A STUDY OF THE GROWTH
REQUIREMENTS OF THE GENUS
BRUCELLA

Thesis for the Degree of M. 3.
MICHIGAN STATE COLLEGE

Thomas O. Roby
1944

TH ES!S

 

A STUDY OF THE GROWTH. REQUIRmENTs OF THE GENUS BRUCELLA

by

Thomas O. geby

A THESIS
Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of

MASTER OF SCIENCE

Department of Bacteriology

April, 19M;

THE

(f:

This study was made possible by a grant
from the Difco Laboratories, Detroit,

Michig an;

bah

r5
J

N

I‘ “-

x. I

t L"

1'.
2.
3.
h.
5.

TABLE OF CONTENTS
page
Introduction ....................... 1
Review of the Literature ........... 3
materials and.Methods .............o 9
Data and Discussion .............oo.13
Summary and Conclusions coo-0000000029

Literature Cited 00000000000000.0000}?

A STUDY OF THE GROWTH REQUIREMENTS OF THE GENUS BRUCELLA
Introduction

The problem of determining the necessary growth requirements for
. bacteria is a complex one. In their evolution bacteria may become
adapted to an environment which differs widely from the original. Not
only are there differences in requirements between families, but also
among members of the same genera. There is also the possible occurrence
of "adaptation" of a particular micro-organism; it may acquire the
ability to gradually overcome an unfavorable environment when grown in
a certain medium and still perpetuate itself.

Knowledge of the nutritional requirements of bacteria serves many
purposes. Among these are, (1) the perfection of a suitable culture
medium, (2) a better means of studying metabolic activity, (3) the
determination of differences in various species, ()4) a means of
determining the factors that promote dissociation, (5) a'method for
obtaining soluble cell constituents for biochemical studies.

This study was directed toward finding the exact nutritional
requirements of the species of Brucella and the development of a
highly satisfactory synthetic medium. A medium that fulfills this
requirement should possess the following properties: (1) promote
rapid growth from a small inoculum of bacterial cells, (2) permit
multiplication of cells and a slow death rate over a maximum period
of time, (3) freedom from factors that promote dissociation, (14) cause
no reduction in the virulence or other change in the characteristics

of the organism.

Although the literature contains several reports of studies
pertaining to the food requirements of Brucella, none of them have
been conducted with sufficient exactness to warrant final conclusions

as to what the requirements are.

-3-
Review of Literature

The early attempts to grow Brucella organisms in synthetic media
indicate that the media then used were deficient in many respects,
because maximum growth, equaling that in a peptone medium, was never
obtained. Zobell and Myer (1) reported that they were unable to find
a liquid synthetic medium in which Brucella organisms would produce
an appreciable turbidity. However, they found that the carbon or energy
requirements of Brucella were best met with lactates, citrates and
cystine: the nitrogen demands were fulfilled with 0.2 per cent cystine
and 0.5 per cent asparagine; the optimum phosphorus concentration was
that supplied by phosphates at 0.02 to 0.15 per cent. Sulphur was
best furnished by oystine at 0.02 per cent. The mineral requirements
were completed by the addition of sodium, potassium, iron and magnesium
ions.

Zobell and Myer (2) in another report on the physico-chemical
requirements of Brucella in synthetic media stated: ”the optimum
osmotic pressure for Brucella is slightly lower than an isotonic
solution; by depressing the surface tension of the medium to 50 dynes
with sodium taurocholate multiplication is expedited, the maxim
multiplication occurs at a pH between 6.6 and 7J4, and cultures of
Brucella grow best at a slightly reduced oxygen tension which may
be accomplished by the addition of 0.2-0.3 per cent washed agar,
retarding the diffusion of gases and giving a medium of semi-solid
consistency."

The composition of the synthetic medium which evolved from their

work follows a

.4-

Sodium ammonium.hydrogen phosphate ------------2.0 g.

Cystine ----------------------------------------0.2 g.

Potassium acid phosphate -----------------------1.0 g.

 

 

Sodium chloride ------------- -------- - -------- --2.0 g.
magnesium sulphate -‘ —====------------0.1 g.
Aspersgin =- — ------------- = ------ 3.0 g.
Glycerol ------------------- ------- -- -------- --20.0:m1.
Ammonium.1actate -------------------- --------- --3.0 ml.

‘Water to make ----------------------------------1,000 0 ml.

After the fifth transfer in.this medium growth began to improve,
indicating an adaptation to the medium. Multiplication was slow but
the death rate was less than in a peptone medium. An inoculum.of
less than 10,000 viable Brucella cells per cubic centimeter of medium
failed to groW.

Kerby (3) found that the addition of 30 mg. of nicotinic acid
and 25 mg. of thiamin hydrochloride to each liter of Bact-Tryptose
Agar resulted in a marked increase in.the growth of Brucella abortus.
This was indicated by an increase in the initial rate of growth and
size of the colonies.

The first detailed report on the accessory growth factors required
by Brucella was made by Koser, Breslove and Dorfman.(h). They employed
a basal synthetic medium of amino-acids, glucose and inorganic salts.
The following substances were studied for their growth promoting effect

on Brucella: nicotinamide, Coenzyme 1., thiamin hydrochloride,

 

diphosphothiamin, beta-alanine, calcium.pantothenate, vitamin B6
hydrochloride, riboflavin, inositol, glutamine, adenine, sodium

pyrophosphate and biotin. The amounts used were 0.2 to 0.5 microgrqms

.5-

per m1. of basal medium. From 8,000 to 20,000 bacterial cells were
added to each ml. of basal medium. Their results indicated that the
significant accessory growth factors involved in.the growth of the
three species of Brucella were thiamin, pantothenic acid (calcium.salt),
nicotinamide and probably biotin. They also found the optimum concentration
of sodium chloride to be 0.6 to 1.0 per cent.

The synthetic basal medium Koser and associates (h) used in the

above work consisted of the following:

Basal Synthetic Medium No. L pit; $113;
Glycine --- ------- -------- ------------------- -0.2 g.
dl-alpha-alanine ----- - ------ -—---------------0.5 g.
dl-valine --—------------ ------- - ---------- ---0.1 g.
dl-leucine ---------- ------------------------ 0.1 g.

(+) 1-lysine dihydrochloride -------- ------ -------o.1 g.

(+) l-arginine hydrochloride ----------- -------- --0.1 g.
dl-serine -----—------------------ ------ ----0.015 g.
dl-threonine ----- ----- -----------------------0.015 g.

(+) d-glutamic acid ---- ------- ----------—- ----- --O.5 g.

(-) l-cystine ----------- --------- ----- --------- --0.15 g.
dlqmethionine ----------- --------- ------------0.1 g.

(-) l-histidine hydrochloride ---—--- ------------ -0.2 g.

(-) l-tyrosine -- ------------------ - --------- ----0.05 g.
dl-phenylalanine - ----- - --------------- ---—---0.1 g.

(-) l-proline ----------- ------------------------- 0.1 g.

(-) 1-hydroxyproline ---—- ----------------- -------o.1 g.

(-) litryptophane ---—--- -------- - ------------- ---0.2 g.

EéflPo‘ --—-——--- -------------------------- ----1.0 g.

.6...

MgSO.I ----- ---------------------- ------- ----- 0.1 g.
NaCI ------------------é-------------------—--6.o g.
Glucose --- ---------------- - ----------------- 3.0 g.
The pH of the medium was brought between 6.8 and 7.0 with N/l
NaOH. It was sterilized by autoclaving at 15 pounds pressure for
15 minutes.
Plate counts made after three days incubation at 37°C. yielded
600 million to 800 million bacteria per ml. when the basal medium
contained the four accessory growth factors.
In a continuation of the studies, Koser and Knight (5) reported
on the effect of biotin as a growth promoting factor for Brucella.
The medium they used consisted of the following:
Basal Synthetic Medium No. b, ---- ----- ------cne liter.
I‘Ticotinamide (0.2 microgram/ml.) ----------0.2 mg./liter.
Thiamin (0.2 microgram/ml.) -- ---------- ----0.2 mg./liter.
Calcium pantothenate (0.2 microgram/ml.) ---0.2 mg./ liter.
Thioglycolic acid (50-100 microgram/ml.) ---50-100 mg./ liter.
They compared the effect of varying the concentration of pure biotin,
biotin concentrate and biotin methyl ester. Either of the three biotin
preparations was effective in promoting growth of three strains of £35.
abortus. A concentration of 0.0001 microgram of biotin per ml. gave
90 per cent maximum growth. They also found that the pyrimidine, but
not the thiazole component of thiamin was required by seven Brucella
cultures.
McCullough and Dick (6) using the basal synthetic medium No. LL and
the growth factors employed by Koser and associates, investigated the

differences in the requirements of the three species of Brucella. Also,

.7-

they determined the optimum concentration of each substance required
for growth. They found that all three species of Brucella required

thiamin for growth, that E. abortus also required biotin, and that

 

2E. suis and 21;. melitensis also required nicotinic acid. It was

 

 

found that while 21;. suis did not require calcium pantothenate for
growth, the presence of this substance initiated earlier growth.
The amount of growth was estimated by visual examination of the
turbidity of the cultures.

