteria.
These developments have been largely the results of re­
search in biochemistry, and particularly the biochemistry of nutrition.
Y.Tien it became apparent that certain chemical compounds essential to the
nutrition of micro-organisms were also essential to the nutrition of plant
and animals, another method of study of these complex chemical sub­
stances became available to the; biochemist.
Thus, the work of Mueller
(1) demonstrated the existence of methionine, which subsequently became
recognized as an essential amino acid in human nutrition.
V/illiams, et al.
(2) found pantothenic acid to be required for growth of the yeast cell,
and subsequently this compound has been shown to be important in animal
nutrition.
The bacteriologist has derived a wealth of information from the ef­
forts of the biochemist, and a new approach to many problems in bacter­
iology has been gained.
There is ample reason to believe that the clas­
sification of bacteria will be essentially strengthened by nutritional
studies.
Furthermore, the bacteriologist now possesses a scientific
method for the preparation of media for the growth of bacteria with com­
plex nutritional requirements.
The usual meat infusion or meat extract
media have been demonstrated to contain both growth-stimulating and
growth-inhibiting substances, and certain growth-stimulating factors are
known to be growth-inhibiting in concentrations greater than optimum.
In the field of chemotherapy, the mechanism of inhibition of growth of
bacterial cells is becoming apparent from the work of Woods (3) and
Rubbo and Gillespie (i).
Finally, the efforts of research in bacterial
nutrition have contributed greatly to a knowledge of the fundamental bi­
ology of the normal cell.
In this thesis, a presentation is made of certain original studies
of the nutritional requirements of a pathogenic organism, Clostridium
sordellii. The investigations herein reported constitute the first
successful attempt to cultivate this species of the gas gangrene group
of bacteria in a medium of known chemical composition.
Since the nu­
tritional requirements of this organism have not been completely satis­
fied by recognized chemical compounds, this work furnishes a basis for
further research along these lines.
It is anticipated, furthermore,
that similar studies with other members of the gas gangrene bacteria
will establish fundamental differences and similarities among the
group.
I.
THE NUTRITIONAL REQUIREMENTS OF BACTERIA
A.
Introduction
The chemical substances essential to the life processes of
bacteria are required to supply food for energy and growth.
There is
a wide divergence among bacteria in respect to the chemical nature of
their nutritional requirements, and certain classifications have been
based upon these differences.
The energy requirements of bacteria are such that two large
groups are generally recognized, namely, the autotrophic and heterotrophic bacteria.
The autotrophic bacteria are those forms employing char­
acteristic oxidations of inorganic substances as sources of energy for
the assimilation of carbon as carbon dioxide, while the heterotrophic
organisms assimilate carbon in more complex and reduced forms.
It is our intention to review the subject of bacterial nutri­
tion by an examination of the general methods employed by the autotro­
phic bacteria in obtaining their energy for the assimilation of carbon
as carbon dioxide.
The heterotrophic bacteria, on the other hand, will
be discussed principally from the viewpoint of nitrogen relationships,
and the need of complex nutritional factors.
A n effort has been made to
describe briefly the chemical nature of the known growth factors, with a
review of the bacteria known to require these factors.
The autotrophic and heterotrophic organisms vary in respect to
the methods employed in obtaining their energy requirements.
Knight (5)
has pointed out that the essential difference between these two broad
groups of organisms is not a qualitative, but rather a quantitative dif­
ference.
The reduction of carbon dioxide by autotrophic bacteria to the
ultimate structure of protoplasm is far more costly than the reduction
of more complex carbon compounds as accomplished by the heterotrophic
organisms•
Several investigators
(6)(7)(8)(9) have recently demonstrated
in studies of nonphotochemical reduction of carbon dioxide by biologi­
cal systems that a number of heterotrophic bacteria assimilate small
amounts of carbon dioxide.
This observation supports the view that car­
bon dioxide reduction is not exclusive to photosynthetic and chemosynthetic autotrophic organisms,
Ruben and Kamen (10) assert that it is
reasonable to suppose the formation of reduced radio-carbon from la­
beled carbon dioxide is not due to simple interchange but rather that
carbon dioxide is used as a specific oxidizing agent in one or more
steps in the respiratory mechanism.
B. Autotrophic Bacteria
Among the autotrophic bacteria one may distinguish between
the photosynthesizing and chemosynthesizing organisms.
The green plant,
by means of chlorophyl and the energy supplied by the sun, converts car­
bon dioxide and water into complex carbon compounds.
By classification,
we exclude bacteria from that group of living organisms possessing
chlorophyl.
However, there exists a transitional group of organisms,
classified as bacteria, which possess pigments similar in function to
chlorophyl.
The chemical nature of these pigments is obscure.
Van Niel
(11) has suggested that two types of pigments can be identified, namely,
a purple carotenoid type of pigment, and a green pigment related to chloro­
phyl.
Roelofsen (12), according to Knight (5) has reported that two types
of pigments exist, one of which is similar to the green pigment described
by Van Niel, and the other being a red pigment, called bacterio-erythrine.
Gaffron (13) has described the existence of several pigments, but concludes
that only the green pigment, bacterio-chlorophyl, is pnotochemically ac­
tive.
The photosynthetic bacteria obtain their energy partially from
sunlight, and the remainder by the oxidation of simple chemical compounds.
The Thiorhodaceae. or purple and green "sulfur" bacteria, obtain some of
their energy from the oxidation of sulfur compounds.
Saffron (13) and
Van Niel ^xl^ have reported tnat no reouctaon of carbon ds.oxs.de takes
place by the photosynthesizing bacteria in the absence of one or both
sources of energy.
However, Ruben, Hassid and Kamen (14) have recently been able to
demonstrate, by means of labeled carbon dioxide, that carbon dioxide
has been "picked-up" by certain photosyntnetic bacteria in the dark.
The chemosynthetic autotrophic bacteria represent a group of
organisms which derive all their energy from the oxidation of compara­
tively simple inorganic substances, such as ammonia, nitrite, hydrogen
sulfide, sulfur, and other oxidizable sulfur compounds.
Among the chemo­
synthetic autotrophic bacteria we may recognize two groups, the obligate
and facultative types.
The obligate autotrophic bacteria are characterized by the
fact that they can use only one specific oxidation process in procuring
their energy.
In 1890, ’.Vinogradsky (15) obtained from the soil two types
of organisms oxidizing nitrogen compounds.
Hitrosomonas and Hitroso-
coccus, the latter being a variant found in this country, oxidize am­
monia to nitrites, and Nitrobacter oxidizes nitrites to nitrates.
organisms have not been known to oxidize another energy source.
These
It is
interesting to note in regard to the nitrifying bacteria that their food
for growth and energy are identical, being either ammonia or nitrites.
The facultative autotrophic bacteria may use a variety of oxi­
dation processes to obtain energy.
They are capable of oxidizing hydro­
gen, thiosulfate, carbon monoxide, and methane (16).
The methane bac­
teria have been shown by Carson and Ruben (7) to reduce carbon dioxide
to methane as a result of the fermentation of methanol.
An appreciable
portion, about ten per cent., of the radio-active carbon appeared in
non-volatile cell material.
Furthermore, they can grow heterotrophically
upon ordinary laboratory media.
These organisms represent, therefore, a
transitional group between the autotrophic and heterotrophic types of
bacteria,
an example of this transitional type of organism has been
described by Starkey (17), and named by him Thiobacillus novelIns.
Starkey was able to cultivate this organism autotrophically in a mineral
medium, but growth was improved by the addition of glucose and asparagine.
The addition of these organic substances did not increase the amount of
thiosulfate oxidized, and in some instances apparently decreased it.
This investigator was furthermore able to show that Thiobacillus novellus
could grow heterotrophically in complex organic media without thiosulfate
present.
C.
The Heterotrophic Bacteria
As has been previously indicated, the heterotrophic bacteria
include that vast group of organisms which do not assimilate carbon as
carbon dioxide.
They obtain their carbon from sources more reduced than
carbon dioxide, and their energy from the utilization of various carbon
compounds.
It is beyond the purposes of this discussion to review ex­
haustively the differences between and within the major groups of hetero­
trophic bacteria.
It is considered sufficient to outline briefly the
major groups of heterotrophic bacteria, and to describe an organism, or
certain relationships existing in each group.
The classification of
heterotrophic organisms, established by Knight (5)> which has been adopted
for characterization of the group include the nitrogen-fixing bacteria;
denitrifying bacteria; bacteria which can use ammonia as a nitrogen
source, but do not require amino acids; bacteria using ammonia, or re­
quiring amino acids as sources of nitrogen; and bacteria with complex
nutritional requirements.
The nitrogen-fixing bacteria may be divided into two groups, namely,
the free-living forms, and the symbiotic nitrogen fixers.
Among
the free-living organisms, we can distinguish between aerobic and anaer­
obic types; the Azotobacter being representative of the former, and
Clostridium pastorianum, the latter.
Concerning the nutrition of the free-living nitrogen-fixing
bacteria, it must be noted that nitrogen is not fixed if combined nitro­
gen, as ammonia, for example, is present in the culture medium.
For nu­
tritional requirements other than nitrogen, these organisms derive energy
and carbon from glucose, sucrose, mannitol and other carbohydrates.
The
inorganic requirements of Azotobacter have been studied exhaustively by
Burk and Horner (18).
The symbiotic nitrogen-fixers, or Rhizobia. have been known to fix
nitrogen only in the root nodules of leguminous plants.
Several at­
tempts have been made to demonstrate nitrogen fixation in vitro, but none
of them have been successful.
West and Wilson (19) have been able to
show that Rhizobium trifolii requires both thiamin and riboflavin for
growth in vitro.
In view of these requirements, and the symbiotic rela­
tionship between this organism and the infected plant, it may be said
that the Rhizobia most probably belong to that group of bacteria possess­
ing complex nutritional requirements.
The denitrifying bacteria represent a group of organisms with
comparatively simple, although interesting, nutritional requirements.
In a medium composed of inorganic salts, nitrate, and a carbon source,
these bacteria grow anaerobically without difficulty.
They obtain their
energy from the reduction of nitrates, nitrites, or ammonia.
Elema,
Kluyver and Van Dalfsen (20) have recently studied the oxidation-reduction potential relationships existing in a grov/ing culture of denitri­
fying bacteria.
These investigators have been able to correlate the
observed oxidation-reduction potential changes vdth the disappearance
of nitrate from the culture medium.
Among the bacteria which can use ammonia as a nitrogen source,
without the necessity of nitrogen as amino acids, may be listed organisms
from various sources.
Among these are Serratia marcescens. one of the
common cnromogenic bacteria, Escherichia coli, Pseudomonas ovocvanea and
fluorescens. and several less important and unfamiliar forms.
These
organisms have been successfully cultured on a synthetic medium composed
of certain inorganic salts, glucose and other carbohydrates as sources of
carbon and energy, and ammonia as the sole source of nitrogen.
The next group of organisms to be discussed are those forms
which are frequently associated vdth animal waste products, and include
those bacteria using ammonia or requiring amino acids as a nitrogen source.
The enteric forms of bacteria are found in this group, since the digestion
of foods involves the hydrolysis of proteins to amino acids and ammonia.
Perhaps the most significant fact about this particular group of organisms
is found in the observation that certain members of a given species have
been found to possess differing nutritional requirements.
One must,
therefore, distinguish between "exacting" and "non-exacting" strains of
a bacterial species, when considering its nutritional requirements.
Fildes, Gladstone, and Knight (21) have studied the problem of
"exacting" and "non-exacting" strains of Eberthella typhi. From the
work of Van Loghem (22), it has been demonstrated that certain strains
of Eberthella. typhi can grow upon a basal medium containing ammonia as
the sole source of nitrogen.
Fildes, Gladstone, and Knight selected four
strains of Eberthella typhi. isolated from epidemic cases.
All four
strains failed to grow in a medium in which ammonia was the only source
of nitrogen, but growth took place in the presence of fourteen amino
acids as sources of nitrogen.
Further work indicated that of this group
of amino acids, tryptophane alone was required for the maximum growth of
the organism.
Upon omission of the other thirteen amino acids, growth
proceeded slower, but eventually attained the maximum observed in their
presence.
It thus becomes apparent from the work of these investigators
that the "exacting" strains of Eberthella typhi differed from the "non­
exacting" strains in their need of tryptophane as a nitrogen source.
The jrdcro-organisms vdth complex nutritional requirements con­
stitute trie final group of heterotrophic bacteria to be discussed.
Among this group, we find many of the pathogenic organisms, and this
fact, among others, probably explains considerable recent interest in
the nutritional requirements of these forms.
The major interest of this
thesis centers about the complex nutritional requirements of bacteria,
and particularly vdth the growth factors, or growth-activating substances.
In discussing the nutrition of bacteria with complex require­
ments, there is no intention to discuss the need of inorganic requirements
the sources of energy, or other considerations not directly associated
XV/.
vdth the need of these organisms for certain specialized grovrth factors.
In developing this subject it is planned, first, to survey briefly the
origin of the grovrth factor conception; secondly, to review the chemical
entities known to be grovrth factors for bacteria; and lastly, to present
an extensive summary of the bacteria known to require these factors for
grovrth.
D,
Origin of the Grovrth Factor Concept
Uschinsky (23) is probably the first investigator interested in
the cultivation of pathogenic bacteria in a chemically defined medium.
He employed a medium containing inorganic salts, sodium aspartate, and
glycerol, and it may be concluded, in view of the subsequent development
of this field, that growth on this medium was relatively weak.
Attempts
to repeat the work of Uschinsky by Hugounenq and Boyon (24) were unsuc­
cessful.
The development of the growth factor concept in bacteriology is
largely due to the work of 7/ildiers (25).
This investigator found that
the synthetic..medium described by Pasteur for the grovrth of yeasts
was unable to support grovrth if the inoculum for seeding was small.
The
medium described by Pasteur vras composed of yeast a3h, presumably to
satisfy the mineral requirements of the organism, ammonium salts, and
a fermentable sugar.
Liebig, while involved in that classical contro­
versy with Pasteur, had also reported failure of yeast to grow in this
medium.
’.'ildiers performed an interesting experiment In an effort to
explain the observation that large inocula grew in the medium described
by Pasteur, while smaller inocula failed to respond.
He obtained culture
medium in which yeast cells had grown, and removed the living cells.
After insuring the sterility of this preparation, various amounts were
added to the synthetic medium of Pasteur.
Another set of control flasks
did not contain the sterile yeast medium.
By progressive reduction of
the size of the inoculum,
ildiers eventually reached a point where grovrth
took place in the synthetic medium fortified with yeast filtrate, but
failed to occur in the control medium,
L'ildiers postulated the necessity
ox a substance which he called "bios" for growth of yeast in the synthetic
medium described by Pasteur.
The necessity of "bios" for the development
of yeast was confirmed by several workers, and fractionation of this sub­
stance by Lucas (26), in 1924, resulted in the recognition of Bios I and
Bios II.
Lucas reported that the yeast crop resulting from the addition
of either Bios I or Bios II to a basal medium is not greatly increased,
but the addition of both yields a large crop.
The identification of Bios I was accomplished by Eastcott (27) in
1928, under the supervision of 17. Lash killer.
Working with tea dust,
Bios I was identified as inactive inositol, and curves showing the repro­
duction rate of yeast in solutions containing salt, sugar, inosite and
crude Bios II have agreed in form with those obtained vdth solutions of
salts, sugar and wort.
The inosite taken up by yeast can be quantitatively
recovered by hydrolysis of the yeast crop; each cell has been demonstrated
to take up 1-2 parts per thousand of inosite on the basis of the dry
weight of the cell.
Eastcott also has shown that yeast grown in a med­
ium containing only sugar and salts yields inosite upon hydrolysis,
which indicates that this substance is partially synthesized by the or­
ganism, if unavailable in the medium.
The conception of grovrth factors for bacteria vras an out-growth of
the work of V/ildiers, Lucas and Eastcott vdth the nutrition of yeast.
Upon the subsequent fractionation of Bios II, many factors important to
the grovrth of a variety of bacteria have been discovered.
Burrows (28), in a review of the general aspects of bacterial
nutrition, has defined the concept of growth factors.
