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0-169
INVESTIGATION OF POSSIBLE FACTORS THAT INFLUENCE THE
DEVELOPMENT OF NON-CARBON DIOXIDE-DEPENDENT
CELLS OF BHUCELLA ABORTUS FROM
CARBON DIOXIDE-DEPENDENT ONES
by
Hamza Keskintepe
A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Bacteriology
195a
ACKNOWLEDGMENTS
The author wishes to express his sincere
appreciation to Dr. I. F. Huddleson for his
guidance and advice throughout this study.
Most sincere gratitude and thanks is ex-
pressed also to Dr. Evelyn Sanders, for her
aid, constructive criticism.and valuable
suggestions.
344(188
INVESTIGATION OF POSSIBLE FACTORS THAT INFLUENCE THE
DEVELOPMENT OF NON-CARBON DIOXIDE-DEPENDENT
CELLS OF BRUCELLA ABORTUS FROM
CARBON DIOXIDEADEPENDENT ONES
by
Hamza Keskintepe
' AN ABSTRACT
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Bacteriology
Year 195M
Approved by' ‘//3j;§2?"jfgfziKg54jzzf?
The main objective of this study was to determine the
effect of glucose and pH of various mediums on the growth
of carbon dioxide-dependent strains of B3. abortus in a
normal atmosphere.
Bacto-tryptose and M-peptone agar were the base mediums
employed. The strains used throughout the experiments were
cultures of carbon dioxide-dependent‘gg. abortus.
Killed £3. abortus strain 2308 and yeast extract were
used as growth promoting factors in tryptose agar for strain
3029.
The colony plating method was employed in order to
demonstrate the presence of aerobic cells of fig. abortus which
require carbon dioxide. As a source of inoculum for these
plates M-peptone agar slants were streaked with cells from
the stock culture and incubated for h8 hours at 37° C. in an
atmosphere of 5 percent carbon dioxide.
The brucella cells from the M-peptone agar slants were
suspended in diluting.fluid which was composed of 0.05 per-
cent Baoto-tryptose and 0.5 percent sodium chloride in dis-
tilled water. The suspension was adjusted to a standard
turbidity of 28 as measured by the photoreflectometer. One
ml of the suspension contained 1.5 x 109 cells. The plates
were inoculated with one m1 of suspension, and incubated,
aerobically at 37° C. for four days. At the end of a four-day
incubation period, the colonies on each agar medium were
counted and the size of the colonies was measured. Form of
growth and colonial type was determined by Huddleson's
method (18).
The two mediums used were generally inadequate for the
cultivation and isolation of fig. abortus in the absence of
carbon dioxide. The absence of carbon dioxide (5%) had
bacteriostatic rather than bacteriocidal effect upon natur-
ally occurring strains of 2;. abortus.
Tryptose-agar medium, containing 0.1 percent glucose as
a source of carbon and energy effected better development
of non-carbon dioxide-dependent cells of fig. abortus at pH 7.2.
When glucose was increased from.0.l percent to 0.5 percent
in tryptose agar the number of non-carbon dioxide-dependent
colonies of fig. abortus was not increased.
Strain 3039 showed that the loss of the requirement
for increased carbon dioxide was gradual and occurred in
individual cells rather than in all the cells in the culture.
It is not possible to state from the data presented that
one medium supports the development of non-carbon dioxide-
dependent cells of B3. abortus significantly better than the
other mediums.
Experiments concerning the pH of the medium showed each
strain under investigation to yield a few colonies at any pH.
In this connection, differences in pOpulation (from 1 to 8
colonies) are associated with differences in pH or the
various mediums. The results showed that a larger number of
colonies may be obtained at pH 7.2 on all mediums in question
than at any other pH.
The size of colonies varied from 0.2 mm to 2 mm.and all
were of the smooth type. In general, the colonies of largest
size (2 mm) were developed at pH 7.2. No dissociation to
non-smooth colonial types occurred during incubation at 37° C.
for four days.