McCullough and Dick (6) suggested that the amounts of the accessory
growth factors necessary for growth of Brucella in the basal medium were:

Thiamin -—----------------0.2 microgram per m1.
Nicotinic acid -----—-----0.2 microgram per m1.
Calcium pantothenate ---------0.d.'r microgram per m1.
Biotin ------------------0.001 microgram per ml.

The addition of these substances failed to initiate growth when
only 200 bacterial cells were added to each m1. f medium.

In a second paper, McCullough and Dick (7) analyzed the critical
growth factor requirements of recently isolated strains of 2:. abortus.
The cultures that required carbon dioxide failed to grow in the complete
medium even with increased carbon dioxide tension. After becoming
adapted to grow aerobically, 50 of the I41 original strains grew in the
basal medium plus the four accessory growth factors. None of the strains
grew if either thiamin or biotin were absent. 0f the 30 strains which
grew, 25 had an absolute requirement for thiamin and biotin. However,
10 of these strains grew more quickly if nicotinic acid was present,
end 11 strains grew more quickly if calcium pantothenate was present.
The final growth, however, was no greater than in a medium without

nicotinic acid and calcium pantothenate. It was noted that when the

-8-

inoculum was less than 25,000 cells per ml. of’medium, growth did not occur.
NbCullough and Dick (6) and (7) based the quantitative measurement of
growth on the turbidity of the culture medium.as determined by visual
examination. No plate counts were made to determine the number of viable
cells present at the time measurements were taken. The viable cell count
must be taken into consideration in the development of an ideal medium
for any'bacterium. For, while the turbidity-oi’the medium.is a comparative
index of growth it does not give any information as to the prOportion of

live to dead bacteria during the growth phase.

-9-

Materials and methods

A. Preparation of Media.

The basal material used in this study was Casamino acids, a
commercial product prepared by DIFCO Laboratories. It consists
of the amino-acids resulting from the strong acid hydrolysis of
commercial casein. The amino-acid analysis of Casaminc acids as

submitted to us by the manufacturer is given below:

Average percentage of several lots

 

Cystine - --------------------------- less than 0.1
Methionine ---------------------- - ----------- 2.0
Arginine ---- ----------------------------- --- 2.1
Histidine -- ------------- - ----------------- -- 1.6
Lys ine ----------- --------------- - ----- ------ 14.1;
Tyrosine -------------------- -------------- -- 0.8
Tryptophane - -------------------------- ----absent
Phenylalanine - --------- - ------- ------------- 1.2
Threonine ------ -------- - ------------------ -- 2.7
Valine - ----------------------------------- -- 5.0
Leucine -- -------------------------------- -- h.2
Isoleucine --- ------------ -------- ----------- 2.2
Glycine -- ----- - -------------- - ----- - ------- - 0.35
Serine -------------- ------------------------ 5.5
Praline -- ---------- - ----------------------- - 5.h
OH-proline ---------- --------------- ---none found

Alanine ------—----------------------------- 2.5

Glutamic acid ---—-- ----- ------------------- 5.5

Aspartic acid -- ----------------------------- 2.3

-10-

The glassware in which the media was dispensed was cleaned in
acid-chromate cleaning solution, washed several times with distilled
water, and then placed in the autoclave in distilled water and
autoclaved for one-half hour at fifteen pounds pressure. This last
step was done to assure complete removal of foreign material from
the glass surfaces. It was found in preliminary trials that growth
of the bacteria was not uniform in different tubes of the same
medium if this step was omitted. After the cleaning process, the
glassware was dried in a gas oven at 65°C., then plugged with
absorbent cotton and sterilized by dry heat.

The preparation of the media was accomplished by making a
solution of Casamino acids in distilled water. The other ingredients
were added subsequently and all mixed thoroughly. Throughout most
of the study 3/1.; by 6 inch test tubes (pyrex) were used. Fifteen
milliliters of media was placed in each tube.

Sterilization was accomplished by autoclaving for ten minutes

at twelve pounds pressure.

B. Inoculation of the Media.

The strains of Brucella used were representative cultures which
had been maintained on liver infusion agar. They were periodically
checked for dissociation and only smooth cultures were employed in
the preparation of the inoculum. The major part of the study was
done with Brucella abortus No. 1257 (aerobic); Brucella _s_u_i_._s_ No. 1722

(aerobic); and Brucella melitensis No. 21469 (aerobic). These were

 

stock cultures maintained in the collection at the Central Brucella

laboratory.

-11-

In order that the data obtained throughout the investigation
might be comparable, an attempt was made to inoculate the same number
of organisms into constant volumes of media. Each tube of medium
(15 ml.) was inoculated with approximately 100,000 viable organisms.
This was done. by first making a suspension of Brucella cells in sterile
distilled water. Enough cells were added to produce a turbidity of
28 as measured by the Photronreflectometer described by Libby (8),
when the initial setting of the galvanometer was at fifteen. Before
the turbidity was measured in the reflectometer the suspension was
thoroughly shaken on a shaking machine to achieve complete dispersion
of the cells. Forty-eight hour liver agar slants of a smooth culture
of Brucella were used as the source of organisms.

When the suspension was adjusted to a turbidity of 28 by the

reflectometer method, each ml. contained approximately 15:108

organisms.
Hence one-half a m1. of a 1-5000 dilution of the original suspension
contained approximately 15x10‘.

In making the dilution to obtain the inoculum, one m1. of the
original suspension (turbidity-28) was placed in 99 milliliters of
sterile distilled water and thoroughly shaken. Then one m1. of this
solution (1-100) was added to 149 ml. of sterile distilled water,
resulting in a 1-5000 final dilution. One half of a m1. of this
suspension was used to inoculate each tube of medium. After the
inoculum had been added, the tubes were rotated by hand to obtain
complete mixing.

The inoculated media were incubated at 57°C.

-12..

0. Methods of Measuring Growth of Cultures.

1. Turbidity Evaluation: Using the Photronreflectometer as
described by Libby (8) the turbidity of each set of inoculated liquid
medium was determined on the 3rd, 7th, and 15th day following the
date of inoculation. The scale of the reflectometer was first set
at 50 and then the culture medium, after thorough shaking, was
placed in the glass cell and the turbidity value read directly from
the galvanometer scale. The final turbidity value was that of an
uninoculated tube subtracted from that of the inoculated tube.

2. Plate Count Method: Using the dilution method, plate
counts were made to give an accurate index of the number of viable
organisms contained in the medium at 3, 7, and 15 days. The diluting
fluid was composed of 0.05 per cent Bactc-Tryptcse and 0.5 per cent
sodium chloride in distilled water. Bottles containing 100 m1. of
diluting fluid were prepared and sterilized by autoclaving. Using
3' of these bottles and transfering one ml. each time, a 1:1,000,000,000
dilution of the culture was made. One ml. of the final dilution was
placed in a sterile petri dish and 30 m1. of melted Bacto-Tryptose
agar added. After )4 days incubation the colonies were counted.

Counts were made in this manner in duplicate.

Data and Discussion

The initial part of the study on the growth requirements of Brucella
consisted of adding each of the available known bacterial accessory
growth factors in turn to the basal ingredient, Casamino acids. In
addition to daily observation of the cultures, quantitative turbidimetric
readings were made on the 3rd, 7th, and 15th day after inoculation.
Bactc-Tryptose broth was used throughout the study as the control medium.
The Brucella organism grew readily in it. Comparison of growth in the
control meditm afforded a means of evaluating the growth promoting

property of the experimntal synthetic media.

Proliminary Growth Factor Tests

Although the accessory growth factor requirements for the genus
Brucella had been determined for several strains by Koser and his
associates (1;, 5) and that knowledge had been expanded by McCullough
and Dick (6, 7) , it was thought advisable to make some preliminary
studies with Casamino acids as the basal medium. is Casamino acids
was a recently developed commercial product, its use in a bacterial
medium has not been extensively investigated. The work of Koser and
Wright (9) had shown that the growth response of bacteria to vitamins
depends considerably upon the basal material used.

In the early trials it was found that in 3 days no growth
occurred in a medium consisting of Casamino acids, sodium chloride,
and s..m=c.'. The initial pH of the medium was 6.8, which is considered
optinmm for the growth of Brucella (2). After 7 days incubation a very
slight turbidity developed, indicating definite but greatly retarded

grmrth.