He defines a
growth factor for bacteria as a vitamin-like substance, differentiated
from essential amino acids, energy sources and mineral requirements on
the basis of a smaller quantitative requirement.
This definition has
been generally accepted, and it is in this sense that subsequent ref­
erence is made to growth factors.
The study of the nutritional requirements of pathogenic and other
bacteria with complex needs has developed rapidly within the past few
years due to the recognition that certain chemicals are essential to
the metabolic activities of the normal cell.
The number of growth fac­
tors known to be necessary for the growth of bacteria has increased
through the efforts of workers in many fields.
It may be anticipated,
furthermore, that research in the future will reveal the identity of
many more important chemical factors.
At this point, it may be emphasized that the study of bacterial
nutrition involves more than a determination of the complex chemical
requirements of an organism.
After growth has been obtained on a basal
medium of known chemical composition, further studies must be under­
taken to discover the response of the species under investigation to a
variety of substances known to be essential for other biological en­
tities.
The recognition of growth factors as essential requirements for
bacteria with complex nutritional demands has enabled the bacteriolo­
gist to cultivate these organisms upon a medium of known chemical com­
position, and thus made it possible to study various biological phen­
omena in the light of more critical and exact methods.
E* Chemical Compounds Recognized as Growth Factors For Bacteria
The chemical compounds that have been recognized to be growth
factors for bacteria are presented in this section.
It has been con­
sidered beyond the scope of this presentation to describe the histor­
ical aspects of the chemical isolation of each factor, and their biologi
cal importance in fields other than bacteriology.
An effort has been
made, rather, to give a general description of the more important chem­
ical and physical properties essential to the bacteriologist in studies
of bacterial nutrition.
Biotin
The empirical formula for the methyl ester of biotin has been re­
ported by K8gl (29) as C 1 1 H 1 8 O 3 N 2 S, and the molecular weight of this
compound is 258.22.
The structural formula of biotin is not known.
Biotin is dialyzable, heat stabile and resistant to treatment with
acid and alkali.
It is soluble in water and alcohol, but insoluble in
chloroform, ether, and petroleum ether.
Hence, the bacteriologist can
sterilize a biotin solution by means of the usual autoclave temperature.
At this point, it may be well to point out that the methyl ester
of biotin is the closest approach to the pure substance isolated at
this time.
Many preparations are recognized to contain biotin, and the
amount of it in these preparations can be assayed accurately, but it is
well to keep in mind that actual proof of the need for this substance
by a given micro-organism must be furnished b y use of the pure substance
Choline
The empirical formula for choline is CgH^gC^N, having a molecular
weight of 121.13.
Choline is tri-methyl, 2-hydroxyethyl ammonium
hydroxide, and for use in bacteriological media, choline hydrochloride
is most commonly used.
The structural formula for choline may be
written as follows:
gh3
I
ch3 \
I
CH^— N — CH2 .CH20H
OH
Choline hydrochloride is very soluble in water and absolute ethyl
alcohol, is stable to autoclaving at 120° C. for 15 minutes.
It can,
therefore, be sterilized in this manner for bacteriological purposes.
Co-dehydro genas es
Di- and tri-phospho-pyridine nucleotides belong to that group of
chemical compounds known as co-dehydrogenases.
Di-phospho-pyridine nu­
cleotide has been described as Harden and Young1s cozymase, and triphospho-pyridine nucleotide has been recognized as Warburg's coferment.
Upon hydrolysis both of these substances yield adenine, two molecules
of ribose, nicotinamide, and two or three molecules of phosphoric acid.
both the di- and tri-phospho-pyridine nucleotides are soluble in
water, sensitive to heat, being destroyed in alkaline, but not in acid
solution, when existing in the oxidized form.
The reverse lability to
heat exists when these compounds are in the reduced form.
A solution
containing either of these compounds can be sterilized by passage through
a Berkefeld filter.
Glutamine
The empirical formula for glutamine is
weight is 146.09.
and its molecular
It has a melting point between 184-5° C.
Glutamine
is the monoaraide of glutamic acid, and its structural formula may be
represented as follows:
Glutamine is soluble in water, but very sparsely so in ethyl al­
cohol.
It is furthermore stable to the temperatures required for ster­
ilization in the autoclave.
Mcllwain (30) in a note on the stability of glutamine makes the
following remarks:
"(a)
Solid glutamine prepared from beet and containing a little
moisture was found to have decomposed to the extent of 66%
(as judged by labile arnide-M) in the course of keeping for
3 months at room temperature.
Later specimens have been
kept over CaCl^at 0° C.
"(b)
In certain bacteriological culture media containing agar,
glutamine was stable at 120° C. and pH 7.2 for 20 minutes
....
In peptone infusion not containing agar, some des­
truction of glutamine occurred."
Hematin
Hematin has also been referred to as ferriprotoporphyrin hydroxide
to distinguish it from hemin, which is Imown as ferriprotoporphyrin
chloride (31).
Hematin has an empirical formula of C^I^-jO^N^Fe, and
is found in nature combined vdth globin to form the respiratory pigment,
hemoglobin.
follows:
The structural formula for hematin (32) may be written as
16.
CII
c.
CH-
■c
CH? = C H — C
-CH z CH,
:
\
c—
c — ch.
I//
A]....... F e ..... N-^
CII^-C
C — C — C^HjCOOH
\
/
\ w j /
\ / " \ /
U'C.\
W
y-^'M
I
I
;C —
c h 3~ c
C2H^COOH
Solutions of hematin for bacteriological use may be prepared dis­
solving hemin in water at pH 9.0.
At this reaction hematin is stable
to heat at 120° C. for 10-15 minutes.
After sterilization the reaction
of the solution may be adjusted to pH 7.0 — 7.5, or used at the reaction
of the original solution,
Hematin decomposes at 200° C. without melting.
Nicotinic Acid
The empirical formula for nicotinic acid is 0^ 502^’, its molecular
weight is 123.05, and its melting point is between 235° and 237° C.
The structural formula may be written as follows:
H
H— C
II
H— C
A
N
✓
COOH
-H
Nicotinic acid is soluble in water to the extent of 1.67 gram in
100 ml. at 25° C.
It is very soluble in alcohol, and in the alkali
carbonates and hydroxides.
It is not decomposed by autoclaving an
aqueous solution at 120° C. for 20 minutes.
In the solid form it is
stable in air and light, and is not hygroscopic.
Both nicotinic acid and nicotinic acid amide have been demonstrated
to be essential growth factors for micro-organisms, but the optimum con­
centration of each for a given species vafies.
In some cases, it has
been observed that nicotinic acid is more effective than the amide (33) t
and in other instances the reverse situation has been found to exist
(31).
Nicotinamide is important in biological reactions because of its
relationship with cozymase from yeast and coferment from horse blood*
A molecule of cosymase, otherwise known as diphosphopyridine nucleo­
tide, and one of coferment, also known as triphosphopyridine nucleo­
tide, each, contain a molecule of nicotinamide.
Both diphosphopyridine
nucleotide and triphospopyridine nucleotide enter into certain biologi­
cal oxidations by reversiblv taking on or giving up hydrogen(35).
Pantothenic Acid
The empirical formula for pantothenic acid is CgH-j^O^N,
molecular weight is 219*14.
Pantothenic acid is a viscous oil.
In its
synthesis, difficulty has been experienced in removing the last traces
of solvents, and Mhence physical constants made on such viscous oils
may vary" (36).
The structural formula of this substance may be writ­
ten as folloY^s:
H
I
H— C— H
H
|
HO 0
H
H
H
n
'
'
•
K
i
1
i
^ 0
HO — C -------- C -------- C — C — N ---- C — C-- C ^
I
H
For use in studies in bacterial nutrition, a calcium or sodium salt
of pantothenic acid is generally employed.
Both of these salts are soluble
in water, and are stable to temperatures used in sterilisation in the
autoclave•
Williams and Major (37) have demonstrated pantothenic acid to be
a compound composed of beta-alanine andot-hydroxybutyrolactone.
-dimethyl-^ -
Each of these compounds may be written structurally
as follows:
Beta-alanine
E
I
H
H
I
C ---- C
I
I
nh2
h
COOH
01- h y d r o x y - - d i m e t h y l - % -butyrolactone
ch3
ch3
oh
c h 2o
---------------
It is very interesting to note that either beta-alanine or
ct-hydroxy-^,$ -dimethyl-J-butyrolactone, or the complete pantothenic acid
molecule, may be required as growth factors for certain micro-organisms.
Mueller (38) has described the necessity of beta-alanine in the
nutrition of 0* diphtheriae.
Woollev f39) has found a hemolytic
streptococcus which required the lactone as a nutritional factor.
The organisms requiring pantothenic acid for their nutrition will be
discussed in a subsequent portion of this paper.
Para-anino Benzoic Acid
The empirical formula for para-amino benzoic acid is C^H^OgN,
with a molecular weight of 137.064.
to 187° C.
It has a melting point from 186°
The structural formula for this substance may be re­
presented as follows:
H
H
/ c\
C
II
C
H
I
C --- H
H— H
I
H
Para-amino benzoic acid is soluble in water, alcohol, and ether,
and is stable to the temperature required for use in sterilization in
the autoclave.
Phthiocol and 2-*aethyl-l:4 naphthoquinone (Antihemorrhagic Vita­
mins .)
It is recognized that several chemical compounds, structurally
related to each other, may be included among the antihemorrhagic vita­
mins.
However, since phthiocol and 2-methyl-1:A naphthoquinone are
the only members of this group that have been demonstrated to possess
growth stimulating properties for bacteria, we shall confine our dis­
cussion to these substances.
Phthiocol
(3 hydro:xy-2-methyl-l:4 naphthoquinone).
Phthiocol has an empirical formula of C^H^O^, and a molecular
weight of 138.06.
It has a melting point of 173°-174° C.
tural formula of phthiocol is as follows:
The struc­
Fhthiocol is soluble in dilute alkali solutions, with the production
of an intense red color.
Phthiocol is stable to the autoclave tem­
perature required for sterilization of its solution.
2-heth.yl-l;4 naphthoquinone
The empirical formula of 2-methyl-l:4 naphthoouinone is C H O .
II 8 2
and its molecular weight is 172.06. It has a melting point of 106 C.
The structural formula of 2-«iethyl-l:4 naphthoquinone is as follows:
0
II
.c
H
c
H-C
/ \ C/ \ C
H-C
C
- CH3
- H
i
h
0
This compound is soluble in benzene, and is moderately soluble in ethyl
alcohol, and but sparingly soluble in water and petroleum ether.
It is
stable to the autoclave temperatures required for sterilization of a
solution.
Pimelic Acid
The empirical formula for pimelic acid is
vreight of 158.08.
with a molecular
It has a melting point of 103° C.
Pimelic acid is
one of the higher dibasic organic acids, vdth the following structural
formula:
H
0*
I
H
H
I
H
I
H
I
I
*o
C - C — C ■«— C — G — C — C
H0X
II
H
\ I I
H
Pimelic acid is comparativelysoluble
H
H
'OH
H
inwater, and is stable at
the temperature required for sterilization in the autoclave.
Uracil
The empirical formula for uracil is 0^11^02
weight is 112.04.
and ats molecular
It has a melting point of 335° C.
Uracil is one of
the pyrimidine constituents of yeast nucleic acid, and its structural
formula may be written as follows:
H— N— —
I
1
0 = C
1
H— N — —
c= 0
I
I
c— ■H
!!
c— H
Uracil is soluble in hot water, but only sparingly in cold.
is also soluble in ammonia water.
It
It can be autoclaved without des­
truction at the usual temperature and length of time required for
sterilization.
Vitamin B-^; Thiamin Chloride Hydrochloride.
The empirical formula for thiamin hydrochloride is ^12^18^4SG^2 *
and its molecular weight is 337.26. It has a melting point of about
o
245 C., with some decomposition. Its structural formula may be writ­
ten. as follows:
CHo
NH0.HC1
C = C —
I
I
2
/
G--- C -------- CH---- N.
!!
!!
2
/V
N— CH
Cl V GH
N = C—
CH0—
3
CH4.CH_0H
2* 2
Thiamin chloride hydrochloride is very soluble in water, and only
slightly so in alcohol, and insoluble in ether.
In N/1000 acid solu­
tion, it is stable at a temperature of 120° G. for 20 minutes, and can,
therefore, be sterilized without destruction in an autoclave prior to
use in bacteriological media.
IVhen dry, the crystals are stable to
light and air, at room temperatures, and heating for 24 hours at 100° C.
in contact with air causes no loss in potency.
From the structural formula, one can note that thiamin hydrochloride
is composed of a pyrimidine and thiazole ring.
It is interesting to note
that some micro-organisms can avail themselves of the pyrimidine and thi­
azole components when supplied separately in equivalent molar concentra­
tions.
Koser (AO) believes that organisms capable of using these com­
ponents separately are able to synthesize the whole vitamin molecule.
According to this view, these two components are not used separately for
differing purposes.
More exacting organisms are able to use only the
complete thiamin chloride hydrochloride molecule, and are apparently un­
able to synthesize the whole molecule from its component parts.
Vitamin B,: Riboflavin.
The empirical formula for riboflavin is
lecular weight is 296.27.
with decomposition.
and its mo­
It has a melting point between 275°-2oO° C.,
It has a structural formula as follows:
CH-,0H
I ^
(CH0H)3
I
CH.
I
N
N
H
C
C
C
N — II
II
0
n
Riboflavin is not very soluble in water, for about 11.0 milligrams
will dissolve in 100 ml. at 25° 0.
It is less solxible In alcohol; 4.0
mgms. dissolving in 100 ml, at 2f>° C.
ordinary fat solvents.
Riboflavin is insoluble in the
In the solid form, this vitamin is not appreciably affected by
diffused light.
In solution, and particularly alkaline solutions, it
rapidly deteriorates on exposure to light.
'Then exposed to visible
light or ultra-violet rays, riboflavin undergoes irreversible decom­
position.
Solutions of riboflavin for bacteriological media purposes
are best prepared by dissolving 5.0 milligrams of the solid form in
100 ml. of N/50 acetic acid, which, ■when kept aseptically in the cold,
gives a stable solution for at least three months.
It may be sterilized
at the usual temperature and length of time required for this purpose.
Riboflavin contains the isoalloxazine nucleus, in conjunction with
ribose, and is a widely distributed yellow substance with a character­
istic green fluorescence.
It has been suggested (41)(42) that ribo­
flavin may function in cell respiration, since it is probably an essen­
tial constituent of the "yellow oxidative enzyme" of ITarburg and
Christian (43)(44)(45).
Vitamin 6^; Pyridoxine.
The empirical formula for Vitamin
is C
weight is 169.1, and it has a melting point of 157
Its molecular
o
C.
The structural
formula of this substance may be represented as follows:
CH_0H
i 2
C
HO-C
\
CH--C
Vitamin
at 26.5° C.
C-CHo0H
i
2
C-H
is soluble in water; 15 grams will dissolve in 100 ml.
It is somewhat less soluble in alcohol, and only slightly
in ether, chloroform and in oils.
Vitamin
hydrochloride is stable
in the solid form, and aqueous solutions can withstand autoclaving at
120° C. for 20 minutes, if the reaction of the solution is below pH 6.
Because of the comparatively recent isolation of Vitamin B ,
6
there is not available a great amount of experimental work on the
function of this vitamin in the living cell.
It has been reported,
however, that fatty acids are essential for its complete utilization
(46).
F.
Bacteria Requiring Known Growth Factors
1
_ J- j
i>J.U 0x11
KOgl and van TVagtendonk (47) were the first investigators to employ
biotin in the study of bacterial nutrition.
Upon the addition of
small amounts of the methyl ester of biotin to a suitable basal medium,
the growth of Staphylococcus aureus was increased.
’Then the optimal
concentration of biotin was used, which these workers found to be 0.005
microgram per milliliter of medium, a three-fold increase in growth of
the organism was observed.
Subsequently, Sartory, Beyer and Better (43) have confirmed the work
of KOgl and van Wagtenionk with a purified biotin preparation.
Further­
more, they observed that the addition Ox £11* ether-alcohol soluble sterol
greatly increases the response of the organism to the presence of biotin
in the basal medium.