TABLE OF CONTENTS
Page
INTRODUCTION................... 1
REVIEWOFIITERATURE................ 3
MATERIALSANDMEI'HODS............... 9
RESULTS........._.............13
DISCUSSION . . . . . . . . . . . . . . . . . . . . . 27
SUMMARY......................31
HTERATURECITED..................32
LIST OF TABLES
TABLE Page
I. Growth of COZ-dependent strain 3029 in normal
atmosphere.................. 18
II. Growth of Goa-dependent strain 3028 in normal
atmosphere.................. 19
III. Growth of COZ-dependent strain 3051 in normal
atmosphere.................. 20
IV. Growth of Goa-dependent strain 3051 in normal
atmosphere.................. 21
V. Growth of Goa-dependent strain 119 in normal
atmosphere.................. 22
VI. Adaptation of carbon dioxide-dependent strain
3039'to normal atmosphere . . . . . . . . . . 23
VII. Growth of CO -dependent strain 3029 in tryptose
medium cogtaining killed cells . . . . . . . 2h
VIII. Growth of Goa-dependent strain 3039 in normal
atmosphere before serial transfers . . . . . . 25
IX. Growth of strain 3029 on tryptose-agar con-
taining yeast-extract . . . . . . . . . . . . 26
INTRODUCTION
It has been conclusively demonstrated that carbon dioxide
is a vital factor in the growth of practically all bacteria.
In their natural environment, bacteria are constantly being
exposed to carbon dioxide. However, the growth of some fas-
tidious bacteria is greatly increased by small amounts of
carbon dioxide in addition to that found in the atmosphere.
The Brucella is one of those organisms which are
characterized by their microaerophilic properties. Particu-
larly the recently isolated Brucella abortus, if it is a
naturally occurring one, needs 5 percent carbon dioxide for
growth.
Since Huddleson (3) found that carbon dioxide is an
essential factor in the isolation and cultivation of‘gg.
abortus, much work by various investigators has been done
to explain the metabolic basis for the increased carbon
dioxide requirement.
So far, the experimental evidence has been inadequate
to offer a definite solution to the problem of carbon dioxide
requirement. However, knowledge of the physiological and
nutritional requirements of Brucella is essential in at-
tacking many of the problems which are encountered.
The main objective of the present study was to develop
non-carbon dioxide-dependent cells of B3. abortus in tryptose
agar and M-peptone agar, at various pH levels and with dex-
trose present.
REVIEW OF LITERATURE
When Bang (1897) first isolated Brucella abortus from
the aborted fetuses of cows, it failed to grow aerobically.
This problem was overcome by transplanting it many times on
artificial culture media. Bang thought the chief factor
responsible for this was an oxygen pressure above or below
that of the atmosphere (A).
Nowak (13) in 1908, observed that the primary isolation
of B3. abortus was greatly enhanced when a culture of
Bacillus subtilis was set in a closed jar. He attributed
the success of his technique to the reduced oxygen tension
in the cultural environment.
Huddleson (3) in 1920, made a study of the atmosphere
in tubes containing agar slant cultures of B. subtilis
and pg. abortus. This study showed that the growth of 211;.
abortus was due to an increase in carbon dioxide given off
by‘fi. subtilis, instead of decreased oxygen tension as for-
merly believed.
Huddleson (3) also found that increased carbon dioxide
tension was an important factor in the successful isolation
of the organism. The correct percentage, by volume, neces-
sary for the growth of fig. abortus was found to be 10 percent.
Valley (18) clearly showed that carbon dioxide, even in
small amounts, was necessary for the growth and activities
‘of bacteria, instead of being merely a waste product.
Wilson (19) in a study of the growth of B3. abortus in
sealed tubes, proved that carbon dioxide was present as a
result of flaming the cotton-wool plug and the paraffined
or rubber corks. He confirmed the results of Huddleson
showing that under raised pressure of oxygen from no to
100 percent no growth occurred unless carbon dioxide was
present.
In another paper Wilson (20) demonstrated that growth
occurred under any pressure of oxygen from.0.5 percent to
99 percent, when furnished a minimum.carbon dioxide (0.5%).
Good growth was obtained in any increased carbon dioxide
tension from 0.5 percent to 98 percent when furnished a
minimum of oxygen (0.5%). He concluded that, in order to
obtain maximal growth of fig. abortus, the atmosphere should
contain 20 percent oxygen and 5 to 10 percent carbon dioxide.
Wilson (21) also found that sodium bicarbonate could
not be substituted for added carbon dioxide in a solid medium,
whereas, when bicarbonate solid medium.was subjected to
carbon dioxide (5-10%) growth occurred.