-11.,

The next step was the addition in turn, of each of the available
accessory growth factors to the basal medium. The resulting growth
in each case was slight. In fact no greater than in the Casamino
acids and salt alone. The accessory growth factors used in.these
trials were nicotinic acid, nicotinamide, calcium.pantothenate,
thiamin.hydrochloride, riboflavin, biotin, para-amino benzoic acid,
ascorbic acid, inositol, pimelic acid, pyridoxine hydrochloride,
menadione, methionine, glutamic acid, cystine and choline. Growth
in'the Tryptose control medium was rapid and reached a maximum.in
about 5 days.

Aeoording to Mueller and Johnson (10), the tryptophane content
of casein is destroyed by the strong acid hydrolysis used in the pre-
paration of the hydrolysate. Hence, it was expedient to make a
supplementary addition of tryptophane and to repeat the experimental
trials. With this addition it was seen that growth'was appreciably
improved. Greatest growth acceleration occurred'when'both
tryptophane and nicotinic acid were added.

The fat-soluble vitamins A.and B were also added to the basal
medium of Casamino acids. Glycerin‘was used as a vehicle to
incorporate these vitamins into the medium. The resulting turbidity
in 3 dayw'was equal to that in.the control Tryptose broth. However,
the addition of glycerin alone‘was followed by an equally stimulating
effect. In his'work on dissociation, Henry (11) used a 2 per cent
glycerin-dextrose broth for growing bovine and porcine strains of
Brucella.

The preliminary studies indicated that'when.using Casamino acids
as a basal material, the growth of £5. abortus was enhanced by the

addition of‘tryptophane, nicotinic acid, and glycerin, as is shown in

Table I.

m:
m:
mm
mm
HH

«hoolma

mm
mm

5

choclh

acceapcsa

ma

o.o
m.m

whocnm

.gvoup oucuhhuyuaoapnco

.HE moo .wE N.o .mfi 04H .m NN.O 4w 0.0

0? N00 03 00H ow NNeO ow CeO

.3 on .m $6 4» e6

OM FNeO em $00 .

ease

nfihoohau oadeOOfiz ondzmovmhha eommmoz H062

 

weakens .

anacoa Ho .flfi OOH hem manoovanmfloo

Mm.u0h muovcom satchm hhocuccod Ja capoa

4w o.H
.w o.H
4» etc
to on”

mowed
onweocoo

-15-

Optimum Quantities of Growth Stimulating Factors

To determine the most effective amount of each of the above
ingredients, a series of tubes was set up containing constant amounts
of two materials and varying concentrations of the other one. This
was done with tryptophane, nicotinic acid and glycerin. It was
observed that tryptophane in a concentration of one mg. per 100 m1.
of medium gave maximum growth. Glycerin produced as much growth at
a concentration of 0.5 ml. per 100 m1. of medium as did larger amounts.
Nicontinic acid in quantities of 0.2 mg. per 100 ml. of medium was the
most effective concentration. The protocols for these observations are

shown in Table 2.

Optimum Quantity of Casamino Acids

Host synthetic media used by previous workers contained from
one to 1.5 per cent casein hydrolysate. Hence, it was considered
most likely that the effective concentration of Casamino acids would
also be within that range. By using Casamino acids in concentrations
of one to 2.5 per cent, it was found that a one per cent solution of
this material when combined with glycerin, nicotinic acid and

tryptophane gave optimum growth. Table 3 illustrates this finding.

The Most Suitable Type of Casamino Acids

The composition of a casein hydrolysate will vary slightly
with the procedure employed in its manufacture. However, it can be
standardized for making a uniform basal medium by adjusting its
nitrogen content.

Three different lots of Casamino acids were obtained from the

Difco Laboratories with their analyses. A medium made from each Lot was

Casamino

acids

g.
{5.
go
g.
g.
g.

g.
g.
g.
80
89
$0

89
g.
g.
g.
g.

00
OO

. .

Table

Optimum quantities of accessory factors for Br. abortus

Constituents per 100 ml. of medium

NaCl

0053000
O\J\3\3\3\3\

O O OO 00
O
3\U\3\3\3\0\

0
00000

0‘3
0

NazHPO.

0.25
0.23
0.23
0.23
0.25
0.25

0.25
.25

g.
g0
80
g.
g.
g.

0.23 0

0.25
0.23
0.25

0.23
0.25
0.23

0.25 .

0.25
0.25

0.25
0.25
0.23
0.25

g.
g.
30

g.

o§o\

3\O\J\O\
020909090909
0 0 0 0

O O .'
C\J\O\O\3\
0 0 0

0023 g.
0023 Q0

0.23
0.25
0.25
.25
.25
0.25

0.23
0.25
0.25
0.25
0.25
0.23

0.25
0.23
0.25 -
0.23
0.25

Tryptophane

0025 mg.
0025 mg.
0025 mge
0025 mg.
0.25 mg.

025 mg0

0050 mg.
0050 Inge
0050 mg.
0050 Inge
0.50 mg.
0.50 mg.

1.0‘mg.
100 mg.
1.0 mg.
100 Inge
100 mg.

mg.

22????
000000

0 C

O C

Nicotinic

acid

0005 mge
0.10 mg.
0020 mg.
0.50 mg.
0.00 mg.
0050 Inge

0.05'mg.
0010 mg.
0.20 mg.
0050 mg.
0.110 mg.
0050 mg.

0.05‘mg.
0010 mg.
0.20 mg.
0030 mg.
00).}.0 mge
0050 mg0

0005 mg.
0010 mg.
0020 Inge
0050 mg.

O.h0
0.50

0.05
0.10
0.20
0.30
O.h0
0.50

mg.
mg.

mg 0
mg 0
mg.
mg 0
mg 0

mg.

0005 mg.
0010 Inge
009.0 mg.
0050 mg.
0.h0 mg.
0050 mg.

mg.
mg.
mg.
mg.
mg.

Glycerin

0.5 ml.
005 U110
005 ml.
0.5 ml.
005 ml.
005 ml.

005 mle
005 mle
005 mle
0.5:ml.
0.5 m1.
005 ml.

0.5 ml.
0.5 ml.
005 mle
005 m1.
005 ml.
005 T1110

0.5 ml.
005 ml.
005 17.110
0.5 ml.
005 H110
0.5 ml.

0.5 ml.
005 mle
0.5 ml.
0.5 ml.
0.5‘ml.
0.5 ml.

0.5 File
005 ml.
0.5 m1.
005 ml.
0.5 ml.
005 mle

0005 ml.
0.10 ml.
0050 11110
0050 ml.
1000 1111.

(DO 00% CD\O

H
W

BEEF

E;

Turbidity

7

days

16
15
10
1h
16

1h

15

days

16
ll
ll
12
1h
21

25
£0
22
22
2h
22

55
55
51
51
28
50

22
51
25
28
26

55

31
32
27
2h
27
27

21
19
20
20
22
20

0.23
0.25
0.25

mg. 2.00 m1.
mg. 3.00 ml.
14-000 m1.

g0
g.
g.

O
O
0

oxbxox

O

.. .
000000
0
0

P‘k‘k’k’k‘k’k‘rflm
0
00009000
Ososoqosoaososoe
O
P‘P’F’F’k‘k‘h‘k’
0
00000000

0
O
O
O

Control-Tryptose broth

Note: a. through f. show the effect of varying the concentration of nicotinic acid
and tryptophane. Group g. shows the effect of varying the amount of glycerin
with optimum.amounts of the other two materials.

 

mm
mm
mm
mm

0:

when ma

 

ma oH
Hm m
Hm oH
mm HH
mm NH
050015 whacnm
hpfiefippsa
.uavhond .

MN MO 23.39% no.“

.da m.o
0H5 moo
.fla m.o

.aa m.o

nwpoohaw

.mfi Noo
.ma «.0
.05 «.0
.me.m.o

UHOd

aflqfipoofiz

0w:— 00H
0M5 00H
0mg OOH

0MB 00H

Quanmonnhha

Sacha ouovmhuauaoupnoo

0m E00
0& $00
.w >m.o

0w NNOO

.ommnaz

0w 000
0w Q00
CM 000

0w 000

H002

_fiswwoa mo .dfi OOH pom apnospfivmnoo

mowed onflfidmdo Mo hpwpndsv_adawpmo

.m magma

.m m.m
.m o.m
.w m.H
.w o.H

nuwod
onfiadmdo

Table 1;. The most suitable type of Casamino acid-Ls for the growth of‘ Brucella.