Very recently, Porter and Felczar (49) have published an excellent
report on the nutrition of Staphylococcus aureus.
They have been able
to confirm the need of biotin for only certain strains of Staphylococcus
aureus. Several strains of this organism were observed to show no
response to the growth-stimulatijig capacities of this substance.
Snell and billiams (50) investigated the need of Clostridium
but.ylicum for biotin.
Using a medium composed of hydrolyzed casein, and
certain inorganic salts, these workers were able to demonstrate a
response of this organism to the presence of as little as 0.0000133
microgram of biotin per milliliter of basal medium.
The preparation
employed by Snell and Williams was a sample obtained from Kogl, and
was described as the methyl ester of biotin.
This material was five
hundred times more active than a similar weight of liver concentrate
known to contain biotin.
In 1937, Weizmann and Rosenfeld (51) described the successful cul­
tivation of Clostridium acetobutvlicum in a synthetic medium,
plemented by a yeast concentrate factor and asparagine.
sup­
The factor
supplied by the yeast concentrate could be replaced b y certain
cereal grains.
Riboflavin and cozymase were found to be inactive in
the nutrition of this organism.
In 1939, these investigators reported
(52) the similarity of the factor obtained from yeast concentrates,
and that observed in egg yolk by Kogl and Toimis (29) in 1936, and
called biotin.
Further studies with concentrates from various sources
have enabled Weizmann and Rosenfeld to conclude that biotin is an
essential factor in the nutrition of Clostridium acetobutvlicum.
They
note, however, that another factor is required by this organism, since
only a seventy per cent yield of solvents can be obtained in a
synthetic medium containing biotin and asparagine.
West and Wilson
(53) (54) (55) have pointed out the similarity in response of organisms
of the genus Rhizobium to biotin and Coenzyme R preparations.
These
workers demonstrated that, in the presence of small amounts of betaalanine, both of these preparations gave about the same degree of
stimulation to several members of the Rhizobium group.
On the basis of
these investigations, West and Wilson have concluded that biotin and
coenzyme R are either identical, or that the relationship between them
is so close that available physical, chemical and physiological methods
are not able to distinguish between then:.
hilsson, Bj«lve, and Btlrstrom (56) have confirmed the conclusions
of West and Wilson in regard to the need of Rhizobia for biotin.
Em­
ploying a pure methyl ester of biotin, these workers have been able to
demonstrate that growth of many strains of root nodule bacteria, in a
suitable synthetic medium, was essentially the same as in the presence
of yeast extract.
Iittller (57), in his studies of the nutritional requirements of
lactic acid bacteria, has reported the stimulation of several strains
upon the addition of a highly purified biotin preparation to the
basal medium.
Choline
The use of choline as a growth factor in the nutrition of
bacteria was first indicated by Rane and Subbarow (53) in their study
of the nutritional requirements of the pneumococcus.
These workers
reported the successful cultivation of Types I, II, V and VIII
pneumococcus, and indicate that most of the other types have not
been investigated.
Choline was found to stimulate the growth of the
pneumococcus, combined with pantothenic and nicotinic acids in a
basal medium composed of gelatin hydrolysate, inorganic salts, and
certain amino acids.
The optimal concentration of choline varied some-
vjhat with the various types of pneumococci, and the amounts of panto­
thenic and nicotinic acids present in the medium.
No other instances of the use of choline as a growth factor for
bacteria have been reported.
Codehydrogenases
Lwoff and Lwoff (59) have been able to show that codehydrogenase
is an essential factor in the nutrition of Hemophilus influenzae.
It
had long been recognized as growth Factor "V", and its identification
as codehydrogenase was made by the above workers in 1937.
Codehydrogenase may be found in yeast, blood corpuscles, animal
and vegetable tissues.
Lwoff asserts that the best source is yeast,
and has been able to show that 0.004 microgram of codehydrogenase per
milliliter of basal medium permits growth of Hemophilus influenzae.
It is interesting to note that these workers feel "all freeliving and most parasitic bacteria, with a few exceptions, synthesize
codehydrogenases"•
Rao (64) has just recently reported the need of Bacillus pestis
for cozymase.
Glutamine
Mcllwain, Fildes, Gladstone and Knight (60) have disclosed the
presence of glutamine in meat extracts, and have found this substance
to be essential to the nutrition of the "Richards" strain of Strept­
ococcus hemolvticus.
These investigators have also studied the effect
of glutamine on ten other strains of hemolytic streptococci, and found
that only two of these strains grew as well without the addition of
glutamine to the basal medium.
In a subsequent paper, Mcllwain (61) has studied the specificity
of glutamine on the growth of Streptococcus hemolvticus. and reports
that a large number of glutamine analogues and derivatives have been
found incapable of replacing glutamine.
Fildes and Gladstone (62) have investigated the need of glut­
amine for several bacterial species, and the results of this
investigation are interesting.
Several strains of hemolytic strept­
ococci, as well as several members of Group A, Group B, Group G,
Group E, Group F and Group G streptococci were found to require
glutamine.
A type I pneumococcus, grown on a simplified medium,
showed marked stimulation upon the addition of glutamine to the
basal medium.
Bacillus anthracis was examined, and five out of six
strains showed some acceleration of development vrhen glutamine was
added to the culture medium.
A strain of Proteus vulgaris responded
vigorously to the presence of glutamine in a basal medium.
Certain
strains of Escherichia coli. Hemophilus influenzae. a few strains of
Corvnebacterium diohtheriae. Staphylococcus aureus. and Neisseria
gonorrheae gave either no grovrth increase or a trivial response
upon the addition of glutamine to the medium in which they were
cultured.
Hematin
The need of certain bacteria for blood has long been recognized.
Possibly the first observation, however, that the hemoglobin in the
blood was the essential substance required by a bacterial species was
made by Pfeiffer in 1893 (63)> while working with Hemophilus influenzae.
Pfeiffer obtained hemoglobin in pure crystalline form, and added it as
such to ordinary media.
He observed that hemoglobin was essential to
the continued growth of the organism, while the serum and other
constituents were not.
Recently, Kao (64) found that the plague bacillus requires
heioatin for its growth.
He found that hematin accelerates the rate
of growth of washed plague bacilli in an amino acid medium containing
all the essentials for growth.
Nicotinic Acid
Mueller (33) (65), in 1937, was able to isolate from certain liver
extracts a substance which he identified as nicotinic acid.
He was
able to demonstrate that nicotinic acid possessed growth-promoting ac­
tivity for the diphtheria bacillus.
The optimal concentration of nico­
tinic acid for growth stimulation appears to vary, as Mueller points
out, with the concentration of one or more other substances in the
medium.
By the addition of certain other essential factors to the
medium, the optimal concentration of nicotinic acid increases as great­
ly as tenfold.
Mueller also found that the amide of nicotinic acid
appears to be only about one-tenth as effective, weight for weight, as
nicotinic acid.
This is in contrast to what Knight (66) found to be
true in the nutritional requirements of Staphylococcus aureus, where
the amide of nicotinic acid was found to be five times more active than
the acid.
Relative to the need of Staphylococcus aureus for thiamin, it has
been noted that Knight (66) found this organism to require also nicotinic
acid.
Knight demonstrated that nicotinic acid could replace a growth
factor found in the high-vacuum distillate derived from autolyzed yeast.
He demonstrated that closely allied derivatives of nicotinic acid
could be substituted for the acid with varying responses in growth of
the organism.
Nicotinamide was found to be about five times more active
than the acid in giving traces of growth, while methylnicotinate gave
the same amount of growth as an equivalent amount of nicotinic acid, but
growth proceeded considerably slower.
Knight found that the optimal
concentration of nicotinic acid, in the presence of 0.002 micro­
gram per milliliter of thiamin, was 0.20 microgram per milliliter of
basal medium.
Fildes (67) has succeeded in growing several strains of Proteus
vulgaris in a synthetic medium composed of inorganic salts, ammonium
lactate, and nicotinic acid.
It is of particular interest to note
that Fildes was able to demonstrate that as nicotinic acid was re­
moved from the medium, cozymase was being synthesized at a correspond­
ing rate.
This would strongly suggest that nicotinic acid is used by
this group of organisms to synthesize the cozymase molecule, which, as
other workers have demonstrated, yields nicotinic acid, among other
substances, upon hydrolysis.
Pelczar and Porter (68) have recently confirmed the work of Fildes
with one hundred and eighty-nine strains of Proteus vulgaris and re­
lated species.
Certain similar pyridine compounds which had been
demonstrated by Knight (66) to be active in the nutrition of Staphylo­
coccus aureus we re also shown by these observers to be essential for
the majority of Proteus strains investigated.
Pelczar and Porter
furthermore noted that many strains of ;.organ* s bacillus, which re­
cently has been classified (69) with the genus Proteus, were unable
to grow in a basal medium containing nicotinic acid.
At the time,
these investigators indicated that some additional factor was essential
for the growth of Morgan's bacillus (Proteus morganii). iSubsequently,
however, this conclusion has been confirmed by these workers (70), for
they have observed excellent grovrth in a basal medium containing
nicotinic acid and pantothenic acid.
Kliger and Grosowitz (71)(72) have reported a study of the fer­
mentation of glucose by members of the colon-typhoid group in a semi­
synthetic medium.
These vrorkers disclosed that Salmonella paratyphi A,
Shigella dysenteriae Shiga, Shigella dvsenteriae Flexner. and Shigella
dvsenteriae Y are unable to ferment glucose unless nicotinic acid is
Drsssnt in the medium.
Other strains such as Ebertheila typhosa (H441),
Salmonella paratyphi 13, and Escherichia coli were found to be able to
ferment glucose without the aid of nicotinic acid.
The authors report
tnat the difference thus established between Salmonella paratyphi A,
and Salmonella paratyphi B constitutes another simple means of differ­
entiation between the two species.
Kerby (73) found that nicotinic acid, in conjunction with thiamin
in a suitable basal medium, stimulated the growth of several strains of
Brucella abortus. She observed that thirty nicrograms of nicotinic acid
per milliliter of basal medium, in the presence of twenty-five micrograms
per milliliter of thiamin, produced the optimal growth-stimulating effect.
It is interesting to note that the concentrations of both nicotinic acid
and thiamin required for best growth are considerably higher than the
optimal amounts of these substances reported by other workers for growth
stimulation of various bacterial species.
Kerby used Bacto Tryptose agar
as a basal medium, and it is probable that the use of such a chemically
undefined medium may explain the need for relatively large doses to obtain
optimal growth response.
Koser and his associates (74)(75)(76) found that nicotinic acid was
essential for the growth of the dysentery bacillus.
They found that in a
medium, containing anino acids, glucose and several inorganic salts,
luxuriant growth took place in the presence of 0.10 microgram of nico­
tinic acid per milliliter of basal medium, after 24 hours incubation.
Further, they found that nicotinamide was slightly more active than
nicotinic acid, methyl nicotinate about equally as active, while ethyl
nicotinate was less active than nicotinic acid.
Snell, Strong and Peterson (77)(78) have reported stimulation of
growth of Lactobacillus easel and Lactobacillus arabinosns upon the
addition of nicotinic acid to a basal medium.
The optimal concentration
of nicotinic acid for development of these two forms, in the basal medium
described, was between 0.30 and 0.50 microgram per milliliter.
Further­
more, it was observed that the addition of nicotinic acid to the basal
medium greatly stimulated growth in the first culture, but that growth
failed to take place on subculture.
These workers conclude that this is
due to a lack of other essential growth factors in the medium.
In the
same medium containing nicotinic acid, it was demonstrated that no growth
occurred when inoculated with Leuconostoc mesenteroides. Proprionibacterium
pentosaoeum. and Streptococcus lastis.
Berkman, Saunders and Koser (79) have described the successful cul­
tivation of several strains of Pasteurella in a medium composed of
gelatin hydrolysate, certain inorganic salts, pantothenic acid, and
nicotinamide.
These investigators reported that cozymase could be
substituted successfully for nicotinamide.
Evidently, therefore, these
organisms have not lost their capacity to synthesize cozymase, if
nicotinamide is available in the culture medium.
Thirteen of the seventeen
strains of Pasteurella examined gave an excellent growth response in the
presence of pantothenic acid and nicotinamide, and the four remaining
strains gave a comparatively scant growth.
It was found subsequently
that a "butyl factor for Clostridia", probably containing biotin as
the active component, was essential for more abundant bacterial
development.
The optimal concentration of nicotinamide required in
the basal medium described was found to be 0.10 microgram per milli­
liter of medium.
Rane and Subbarow (58) have reported the successful cultivation
of certain types of pneumococci in a synthetic medium containing nico­
tinic acid, pantothenic acid, and choline.
The types of pneumococci
which grew in this synthetic medium were found to be Types I, II, V and
VIII, and the authors report that most of the remaining types were not
investigated in this medium.
The optimal concentration of nicotinic
acid for development of the types investigated was approximately the
same, and in the presence of an excess of the other factors was
found to be about 2.0 micrograms per milliliter of medium.
Hornibrook (80) has demonstrated recently that Hemophilus pertus­
sis requires nicotinic acid or nicotinamide as a growth factor.
In
a medium containing certain inorganic salts, soluble starch, and various
amino acids, he was able to show that nicotinic acid stimulated the
growth of this organism.
The technique which Hornibrook employed to
demonstrate the need of this organism for nicotinic acid is interest­
ing.
In contrast to the usual method of noting an increase in turbid­
ity upon the addition of a test substance to a basal medium, Hornibrook
diluted out the culture inoculum to a point where growth in the basal
medium failed to take place.
At the lowest dilution of culture fail­
ing to give growth, he added 0.5 to 0.001 microgram of nicotinic acid
or nicotinamide per milliliter of basal medium, and found thereupon
that growth again took place.
Rao (64) has found nicotinic acid is essential for the growth
of the plague bacillus (Bacillus pestis).
Pantothenic Acid
Snell, Strong and Peterson (81) in 1937 described the preparation
of certain concentrates of an essential growth factor for lactic acid
bacteria.
After considerable effort, these workers were unable to
purify this factor sufficiently for identification.
However, they
recognized (78) a similarity in properties between the factor with
which they were working and that which Williams et al. (2) had de­
scribed as pantothenic acid.
From Tdlliame they were able to obtain
two samples of calcium pantothenate, which, in conjunction with nico­
tinic acid, were found to be highly active for several strains of lac­
tic acid bacteria, and one species of proprionic acid bacteria.
Sub­
sequently, these workers enlarged their original work on the lactic
acid bacteria (77), and found that Lactobacillus casei, Lactobacillus
arabinosus. Lactobacillus pentosus. Bacillus brassicae. Bacillus lactis-acidi. Leuconostoc mesenteroides. and Streptococcus lactis required
pantothenic acid for growth.
The proprionic acid organism requiring
pantothenic acid was identified as Proprioni-bacterium pentosaceum.
Lactobacillus delbruckii. and Lactobacillus manitopoeus were found to
be unable to avail themselves of pantothenic acid, and these workers
suggested that possibly additional factors are required for growth of
these organisms.
By the use of Lactobacillus casei. Pennington, Snell and
Williams (82) have described a microbiological method for the assay of
pantothenic acid.
The sensitivity of the test is such that 0.001
microgram of pantothenic acid can be detected in one milliliter of
an unknown preparation.
Berkman, Saunders and Koser (79) found that pantothenic acid
and nicotinamide were essential for the nutrition of several strains of
Pasteurella, including Pasteurella avicida and Pasteurella boviseptica
I.
In a basal medium composed of gelatin hydrolysate, certain inorgan­
ic salts and glucose, the optimal concentration of pantothenic acid for
growth was found to be 0.10 microgram per milliliter of medium.
Pelczar and Porter (08) reported they have been able to obtain
growth of most species of Proteus in a chemically defined medium con­
sisting of inorganic salts, glucose, and nicotinic acid.
They were
unable to culture Proteus morganii in this medium, however, and con­
cluded that this species required additional factors for development.
Recently (70) these workers announced the successful cultivation of
Proteus morganii in the medium supporting the grovrth of other strains
of Proteus, upon the addition of pantothenic acid.