Studying the gaseous requirement of fig. abortus in
semisolid medium.Zobell and Meyer (23) reported that growth
could be obtained by adding 0.1 percent sodium bicarbonate
at either pH 6.8 or 7.2, however, when atmospheric carbon
dioxide was absorbed by potassium hydroxide, in complete
absence of carbon dioxide and carbonate, no growth was
obtained.
Schuhardt'gplgl. (16) found that certain lots of Difco
tryptose had a bactericidal effect on the relatively large
inocula of each of 13 cultures of fig. abortus tested.
Zobell and Meyer (23), utilizing a synthetic medium,
found that three species of Brucella showed the best growth
between pH 6.6 - 7.h. The optimum osmotic pressure for the
cultivation of Brucella was from 2 to 6 atmospheres.
Kerby (6) reported that addition of nicotinic acid
(30 mg/L) and thiamin (25 mg/L) to Bacto-tryptose agar
enhanced the size of colonies of several strains of fig.
abortus.
Koser, Breslove and Dorfman (7), studying the acces-
sory growth factor of the brucella group in a chemically
defined medium, stated that growth of seven of the eight
strains was; successfully cultured. The medium was comp
posed of amino acids, glucose and inorganic salts. Thia-
min, biotin, nicotinic acid, calcium pantothenate were ac-
cessory growth factors for these strains. Thiamin and
nicotinamide were required by all the strains, biotin was
essential for the g3. abortus strains.
McCullough and Dick (10) attempted to grow a group
of recently isolated strains of fig. abortus using the
medium described by Koser (7) and found that carbon dioxide
sensitive strains failed to grow in the same synthetic
medium with the same accessory growth factors even with in-
creased carbon dioxide tension. After becoming adapted to
grow aerobically, 30 of the A1 original strains grew in
the basal medium.plus the four accessory growth factors.
Failure to grow carbon dioxide sensitive strains of B3.
abortus was explained as follows:
1) A lack of unknown accessory factors needed by
these strains.
2) Inappropriate constituents employed in the basal
medium.
3) An unfavorable electrical potential of the medium.
A11 and Werkman (1) showed that in the metabolism of
‘Egcherichia coli or Aerobacter aerogenes, certain compounds
related to the Krebs cycle or their metabolic products can
be substituted for carbon dioxide. When synthetic medium
was aerated with carbon dioxide, free air growth was poor or
absent. When_ordinary air was bubbled through the medium
good growth took place. Growth was obtained in the absence
of carbon dioxide when alphaketoglutaric acid, oxalic acid,
glutamic acid were added individually.
Gerhard'gtlgl. (2) attempted to grow carbon dioxide-
dependent strains of fig. abortus in tryptose broth and
chemically defined mediums without an increased atmosphere
of carbon dioxide. Glutamic acid in amounts of 0.1 percent
did not substitute for an increased carbon dioxide tension,
even though glutamic acid was readily utilized by Brucella.
When added to the chemically defined basal medium as nitro-
gen source, L-glutamic acid again was ineffective. The
compounds tested were aspartic acid, arginine, proline,
malic acid, fumaric acid, succinic acid, and alphaketoglu-
taric acid. The results were negative. The controls,
incubated in an atmosphere containing 10 percent carbon
dioxide, showed good growth.
Sanders and Huddleson (1h), studying the influence of
atmospheric gases on the multiplication of Brucella, found
that there was a demand for large amounts of oxygen by @32-
29215. E. abortus exhibited a much greater tolerance for
high concentration of carbon dioxide than Brucella £21;
and Brucella melitensis.
Sanders and Huddleson (15), studying the influence of
oxygen on the metabolic activities of Brucella, found that
2;. abortus in one percent tryptose medium decomposed more
glucose in stagnant air than in an atmosphere of oxygen.
The slow rate of multiplication of B3. abortus in that
medium exposed to oxygen indicated that the nutrient and en-
vironment were not favorable for metabolism. One of the
important effects produced by oxygen was an increase in the
activity of the oxidative enzyme system that is responsible
for the decomposition of glucose by all three of the species.
McAlpine £3 51. (8) reported that in a liquid medium
without glucose all strains of Brucella produced large
amounts of free ammonia. In the medium containing glucose,
strains which utilized the carbohydrates produced only
slight amounts of ammonia. They were unable to demonstrate
the utilization of glucose by By. abortus.