 

Constituents per 100 ml. of medium. Turbidity
Casamino NaCl Na2HP04 Tryptophane Nicetinio Glycerin 3 days 7 days 15 days
acids acid
(h2521) a
’3 A. ~ , 0‘ L; x 0 1L
(1% 190 go 006 g. 0.6. C5. 100 m5. 002 m‘u‘ 0.: 3“]. j 26 55
.5
“ l”?1q)
o (49—,
f5) 1.0 go 0.6 go 0.2 go 1.0 mg. 0.2 mg. 0.5 ml. lit 28 14.2
‘tl ([1220)
m 4L
1.0 g. 0.6 g. 0.2 g. 1.0 mg. 0.? mg. 0.5 1121. 11.5 25 52
Control-Tryptose broth 9 21.5 32
(h2521) , ,
1.0 g. 0.6 g. 0.? g. 1.0 mg. 0.2 mg. 0.5 m1. 1h 50 29
VJ
~r+ (16219)
3 1.0 g. 0.6 g. 0.? g. 1.0 -.g 0.2 mg. 0.5 ml. 12 16 20
£1 a _
m (Mano) r
1.0 g. 0.6 g. 0.2 g. 1.0 mg. 0.2 Inge 0.5 IP19 16) 27 (Effie.
2)
Control-Tryptose broth 12 28 22
(02521) P
w 1.0 g. 0.6 g. 0.2 g. 1.0 mg. 0.2 mg. 0.5 ml. 12 28 29
0‘ (hEElO)
Ifi! 1.0 g: 0.6 g. 0.2 g. 1.0 mg. 0.2 mg. 0.5 ml. 10 9 9
PH
(1).
1
5*; (M220)
£3: 100 g. 006 g. 002 go 100 mg. 00? mg. 005 ml. 8 19 214‘
nnntrnl-Trvntose broth 11 27 36

Note: When (15) is placed next to the turbidity reading the reflectometer
was first adjusted to a galvanometer reading of fifteen before the
tube of suspension was added. Thus, indicating a higher turbidity
then when the machine was set initially at fifty.

 

exp he madam 05% M%0
h a vow hafldwvaud udi.0dw£o¢fi o
Jammwn WWW“ Wasp: wnwoaou on» pdsv puoum on mok.hpwcwnhavonmw
ma Amav mo nopoaowooflmou on» no wnwppmu Hauvwnw adoMpakw was «0902
we: copay omopmhua o£v_qo wnfivdoh hpfiowpusv.hac spa 9%“

a:
om

Amav
om

Amav
om
H:
o:

mm
mmaeuma

mm

0N

Hm

ma
mhwvub

hpfleapksa

odasooqw Hadfim_50hh usphona .

NH

mhdvlm

 

sweep
owovmhua

snags
omovmhha

£9093
euopmhha

wagon
oqfiadmdo

mcwod
onfiadmao

wowed
onwEdwdo

538:

mm.uo avenue

ooo.ooa
OOOH

0H
000.00H
oooH

0H

easy

you copaasooqw
mHHoo Mo uopfisz

om wands

mm

0H

mafieflpusa

anon pom m.m

pace hem a

vow: cwooaam
odedebd

 

com

mum

mmm

NmN

.dfi 00H you
.wa Onoosam

om Aeopaasoonfi nozv

 

 

acupuoo
a E: .....e
«NS
om neopaflsoogfl pozv
doupnoo
on $3.8. .ue
Ema
oappop
hom_ssfl©oa
no gopaaaaflfiz uaagpm

.dHHoozhm hp anecdaw mo nowvduuaanb ow oupda

aH

mm

mmm

$0
wad
mmm

mam
mom

08
993-3

on»
02
OS

03
0mm

com

oom

0%
0mm

0mm

84

own

one
0%

0R
mhdvlh

3:

men
mam

4mm
mmm

eon
0R
Rm

3m
mom
Rm

mom
mm:

fim
mhdvlm

ods hem dfiuopodn nofiaaw:

outflow onflfid Hdnowwwovd ov endomm0h ##kth

:m ow
mm an
:m mm
an em
on em
mm MN
3 an
N: 4m
3 em
mm mm
Hm mm
m: mm
m: mm
3 mm
.4: mm
whdvnma whaclw
$33.39

ma
ma
0H

mfl

0N

ma

8
mm
3

ON

4N
S

om
mm

9
mhdvlm

afimnmpwaoa
was»

maphopd

nwmnovwaes
mnzn

nsvhopd

mwmqmvfiaofi
undo

uspuond

uwucovwaofi
mwau

”3&2“

namnopwaoa
can.

nspuopd

newcomm

GHSwaa Hahfiaoov a .02
.35acma uvflod onfiaamao

ed: 00H .89
.wmoéufioa 333:3
Imsam H «02
«5305 .323 05530

.Hfi 00H you

.w . Ianfindndahnogm
a0 0 Imsam H 402
fifiwcos ucwoa anadmdo

.dn OOH hem
. ow Ho.0IonaosoA
. Insam H .02
.Esficufi avwodzonflfidmdo

.3 8” fig
.w «0.0.333
Innam H .02
.fizfleoa newou ondadmoo

:3.“on

oh oafida

 

Table 8. Effect of other vitamins.

Added vitamin Species Turbidity Million bacteria per m1.
5-days 7ndays IS-days E-days 7-days lE-days
None abortus 1h 27 52 5&5 550 l
suis 1h. 31 35 589 MOO 1
melitensis 1h 28 29 LAB 770 1
Biotin abortus 1h 29 no 569 550 120
0.03 Tamma per 100 m1.
E‘ suis 16 to 50 1.1.2 750 86
melitensis 1h 5h ho A26 920 296
Calcium. abortus 15 27 32 526 A60 1
pantothenate
0.02 mg. per 100 ml. suis 16 53 Eh. MOS 670 35
melitensis 15 29 33 A27 720 It?
(15)
Thiamin hydrochloride abortus 18 59 16 519 550 198
0.02 mg. per 100 ml. (15) (15) -
suis 22 22 30 319 390 518
(15) (15
melitensis 19 22 26 507 310 142
(15,3)
Biotin 0.03 gamma abortus 19 L0 lb 29h 350 227
per 100 m1. Ca- (15) (15)
pantothenate 0.02 g. suis 27 23 29 520 béo 278
per 100 ml. Thiamin (15) (15)
H01 0.02 a- Per 100 mlo melitensis 25 50 1.2 Lao 1.50 87
Tryptose broth abortus 18 27 hh. 29h 510 7
control medium. (15)
suis 17 57 19 28A 250 h2
melitensis 18 59 A2 27L 150 1

Note: When the

turbidity reading has (15) above it, the turbidity of the culture

was so great that the reflectometer had to be set from fifty down to
fifteen in order to obtain a reaCing on the scale.

 

 

 

 

Amino acid
added per
100 m1 . medim

Leucine 0.01 g.

N

"

Leucine 0.01 g.

N

Leucine 0.01 g.

N

Lysine 0.02 g.

H

N

Lysine 0.02 g.

Lysine 0.02 g.

H

Leucine 0001 go
Lysine 0.02 g.

"

Leucine 0.01 g.
Lysine 0.02 g.
"

Leucine 0.01 g.
Lysine 0.02 E; .
fl

1!

Table 9. Acceleration of growth by agitation.

Vitamin added
per 100 ml.
medium

Thiamin 0.02 mg.

3'

Ca-pantothenate 0.02 mg.

I!

H

Biotin 0.05 gamma

"

Thiamin 0.02 mg.

1'

Ca-pantothenate 0.02mg.

H

W

Biotin 0.05 gamma

fl

Thiamin 0.02 mg.

Ca-pantothenate 0.02mg.

N

Biotin 0.05 gamma

9'

1!

Species

abortus
suis

melitensis

abortus
suis

melitensis

abortus
suis

melitensis

abortus
suis

melitensis

abortus
suis

melitensis

abortus
suis

melitensis

abortus

suis

melitensis

abortus
suis

melitensis

abortus
suis

melitensis

Turbidity
(reflectometer set at 15)
5-days 7-days
25 52
28 D5
29 Lo
15 16
15 15
20 20
15 17
1h 15
l9 15
25 35
no 05
28 Lo
11 11
8 9
12 1h
15 10

9 7

lb 12
17 50
55 A6
50 be
15 20
25 25
22 27
17 20
19 22
22 19

Million bacteria

per ml.
B-days 7-days
2,660 1,880
b.070 b.500
2,660 1,850
1,810 10
1,170 20
2,000 1&0
1,730 20
1,280 to
2,050 180
2,610 1,610
8.950 3.100
2.920 1,9b0
1,530 10
1,150 50
1,890 150
1,980 10
1:550 70
1,780 150
1,8h0 1,520
5.970 b.230
2 J 7h0 3 91320
1,850 10
2,690 260
2.590 250
1,780 10
2,510 190

2,1110

120

 

oppose cap no ates» was» oownv ooh.osHo>
Hoopoo on? .oonom .oodfi no: unoaonsmooEprficwnusP 0:9 ohomon mphom Hoovo vopzadu
ohms nonopfiso moom on. mmeH on» .588 npmfippfigp oz. oumnuimnnmzp .npnooeqmn on» no .