'.Voolley and Hutchings (83) vr re able to show that a calcium panto­
thenate salt, in the presence of "reduced" iron and riboflavin, stimu­
lated the growth of Streptococcus zymogenes.
The calcium pantothenate
preparation used by these workers was not a pure substance, but evidence
is presented which strongly indicates that pantothenic acid is the ac­
tive principle.
The optimal concentration of pantothenic acid in the
presence of 1.0 ;oicrogram of riboflavin per milliliter of medium was
found to be 0.10 to 1.0 microgram per milliliter.
Subbarow and Rane (84) found that a pantothenic acid preparation
derived from natural sources could replace the calcium-alcoholic pre­
cipitate of liver extract in the nutrition of the Dochez IiY 5 strain of
Streptococcus hemolyticus.
With this pantothenic acid preparation,
these investigators were able to provide almost optimal conditions
for the grovrth of this organism in a synthetic medium.
pantothenic acid was found by Schuman and Farrell (85) to be
essential in the nutrition of Streptococcus rheumaticus.
These
workers demonstrated the optimal concentration of pantothenic acid, in
the i-presence of 0.5 micro gran of '/itamin 3/.
and 0.3 microgram of
O '
riboflavin, to be 1,5 micrograms per milliliter of basal medium.
hcllwain (86) briefly reported the successful cultivation of the
"Richards" strain of Streptococcus hemolyticus in a synthetic medium,
kcllwain was engaged in the isolation of an active factor from yeast,
when a publication by Subbarow and Kane (84) suggested that pantothen­
ic acid was the factor with which he was working.
Upon securing a
sample of pantothenic acid, he was able to confirm the data of Sub­
barow and Rane.
Rane and Subbarow recently (58) described the successful cultiva­
tion of a few of the more important types of pneumococci in a chemi­
cally defined medium.
Grovrth was obtained on a medium consisting of
gelatin hydrolysate, certain amino acids, inorganic salts, glucose,
choline, nicotinic acid, and pantothenic acid.
The pantothenic acid
preparation used in this study was derived from natural sources, but
it was found that a synthetic pantothenic acid preparati on successfully
replaced the naturally derived material for the development of Type II
pneuraococcus.
The authors did not attempt to replace the synthetic
preparation for the naturally derived pantothenic acid for the other
types of pneumococci investigated.
The optiioal concentration of panto­
thenic acid for growth was found to vary when the amounts of choline
37
and nicotinic
acid were varied, thus indicating, possibly, that other
factors are important in the nutrition of these organisms.
Mueller and Klotz (87) have demonstrated that the diphtheria
bacillus avails itself of pantothenic acid in its growth.
Mueller (88)
had previously demonstrated that beta-alanine was essential to the
development of certain strains of the diphtheria bacillus.
Previous
to the synthesis of pantothenic acid, it was known that beta-alanine
was a component of the pantothenic acid molecule, combined with a
lactone.
Mueller and Klotz set up an experiment designed to discover
whether beta-alanine was converted to pantothenic acid for use by the
diphtheria bacillus.
It was found that the organism apparently
synthesized pantothenic acid from beta-alanine, for pantothenic acid,
when supplied to the organism, was active in very m uch smaller con­
centrations, and growth was found to proceed more rapidly and reg­
ularly.
Evans, Handley and Happold (89) have reported the need of panto­
thenic acid for certain "exacting" strains of Corynebacterium diphtheriae
gravis, when grown in a synthetic medium.
These w r k e r s report that
the "exacting" strains of Corynebacterium diphtheriae gravis ■were not
able to synthesize pantothenic acid from beta-alanine.
All other
strains of Corynebacterium diphtheriae (mitis, some gravis, and inter­
mediate) examined by these workers were able to grow in a synthetic
medium containing beta-alanine.
Presumably, these strains we re able
to synthesize pantothenic acid from beta-alanine.
The work of
Mueller and Klotz (87) regarding the function of beta-alanine and the
need of pantothenic acid by Corynebacterium diphtheriae has been
confirmed, therefore.
para-amino Benzoic Acid
Para-amino benzoic acid recently has (4) been demonstrated to be
a growth factor in the nutrition of Clostridium acetobutvlicum by
Rubbo and Gillespie.
By use of a yeast extract, these workers were
able to show a definite increase in the grovrth of the organism on a
basal medium composed of certain inorganic salts, asparagine, and
glucose.
Fractionation of the yeast extract demonstrated the pre­
sence of para-amino benzoic acid, which upon addition to the basal
medium was able to replace completely the stimulation of the original
yeast extract.
The optimal concentration of para-amino benzoic acid for
growth was not indicated, but it was shown that a response could be
noted upon the addition of 0.20 to 0.00002 micrograms per milliliter of
basal medium.
The substitution of meta- and ortho-amino benzoic acid resulted
in grovrth, but the concentration of these substitutes required to
yield a response was considerably greater, and the authors conclude,
therefore, that these substances are "probably inactive".
Novocaine,
a local anaesthetic, (para-amino benzoyl-diethyl aminoethanol) was
shown by Rubbo and Gillespie to have practically as much activity in
stimulation of the growth of Clostridium acetobutvlicum as para-amino
benzoic acid.
beizmann and Rosenfeld (52) have demonstrated the need of biotin
for Clostridium acetobutvlicum.
Rubbo and Gillespie (4), in comment­
ing upon this observation, conclude that the biotin preparation used
by IVeizmann and Rosenfeld contained para-amino benzoic acid.
It is
not clear from this statement whether they believe biotin and paraamino benzoic acid both function as grovrth factors for this organism,
or if para-amino benzoic acid alone is active for this species.
Phthiocol, 2-Methyl-li4 Naphthoquinone, and Vitamin K
Woolley and McCarter (90) have been able to demonstrate the
need of a bacterial species for a fat soluble, growth-stimulating com­
pound.
At present, this work is the only reported instance of the
need of such a substance in the nutrition of bacteria.
However, it
has long been recognized that fat soluble compounds are present in
the cells of same bacterial species.
It is particularly interesting
to note that phthiocol, isolated from human strains of the tubercle
bacillus by Newman, Crowder and Anderson (91), is active as a growthstimulating factor in the nutrition of a related species, Mycobact­
erium paratuberculosis (Johne's bacillus).
On a synthetic medium composed of asparagine, glycerol, and
certain inorganic salts, Yfoolley and McCarter have been able to
get a comparatively scant amount of growth of Mycobacterium para­
tuberculosis.
When the basal medium was supplemented with either
phthiocol, 2-njethyl-l:4 naphthoquinone, or Vitamin K, it became
apparent that an increase in growth resulted.
These workers also
noted, however, that the addition of either of these substances to
the basal medium does not completely satisfy the nutritional re­
quirements of the organism.
Certain cell extracts are reported to
further increase the amount of growth.
As Woolley and McCarter observe, it is of interest to consider
whether the utilization of a fat soluble growth factor is in any
way concerned with the very slow growth rate or with the high fat
content of these organisms.
Pimelic Acid
Pimelic acid was demonstrated to accelerate the grovrth of a
certain strain of the diphtheria bacillus.
Mueller and Subbarow
(92) were able to show that a substance obtained from a liver ex­
tract definitely stimulated the development of the diphtheria
bacillus, and concluded that the substance concerned was probably
an organic acid.
Subsequently, Mueller (93) found that -this same substance was
present in the urine of the horse and cow, and by means of a vacuum
distillation column, obtained an active fraction of dimethyl pimelate.
In comparing a pimelic acid fraction derived from natural sources with
a synthetically prepared product, Mueller could observe no difference
in growth stimulation of the diphtheria bacillus.
Pimelic acid exerts
an apparent effect on growth of this organism in a concentration of
0.005 microgram per milliliter of medium, and reaches a maximum
growth-stimulation at a concentration of 0.025 microgram per milliliter.
Further increases in pimelic acid up to 1,0% has no additional effect
on the grovrth of the organism.
Uracil
Richardson (94), in 1936, discovered that Staphylococcus aureus
could be grown anaerobically on a simple medium containing pyruvic acid,
if uracil were added.
It is interesting to note that this organism
fails to grow anaerobically on this medium in the presence of uracil.
However, Richardson has shown that during the anaerobic growth of Staphy­
lococcus aureus and Eberthella t v phl. the only organism tested b y the
author, uracil is synthesized.
This would indicate that uracil may
have an important function in bacterial nutrition in general.
The need of this organism for uracil in anaerobic growth is
z-x
quite specific,
_
Richardson has presented evidence indicating that
several pyrimidines, purines and other cyclic nitrogenous compounds,
mostly synthetic in origin, fail to give the response to growth
characteristic of uracil.
This would indicate that the anaerobic
growth of Staphylococcus aureus may provide a specific test for the
identification of uracil.
Furthermore, the method appears to be
sensitive, since Richardson has observed traces of growth after 48
hours in the presence of as little as 4.4 micrograms per milliliter
of l>2,scii medium*
Vitamin 3^; Thiamin Chloride Hydrochloride
Tatum, Rood and Peterson (95) were the first workers definitely
able to demonstrate the necessity of thiamin for the nutrition of a
bacterial species.
Employing several strains of Proprionibacterium,
they were able to replace various protein hydrolysates with thiamin,
and observe an increase in grovrth comparable to that exerted by the
hydrolysates.
Certain chemical similarities of the stimulating factor
in the protein hydrolysates and thiamin were also demonstrated.
Among
those noted were the similarity of solubility in water, acetone, and
alcohol; insolubility in ether;
stability to acid and destruction by
alkali; and finally, aasorbability on norite charcoal.
These investi­
gators also found that inositol, pantothenic acid, ascorbic acid
(Vitamin C), hepatoflavin (Vitamin
nicotinic acid amide, and
indolacetic acid could not replace the stimulating properties of
thiamin.
The optimum concentration of thiamin for growth stimulation
of the species studied was found to be 0.005 microgram per milliliter
of medium.
In 1937, Knight (34) was aole to show that one of the active com-
ponents in the high-vacuum distillate derived from autolyzed yeast
stimulated growth of Staphylococcus aureus*
component to be thiamin.
Knight demonstrated this
Hughes (96), in 1932, had concluded from his
work on meat extract fractionations that a substance actively stimulat­
ing the growth of Staphylococcus aureus could be classified as a
member of the "Vitamin B group".
Synthetic Vitamin
(thiamin) had
not made its appearance as yet, and Hughes was, therefore, unable to
verify his conclusions with pure substances.
Knight found that thiamin added alone to a basal medium gave very
little stimulation of growth.
However, in conjunction with nicotinic
acid, which was also found by Knight to be essential for the nutrition
of Staphylococcus aureus, an abundant response took place.
In the
presence of 0.2 microgram of nicotinic acid per milliliter of basal medium,
Knight observed that 0.002 microgram of thiamin per milliliter of
basal medium exerted an optimum effect on growth.
Koser and others (97) confirmed the work of Knight regarding the
growth-stimulating capacity of thiamin, and also showed that one strain
of Staphylococcus albus responded to this factor.
Kerby (73; found that thiamin hydrochloride and nicotinic acid
enhanced -the growth of several strains of Brucella abortus in a non­
synthetic basal medium.
She found that some strains grew better in a
basal medium containing thiamin hydrochloride only, while others ap­
peared to require nicotinic acid only.
However, Kerby observed that
the great majority of the strains studied were found to require both
thiamin hydrochloride and nicotinic acid for optimum response.
The
concentration of thiamin hydrochloride found to be optimal for Brucella
abortus, in conjunction with an excess of nicotinic acid, was 25
micrograms per milliliter of medium.
It is interesting to note that
the optimal concentration of thiamin hydrochloride for growth of
Brucella abortus is large in comparison with the needs of other bact­
erial species for this factor.
It is possible, however, that the com­
plex meat broth medium used by Kerby as a basal medium contains factors
inhibiting the response of the organism to lower concentrations of
the vitaruin#
Kerby further reported that the development of one strain of
Brucella melitensis was inhibited by the addition of thiamin hydro­
chloride or nicotinic acid.
Strains of Brucella suis were not in­
vestigated for response to either thiamin hydrochloride or nicotinic
acid.
best and Wilson (19) have been able to demonstrate that Rhizobium
trifolii requires both thiamin hydrochloride and riboflavin for growth.
These authors find W a t the optimal concentration of thiamin hydro­
chloride for stimulation depends upon the amount of riboflavin present,
and vice versa.
Furthermore, it was pointed out that if sub-optimal
concentrations of either of these compounds were added to a basal
medium separately, growth resulting from the addition of either was
frequently greater than when the same concentration of both factors
was added to the medium.
'Vest and Wilson report that 0.02 microgram
of thiamin hydrochloride, in conjunction with the same amount of ribo­
flavin, produces the optimal growth effect.
The above workers have also pointed out that Rhizobium trifolii
synthesizes rather large amounts of both thiamin and riboflavin during
development in a synthetic medium.
It is probable, therefore, that in
the root nodule these vitamins are also synthesized, and it would
be interesting to know if these substances are utilized nutrition­
ally by the infected plant.
Nilsson, Bjalve, and Eurstrom (98) have been able to demons­
trate a need for thiamin hydrochloride by Bacterium radicicola.
These
investigators have reported that in a synthetic medium, composed of a
casein hydrolysate, and certain inorganic salts, thiamin hydrochloride
will stimulate this organism in concentrations as low as 0.006 microgram
per milliliter of basal medium*
It was also pointed out in the above
report that growth in a synthetic medium was not as luxuriant as that
obtained when certain yeast or molasses extracts were added to the
medium.
This would indicate definitely that other factors plan an
important part in the nutrition of this organism.
Rao (64) has recently described the need of Bacillus pestis for
Vitamin Bp, in a synthetic medium composed of amino acids and other
essentials for growth.
Vitamin Bp; Riboflavin
Orla-Jensen, Otte, and Snog-Kjaer (99), in 1936, found that
growth activation of certain lactic acid bacteria took place in a
basal medium supplemented with a yeast fraction known to contain ribo­
flavin.
These investigators found that all the true lactic acid
bacteria responded to fractions known to contain riboflavin, and were
unable to cultivate these species in a riboflavin-free medium.
How­
ever, it must be noted that they were not working with pure riboflavin,
and consequently could not offer conclusive evidence for their
observations.
Wood, Anderson and Y/erkman (100) (101) confirmed the work of OrlaJensen et al. (99) regarding the need of certain lactic acid bacteria
for riboflavin, but found one species of lactic acid fermenters, Lacto­
bacillus c entoaceticus, which did not require riboflavin for growth.
They also demonstrated that Lactobacillus delbruckii and Lactobacillus
helveticum cannot grow on a basal medium containing hydrolyzed casein,
tryptophane and riboflavin, unless an alcohol-soluble fraction of malt
sprouts is added to the medium.
Subsequently, Snell, Strong and Peter­
son were able to show that this factor was pantothenic acid (84).
V/ood,
Anderson and Y/erkman also reported the need of riboflavin for several
species of Proprionibacterium. and thus extended and confirmed the work
of Tatum, ’hood and Peterson (95).
Snell and Strong (102) investigated the riboflavin requirements of
a number of lactic acid bacteria, and found that seven of the eleven
species examined did not require riboflavin for growth and acid produc­
tion.
Three species of true lactic acid bacteria, Streptococcus lactis,
Lactobacillus pentosus, and Lactobacillus arabinosus, are able to syn­
thesize riboflavin in a riboflavin-free medium.
These results do not
agree with the conclusions of Orla-Jensen et al. (99 )» who reported
that all true lactic acid bacteria require riboflavin for growth.
Orla-
Jensen et al. did not use pure riboflavin in their investigations, and
it is conceivable, therefore, that another factor complicated their
interpretations.
Snell and Strong also presented evidence showing that
the response of Lactobacillus casei and Bacillus lactis to a number of
synthetic flavins parallels rather closely the effect of these same
flavins on the growth of rats.
7,’est and Wilson (19) have been able to show that riboflavin, in
conjunction with thiamin, is required b y Rhizobium trifolii.
As
indicated in the discussion of those organisms requiring thiamin, a
very definite inter-relationship exists between riboflavin and thiamin
concentrations for optimal growth response of this organism.
The
optimal concentration of riboflavin, in conjunction with the same
amount of thiamin hydrochloride, was found to be 0.02 microgram per
milliliter of basal medium.