MATERIALS AND METHODS
Cultures
The strains used, throughout the experiments, were
stock cultures of carbon dioxide-dependent‘gg. abortus,
strains 3028, 3029, 3039, 3051, and 119, maintained as part
of the collection of the Brucella Laboratory. Each cul-
ture was identified as 23. abortus by Huddleson. Strains
3028, 3029, and 3039 were described as typical, and 3051
and 119 as atypical £5. abortus strains. {The atypical
strainstvere designated as such in that they require an
increased carbon dioxide tension for isolation and subse-
quent cultivation, and produced H28 like typical £3.
abortus, but failed to grow on agar medium containing either
basic fuchsin or thionin.
Preparation of Mediums
In order to make a quantitative study of the effect
of pH of the medium and glucose on the viability of the
organisms under investigation, the following technique
was employed.
Three solid mediums were used, namely Bacto-tryptose
agar (Difco Laboratories), 0.5 percent glucose-tryptose
agar, and M—peptone agar (Albimi Laboratories). These
10
mediums were adjusted to pH 6.5, 7.2, 7.6 respectively.
Tryptose agar and Mppeptone agar have been successfully
used for isolation, cultivation and for colonial growth
studies.
Each.medium.was prepared by suspending the ingredi-
ents in 300 ml of cold distilled water and heating to
dissolve completely. It was then dispensed equally in
three flasks and sterilized in the autoclave for 15 minutes
at 121° C. The medium in each of the flasks was adjusted
to a different pH: one 6.5, one 7.2, and the other 7.6.
The pH of the mediums was adjusted to the desired level with
10 percent sodium carbonate and 1/10 N HCl after autoclaving.
Sodium carbonate (10%) was filtered through a D-8 Hormann
pad as a means of sterilization. The contents were then
poured into four Petri plates, thus giving four plates of
'each pH for each medium. The poured plates were dried in
a 37° 0. incubator for H8 hours.
Killed §;. abortus strain 2308 and yeast extract were
used as growth promoting factors in tryptose agar for
strain 3029.
Inoculation of the Mediums
The presence of aerobic cells, among the strains of
£3. abortus which require carbon dioxide, can be best demon-
strated by employing the colony plating method.
11
In order that the data obtained throughout the experi-
ments might be comparable, an attempt was made to inoculate
the same number of organisms on constant volumes of mediums.
As a source of inoculum for these plates, M-peptone
agar slants were streaked with cells from the stock culture
and incubated for h8 hours at 37° C. in an atmosphere of
5 percent carbon dioxide. The brucella cells from the M-
peptone agar slants were suspended in diluting fluid which
was composed of 0.05 percent Bacto-tryptose and 0.5 percent
sodium chloride in distilled water. The suspension was
thoroughly shaken to achieve complete dispersion of the
cells and adjusted to a standard turbidity of 28 as measured
by the photoreflectometer. The suspension then contained
1.5 x 109 cells per ml. 0ne:ml of the suspension, 1.5 x 109
organisms, was aseptically pipetted on each of four plates
of each medium. The plates were then rotated to obtain
an even spread of organisms over the surface. The inocu-
lated mediums were incubated, aerobically at 37° C. for
four days.
Examination of Plates
Plgte count. At the end of a four-day incubation
period, the colonies on each agar medium.were counted.
Colony size. The size of the colonies that developed
on each.medium was measured on the fourth day of incubation.
12
Form of growth and colonialitype was determined by
Huddleson's method (18):
a) Color
b) Consistency and texture
c) Acriflavine spot test
d) Staining with crystal violet.
RESULTS
A. The growth of five carbon dioxide-dependent strains
of £3. abortus was studied on three agar mediums in a normal
atmosphere.
Tables I, II, III, and IV show the number and size of
the colonies which developed from cells of strains 3028,
3029, 3051, and 119 after incubation for four days at 37° C.
in a normal atmosphere. All four strains gave rise to very
few colonies on all mediums. The size of the colonies
which developed on each medium, varied in diameter, and
is indicated as one plus to five plus and measured approxi-
mately 0.2 mm to 2 mm on the fourth day of the incubation
period.
The data in Table I show that B3. abortus strain 3029,
incubated h days, gave rise to one or two colonies at pH
6.5 to 7.2 and no colonies at pH 7.6 on each of the mediums.