Jammy noovmfih Mo afloow o no new haadapfinfi was hovoabvooamoh onv .Aomv coaopda monnD
owed omoa ooom somom omea oqoa :oH .om .mm .mm an em om m

comm comm ommm oomm omefi omma om .m: .mm .om Hm om om m

38
com omHH ommH coma ooHH oqqa o :m :m m: mm ma mm o
83 83
on mm ma ma a” e m on mm mm ma H” e m
whdv
E3305 mo .Hfi you 23:38
gauche mace spoofingso

 

Jonavdhomfiop soon um oHHooshm mo spaohw 40H canoe

.opoz

nwmaovwama

maze

msvhopd

uofioomm

22111011 1'11. inflow of ”vol-o1 agents on tho growth of tho opooios of Bruoollo

 

Turbidity (I)

Colony count

 

 

 

 

mm: ‘ cmutuwzdi’: 100 .1. PI!“ «It 118*“ million p.1- :1.
1 ext.
3 . .5 a .3 . Day: incubation
g b. g. . é . g: . :3 . :3 3 "' 3 7 15 3 7 15
s 2 so 5 2 a: 8 :2 2 £2 35,
won-1m o 0.5 0.01 1 0.02 0.2 0.6 0 0 0 o o 0
1 ' ' ' -' ' ' 67 73 78 2380 1700 52
I I I I I 0 I 62 65 63 2680 860 32
I I I I 0 0.2 I 51 52 ‘58 1320 1 2
I I I 0 0.02 I I 52 _ 61 63 1550 90 25
I I 0 1 I I I 66 69 72 2620 11.60 1
. I 0 0.01 I I I I 67 69 86 2620. 1890 0
m1. 2 0 0.5 0.01 1 0.02 0.2 0.6 0 0 0 0 0 -0
1 a o a a a a 70 82 90 3270 h220 1500
I I I I I o I 66 68 72 1610 1900 10
I I I I 0 0.2 I 39 39 1.2 190 20 2
I I I 0 0.02 I I 71. 81. 88 3870 11080 1070
' ' 0 1 ' ' ' 6h 77 8h 3390 h600 310
I 0 can I I I I 75 85 _89 3970 Moo aho
301115011010 0 0.5 0.01 1 0.02 0.2 0.6 0 0 0 0 0 o
1 I I I I I I 61 77 81 2160 2111.0 20
1 I I I I 0 I 60 71 71. 1870 2390 10
1 I I I 0 0.2 I 1.2 1.9 LS 1530 ho 2
1 I I 0 0.02 I I 61. 78 81 2500 2270 10
1 I 0 1 I I I 52 78 79 1810 200 2
1 0 0.01 I I I I 62 75 76 2210 21.30 10

 

 

 

 

(a) Light extinction measured by Canoe-Shear photelometer.

Conco filter #2 (green)

Medium adjusted to pH 6.8 with 10,8150.
Sterilization-autoclave 115'C for 12 min.

 

during growth in two liquid media.

Table 12 Showing daily colony counts of the specie: of Brucella

 

 

 

 

 

 

 

 

 

 

flay: l
incubation Br. abortus [ Br. Nil 7 Br. melitensis
7 7 Medium 7
Synthetic l Tryptoso l Synthgtio l tryptcccl Syllthctic I Tryptocc
7 Colony count, million: per I1. 7
not Shaken 2.3 21. 8 81 2.5 15
2 Shaken 1.55 1010 2150 581 951 1920
Not Shaken 550 255 21.7 259 260 275
5 Sheken 1.900 5720 SL110 51.140 1.11.0 1.350
1101: Shaken 1.05 550 558 299 1150 360
1. Shaken 5550 5000 7200 8120 7210 1.560
Not Shaken 560 1.02 518 556 510 1.98
5 Shaken 2650 5270 5860 721.0 1890 581140
Not Shaken 551. 521. 521. 227 626 590
6 Shaken 11.70 5980 5860 5900 770 2050
Hot Shaken 1.50 552 560 321. 780 560
7 Shaken 850 1780 1.810 5080 21.8 1.70
Not Shaken 280 250 1‘30 250 1481 312
8 Shaken 51.8 710 1.510 1590 19 229
Not Shaken 312 2.25 625 233 525 2%
9 Shaken 220 192 2750 870 1. 10
Not Shaken 500 161. 586 219 1.21. 262
10 Shaken 55 275 1500 1860 2 1
Not srnken 251. 108 112 287 1.50 87
11 Sheken 55 192 51.5 1.68 1 1
Not Shaken 187 125 565 150 586 0
12 Shaken 21. 119 188 572 1 0
Not Shaken 180 62 560 125 1.12 75

 

 

an... \SIVIHUV

1.34! I- II Jli

 

II P .1 etc-#1....

 

{‘0

'IV o C- at... I! 31!.“ “at

De; n!~.°° \nanaafisvc

uncN: I .9th

h\fih.~.>)|1-§MU.

In!”

5.. ‘ Ad QSPN.

 

mm
o
H

um
H

mom
0H

Jam
mmm

man
on:

com
omom

com
osom

mm:
com:

com
0mm:

mam
omma

ma
o.m

cmopmhua

 

HQJ

om»

envennnnm

uwmdcvfiaca_nmm

 

m o m N can doxdgm #02
WWW me mmw om noxnnm NH
omH mom mmH and nonnnm 962
we: mom moH mm nonenm Ha
emm mad mod 4mm nonenm pea
coma oomH mam mm nonenm oH
mam mom 30H oom nexenm pez
o3 omem m9 omm noggin m
mmm mmm mmm mam nexenm pez
coma can: can mom nemenm o
omm om: omm omm neunnm pea
ooom ode: one“ omm nonenm a
2mm com mmm ems. nonenm pea
ooo comm comm oeaa neuenm w
emm :mm :mm :mm neuenm pea
come comm 2mm omom .8298 m
0mm mam mo: oom nonenm pez
omam come ooom ommm nexenm :
mmm mmm omm no: nonnnm pea
024m 644m omem com: neunnm m
mmm hem mmm omm nemenm pen
”mm omam oHoH mm: neuenm m
an m 3m m.m nexenm pez
am m m.e 04H nexenm H
.Ha hem oQOfiHHHS .Hnsoo.hnoaoo
onopmhna owponpnmm cmopmhha owponpnhm
gmcomml Isl flow Pd @503
own» ohm cophonc .hm .- shun

u

 

Jedcofi weaved at» :w avabhw wfldhav cHHoOShm ho newcomu on? Ho cvnzoo hQOHoo hflwct wnwkbnm JMH canoe

-16..

used for each of the three species of Brucella. The composition of

the three Lots is as follows:

Casamino acids Rat-1.2521

(Regular) Moisture 3.12 per cent

A811 - 16.56 per cent
Sodium chloride - 13.1 per cent
Nitrogen - 10.6 per cent
P as P0,, - 1.6}... per cent
Copper - 100 parts per million.

Casamino acids Ric-1.11220

(Regular) Moisture 5.98 per cent

Ash - 18.110 per cent
Sodium chloride - 11.9 per cent
Nitrogen - 9.88 per cent
P as PO. - 3.90 per cent

Copper 18 parts per million.

Casamino acids Rat-14.3219

(Technical) Moisture 5.01.. per cent

Ash - 1.10.92 per cent
Sodimn chloride - 36.70 per cent
Nitrogen - 6.91. per cent

The effect of each of these lots upon the growth of members
of the genus Brucella is shown in Table 11. Casamino acids Rat-1.3219
was used in a 1.5 per cent solution because of its lower nitrogen
content in relation with the regular grades. Also, less sodium
chloride was added to the medium prepared from Rat-113219 as its
original sodium chloride content was very high.

Growth in media made from the different lots of Casamino acids
indicate that the Technical grade, Rxeh5219 did not have the growth
promoting property of the other two regular grades. Also, of the
two regular grades, the one which was lower in its copper content,

Rat-1.1.220, gave no improvement in growth over the other regular grade,

Rxeh2521.

-17-
Growth of Brucella in Casamino Acids Medium From Small Inocula

Previous work was done with an inoculum of approximately 15x10‘
organisms in 15 m1. of medium. If the medium devised was capable
of promoting rapid and good growth from an inoculum of a smaller
number of organisms, its value would be greatly increased. To test

this a Casamino acids medium of the following composition was made:

per 100 ml.
Casamino acids - 1.0 gm.
Sodium chloride - 0.6 gm.
Disodium hydrogen phosphate - 0.2 gm.
Niootinic acid - 0.2 mg.
Tryptophane - 1.0 mg.
Glycerin " Oe5 m1.