Rane and Subbarow (103) reported the need of riboflavin for the
growth of the Dochez NY5 strain of Streptococcus in a basal m edium to
which a calcium-alcoholic precipitate of liver extract had been added.
They reported that glutathione, thiochrome, riboflavin, nicotinic acid,
betaine, and glucosamine are necessary for the development of this
organism, and the omission of any of the above factors resulted in a
failure of growth.
The optimal concentration of riboflavin w a s found
to be 0.10 microgram per milliliter of basal medium.
Subsequently,
Subbarow and Rane (84) have demonstrated that pantothenic acid can
replace the calcium-alcoholic precipitate of liver extract,
Woolley and Eutchings (83) treated a complete medium for the
growth of Streptococcus epidemicus. strain X40, with alkali and noted
that a response no longer occurred after such a procedure.
Upon the
addition of pantothenic acid and riboflavin to an alkali-treated
medium, in the presence of "reduced” iron, development was again
observed to take place.
The optimal concentration of riboflavin re­
quired for growth was found to be 0.1 microgram per milliliter of basal
medium.
A number of other hemolytic streptococci were demonstrated to have
requirements similar to those of Streptococcus epidemicus. strain
X40.
Three additional strains of Streptococcus epidemicus, namely,
C108, X32 and W116-7 responded t o the same growth stimulants.
Strepto­
coccus pyogenes, strain J17A4 (Lancefield group A) and Streptococcus
equi, strain F132 (Lancefield group C) behaved as the strains of
Streptococcus epidomicus.
A Lancefield group B organism, Streptococcus
mastitidis, strain 0-90R, and a Lancefield group D organism, Strepto­
coccus zvmogenes. strain H-6905, failed to develop on the alkalitreated medium, but responded satisfactorily when pantothenic acid and
riboflavin were added.
Schuman and Farrell (85) have reported that Streptococcus rheumaticus could grow in a synthetic medium composed of glucose, an inor­
ganic salt mixture, and seven amino acids, in the presence of panto­
thenic acid, riboflavin and Vitamin Bg.
The optimal concentration of
riboflavin, in the presence of 1.5 micrograms of pantothenic acid and
0.5 raierogram of Vitamin Bg per milliliter of medium, was found to be
0.3 microgram per milliliter.
Vitamin Bp
j Pyridoxine
In 1938, HSller (104) discovered another growth-promoting factor
for bacteria, namely. Vitamin Bg, when he demonstrated that this
substance was essential for certain lactic acid bacteria.
Moller,
previous to this discovery, observed that a yeast fraction w a s able to
further stimulate the development of certain lactic acid bacteria in a
synthetic medium.
This yeast fraction was replaced by a preparation
obtained by him from the I.G. Farbenindustrie.
The Farbenindustrie
fraction was known to contain 0.4 to 0.6 microgram of Vitamin B6 per
milliliter.
After certain high vacuum distillations, a pure cry­
stalline Vitamin Bg preparation was obtained, with a melting point of
204^-205° C.
MSller showed that Vitamin Bg was the active substance
for developmaxt of these organisms, and that the optimal concentration
for growth was 0.5 to 1.0 microgram per milliliter of medium.
Woolley and Hutchings (105) (106) demonstrated the necessity of
Vitamin Bg for a Lancefield group D streptococcus, namely Strepto­
coccus zymogenes. strain H-6905.
Streptococcus zvmogenes had previous­
ly been shown by these investigators (85) to be able to grow on a
synthetic medium containing riboflavin, pantothenic acid, and a suitable
reducing compound.
The addition of an aqueous liver fraction to this
medium further stimulated this organism in the synthetic medium
described by them.
Fractionation of the liver extract suggested the
possibility that Vitamin Bg was the active constituent, and upon
testing, found this to be true.
The addition of 0.5 microgram of
Vitamin Bg provided an optimal concentration following incubation for
72 hours.
In a brief report, Vilter and Spies (107) have described the
growth stimulating effects of Vitamin B e on a strain of Staphylococcus
albus.
They have used essentially the same basal medium to demonstrate
this effect as that employed by Koser and associates (97) in confirm­
ing the w o r k of Knight.
These workers have demonstrated that Vitamin
Bg in concentrations of 0.3 to 1.2 micrograms per milliliter of basal
medium enhances the growth of Staphylococcus albus.
These observations
were made, apparently, upon one strain of Staphylococcus albus. and it
would be indeed interesting to know if this need for Vitamin B c is
6
consistent for all or most strains of Staphylococcus albus.
This
might prove to be a valuable means of differentiating Staphylococcus
albus from Staphylococcus aureus, since no report of the need of
Vitamin Bg for Staphylococcus aureus has appeared.
Snell and Peterson (108) have confirmed the work of Miller (104)
regarding the necessity of Vitamin Bg for certain lactic acid bacteria.
These workers used a strain of Lactobacillus helvetieus, and upon the
fractionation of a yeast preparation found that yeast contained two
separate factors important to the nutrition of this organism.
One of
these substances was found to be readily adsorbed on norite, while the
other material was less readily adsorbed; the two preparations were,
as a result, referred to as the norite eluate and norite filtrate
factors, respectively.
Snell and Peterson substituted Vitamin Bg for
the norite filtrate factor and found that it replaced this substance
almost completely.
There was slightly more total acid produced in a culture containing
the norite filtrate preparation as compared with that produced by
Vitamin Bg, which, these workers state, may be due to the presence of
another factor or factors in the norite filtrates.
Schuman and Farrell (85) have demonstrated that Streptococcus
rheumaticus requires 0.3 microgram per milliliter of basal medium for
optimal growth.
In contrast with the work of Woolley and Hutchings
(106), however, they have not found a reducing substance essential for
maximum growth of their strain of streptococcus.
Mcllwain (109) found that a strain of Streptococcus hemolyticus,
growing in an acid-hydrolyzed egg albumin medium, containing certain
additional amino acids, glutamine and pantothenic acid, required a
yeast factor for optimal growth.
After several fractionation pro­
cedures, Mcllwain concluded that a similarity existed between the
active yeast factor, end Vitamin E g .
This observation was confirmed
experimaatally, and it was found that Vitamin Bg almost completely
replaced the active yeast factor.
The work of Woolley and Hutchings
(106), who employed another strain of streptococcus, was thus con­
firmed.
Summary of Bacteria Requiring Knovjn Growth Factors.
51,
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II.
EXPERIMENTAL WORK
A.
Experimental Methods
Sterilization
The sterilization of meat infusion, meat extract, or
other protein-containing bacteriological media involves the use of
steam, at atmospheric or higher pressures, or passage through steril­
izing filters of one type or another.
It has been known for some
time that certain of the nutritional reoxuirements of bacteria are
heat labile, and a method of sterilization employing heat must as a
consequence be abandoned in most instances.
In such a circumstance,
a medium nay be filtered to render it sterile. However, it is known
that in some instances certain mechanical or physical difficulties
make the use of a filter impossible.
Further, a filter may adsorb
or inactivate certain other preparations and thus prohibit its use.
Under those conditions, the bacteriologist is obliged to obtain the
required growth factors, which may be contained in blood, serum, or
plant and animal extracts, by sterile procedures, and upon addition
to the basal medium, observe the newly prepared medium at intervals
for sterility.
The recent developments in the study of bacterial
nutrition have presented basic principles regarding the sterilization
of bacteriological media.
It is possible to prepare a medium specif­
ically for use by a certain organism or group of organisms with com­
plex nutritional requirements, and understand the problems connected
with, the sterilization of that medium.
It has been demonstrated, for
example, that a given organism requires a growth promoting factor
which is not stable to heat at an alkaline reaction, but is stable to
heat at an acid reaction.
With this knowledge such a growth factor
can be sterilized separately arid subsequently added to the medium
aseptically.
Regarding the sterilization of synthetic media, it is general
practice to combine the heat stabile preparations before steriliza­
tion, while those substances which are stable to heat only under cer­
tain conditions are sterilized separately.
The heat-labile factors
are sterilized by means of bacteriological filters, and added to the
bulk of the medium aseptically later.
Sterilization by heat involves the minimum temperature and time
compatible with its purpose, and a temperature of 120° C. for ten to
fifteen minutes is sufficient to sterilize most synthetic medium
preparations.
Furthermore, it is important to cool the medium immed­
iately after sterilization, since undue prolongation to heat may par­
tially destroy certain of the growth factors, and thus lower the ac­
tual concentration of this substance or substances in the medium.
In work involving the fractionation of growth promoting sub­
stances, it is of prime importance to know the response of the un­
known substance to heat under all variations of circumstances, before
assay procedures can be followed intelligently.
Methods of Assay of Growth Response
The determination of the response of a micro-organism
to the omission of or addition to a basal medium of a given chemical
compound may be measured by various means.
Investigators in the field
of bacterial nutrition have not adopted one particular method of
growth assay, since each problem may present demands peculiar to it­
self.
In the following discussion, the more generally employed me­
thods of determination of growth response, with the advantages and
disadvantages of each method, are presented.
Several workers, and particularly the European investigators, have
employed visual inspection of the culture tubes for turbidity.
observations are recorded as / to / / / / ,
The
and indicate progressive
differences in relative turbidity resulting from growth.
It is ap­
parent that this method of response measurement is economical and
rapid, but its accuracy is very limited.
Furthermore, the response
obtained in one test cannot in all. instances be transposed to another,
since growth measurements are relative and not absolute.
Another method of assaying growth response by means of turbidity
involves the use of an instrument described by Gates (110), and sub­
sequently redesigned by Feemster, et al. (ill).
This apparatus in­
volves the use of "a disappearing loop" and at the point of disap­
pearance of the loop into a turbid bacterial suspension, a relatively
accurate estimation of the turbidity can be made.
The use of this in­
strument provides a rapid, economical, and absolute method of estimat­
ing the turbidity of many bacterial cultures.
Certain instances exist,
however, which make turbidity measurements by this instrument imprac­
ticable.
In bacterial cultures where growth responses are slight,
or the total number of cells few, one is unable to make estimations
of response by employing this apparatus.
A most satisfactory method of determination of turbidity of bac­
terial cultures involves the use of an instrument employing the photo­
electric cell.
Several types of instruments have been manufactured
using this principle, and each has its salient features.
Possibly,
the Evelyn colorimeter (112) has been more generally employed in bac­
terial nutrition studies than any other instrument.
Since the Evelyn
colorimeter was used in the studies reported in this thesis, we shall
briefly discuss some of the principles involved in its operation. When
light falls on a self-generating photo-electric cell connected in a
circuit of low resistance, a current is produced which is directly
proportional to the intensity of the light beam.
If a galvanometer of
very low interval resistance is included in the circuit, the deflec­
tion of the galvanometer is proportional to the intensity of light
falling on the photo-electric cell.
In the Evelyn colorimeter, a beam of filtered light is directed
upon the photo-electric cell, through a glass absorption tube in which
the turbid bacterial suspension under examination may be placed.
For
each determination, or series of determinations at one time, a blank
reading is obtained by introducing the uninoculated basal medium in a
glass absorption tube, and adjusting the galvanometer reading to 100.
If, without altering the intensity of the light beam, the blank is
replaced by the sample to be measured, the galvanometer deflection will
afford an accurate measurement of the percentage light transmission of
the sample suspension.
This, in turn, is a definite function of the
concentration of turbid substance in the suspension.
The galvanometer
reading may, therefore, be converted by means of a suitable calibration
into a measure of the concentration of the turbid material under inves­
tigation.
However, it is common practice among workers in bacterial
nutrition to report only the galvanometer readings in describing the
results of their work.
Since the galvanometer scale readings range from 0 to 100, or
from complete opacity to entire transmissability, the interpretation
of the galvanometer reading in terms of growth response is important.
In the tables describing the experimental work accomplished, it will
be noted that the basis for the determination of the necessity of a
given chemical compound in a basal medium depends upon the galvanometer
reading obtained.
If we adjust the galvanometer to read 100 with a
sample of uninoculated medium, it follows that any reading lower than
100 gives an index of the turbidity.
The lower the reading of the
galvanometer, the greater its turbidity, and, as we assume, the growth
response.
The use of color filters in the Evelyn colorimeter confines the
light used for the transmission measurements to the particular band
of wavelengths most strongly affected by the substance under test,
and thus increases considerably the sensitivity^ and accuracy of the
determination.
Since the Evelyn colorimeter is adapted to a variety
of uses in colorimetric quantitative chemical analysis, it is impor­
tant that the correct filter is used in the light beam. Because most
of the media employed for bacteriological studies have a yellow
color of greater or lesser intensity, a green filter (No. 54-0) is
employed.
The Evelyn colorimeter is rapid and extremely accurate.
However,
the cost of this instrument is greater than that of most photo-electric
colorimeters, but the variety of tests for which this instrument may
be used should not make it prohibitive in a well equipped laboratory.
Another accurate method of measurement of growth response involves
the estimation of the bacterial nitrogen.
This means of determination
has been developed by Mueller (113) in his studies of the nutritional
requirements of the diphtheria bacillus, and varied by others to ac­
commodate their immediate needs.
Essentially the method consists in
culturing the organism in a test tube with the end drawn to a blunt
point like a centrifuge tube.
The tubes are autoclaved after incuba­
tion has been completed, and then centrifuged to throw down the solid
material.
The supernatant is removed, and the sediment washed with
0.05 per cent acetic acid.
A second centrifugation is then carried
out, and the wash fluid drawn off.
For the actual determination of
nitrogen, Hueller employed the micro-kjeldahl method of Fregl (114),
but mentioned that any other micro-kjeldahl method would prove equally
satisfactory.
Koser and Saunders (40) feel that the method is subject
to some serious errors due to the possibility that nitrogenous material
from the medium might be included with the organisms, or if the organ­
isms are washed too thoroughly, nitrogen may oe lost.
The estimation of growth response by titrating the acid formed
during growth is a method that has yielded consistent results vdth cer­
tain groups of micro-organisms, notably the lactic acid bacteria.
The
method involves titration vdth 0.10 normal sodium hydroxide, and
a close correlation between acid formed during growth and turbidity
has been found.
Snell, Strong and Peterson (81) have employed this
method of assaying growth response in their studies of the nutritional
requirements of the lactic acid bacteria.
It may be said in conclusion to this discussion of assay methods
of growth response, that the methods presented do not involve any
consideration of the numbers of living cells present in the cultures
under examination.
The fact that all measurements of growth response,
irrespective of the method followed include a unit of time for ref­
erence obviates the necessity ox determining the numbers of living
bacteria in a given sample.
The Determination of Biotin in Biological Materials
One of the more important contributions to the study of
bacterial nutrition involves the conclusion that one of the butylalcohol-producing anaerobes, Clostridium butylicum, requires biotin
as the only accessory substance for growth in a synthetic medium (115)•
Clostridium butylieum is usually cultured in a corn mash in
yeast-water glucose, or in an inorganic salts-peptone-glucose medium
(116).
Tatum, Peterson, and Fred (117) found that 1-asparagine, or
related dicarboxylic acids, were necessary for a normal fermentation
in corn mash.
In 1938, Brown, Wood, and Werkman (118) demonstrated
that a normal yield of solvents could be obtained by growing Clos­
tridium butylieum in a medium containing hydrolyzed casein, ammonium
sulfate, tryptophane, glucose, inorganic salts, and an acidic, ethersoluble fraction of Difco Yeast Extract.
McDaniel, Woolley, and
Peterson (119) in 1939, reported that the acidic, ether-soluble frac­
tion of yeast extract could not be replaced by riboflavin, indoleacetic acid, the "sporogenes” growth factor, nicotinamide, Vitamin
B.. , Vitamin B,, pimelic acid, inositol, beta-alanine, or pantothenic
1
6
acid.
Soon after the appearance of this publication, Snell and Williams
(50) demonstrated that biotin could replace the acidic, ether-soluble
fraction described by the Wisconsin investigators as essential for
grow th.
They were able to show that biotin is the only accessory sub­
stance required for growth of Clostridium butylieum in a medium com­
posed of glucose, asparagine, and certain inorganic salts,
Snell, Eakin and Williams (120) have described a sensitive and
accurate assay for biotin, involving the growth of a strain of Saccharorayces cerevisiae from a small seeding.