0n the fifth day of incubation, the number of colonies on
tryptose agar at pH 6.5 had increased to four. All colonies
which developed were of the smooth colonial type and their
size varied from.0.2 mm to 1 mm. The colonies on Bacto-
tryptose agar were larger at pH 6.5 than at pH 7.2 or 7.8.
Table II shows the results obtained with strain 3028.
On tryptose agar (0.5% glucose) at pH 5.5, H, 7, and 8
colonies developed on the fourth, fifth and sixth day of
the incubation period respectively, whereas, on the same
mediums at pH 7.8 there developed one colony on the fourth
day and three colonies on the sixth day of incubation. The
size of these colonies was 0.2 to 0.3 mm in diameter. No
growth was obtained on M-peptone agar at pH 6.5 and only
one colony on tryptose agar at pH 6.5 throughout the incu-
bation period. Larger colonies developed at pH 6.5 and 7.2
in tryptose agar than on any other medium used. All colon-
ies were smooth.
Strains 3051 and 119 are the two atypical forms of fig.
abortus. The results obtained are shown in Tables III and
IV. In the case of both strains, the largest number and
size of colonies were obtained at pH 7.2 on all three mediums
employed. No colonies of strain 119 were observed at pH
6.5 on any medium.
E. An experiment was conducted to find out whether
killed brucella cells and yeast extract in tryptose agar
medium.would have any effect on the development of non-carbon
dioxide-dependent cells of strain 3029 B2. abortus. JFor
this purpose, a suspension of strain 2308 was added to
tryptose agar medium before autoclaving.
15
By analyzing TableaI and VII comparatively, one may
note that certain differences are evident. Strain 3029,
on Bacto-tryptose agar at pH 7.2 gave rise to one colony,
and none at pH 7.6, whereas, on tryptose agar, containing
killed fig. abortus cells 7 colonies developed at pH 7.2
and h colonies at pH 7.7. Furthermore, the size of these
colonies on the medium containing killed cells was larger
than that of the others. Smooth type colonies were ob-
served. 8
Yeast extract was employed as a growth promoting
factor in tryptose agar at concentrations of 0.1 and 1
percent. In this experiment also, strain 3029 of fig.
abortus was used. Table DC shows that only one colony
developed at pH 7.2 and none at pH 6.5 and 7.6 on both
0.1 and 1 percent yeast-tryptose agar.
C. Strain 3039 had been employed for some early in-
vestigations. In these studies, this strain had given rise
to only two colonies at pH 7.32 (Table VIII). During the
following three months the strain was transferred five or
six times on tryptose agar slants and incubated in an at-
mosphere of 5 percent carbon dioxide. Growth of strain 3029
in a normal atmosphere was then studied again to determine
the influence of frequent transfers on the development of
non-carbon dioxide-dependent cells. Thirty-seven to 137
16
colonies were observed on the plates (Table V). This is a
marked increase over the two colonies observed in cultures
of this strain before the period of frequent transfers and
the 1-8 colonies which developed in cultures of the other
four strains of fig. abortus not subjected to the period of
frequent transfer and growth in 5 percent carbon dioxide.
A larger number of colonies was obtained on the three
mediums at pH 7.2 than at pH 6.5 or pH 7.8. Tryptose agar
at pH 7.2 effected better development of non-carbon dioxide-
dependent colonies than the other mediums under investigation.
The size of colonies that developed on each medium.varied
in diameter. However, pH 7.2 gave rise to the largest
colony size (1.7 mm to 2 mm). All colonies were identified
as the smooth type.
D. It was estimated that the inoculum (1 ml) placed
on each plate contained 1.5 x 109 viable cells. Only a few
colonies developed in a normal atmosphere. To determine
whether the remaining cells in the original inoculum were
killed after incubation at 37° C. for four days, or merely
suppressed due to the absence of carbon dioxide, a wire loop
was streaked across a clear area of a tryptose agar plate
culture of strain 3039 and then streaked on an M-peptone
agar slant. Good growth was obtained when this slant was
incubated in the presence of 5 percent carbon dioxide for A8
17
hours. Using this growth, tryptose agar plates were then
inoculated with 1.5 x 109 viable cells. The data obtained,
recorded in Table VI, show that pH 7.2 gave rise to E9
colonies which were larger than the colonies which developed
at pH 6.6 and 7.6. Twenty-five colonies developed at pH
6.6 and 17 colonies at pH 7.6. All colonies were the smooth
type.
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