Distilled water, to make 100.0 ml.

Tryptose broth was used as the control medium. The size of
the inoculum was varied so that tubes of each medium received approximately
15x10“, 15x10’3 cells and 15 cells per tube. Turbidity measurements were
made to indicate the amount of bacterial growth. The results are
recorded in Table 5. Growth occurred in both media from small inocula.
Although the initial growth was less in tubes inoculated with fewer cells,
the final growth, as indicated by the turbidity measurements, was equivalent
after 15 days incubation. The final bacterial concentration in the Tryptose

broth medium exceeded that obtained in the Casamino acids medium.

Glucose Utilization in Casamino Acids Medium

In their studies on Brucella metabolism, Zobell and Myer (1)
found that. there was very little difference in the dextrose utilization
of the three species of Brucella. A glucose utilization test was
devised by Plastridge (12) for the differentiation of the species of
Brucella. To determine if glucose was utilized when added to the
Casamino acids medium a similar test was made with 31;. abortus and

Er: suis.

It had already been observed that the growth of Brucella was
less in the Casamino acids medium when 0.5 per cent glucose was added.
A medium was prepared consisting of Casamino acids, salts, tryptophane,
nicotinic acid, glycerin and glucose. Duplicate bottles of medium
were prepared. One was inoculated and the other left as an uninoculated
control. After two weeks incubation, glucose analyses were made on
both bottles according to Benedicts method (13). The original concen-
tration was not determined as a decrease was expected to occur upon
autoclaving and during the two weeks incubation period.

The resulting glucose concentration after a 2 weeks period showed
that in the case of Brucella abortus there was one per cent utilized
and that Brucella in}: used 8.3 per cent of the available glucose.

The results are set forth in Table 6. However, the turbidity of
the cultures were approximately 50 per cent less than that which had
been obtained with the same medium minus glucose when inoculated with

the same number of organisms.
Effect of Other Materials on Medium

On several occasions the turbidity in Tryptose broth had greatly
exceeded that in the synthetic medium when the turbidity reading was
made on the 15th day. This necessitated a lower initial setting of
the reflectometer when the controls were tested in order to have the
galvanometer reading within the scale limits. It was obvious that
maximum growth was not occurring in the synthetic medium. Dilution
plate counts made at the time of the turbidity measurements, indicated
that there was no correlation beheen the total count of viable cells
and the corresponding turbidity. Although the turbidity was greater
at 15 days, the number of viable cells per ml. progressively decreased

after the first few days. It was considered possible that the Brucella

-19-

organisms might be gradually developing anaerobic tendencies, or becoming
adapted to the synthetic medium and not growing on Tryptose-agar when
plate counts were made, or else they were dying off rapidly after the
first several days of incubation. If the latter explanation is correct,
the death rate would be greater than the growth rate. Hence, while
the turbidity was gradually increasing, the total viable cell content
was decreasing.

Upon incubation under anaerobic conditions in 10 per cent carbon
dioxide atmosphere there was no increase in the growth as indicated
by turbidity or plate count.

The use of the Casamino acids medium in solid form for making

plate counts gave no increase in the total cell count over the usual

Tryptose-agar plates. Hence, it appeared that failure to obtain

maximum turbidity and constant cell counts over the 2 week period
was caused by a high death rate of the bacteria in the synthetic

medium,
Various materials which might have growth promoting qualities

Were added to the ingredients already in use.

In a medium of amino acids, salts and vitamins, Koser ()4) had
1‘tmnd that some strains of Brucella required the addition of
thioglycollic acid before growth would occur in the synthetic medium.
This material and other substances with reducing properties were
added to the Casamino acids medium. Thioglyccllic acid, sodium

1‘hi‘r’BuIlphate , thiourea and glutathione were used individually at

Various levels of concentration. In no instance was the growth

°f Erueene improved.
The amino acid content of Casamino acids afforded the bacteria

an available supply of carbon to meet their energy requirement. How-

ever. in case there might be other organic compounds containing a

.20-

source of carbon more readily utilized, other materials were tried.
Glucose had already been eliminated as having this quality. Zobell
and Myer (1) found that the lactates and citrates supplied carbon
most effectively. The ammonium form of these compounds were added
to the Casamino acids medium. Neither ammonium citrate, ammonium

lactate, nor ammonium malate produced any increased growth response.

The Influence of Additional Amino-Acids

The presence of one amino acid has a direct influence on the
action of others in promoting or preventing bacterial growth. This
was demonstrated by Gladstone (11;). Using _B_. anthracis, Gladstone
observed that "the toxic effect of one may be counteracted by one
or more of others.” At the time these studies were initiated, very
little information was available on the quantitative amino-acid
requirement of Brucella. Although the constituents of the casein
hydrolysate had been determined, the optimum quantity of each amino-
acid for Brucella was unknown.

An addition of several amino-acids, except tryptophane which
was already in the medium, was made to the Casamino acids medium.
hch of the species of Brucella was grown in each combination of
the ingredients. The purpose was to see if the Casamino acids
lacked any particular amino-acid necessary for the maximum growth
of Brucella. The quantities used were those employed by loser (h)
in his synthetic medium.

The amino-acids used were glycine, dl-valine, dl-alpha-
alanine, dl-leucine, d-lysine-HCl, d-arginine, dl-serine, dl-
threonine, dl-glutamicacid, l-cystine, dl-methionine, 1-histidine-

di-ECl, l-tyrosine, dl-phenyl-alanine, l-proline, l-cystine and

-21-

1-hydrcxy-proline. These were added singly to the following combination

of ingredients 8

(Casamino Acids Medium No. 1)
Casamino Acids ---------------------1.0 gm.
Sodium chloride --- ------- - ------- "0.6 gm.
Disodium hydrogen phosphate -------0.20 gm.
l-tryptophane --------------------1.0 mg.

Niootinic acid --------- -------- ----0.2 mg.
Glycerin -------—-----------------0.5 m1.

There was no striking growth response in any of the cultures.
However, there was a slight increase in turbidity on the 15th day
in the tubes containing dl-leucine, d-lysine-HCl, d-glutamic acid
and dl-phenyl-alanine. The greater turbidity was consistent in
each strain under study, 21;. abortus (1257), 13:. 3.123. (1722), and

£5. melitensis (21469).

 

The next step in evaluating the efficiency of the amino-acid
supplement was to repeat the experiment and to make plate counts
along with turbidity readings. The results are shown in Table 7.

The addition of glutamic acid failed to produce an increase
in the growth rate. The other three amino-acids did cause a
greater growth response. This is most apparent after the first
week of incubation. With phenyl-alanine, lysine, and leucine,
the turbidity at 15 days was greater than in the control medium.
Most significant was the number of viable organisms Lin each group
at the termination of the incubation period. The control medium
drapped sharply to a very few million living bacteria per m1. of
medium. In the media containing the 3 stimulating amino-acids the
plate counts indicated a high proportion of living bacteria.

All possible combinations of these )4 amino-acids were added

to the Casamino acids medium No. l. The group containing both

&-

lysine and leucine held the viable cell count at a high peak more
consistently than did any other combination. These 2 compounds
alone, as seen in Table 7 were also quite efficient in maintaining
viability over a 2 week period in addition to producing maximum

turbidity.

Thiamin Hydrochloride As A Growth Stimulating Factor

The growth resulting from the addition of either leuc ine or
lysine was better than any previously obtained. However, the
turbidity in the Tryptose broth tubes occasionally exceeded that
in the synthetic medium. A s the nutritive requirements were
apparently being fulbilled, it seemed that some stimulative substance
was still lacking. Other investigators (3, h. 6) had found thiamin
to be essential to the growth of Brucella in synthetic media. Bacterial
growth in the Casamino acids medium was proceeding without the addition
of this vitamin. It appeared possible that a trace of thiamin might
be in the casein hydrolysate. Biotin and panothenio acid were also
members of the vitamin B-oomplex which had been reported (5, 6) as
accessory growth factors for Brucella. These vitamins were added
to the Casamino acids medium No. l which was then inoculated with
Brucella to see if any stimulation of growth would occur. The results
are shown in Table 8.

The turbidity and plate count methods of recording bacterial
growth offer an interesting contrast in their results. The
turbidities of the cultures containing thiamin at the 7th and 15th
day exceeded any which had been obtained previously in any medium.

The turbidities resulting after adding biotin and calcium pantothenate

were not much greater than those obtained without these materials ..

.23-

Iith these 3 substances together, turbidities were no greater than
in the medium containing thiamin. Judging from the turbidities alone,
thiamin appeared to be the factor necessary for maximum growth of
Brucella.