Under the test conditions,
the response of this organism to biotin is reported to lie within a
range from 0.00002 to about 0.001 microgram per milliliter of medium.
The effectiveness of the biotin response under the conditions of this
test depends to a large extent on the presence of Vitamin B, and beta6
alanine.
Peterson, McDaniel, and McCoy (115) confirmed the investigations
of Snell and Williams regarding the need of Clostridium butylieum for
biotin.
These workers suggest that since biotin is the only growth
factor required by this organism, its growth in a synthetic medium
may be employed for assay purposes.
The response of Clostridium
butylieum to biotin is such that 0.00001 microgram per milliliter of
basal medium can be detected.
In this thesis, in the determination of the presence of biotin
in various biologies]- materials, the method suggested by Peterson,
McDaniel and McCoy has been followed.
Since Clostridium butylicum
requires biotin as the only accessory factor for growth in a syn­
thetic medium, and since the method of assay described by Snell,
Eakin and Williams (120) is complicated by variations in response due
to Vitamin B, and beta-alanine, it has been felt that the procedure
6
of Peterson, McDaniel and McCoy was the preferable method.
The method of biotin determination used in this investigation has
not been developed quantitatively.
Peterson, McDaniel, and McCoy have
reported, however, that good growth of Clostridium butylieum is ob­
tained with 0.00001 microgram of biotin per milliliter of medium.
In this study, therefore, it is not possible to report the concentra­
tions of biotin required for optimal growth of the organisms studied.
The description of the method presented is essentially that developed
by McDaniel, Woolley, and Peterson (119) in their investigations of
the nutritional requirements of Clostridium butylieum. and that re­
commended by Peterson, McDaniel, and McCoy (115) in the determination
of biotin in various biological preparations.
The organism employed for test purposes was Clostridium butylieum.
strain 21, described by Langlykke, Peterson, and Fred (121), and re­
ceived from Dr. W. H. Peterson of Madison, Wisconsin.
The stock cultures
of the organism were prepared by introduction of a well-sporulated
culture suspension into sterile soil, and allowing the soil to dry
thoroughly at 37° C.
After the soil had dried, it was mixed thor­
oughly to distribute the spores, and introduced aseptically into
sterile glass ampoules, which were then sealed.
For growth of culture inoculum, a medium containing an excess
of biotin was used.
This consists of 0,25 per cent. Difco Bacto pep­
tone, 0,10 per cent. 1-asparagine, 2.0 per cent, glucose, Speakman's
salts (K^HPO^, 0,5 gram; KHjPO^, 0,5 gram; MgS0^,7H^0, 0,2 gram;
NaCl 0.01 gram; FeSO ,7H 0, 0,01 gram; and MnSO ,3H 0, 0,01 gram per
A
2
A
2
liter), distilled water, and approximately 20 milligrams of "reduced”
iron per 35,0 milliliter of medium per tube.
8 X 1 inches in size.
The test tubes used were
This medium was sterilized at 120° G. for 15
minutes in the autoclave.
Before inoculation of the above medium, the tubes were heated in
a boiling water bath for ten minutes to drive out absorbed air, and
then cooled carefully.
The medium was inoculated with the soil prep­
arations containing spores, and again heated in a boiling water bath
for two minutes, and cooled immediately afterward.
The inoculated tubes were incubated anaerobically in an oat jar,
at 37° C.
An oat jar consists of a wide-mouth container with a cover,
with about two inches of moistened oats at the bottom.
After the in­
oculated medium has been placed in the container, the lid of the jar
was effectively sealed to prevent an exchange of gases.
After 2U
hours incubation, the culture was centrifuged, and the supernatant re­
moved,
The cells were resuspended in 0.90 per cent, saline, and again
centrifuged.
After a second washing with saline, the cells were re­
suspended to their original volume in saline, and the suspension was
considered adaptable to inoculation of the test solutions.
For the determination of biotin, the medium was prepared in the
same manner as described for the growth of culture inoculum, except
that no peptone was added.
To the test medium, various concentra­
tions of materials under investigation for the presence of biotin were
added so that the final volume of each tube was 35.0 milliliters.
The
medium can be autoclaved with the supplement present, since biotin is
stable to heat.
When possible, the medium was autoclaved just prior
to inoculation, at 120° C. for 15 minutes.
Upon cooling, each tube
was inoculated with 0,35 milliliter of washed cell suspension, intro­
duced into the test medium with the pipette below the surface.
The
tubes were then placed in an oat jar, and incubated at 37° C. for 72
hours.
After incubation was completed, the tubes were read for turbidity
with the Evelyn colorimeter, using a No, 540 color filter.
Some of
the tubes have been found to be gassing after 72 hours, and the gas
may be removed ouickly by means of a vacuum pump attachment.
The
presence of gas in a tube to be read can interfere considerably with
results.
The reading of turbidity consists in adnusting the light in­
tensity of the instrument so that the uninoculated basal medium gives
a galvanometer reading of 100.
A tube which gives a turbidity read­
ing on the Evelyn colorimeter of 50 is considered to have had a
growth-stimulating quantity of biotin originally present.
B. Experimental Study of the Nutrition of Clostridium Sordellii
Historical Survey
Sordelli (122)(123) reported the isolation of a hitherto
unrecognized micro-organism from cases of human gas gangrene, which was
found in two of eleven cases that came to his attention.
He was un­
able to identify these strains with any known species, and was impressed
by the fact that although their morphology and cultural characteristics
resembled those of the "bacilo del edema h'aligno (II Tipo de Fraenkel)",
their pathogenic properties resembled those of Clostridium novyi.
It
was found possible to produce a toxin from these organisms, which was
not neutralized in experimental animals by the antiserums produced by
the toxins of Clostridium welchii, Clostridium oedematis-maligni, Clos­
tridium novyi, or Clostridium histolyticum.
The cultural resemblance
of this new organism to Clostridium sporogenes suggested to Sordelli
that this organism might possibly be a pathogenic strain of this com­
monly non-pathogenic anaerobe.
On the basis of agglutination reac­
tions, there were no definite differences between the new strain and
those of Clostridium sporogenes. By the use of potent anti-sporogenes
serums, however, no protection was afforded against the toxin elabor­
ated by the organism under examination.
Upon the basis of these ob­
servations, Sordelli assigned the trinomial, Bacillus oedematis sporogenes, to the newly discovered pathogenic anaerobe.
Hall and Scott
(124) subsequently suggested the binomial, Bacillus sordellii, for
this organism, and with the revision of the nomenclature, this desig­
nation became Clostridium sordellii.
Lleleney, ffilumphreys, and Carp (125) reported the isolation of a
pathogenic anaerobic bacillus from a fatal wound infection.
These
workers were, at the time, unable to find any similarity between their
organism, and other pathogenic anaerobic bacteria previously described.
They found their organism to be pathogenic in small doses for eight
different species of laboratory animals.
Lesions were produced in
these animals resembling somewhat those produced by Clostridium oedematiens and Clostridium oedemat5 s-ma.li p-ni .
However, they found str ik ­
ing d ifferen ces in certain cu ltu ra l c h a r a c te r istic s , and se r o lo g ic a l
rea ctio n s.
On the b a sis o f th e ir observations, they proposed B a cillu s
oedematoides as a name for t h is sp e c ie s.
Immediately upon pu blication o f the report o f Meleney, Humphreys
and Carp (1 2 5 ), H all and S cott (121) reported the r e su lts of experi­
mental stu d ies with cu ltu res of the pathogenic anaerobe described by
S o r d e lli.
In general, th ese workers were able to confirm the observa­
tio n s of S o r d e lli, and furthermore suggested a c lo se r ela tio n sh ip to
or probable id e n tity with the organism discovered by Meleney, Humphreys
and Carp.
In a subsequent report, Humphreys and Meleney (127) presented
evidence to demonstrate th at the organism previously described by
them as B a cillu s oedematoides was id e n tic a l to the organism named Ba­
c il l u s pedematis sporogenes by S o r d e lli (12p)
redescribed as Ba­
c il l u s s o r d e llii by H all and S cott (121).
Morphological C h aracteristics
The morphology o f Clostridium s o r d e llii has been studied
completely by H all and S cott (I 2 i)*
These in v e stig a to r s have described
the organism as a gram -positive rod, 1 .2 to 1 .5 microns in diameter,
and 1 .5 to 8.0 microns in len gth , with rounded ends.
Filaments as long
as 15 macrons have o cca sio n a lly been associated with the organism.
S o rd elli (123) has observed m o tility of th is sp e c ie s, but H all and
Scott (124) have fa ile d to observe th is featu re except in young cu l­
tu res, and then only among very few organisms.
In brain medium the rods
are e ith e r sin g le or paired, and may form chains of three or four c e l l s .
In cultures three to five days of age more chains may be seen, with
many of the forms containing subterminal spores.
Cultural Characteristics
Clostridium sordellii grows well in all the common lab­
oratory media under strict anaerobic conditions.
The cultural charac­
teristics of growth in various media has been described by Hall, Rymer
and Jungherr (127), and several of these more important observations
will be discussed.
Growth in deep brain medium, at 37° C., is comparatively rapid.
Turbidity occurs in 6-8 hours, and gas formation within 24 hours.
Darkening of the brain medium takes place, and is increased by the
presence of an iron wire in the medium.
The odor produced by Clos­
tridium sordellii is distinctly putrefactive, and very suggestive of
boiled onions,
l.'hite crystals of the amino acid tyrosine are pro­
duced in this medium after about one week incubation.
In deep agar, marked variations in colony form have been observed.
With suitable dilution of the culture inoculum, well separated colonies
have been secured v/ith a diameter of 2-3 millimeters.
Sordelli (123)
observed some colonies that measured 8 millimeters in diameter.
Humphreys and Eeleney (126) have described the appearance of colonies
as "mossy".
Hall, Rymer and Jungherr (127) have observed them "to be
usually opaque, discreet, and with lobulated multiple discs disposed
often at angles v/ithin a single colony, and at other times, feathery
globular masses'.'.
Blood agar slants, incubated anaerobically, gives rise to colonies
of Clostridium sordellii which appear as tiny dew drops, increasing in
size and then spreading upon the surface.
The blood frequently fails
to hemolyze, or if so, only slightly.
Hall, Rymer and Jungherr (12?)
further examined the inability of Clostridium sordaTH-i to hemolyze
blood by using the filtrates of glucose broth cultures on washed and
unwashed citrated human red blood cells, citrated sheep cells, and defibrinated rabbit cells.
A mixture of equal parts of the culture fil­
trates and 5 per cent, cell suspensions in 0.85 per cent, sodium chlor­
ide solution wore incubated for one hour at 37° C., but little or no
hemolysis was observed for any strain of Clostridium sordellii.
Liquefaction of gelatin has been observed by Hall, Rymer and
Jungherr (127) with all strains of Clostridium sordellii investigated.
These workers found that moderate gas production occurred with the for­
mation of a black precipitate, if the cultures were grown anaerobically.
In a milk powder medium, Clostridium sordellii produced increasing
quantities of gas during the first five days of growth.
The casein
was precipitated as a soft coagulum in 7 or 8 days, and the appearance
of this coagulum was followed almost at once by slow liquefaction, but
even after six months the clots were not completely digested.
Sordelli (123) found that all of his strains fermented glucose,
levulose, and maltose, but not galactose, saccharose, mannitol, arabinose, or inulin.
These observations were confirmed by Hall and Scott
(124), who furthermore failed to find any fermentation of salicin and
glycerol.
Serological Reactions
Sordelli (122), in his first studies with this organism,
made the observation that the toxin elaborated by the newly discovered
pathogenic anaerobe was not neutralized by the specific antitoxins of
Clostridium welchii, Clostridium oedematis-maligni. Clostridium histol—
yticum, or Clostridiuar*novyi. This fact contributed substantially to a
conviction that he was dealing with a previously unrecognized patho­
genic organism.
Subsequently, Sordelli (123) immunized a horse to the toxin, and
obtained potent antitoxic serum after an appropriate period of immun­
ization,
With only 0,001 milliliter of this serum, he was able to
neutralize completely, fifty minimal lethal doses of toxin injected
into a guinea pig, while 0,0003 milliliter retarded deatil of the GS-TUG
animal for ten days,
Sordelli calculated that one milliliter of serum
neutralized between 50,000 and 150,000 minimal lethal doses of toxin.
He observed, furthermore, that the toxin-antitoxin reaction followed
the law of multiple proportion; increasing the amount of toxin ten
times required a ten-fold increase in the quantity of serum required
for complete neutralization.
Melenejr, Humphreys and Carp (125) also
noted that potent Clostridiiun novyi and Clostridium oedematis-maligni
antitoxic sera had no protective effect against the toxins of the or­
ganism described by them as Bacillus oedematoides. Later, Humphreys
and Meleney (126) succeeded in producing serum in a rabbit wit}: one of
Kali’s strains of Clostridium sordellii. and that of their organism.
These workers demonstrated that sera prepared from Clostridium sordel­
lii and Clostridium oedematoides protected against the toxins of either
strain.
Hall, Rymer and Jungherr (127) prepared antisera from the toxin of
Clostridium sordellii by subcutaneous injection.
They have advised
this method of preparation in contrast to the intravenous injection of
whole culture because of more effective response from the treated ani­
mal,
They observed frequent death of animals injected intravenously
with whole culture, presumably due to the presence of living spores in
the vaccine.
For the production of agglutinative serum, these investi­
gators first injected rabbits subcutaneously with toxin, and after the
development of some degree of immunity, the intravenous injection of
whole culture was begun.
produced.
In this manner potent agglutinative serum was
By means of the agglutination reaction, Hall, Rymer and
Jungherr (127), and Hall and Scott (128), were able to establish fur­
ther the similarity of Clostridium sordellii and Clostridium oedema­
toides. Weinberg, Davesne and LeFranc (129), however, found that a
serum made from Sordelli’s strains, with a homologous titer of 1:5000,
agglutinated their strains of the organism only as high as a 1:50 dil­
ution.
Conversely, a serum prepared from cultures of their strain failed
to agglutinate the Sordelli strains in a dilution as high as was demon­
strated with the homologous strain of organism.
This would suggest
that, serologically, certain definite differences exist between the
various strains of Clostridium sordellii.
Hall (130) reported briefly on the maternal transmission of immunity
to Clostridium sordellii. A rabbit, which was under immunization treat­
ment for the production of antiserum, was accidently bred.
In spite of
the continued inoculations of toxin and whole cells, this animal gave
birth to ten healthy young.
Experiments devised to show the maternal
transmission of immunity clearly indicated that protection against the
toxin of Clostridium sordellii may be transmitted from female rabbits
to their offspring, but Hall was unable to state whether this trans­
mission was placental or by lactation, or by both.
Pathogenicity
Sordelli*s strains of this organism were obtained under
conditions that suggested their etiological responsibility for the fa­
tal results in the cases of human gas gangrene which he described (122).
On the basis of a small series of human cases of gas gangrene in South
America, Soi’delli (123) found this organism present in 18 per cent, of
the cases examined bacteriolcgieally.
He also demonstrated (123) the
pathogenic capacity of this organism for rabbits and guinea pigs.
A
rabbit weighing two kilograms died within 24 hours after an injection
of 0.10 milliliter of toxin, and guinea pigs weighing approximately 300
grains succumbed to doses of 0.01 milliliter of toxin, when injected
intramus cularly.
In experimental animals, both Sordelli (123) and Hall (131) have
described the lesions observed upon the injection of the toxic filtrates
of this organism.
There is little or no superficial modification of
the skin, no digestion of the tissues, and little odor, but usually an
abundant edema of the subcutaneous tissues upon the injection of the
toxin, according to these workers.
When the organism is found in the
tissiies, the formation of gas may occur.
It has also been noted by
these investigators that the symptoms are aggravated in instances where
the amount of toxin injected into the test animal fails to kill promptly.
As may be anticipated, in cases under observation where large doses of
toxin have been administered, few symptoms have appeared before death
occurs.
Meleney, Humphreys and Carp (125) found Clostridium sordellii path­
ogenic for white nice, white rats, guinea pigs, rabbits, pigeons, chickens,
cats and dogs.