However, the plate counts, showing the number of viable cells
in the media gave more pertinent information. The counts at 7 days
were higher in the medium without thiamin than in that containing it.
Although there were more living organisms at 15 days in the medium
containing thiamin, the number of living bacteria was not at all

proportional to the turbidity. In fact, the biotin medium had nearly

 

as many living cells on the 15th day as did the thiamin medium. But
in the cultures with biotin turbidities were far less than those
containing thiamin. This would seem to indicate that the death rate
in the medium with thiamin was very high and although growth was
abundant the bacteria lived only a short time.

In the Casamino acids media without the vitamins there were
very few viable cells present after 15 days incubation. The addition
of biotin alone increased the final count for each of the 3 strains.
Calcium pantothenate failed to maintain a high level of viable cells
for strain 1257, (31;. abortus), but did increase the 15th day count

for strain 1722 ( 2:. suis), and for strain 21469 (333;. melitensis).

 

The combination of all B vitamins was no more satisfactory than thiamin

alone in maintaining viability.

Acceleration of Growth by Agitation

Two years ago Favorite and Hammon (15) found that slow rotation
of growing cultures of staphlococci produced a higher toxin titre on
casein hydrolysate medium. A similar procedure was applied to the

growth experiments of Brucella in Casamino acids medium.

.21,-

The media were prepared so as to contain all possible combinations
of lysine, leucine, thiamin, biotin and calcium pantothenate in addition
to the Casamino acids medium No. 1. After inoculation the tubes were
kept in continuous motion during the period of incubation. Measurements
of turbidity and plate counts were made on the 3rd and 7th days. The-
results are set forth in Table 9.

In every series of agitated cultures, the growth in 3 days was
far greater than previously observed without agitation. More than
lelO‘living cells per m1. of medium were produced in each combination
of the 2 amino-acids and the 3 vitamins under study. The turbidity was
visible at 36 hours and increased rapidly thereafter.

The medium containing both leucine and lysine, when thiamin was
present, produced a somewhat higher total viable cell count than did
either alone with thiamin. However, the addition of leucine and thiamin,
or of lysine and thiamin produced not only a very high turbidity and cell
count but also succeeded in maintaining the elevated state of growth
through the 7 day period.

The turbidities of the cultures containing biotin and calcium
pantothenate with the amino-acids never were as great as in those
with thiamin plus lysine or leucine. The number of living bacteria
was also low. This is shown by the lowered plate colony count made
at 7 days. Several of the turbidity readings of the biotin cultures
were lower at 7 days than at 3 days. This would appear to indicate
lysis of the bacteria.

The greatly increased growth of all 3 strains of Brucella was
obviously due to the constant agitation plus the growth promoting
effect of leucine, lysine and thiamin. It was not due to thiamin

alone. For when thiamin was added to the medium and either leucine

FL‘H‘JI' ____- i

.25-

or lysine omitted, even with agitation the viable cell count drapped
sharply after 3 days incubation.

When each growth measurement was made, a loopml of the culture
was streaked on a Tryptose-agar plate. The plate was incubated for
)4 days. The appearance of colony variation was studied under low
power magnification with reflected oblique light according to the
method prescribed by Henry (16). The colonies of Brucella were
typically smooth in all of the cultures. In no instance was dissociation
seen.

Growth at Room Taperature

The optimum incubating temperature for Brucella is approximately
37’0 according to Topley and Wilson (17). At this temperature, reproduction
proceeds rapidly. After maximum growth is reached the death rate is
preportional to the multiplication rate until growth begins to diminish.
It was not known whether Brucella would grow measurably in liquid cultures
at room temperature.

A medium was prepared which had the following composition;

Casamino Acids = ------------1.0 gm.
Sodium chloride ---------------—----0.6 gm.
Dipotassium hydrogen phosphate -----o,m gm.
Tryptophane -----------------------1.0 mg.
Nicotinic Acid ------------ ~ “-0.2 mg.
Glycerin ---------------------------0.5 ml.
Leucin -------- ----- --------- ------ -0.01 gm.
Lysine -------- --------- -------------0.02 gm.
Thiamin ----------------------------0.2 mg.
Calcium pantothenate -----—----------0.02 mg.
Biotin ----------- ---------- - ------ «0.05 gamma.
Distilled water to make -----—------100.0 m1.

 

 

This medium was bottled in 20 ml. quantities in 50 ml. bottles.
It was inoculated with _1_3_x_-_. abortus (1257), 25. suis (1722), and p5.

melitensis (21469). Each bottle received approximately 150,000 cells,

 

or 7,500 cells per ml. of medium. They were then held at room

.26-

temperature for 30 days. The bottles were rotated by hand for 10 to
15 secondsseveral times daily. Growth recordings were made at regular
intervals during the 30 day period. The average of the daily low and
high temperatures for the period was 23'0. Table 10 shows the results
of this experiment.
Between the Lth and 5th day an appreciable turbidity developed.
After maximum growth was established, it proceeded on a constant level
for almost 30 days. While the cell count of the £5. abortus strain _
was lower at 30 days, that of 33;. 3333. and 21;. melitensis had not
started to decline. Growth at room temperature with occasional agitation up
was slower than at 37°C. However, growth was persistent and ccnti nuous.

There was no dissociation. All 3 cultures remained smooth.

Influence of Casamino Acids Medium on Pathogenicity

A medium containing Casamino acids plus additional growth promoting
substances was inoculated with 31;. suis (1722). The composition of the

medium was 3

 

Casamino Acids - ——————— —— 1.0 gm.
No.01 -------------------------o.6 gm.
Ksfipoq, “““'"'""""”"""'Oeni we
Leucine -- ------------------ ---0.01 gm.
Lysine -------------------------0.02 gm.
Tryptophane -------------------l.0 mg.
Nicotinic acid ----------------0_.2 mg.
Thiamin —— -~--- -------------'--0.2 mg.
Glycerin ----------------------0.5 ml.
Distilled water to make --------100 ml.

 

The culture was then incubated at 37.5%., with continuous agitation
for 6 days. A 131,000,000 dilution was made of a portion of the culture.
Each ml. of this dilution contained approximately 14,000 living organisms.
Two adult guinea pigs.were inoculated intraperitoneally with one m1. of
the diluted culture. Thirty days later they were both sacrificed and an

autopsy performed. Brucella suis was isolated from typical lesions

.27-

throughout the body. Both of the animals showed extensive characteristic

gross lesions of brucellosis, as described by Huddleson (12).

Absolute Essentials for Brucella

To determine if any of the ingredients now used in the synthetic
medium could be omitted, studies were made omitting each material.
The procedure followed heretofore had been one of accumulating essential
materials that promoted a heavier growth of the organism than had been
obtained with any previous combination. However, it might be possible
that the presence of certain factors now used would eliminate the
beneficial effect of others.

The media were prepared with all of the materials, and also a
series omitting one ingredient in each set. The pH was adjusted to
6.8 with KgHPO‘e It was sterilised at llS'C. for 12 minutes. The
turbidity measurements were made with a Genoa-Shear photolometer,
using a Cenco filter #2 (green), and are expressed as percentage
of light extension.

The results are shown in Table 11. It is seen that glycerin
is not necessary for growth. The emission of thiamin greatly retards
the amount of growth and also results in a rapid death rate of the
bacteria. The amino-acid, leucine is not needed for the growth of
21;. abortus or 11:. £122, but is required by Br. melitensis. Tryptophane
is essential for 13:. abortus, but is not required by E5. 3113 or

21;. melitensis.

Daily Colony Counts 0f Brucella In Casamino Acids and Tryptose Hedium

Using the complete synthetic medium with the three species of

Brucella, daily counts were made of the number of living organisms.

.28..

One set of tubes was incubated under continuous agitation and another
identical set was incubated on the shelf and not shaken. Plate counts
were made for 12 consecutive days after inoculation. Tryptose broth
mediumwwas examined in the same manner to compare the results in the
Casamino acids broth.

As seen from.the tabulated data in Table 12, more rapid multiplication
took place in the unshaken tubes than in those that were shaken airing the
first 2h hours. The multiplication rate was very rapid up to h.days in
those tubes which were agitated. All species of Brucella began to decline
in number of living organisms after 6 days. With 21;. 3.9.3. the decline
is more rapid in the Tryptose medium.

Growth curves of each species are shown on the following pages in

Figures 1, 2, and 3.

s.

c.

D.

E.

F.

G.

H.

I.

J.

-29-
Summary and Conclusions

Er: abortus (1257) develoPed practically no turbidity in broth

medium consisting of Casamino acids and necessary salts. The

addition of tryptophane, nicotinic acid and glycerin enhanced

the growth.

The optimum quantities of each of these three growth promoting

substances per 100 ml. of medium were: tryptophane, one mg.,

nicotinic acid, 0.2 mg., and glycerin, 0.5 ml.

In combination with tryptophane, nicotinic acid and glycerin,

Casamino acids equalling one per cent of the medium produced

the best growth.

The technical grade of Casamino acids, which was high in sodium

chloride, did not produce as much growth as did the regular grade.

The growth promoting ability of Casamino acids was not increased

by lowering its copper content.

When the Casamino acids medium was inoculated with a very small I

number of organisms abundant growth occurred.

The addition of glucose to the Casamino acids medium had a

depressant effect on the growth of Brucella.

Various reducing substances did not facilitate a greater growth

of the organisms. The addition of organic carbon-furnishing

compounds were also without beneficial effect.

The addition of dl-leucine and d-lysine-HCl either singly or

together, resulted in prolonging the viability of the organisms.

When thiamin hydrochloride was added to the Casamino acids medium

' it initiated very rapid multiplication of the bacteria. However,

the death rate in these cultures was also high.
Casamino acids medium containing leucine, lysine and thiamin in
addition to tryptophane, nicotinic acid and glycerin when subjected

to constant agitation produced a very good growth of Brucella.

K.

L.

M.

N.

O.

-30.
Growth of Brucella in Casamino Acids medium at room temperature,
with occasional shaking, was good and was maintained over a 50

day period.

Br. suis (1722) remained pathogenic for guinea pigs after growth
' in Casamino Acids medium.

Brucella organisms grown in Casamino Acids medium developed no

increased tendency toward dissociation.

Al seen throughout this study, there is not necessarily any direct
correlation betwmen the turbidity of a culture and the number of
living organisms it contains. For the maintenance of a high pro-
portion of viable organisms, the growth requirements of the parti-
cular bacterium must be fully met. An ideal medium should contain
those substances which will cause a continued multiplication without
decline in the total number of living cells. Therefore, in deter-
mining the exact accessory growth factors the tctal number of
viable cells after growth has been initiated is a more important
criterion than comparison of turbidities.

Under the conditions employed in this study, a synthetic medium

capable of originating and maintaining growth of strains, 25.

abortus (1257), 21;. suis (1722) and 21;. melitensis (21469) was

 

found to consist of the following ingredients:

 

 

Casamino Acids (Difco) — — *--- -—l.0 gm.
Sodium chloride ------ ------ --------0.6 gm.
Dipotassium hydrogen phosphate «nu-0.111 gm.
51-19110an -m-"' ............. ’--0001 g0.
d-lysine-Hcl ------ --------- ---------o.02 gm.
Tryptophane -----------------------------1.0 mg.
Nicotinic acid ------------— = — =0.2 mg.
ThiaminéHCl ----------------------- .2 mg.
Glycerin ----------------------------e.5 ml.

Distilled water to make -----------lO0.0 ml.

-31-

P. Glycerin may be omitted without reducing the growth promoting
qualities of the medium. Tryptophane is essential for 2:. abortus
and leucine is essential for 21:. melitensis.

Q. Growth of all species of Brucella in Casamino Acids medium equaled
that in Tryptose broth. The bacteria multiply very rapidly after
the first day of incubation when the cultures are shaken. A

decline in growth occurs after 6 days in both media.

1.

2.

3.

14.

5.

7.

8.

9.

-52..

Literature Cited

Zobell, C. E. and Myer, K. F. Metabolism studies on the Brucella
group. 8 Nutritional requirements in synthetic media. The
Journal of Infectious Diseases, 21, 31414-360 (1932).

Zobell, C. E. and Myer, K. F. Metabolism of the Brucella graip.
9 Physiochemical requirements in synthetic media. The Journal
of Infectious Diseases, 2, 361-381 (1932).

Kerby, G. P. Nicotinic acid and thiamin hydrochloride as growth
promoting factors for Brucella. The Journal of Bacteriology,
21. 195-1499 (1959).

Koser, S. A., Breslove, B. B., and Dorfman, A. Accessory growth
factor requirements of some representatives of the Brucella
group. The Journal of Infectious Diseases, £2, 1114-1214 (19111).

Koser, S. A., and Knight, M. H. Further experiments on accessory
growth factor requirements of the Brucella group. The Journal
of Infectious Diseases, 11, 86-88 (19142).

McCullough, N. B. and Dick, Leo A. Physiological studies of
Brucella. 1 Quantitative accessory growth factor requirement
of certain strains of Brucella. The Journal of Infectious
Diseases, ll, 193-197 (19142).

McCullough, N. B. and Dick, Lee A. Physiological studies of
Brucella. 2 Accessory growth factor requirements of recently
isolated strains of Brucella abortus. The Journal of Infectious
Diseases, 11, 198-200 (19142).

Libby, R. L. A modified photrcnreflectometer for use with test
tubes. Science, 23, 1459-1160 (19141).

Koser, S. A.. and Wright, M. H. Vitamin requirements of tarula

cremoris. Proceedings of the Society for Experimental Biology
and Medicine, .52, 2149-251 (191a).

10.

ll.

12.

13.

15.

16.

17.

-33-

Mueller, J. H., and Johnson, E. R. Acid hydrolysates of casein
to replace peptcne in the preparation of bacteriological media.
The Journal of Imlmnology, 112, 33-38, (19141).

Henry, B. S. Differentiation of the bovine and porcine strains
of Brucella abortus based on dissociation. The Journal of
_ Infectious Diseases, 22, 1103-1106 (1933).

Huddleson, I. F. Brucellosis in Man and Animals. The Common-
wealth Fund, 110-111, 188-190 (1915).

Hawk, P. 3., and Bergeim, w. Physiological Chemistry, 11th
edition; Philadelphia, P. Blakiston's Sons and 00., 1133,
(1937).

Gladstone, G. P. Inter-Relationships between amino-acids in
the nutrition of B. anthracis. The British Journal of
Experimental Pathology, 39, 189-200 (1939).

Favorite, G. 0., and Hammon, W. M. The production of staphlococcus
enterotoxin and alpha hemolysin in a simplified medium. The
Journal of Bacteriology, £11, 305-316 (19141).

Henry, B. S. Dissociation in the germs Brucella. The Journal
of Infectious Diseases, 22, 3714-1402 (1933).

Topley, w. w. 0., and Wilson, 6. s. The Principles of

Bacteriology and Immunity, 2nd edition. 1'i'wfm. Wood and Co. 635 (1938).

 

'00

7‘0 '-

.7:0 :-

300 '-

600 r-

600 F

500 r

u
N
G

T

400 *'

COO r

400 '

'-
Q
§

1

PM" Colon; Ce." 0 lo'h
I:
Q
T

['0 P
(40 '-

200 F

[60 r

[:0 #-

COP

 

 

 

 

/‘\
\\
I, ‘
I \
' \
: I"
I
! I
, I
I I
g I
: I
: I
I ‘\
g 014 \ '
' \
I I \
I
1 \
p \
y \
I \
’ \ e
I, \"
\
y \
\
\
\
\
\o
\\
C ’ s
[I ' O ----Z-~ \“
I: -“-“'o ~~“'---o
n 4 1 L L g l ‘1,
a o a s r e 0 IO u I:
00]:
Fig. 1. Growth curves of Br. abortus.

-8ynthetio medium. agb—cultures fiubated on shaker.

--Tryptose medium.

cgd—cultures incubated on shelf.

Gales; Cece! : Io’lnl.

PIO'O

 

 

000
7'0 '-
780 I-
600 '-

sect

Q
Q
Q
T

‘
Q
Q

T

h
N
G

Y

B
Q
Q
T

3:
fi
I

sea-
son I-

100 "

 

um L

no "

00r-

 

‘0

 

 

 

 

Fig. 2, Growth curves Of.§£f suis.

-Synthetic medium. a;b-—cultures incubated on shaker.
--Tryptose medium. c;d-—cultures incubated on shelf.

 

 

 

 

L F »

,Growth curves of Br. melitensis.

 

 

sec
rec» '
rack
3001
:40-

o
eco-
Jso~

20-

mm”

.s‘\a°s - \Q‘IU aceseu {3‘

560 "

no -
soar
Ito -

 

a;b--cultures {EEhbated on shaker.
c;d-cultures incubated on.shelf.

Fig. 3.

--Synthetic medium.
--Tryptose medium.

AC KN OWLEDGI. VENT

The author wishes to express his appreciation
to Dr. I. F. Huddleson, Research Professor of
Bacteriology, Michigan State College, for his
wise advice and patient counsel which.made

this work possible; to Dean'Ward Giltner and
the workers at the Central Brucella Laboratory
for their many considerations; and to the Difco

Laboratories for their cooperation.

I mun—rm v“- ’

ill

fin
5*i1

(Z?

_ 1'1»

"'9 (’2

““ ~~ . >

J8 112

INTER-mm}

 

Y LOAN

091 1 ’55

 

 

 

 

-
.—..-_‘.._