Paralysis of the muscles was noted for some time before
death in pigeons, guinea pigs, rabbits and mice, and sometimes clonic
convulsions were observed immediately preceding death.
All investigators have agreed that the pathogenic activities of
Clostridium sordellii are the result of the elaboration of a -potent exo­
toxin,
The preparation of this toxin has been the subject of many re­
ports, and recently Walbum and Reymann (132), working at the Serum
Institute in Copenhagen have presented a paper on the production of
toxin by this organism in comparison with that produced by other
pathogenic gas gangrene organisms.
Experimental Work
A study of the growth requirements of the pathogenic
gas gangrene bacteria was undertaken as an introduction to a subse­
quent study of the factors involved in the production of their toxins
in synthetic media.
In the following description of the experimental
work accomplished in the study of the nutritional requirements of
Clostridium sordellii« it has been considered appropriate to discuss
the results obtained in progressive steps.
Details of procedure will
be repeated only when considered essential to the development of the
subject
Preparation of a Basal Medium
To initiate a study of the nutritional require­
ments of any micro-organism, it is necessary to obtain some measure
of growth response in a simple medium, preferably, chemically defined.
This medium is referred to as a basal medium, and is employed as a
starting point for further investigations.
For a study of the gas
gangrene group of bacteria, it was obvious that certain, well estab­
lished observations must be considered.
The need of anaerobiosis,
carbohydrate and protein have been sufficiently recognized to include
these requirements in the construction of a basal medium.
In view of the protein needs of some members of
this group, a gelatin hydrolysate was prepared.
A good grade of gela­
tin, Eastman's de-ashed, was employed, and sufficient material hydro­
lyzed for use throughout the course of these studies.
A quantity of
500 grams of gelatin were mixed with 2.5 liters of 6 1! hydrochloric
acid, and refluxed for 20 hours.
The hydrolysate was then vacuum dis­
tilled to remove as much hydrochloric acid as possible, and decolorized
with charcoal.
The volume of the hydrolysate was adjusted so that 3.0
milliliters contained the equivalent of 1,0 gram of original gelatin.
In addition to the amino acids supplied in the g e la tin hydrolysate, the
medium was further supplemented with 1 -c y s tin e , d l-n eth io n in e, and 1tryptophane.
According to Calvery (133), g e la tin contains but l i t t l e
c y stin e , and no methionine or tryptophane.
The add ition o f cy stin e and
methionine \ras based on an attempt to s a t is f y p a r tia lly the su lfu r re­
quirements of the organism, and the in clu sio n o f tryptophane was founded
upon the conclusion o f Knight (5) "that most, i f not a l l b a cteria , re­
quire tryptophane for growth".
The amino acids used to supplement the
g e la tin hydrolysate were purchased from Hoffman-La Roche Co., and i t
was assumed th at they were prepared from natural sou rces.
The addition of potassium dihydrogen phosphate was considered im­
portant for buffering of the medium, and as a means of providing potas­
sium and phosphorus for growth.
Magnesium sulfate was added as a fur­
ther attempt to provide another inorganic chemical requirement.
Glu­
cose was supplied as a readily available source of energy for the bac­
teria.
The addition of "reduced" iron to each tube served to provide
a favorable oxidation-reduction potential to initiate growth of these
anaerobic organisms; its presence also provided this inorganic need.
The addition of a "shotgun" mixture of six chemical compounds,
which have been demonstrated to be growth stimulating factors for bac­
teria, was an attempt to further stimulate, if possible, the growth of
the organisms under investigation.
The mixture of growth factors con­
tained Vitamin B-, Vitamin B , Vitamin B , pantothenic acid, nicotin1
2
6
amide, and ch o lin e, in such a concentration th at the addition o f one-
tenth milliliter of each gave a final concentration of one microgram
per milliliter of basal medium.
All the growth factors were synthetic
preparat iotas.
The basal medium was prepared as follows:
Gelatin Hydrolysate
6.0 grams
K H2 P0^
A. 5
"
1-cystine
0.10
"
dl-methionine
0,20
"
Distilled water to
900.0 milliliters
Thereaction of the medium was adjusted
to pH 7,A with 5 N sodium hy­
droxide, and the medium filled into 6 x 5/8 test tubes, to a volume of
9.0 milliliters, each containing about 10 milligrams of "reduced" iron.
The medium was sterilized in the autoclave at 120° C. for 10 minutes.
The tryptophane solution, containing A0 milligrams per milliliter,
and a ten per cent, magnesium sulfate solution wex'e sterilized separ­
ately by autoclave, and added aseptically in one-tenth milliliter vol­
umes, to all tubes of the basal medium at the time of inoculation.
A
50 per cent, glucose solution, and the six growth factors individually,
in the concentrations previously described, were also sterilized sep­
arately by autoclave at 120° C. for 10 minutes, and added in one-tenth
milliliter volumes to the indicated tubes of medium.
The final volume,
after inoculation with culture, of all tubes was 10,0 milliliters.
The cultures used in this investigation were Clostridium novyi.
strain N 21, Clostridium sordellii strain S A, and Clostridium oederaatismalixni strain VS 23.
The first two species, Clostridium novyi and
Clostridium sordellii. had been recovered from pigeons on the sixth
passage previous to this work, and had been subcultured anaerobically
TO
on a meat infusion broth for the five following cultures.
The culture
of Clostridium oedematis-maligni had not been passed through a pigeon
for three months, and was removed from the ice box and subcultured once,
anaerobically, in meat infusion broth.
All three cultures were twenty-
four hours old, and had grown luxuriantly at the time of use.
The cultures were prepared for inoculation purposes by placing a
10.0 milliliter volume in a centrifuge tube and throwing down the cells
at a speed of 3000 r.p.m. for 10 minutes.
The supernatant was removed
aseptically, and the cells were re-suspended in physiological saline
solution.
After another centrifugation at the same speed and length
of time, the supernatant was again removed, and the cells re-suspended
in the identical manner.
After this treatment, the cells were consid­
ered free of any chemical contamination from the medium in which they
had grovm.
The inoculum for each species was standardized by dilution
of the saline suspension of culture to the point where a galvanometer
reading of 85 was obtained on the Evelyn colorimeter.
As a control of
the viability of the culture inoculum of each species, a meat infusion
broth was prepared in the usual manner, and was inoculated and incubated
in the same fashion as the basal medium tinder investigation.
To provide anaerobic conditions for growth, ’’reduced" iron was
placed in each tube of medium, as has been previously described, and the
tubes further incubated in an oat jar.
An oat jar consists of a wide-
mouth container, the bottom of which is covered to a depth of about two
inches with moistened oats.
After the tubes of medium were placed in
the oat jar, the lid of the container was sealed firmly to prevent an
exchange of gases.
The results of this experiment are presented in Table I, and it is
apparent that definite differences in response have been obtained with
.
77
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variations of the medium and species of organism.
It was found that
the mixture of gelatin hydrolysate, supplementary amino acids, potas­
sium dihydrogen phosphate, inorganic salts and glucose gave a growth
response, in the presence of the "shotgun" mixture of six growth fac­
tors, with all three species of bacteria investigated.
In the same
mixture, but without the six stimulating substances, it was found that
only Clostridium oedemails-mallgni failed to grow, while Clostridium
novyi and Clostridium sordellii gave indications of a weak response.
Further, it was noted that Clostridium sordellii produced a complete
blackening of the synthetic medium in those instances where growth had
been vigorous, while this phenomenon failed to occur in the control
meat infusion tubes.
As a consequence, it vras impossible to obtain a
galvanometer reading of value in these tubes.
It was assumed that the
blackening of the medium was due to the production of iron sulfide, and
there was no attempt to establish this tentative conclusion experiment­
ally.
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which had recently undergone passage through an animal responded to
growth in the synthetic medium more vigorously and after a shorter time
interval than the one species which had not been cultured in this man­
ner.
Since the inoculum employed for each species had been standardized
for turbidity, it does not appear probable that an appreciable variation
in total cell count existed.
It appears more reasonable to assume for
the present that a relationship betv/een the infectivity of these organ­
isms and their capacity to grow upon the described synthetic medium was
observed.
Gas production was observed to occur in the synthetic medium only
in the presence of glucose.
It can be noted from Table I that growth
took place in the absence of glucose, however, in the case of Clostrid­
ium novyi and Clostridium sordellii. but not in those tubes inoculated
with Clostridium oedematis-maligni.
All of the above cultures were examined microscopically at the time
of turbidity measurement, and no contamination were found among them.
In summary, it may be said that a basal medium has been constructed
which is capable of supporting the grovrth of three, pathogenic, gas
gangrene organisms, namely, Clostridium n o w i . Clostridium sordellii.
and Clostridium oedematis-mali.vni.
The addition of a "shotgun" mixture containing six chemical com­
pounds has further stimulated the growth of these organisms.
In view of the results obtained, it was decided that further work
was to be abandoned temporarily on two of the three species investigated.
Subsequent efforts were directed, therefore, to a study of the nutri­
tional requirements of Clostridium sordellii.
Method of Anaerobiosis
> i i V-'C-*.-J-
ditions were obtained by use of "reduced" iron, and the oat jar.
In
view of the observation that growth of Clostridium sordellii resulted
in a blackening of the basal medium, it became necessary to alter the
methods employed in maintaining favorable anaerobic conditions.
Several procedures and various types of apparatus
have been designed to effect anaerobic growth conditions.
The means
available to conduct this work included the use of reducing agents,
the phosphorus jar, the oat jar, the paraffin seal, or a combination
of these methods.
The reducing agents available were glutathione,
cysteine hydrochloride, and thioglycolic acid.
The phosphorus jar may
.
81
be described as a glass jar with a cover, containing a platform which
stands in about an inch of water.
The platform is so constructed that
it can hold either petri dishes or test tubes.
Provision is also made
within the jar for a dish, which when gently wanned provides sufficient
heat to ignite a piece of phosphorus.
The combustion of the phosphorus
removes oxygen from the atmosphere within the jar as phosphorus pentoxide, and this gas is slowly absorbed by the water at the bottom of the
apparatus.
cribed,
The construction of the oat jar has been previously des­
The paraffin seal has been generally accepted as an excellent
means for the prevention of the absorption of molecular oxygen at the
surface of the medium, and is recommended particularly after the medium
has been heated to drive out absorbed oxygen.
An experiment was arranged to determine the most rapid and effec­
tive method of obtaining anaerobiosis for further studies with Clos­
tridium sordellii. The basal medium was prepared in the same manner as
that described in the preceding experiment, and was supplemented with
glucose and the growth-stimulating compounds, In addition, one-tenth
milliliter of Td/500 ferrous ammonium sulphate vras added to each tube
of medium, to replace the "reduced” iron that may have been previously
utilized by the organism.
The reducing agents, sterilized by filtra­
tion, were employed in a concentration of one-tenth milligram per tube
of basal medium for glutathione, and cysteine hydrochloride, and onetenth milliliter of a one per cent, solution of thioglycolic acid was
added per tube of medium.
The same strain of Clostridium sordellii previously used was re­
covered from a pigeon before serving for inoculum purposes.
It was
prepared for this purpose by centrifugation and washing with physio­
logical saline, and standardized to such a turbidity that the galvan-
oraeter of the Evelyn colorimeter gave a reading of 85.
From the results of this experiment, recorded in Table II, it may
be observed that variations in the method of attaining anaerobiosis
had an effect upon the growth of Clostridium sordellii in a s^mthetic
medium.
It was interesting to note that grovrth development in all in­
stances was greater when a combination of methods was emplo3red.
The
reducing agents, alone, provided the more effective single means of
obtaining anaerobiosis.
The blackening effect produced in the previous
experiment was successfully avoided by the alterations provided in this
investigation.
On the basis of the results found in Table II, the method of ob­
taining anaerobiosis selected for subsequent studies of the nutrition
of Clostridium sordellii involved the use of thioglycolic acid and
the paraffin seal.
Thioglycolic acid was as effective as either cys­
teine hydrochloride or glutathione, and its cost was less.
The paraf­
fin seal, although possessing the disadvantage of being messy to
handle, was considered a better choice than either the oa.t jar or the
phosphorus jar.
The phosphorus jar, even in experienced hands, is
dangerous and only a limited number of tubes can be handled at one
given time.
The oat jar failed to show as good a response as the other
alternatives.
Determination of Factor or Factors in the "Shotgun"
llixture Stimulating the Growth of Clostridium sordellii.
As was previously observed, the "shotgun" mixture,
which contained six chemical compounds known to be growth factors for
other species of bacteria, stimulated the response of Clostridium sor­
dellii in the described synthetic medium.
The next problem, therefore,
involved an examination of this mixtixre to determine the factor or
.
83
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factors important to the nutrition of this pathogenic anaerobe.
Two methods of procedure for the resolution of the active and in­
active components were considered.
First, it was possible to make all
combinations of the six compounds, and test each arrangement for its
growth response.
However, this method was not considered practical,
since a great number of tubes were required.
The alternative scheme,
Xsss matsriHl s.nd ©fjTort witn conu?s.x*s."bls sc c m c o n s i s t s d
of an experiment carried out in two stages.
The first step involved
an examination of the effect upon growth of the separate omission of
each factor in the "shotgun" mixture.
Those factors which, when omit­
ted from the basal medium, decreased the extent of growth were consid­
ered to be essential.
The next step consisted in combining the essen­
tial compounds, and an observation made of their growth stimulating
capacity as compared with that of the original mixture containing all
six compounds.
Accordingly, an experiment was organized to discover which compounus, noon omission ircm xtis tusqi'uuIj iislci
compared with the control.
0i*j?p?cx? ’
upon pi^owLr/i
The basal medium employed for this purpose
was constructed according to previous descriptions, and the culture
inoculum prepared and standardized in the usual manner.
The inoculated
tubes were incubated anaerobically for 36 hours, and the readings vrere
made on the Evelyn colorimeter.
An examination of Table III reveals that the omission of Vitamin
and choline had no perceptible effect on the growth of this organism.
Furthermore, it nay be observed that the absence of Vitamin B^, Vitamin
B^, pantothenic acid, and nicotinamide definitely decreased the extent
of growth in each instance.
It may be tentatively concluded, therefore,
that these four compounds are essential in the nutrition of Clostridium
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sordellii. However, this con.clr.sion must be checked by another experi­
ment designed to show that all possible arrangements of these substances
in a basal medium fail to five as great a growth response as a combina­
tion of the four.
Further, it must be demonstrated that these combined
substances give as much stimulation to growth as the original mixture
of six compounds.
A further experiment, the results of which may be found in Table
TV, was performed to establish the necessity of Vitamin B.,, Vitamin B, ,
1
D
pantothenic acid, and nicotinamide in the nutrition of Clostridium sor-
accessory substances alone and in various arrangements with each other,
and it was found that a combination of all four substances was required
for maximum growth.
This combination was capable of stimulating growth
to the same extent as the original "shotgun" mixture.
It may be def­
initely concluded from the results recorded in Table IV that Clostridium
sordellii requires Vitamin B^_, Vitamin 3^, pantothenic acid, and nico­
tinamide for growth.
Determination of the Optimal Concentration of Vitamin B-^,
Vitamin B^, Pantothenic Acid, and Nicotinamide.
After establishing the necessity of the four indicated
substances for the nutrition of Clostridium sordellii in a synthetic
medium, it was of interest to find the optimal concentration of each
of these compounds. In the previously described investigations, the
concentration of each compound employed was 1.0 microgram per milli­
liter of basal medium.
To determine the maximum level of activity of each sub­
stance, it was necessary to find the range of greatest stimulation for
each factor in the presence of an excess of the other three -compounds.
88.
EFFECT OF VARIOUS
GROWTH
FACTORS UPON THE GROWTH
OF CLOSTRIDIUM
SORDELLII
IN A BASAL MEDIUM
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After the levels of most effective response had been established for
each growth stimulant, all possible variations of these points were
tested for the optimal concentrations for growth.
In order to find the concentrations of each substance stimulat­
ing growth most effectively in the presence of the remaining compounds,
a level of 5.0 micrograms per milliliter of basal medium was adopted
as an excess.
The accessory factor under investigation was tested over
a range extending from 0.0001 microgram to 5.0 micrograms per milli­
liter of medium.
Other procedures of the test were conducted in a manner identical
to that of previous experiments, and no variables other than the con­
centrations of the growth stimulating factors were present.
An examination of Table V will disclose that each growth factor
produced various responses within the range of concentrations tested.
The zone of maximum activity for Vitamin B^ was found to lie between
0.05 and 1.0 microgram per milliliter of medium; for Vitamin B^, panto­
thenic acid, and nicotinamide, between 0.10 and 2.5 micrograms.
With
this data on hand, a further study was undertaken to determine the op­
timal concentration for growth of each factor in the presence of an
optimal amount of each of the other three compounds.
The arrangement
and results of this experiment can be found in Table VI, and the me­
thods employed were identical in all respects to those of the previous
investigation.
In examining the results described in Table VI, it may be con­
cluded that the greatest stimulation of growth occurred in the presence
of 1.0 microgram of Vitamin B^, Vitamin B^, and pantothenic acid, and
2.5 micrograms of nicotinamide per milliliter of medium.
It must be
emphasized in connection with the determination of the optimal concen-
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Micrograms of
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Micrograms of
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Evelyn Galvanometer Readings
Micrograras of Vitamin Bj/ml. Medium
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Micrograms of
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Micrograras of
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Micrograms of
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trations of growth factors, however, that the levels found to be opti­
mal are such only under the conditions defined in the investigation.
Thus, the subsequent recognition of an additional substance as an es­
sential nutritional requirement theoretically invalidates previous
determinations.
Growth Factor Requirements of Other Strains of Clostridium
sordellii.
As has been previously stated, the experimental growth
factor studies described were performed with Clostridium sordellii
strain SA.
This strain has been in use for several years in the pro­
duction of toxins for horse immunization, and has been passed through
a susceptible animal at intervals of three months or less during this
time.
After the determination of the importance of Vitamin B^,
Vitamin B , pantothenic acid, and nicotinamide in the nutrition of
Clostridium sordellii strain SA» it was thought possible that other
strains of this organism might differ in their response to these sub­
stances.
A strain of Clostridium sordellii was received from Dr. L. S.
McClung, of Indiana University, labeled strain 102, and had been pre­
viously received by him from Dr. Vawter, as the Questa strain.
Two
strains of the same organism were received from Dr. R. S. Spray, of
West Virginia University, labeled strains 72 qnd 212, according to his
Key Numbers.
It was assumed that these cultures had not been passed
through a susceptible animal for some time, and after four transfers
into beef heart infusion broth at twenty-four hour intervals, each
strain was injected into a pigeon.
After the pigeons had died, each
culture was recovered from the infected pectoralis major muscle tissue,
transferred to a tube of beef heart infusion medium, and allowed to
incubate for twenty-four hours at 37° C.
Each culture was examined
microscopically for contaminants, and on this basis considered to be
a pure culture.
The characteristic odor of Clostridium sordellii. and
typical edema at the site of injection in the pigeon further confirmed
this conclusion.
To conduct an investigation of the necessity of the four chemical
compound^ previously found essential in the nutrition of Clostridium
sordellii strain SA, for other strains, it was considered sufficient to
add three factors to the basal medium, and omit the fourth.
A compari­
son of growth without each factor, as contrasted with a control con­
taining all factors served as an indication of the need for each sub­
stance.
The concentrations of each compound employed for this study
were those previously indicated as optimal for strain SA.
The medium employed in these studies was identical in composition
and manner of preparation to that previously outlined.
The inoculum
was obtained by centrifugation of the culture and washing with physio­
logical saline.
The turbidity of the inoculum for each strain was stand­
ardized by dilution to a point where a reading of 85 was obtained with
the Evelyn galvanometer.
After inoculation, the tubes were incubated
in the usual manner.
In Table VII the results of this investigation have been recorded.
A difference in the extent of response of each strain was noted, but
the same factors are essential for the gro?/th of all strains of Clos­
tridium sordellii investigated.
It is possible, therefore, that most,
and perhaps all strains of Clostridium sordellii require Vitamin B^,
Vitamin B , pantothenic acid, and nicotinamide for growth.
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Effect of Biotin upon Growth of Clostridium sordellii.
The need of biotin in tbe nutrition of some members of
the genus Clostridium has been indicated (47)(£9).
Peterson, McDaniel,
and MeCoy (115) have investigated the biotin requirements of several
species of Clostridia, and have been unable to elicit a response to
its presence with twenty members of this group.
Among these were
Clostridium acetobutylicum, Clostridium botullnum, Clostridium welchii
andClostridium histolyticum. These
workers assert, however, that many
of the species investigated probably require biotin, but failed to
show a response because of the absence of other essential constituents
in the basal medium.
In view of the above work, it was of interest to deter­
mine whether biotin was essential in the nutrition of Clostridium sor­
dellii. Since biotin as a pure chemical substance is not generally
available, it was necessary to conduct preliminary investigations on
crude preparations known to contain biotin.
For this purpose, a liver
protein preparation, which had been hydrolyzed with 1.0 N sulfuric acid
at 125° C. for four hours, was employed.
The preparation was submitted
to the test suggested by Peterson, McDaniel, and McCoy (115) for the
determination of biotin in biological materials.
From Table VIII, it
may be noted that the presence of biotin in the sample has been confinned,
The liver hydrolysate, in a concentration of 0,05 milligram,
contained sufficient biotin to definitely stimulate the growth of Clos­
tridium butvllcum.
An experiment was, therefore, organized to determine the
response of Clostridium sordellii to the liver protein hydrolysate.
The growth factors previously found to be important in the nutrition
of this organism were added to the basal medium in optimal concentrations.
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All procedures concerned in the execution of the experiment were iden­
tical to those previously discussed.
The results of this study have
been recorded in Table IX, and it may be observed that the addition of
the liver protein hydrolysate to the basal mixture in concentrations
approximating those yielding a response when tested with Clostridium
butvlicum. also stimulated the growth of Clostridium sordellii.
It
must be understood, however, that the liver protein hydrolysate rep­
resented a preparation containing many substances other than biotin.
It is impossible to conclude, therefore, on the basis of this experi­
ment that the chemical entity in the liver protein hydrolysate respon­
sible for the stimulation of growth of Clostridium butvlicum was iden­
tical to that involved in the response of Clostridium sordellii.
To substantiate the possibility that biotin was required for the
growth of Clostridium sordellii. another biotin-containing preparation
was tested for activity.
This material had been demonstrated by assay
to contain from five to ten times the amount of biotin found in the
jLzver protean nyciroxyBat-e, aiiu. cijiitaj.ned copper#
Upon testing this
preparation for stimulation of growth of Clostridium sordellii, a def­
inite response was observed, although, it was noted that those tubes con­
taining the greatest amount of material were inhibited by the presence
of copper.
Fortunately, it was possible to obtain a sample of the pure methyl
ester of biotin through the kindness of Dr. 77, H, Peterson of the Uni­
versity of Wisconsin,
This material was treated 1,0 II alkali at 100° C.
for one-half hour to hydrolyze the ester linkage, and both the methyl
ester and the hydrolyzed ester were treated for growth stimulation of
Clostridium sordellii. The amount of each; added to duplicate tubes was
such that a final concentration of 0,002 microgram of biotin was present
102.
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per milliliter of basal medium.
As can be noted from Table X, no
growth response was observed for this organism upon the addition of
these preparations.
Thus, it is impossible, on the basis of these
investigations, to conclude that biotin is essential to the nutri­
tion of Clostridium sordellii.
The Effect of Other Factors on the Growth of Clostridium sor­
dellii.
The response of Clostridium sordellii to several other
chemical factors known to stimulate the gro?rth of bacteria was in­
vestigated, with the result that no other substance was found essen­
tial to the nutrition of this organism.
An alcohol soluble liver
extract was also submitted to examination, and it was observed that
this preparation very definitely increased the growth of this patho­
genic organism.
Thus, it may be concluded that an unidentified com­
pound or compounds may be required in the nutrition of Clostridium
sordellii.
The substances tested for growth activity were glutamine,
uracil, pimelic acid, and para-amino benzoic acid.
Creatine, cadav-
erine, and betaine, which have not been known to stimulate bacterial
growth, were also examined for response.
Each of these substances was
tested at a level of 1.0 microgram per milliliter of basal medium, with
the exception of para-amino benzoic acid, which was tested in smaller
amounts.
The usual procedure was followed throughout this experiment,
and the only variables introduced into the work were the growth factors
under investigation.
From the results recorded in Table XI, it may be noted that the
alcohol soluble liver extract was alone capable of increasing the growth
of this organism.
It is of interest, furthermore, that none of the
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substances tested inhibited the growth of Clostridium sordellii in the
concentrations employed.
Some Amino Acid Requirements of Clostridium sordellii
The composition of the protein known as gelatin, re­
ported by Calvery (133), reveals the presence of fourteen of the known
amino acids.
Since the basal mixture upon which Clostridium sordellii
has been successfully cultured included hydrolyzed gelatin, it was of
interest to know which of these amino acids were indispensable to the
growth of the organism.
The basal mixture upon which Clostridium sordellii has
responded contained the equivalent of six grams of gelatin per liter
of medium.
The concentration of each amino acid in 10.0 milliliters of
this medium was calculated, and recorded in Table XII.
An experiment was devised in which each emino acid was
withheld from a mixture containing those recognized to be present in
gelatin, and the effect upon growth observed.
In the original basal
mixture, it should be noted that dl-methionine, 1-cystine, and i-tryptophane have been added as supplements.
In this experiment, obviously,
it was necessary to omit these amino acids from the basal mixture, with
the exception of 1-cystine, which was used in the concentration usually
present in gelatin.
After each arrangement of the various amino acid
mixtures had been completed for this investigation, 45 milligrams of po­
tassium dihydrogen phosphate was added to each tube, and the volume of
the medium brought up to 9.0 milliliters.
pH 7.4.
The reaction was adjusted to
The medium was sterilized at 120° C. for 10 minutes, and cooled
immediately thereafter.
Every tube was then fortified with one-tenth
milliliter each of li/500 ferrous ammonium sulfate, 50 per cent, glucose,
10 per cent, magnesium sulfate, and one per cent, thioglycolic acid.
.
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These substances had been sterilized either by heat or filtration.
The
culture inoculum, prepared as previously described, was added in a onetenth milliliter volume, and all tubes were then adjusted to a final
volume of 10.0 milliliters with distilled water.
Incubation took place
in the usual manner.
The results of this study have been recorded in Table XIII, and
it can be observed that 1-cystine, d-glutamic acid, and d-alanine were
found to be indispensable for growth of Clostridium sordellii.
With­
out these individual amino acids in the basal mixture, no gas produc­
tion occurred, and growth was very slight.
Another experiment was performed, subsequently, to determine if,
in the presence of 1-cystine, d-alanine, and d-glutamic acid only, growth
of this organism could take place.
A medium was constructed contain­
ing these three amino acids, and additional nitrogen, as ammonium ni­
trate, was added t o the medium in excess to minimize the absence of
nitrogen previously supplied by other amino acids.
Under these condi­
tions, Clostridium sordellii failed to grow, even after an extended
incubation period.
w... .i .a
It was apparent, therefore, that other amino acids
+-.n +-.Vi« H a v a InrHnflnt of this organism.
The amino acids employed in this study were obtained from Hoffman—
La Roche Co., and, as previously indicated, they were assumed to be
naturally derived products.
About this time, it came to our attention
that several synthetic amino acids were available, and it was considered
advisable to abandon further work at this point until the synthetic
products could b e obtained.
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C5
Studies in Toxin Production of Clostridium sordellii in a Synthetic
Medium.
Since the original purpose of the investigation of the nu­
tritional requirements of Clostridium sordellii was based upon a de­
sire to produce the toxin elaborated by this organism in a chemically
defined medium, it was considered appropriate, after the completion of
the previous studies, to ascertain to what extent toxin formation oc­
curred in the described synthetic medium.
For this experiment, a medium was prepared in essentially the
same manner as that previously described in other studies.
It consisted
of gelatin hydrolysate, potassium dihydrogen phosphate, dl-methionine,
1-cystine, 1-tryptophane, M/fSOO ferrous ammonium sulfate, magnesium
sulfate, glucose, and thioglycolic acid.
The growth factors found to
be essential in the nutrition of this organism were added in optimal
concentrations.
The synthetic medium was placed in 250 milliliter
Erlenmeyer flasks in 150 milliliter quantities, and sterilized at 120°
C. for 15 minutes. These flasks were subsequently inoculated with a
washed culture, prepared from a 21-hour growth of Clostridium sordellii.
strain SI, previously recovered from an injected pigeon.
For control
purposes, the meat broth generally used for toxin production was run in
parallel with the chemically defined preparation.
It was made from
beef heart infusion, and contained 1.0 per cent. Difco Bacto peptone,
and 0.50 per cent, sodium chloride.
This medium Wg.s submitted to the
identical experimental conditions described for the synthetic mixture,
and its unknown composition constituted the sole variation.
The titration of the toxin formed in these two groups of
flasks was accomplished by the subcutaneous injection of the sterile
filtrates into white mice weighing 18 to 22 grains.
Three mice were in­
jected at each titration level, and the death of at least two within
60 hours constituted the end-point.
The results of this study have been summarized in Table XIV,
and it may be noted that practically no toxin was formed in the syn­
thetic medium after 96 hours incubation.
It is apparent, therefore,
that certain factors important in the formation of toxin remain to be
determined.
This experiment also confirms an accepted conception
among bacteriologists, namely, that growth and toxin production freauently do not parallel each other.
Summary and Conclusions.
A basal medium has been described, which has been demonstrated
to support growth of Clostridium sordellii in the presence of Vitamin
B^, Vitamin B^, pantothenic acid, and nicotinamide.
It was found that
four different strains of this organism responded to development upon
the chemically defined medium, and it was, therefore, considered pos­
sible that all strains of ClostrIdiuia sordellii could grow on this med­
ium.
Under the conditions described in these investigations, it was
observed that biotin, uracil, para-amino benzoic acid, pimelic acid,
glutamine, creatine, cadaverine, and betaine failed to stimulate growth.
An alcohol soluble fraction of liver extract greatly increased growth
s
I
when added to the synthetic medium.
The amino acids necessary for de­
velopment of this member of the gas gangrene group have not been deter­
mined completely, but it has been indicated that d-glutamic acid, d4
alanine, and 1-cystine are required for optimal growth.
Toxin produc­
tion does not take place in the described synthetic medium, and the
conditions involved in its elaboration remain to be investigated.
.
112
'|ggS§
TABLE XIV
TOXIN PRODUCTION IN A SYNTHETIC AND BEEF HEART INFUSION MEDIUM.
Medium
Hours
Incubated .
pH of
Sample
Minimal Lethal Dose of
Toxin per ml. of Medium
2U
6,6
-
m
6.5
-
ii
72
6 .7
10
ii
96
6,6
10
21,
6.6
100
A8
6.8
500
n
72
6 .9
1000
ii
M
/
M✓
h
v
/ A
O .U
-1Ai^A
H.A.yu
Synthetic
n
Beef Heart Infusion
ii
The work presented in this thesis constitutes a basis for
further studies of the nutritional requirements of Clostridium sordel­
lii, a pathogenic anaerobe, and it is anticipated that nutritional in­
vestigations among other species of the gas gangrene group will estab­
lish fundamental similarities and differences among these bacteria, and
eventually yield a more complete knowledge of the relationships of these
pathogens to their environment.
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Am. J,
Henry Dirk Piersma was born on July 2, 1908, in Grand Rapids,
Michigan, where he attended grammar and high school.
Three years
of undergraduate work were completed at Calvin College, Grand Rapids,
Michigan, from which institution an A. B. degree was conferred in 1932,
after the termination of the first year of study at the University of
Michigan Medical School, Ann Arbor, Michigan.
He was employed by the
Lederle Laboratories, Inc. of Pearl River, New York, from 1931 to
1936, and finished graduate studies at Purdue University, Lafayette,
Indiana, in 1930.
Laboratories, Inc,
Since that time he has been employed by the Lederle
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