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AUTHOR.

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PUBLICATION NO.

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! UNIVERSITY MICROFILMS
ANN ARBOR • MI C H I G AN

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3 hour inter’/a' s.

.w .w . . w. m. m. m

3. yu Ior .ir

3.

"4

« t 3 lovr interval? of h strut’ ' of

3. on I lorur. trowi; it. nutrient broth
-

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strains of

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vi
LI..-T

7AFI.ES (cent ir u e d )

opulatior! at 3 hour i r.tervals of 3 strains of
3 . pu 1 1 orur. ••*rowr in .1
(Experiment

cant *ry~'tose-'rater

y I17) ................................

7.3

opulatior. at 3 hour intervals of b strains of
3.

pul I orur, vrov;r. ir 1 per cent tryptos e-water

(Experiment

;f 2 0 ) ................................

74

opulatior, at 3 hour intervals of 5 strains of
S. pullorur r v own
(Sxperi:. eL t

tov.
t

(St r>erter.t // 4f)
o; if-ith:

at 3 hour

3. pel ior\r

tryptose—water

t fo ) ................................

o :u Vi t;ior at 3 : ■v >

7’. ot.r Peru;

in 1 per cent

i: re'••.-air

f s ‘rnins ef

in 1 re- o.-»r 4 t r y 't* -.-•o—val ■=•■»»
.....

. ....................

7^

* i terval s ef f, strains of

-rowr ir nutrient broth

(Experiment.“ 9 ) .................................
oru 1at !O!

73

nt 7 hour

in terva 1 r

7?

of 3 strains of

3. pul I orur. nr*own in nutrient

broti

(ExoeT'i er.t./ 1 3 ) .................................

73

-'ooulot ion at 3 hour intervals of 3 strains of
3.

mm.

r-.uLioru?r
n . .— — —— r r o v m

1:; nutrient broth

(Ex per i...er.t., 7 1 1 ) ..................................
ovulation at 3
3.

m l lorur

>'vr

ir. terval:? of h strains of

'-rovn ir. nutrient

hr' th

70

vi i
LI8T Or TABLES
Population at 6 hour

(continued)

intervals of 5 strains of

£.• FU 1 lorum c-rovm in a synthetic medium
(Experiment

7 8 ) .................................

81

Popu la tio n at 6 hour intervals of 5 strains of
S.

pulloruir. rrown in a synthetic medium

(Experiment

,r7 8 a ) ............

82

Population at C hour intervals of 8 strains of
3. oullcruir. trown in a synthc t ie medium
(Experiment
Effect of sine

ff7 8 b ) ...............................

of

inecrlur

on action of r.eo; .re ir. .

83
34

LIST fF FIGURES

tare

FI GURES
Growth curves of 5 strains of 3. pullorurr grown
in 1 per cent t r y p t o s e - w a t e r ....................
o

23

..........................

26

Growth curves of 5 strains of 3. pullorum grown
in nutrient broth

3.

.

Growth curves of 5 strains of 3. pullerum grown
in a synthetic m e d i u m ...........

4.

Effect of pi: on
neomycin

o•

29

the antibacterial activity of

................

42

Effect of sice cf inoculum or. action of neomycin
(Strain

.11)

50

Effect of size of inoculum on action of neon.ycin
(Strain

12)

51

Effect of size of inoculum on action of neomycin
(Strain 1 3 ) ...........................................
Q
—•

52

Effect of size of inoculu.. on action of neomycin
(Strain 1 7 ) ........................................

53-

Effect of size of inoculum or, action of neor .ycir.
(ntrain

29)

54

INTRODUCTION
On more than 85 per cent of the farms in ths United States
poultry is a partial, if not the chief, source of income (Tucker,
1950);

it is used more by the food consumer than it has been in

past years.

As a result, the control of diseases of poultry is

important economically to the farmer, and hygienieally to the con­
sumer.

Among poultry diseases, according to Simms (1950), three of

the more important are:

Newcastle disease or pneumoencephalitis,

pullorum disease, and avian leucosis.

Although pullorum disease is

losing ground among our poultry flocks, it is still widespread and
potentially very destructive.

This dissertation is concerned with

the laboratory study of Salmonella pul lor van, the organism which
causes pullorum disease.
Constantly being searched for is a chemotherapeutic agent which
is destructive to the pathogen and harmless to the host.

Preliminary

investigations by Waksman, Frankel, and Graessle (1949), and ?/aksman
and Lechevalier (1949), have indicated that a new antibiotic, neomycin,
is more potent in vivo against S. pullorum than streptomycin, and has
little or no toxicity for animals.
It is recognized that there are a number of factors, such as life
phase,

size of inoculum, hydrogen ion concentration,

interfering sub­

stances, etc., which may or may not modify the antibacterial action
of an antibiotic

lii v lt r o .

A knowledge of these factors is of great

importance in laboratory studies and in the clinical treatment of

2
infections.

Although the same general factors apply to the considera­

tions of the activity of any antibiotic,

the relative importance of a

given factor may vary from one antibiotic to the next.

A review of the

literature indicates that there is need for a study in which data are
collected about these factors in relation to neomycin, using S. pullortgn
as the test organism.

Purpose of the study.

The purpose of this study was two-fold:

first, to determine the bacterial culture cycle of five strains of S.
pullorum; second, to study some of the factors which may or may not
influence the antibacterial action of neomycin.
The bacterial culture cycle was compared when the organisms were
grown in three different culture media; nutrient broth, 1 per cent
tryptose-water, and a synthetic medium.

The complete culture cycle

was not studied, but it was limited to the initial stationary phase,
lag phase, logarithmic phase, and part of the stationary phase.
The factors studied that may possibly influence the antibacterial
action of neomycin were:

the sensitivity of five strains of S. pullorum

which had been grown in three different culture media,

the cultural or

life phase, temperature and time of incubation, size of inoculum, pH of
the medium,
media.

sodium chloride, and some of the constituents of two culture

REVIEW OF LITERATURE
As stated in the problem, one of the factors which is capable of
influencing the action of an antibiotic is the cultural or life phase
of the test organism*

When the literature was reviewed for information

about the culture cycle of S* pullorum, little data were found*

The

only reports were those of Huntington and Winslow (1937), and Mooney
and VJinslow (1935), who studied the culture cycle of S* pullorum in
three different media, l*e*, peptone water, peptone-glucose water, and
peptone-lactose water.

The media chosen for this study were: nutrient

broth, 1 per cent tryptose-water, and a synthetic medium designed for
the strains being used; therefore, the work that had been done with S.
pullorum was useful only a 3 a guide in technique.
The latter medium, a synthetic one, might serve as a useful means
of attempting to study the mode of action of an antibiotic*
edge of such a medium for S. pullorum is restricted*

The knowl­

Davis and Solowey

(1950) found that the one strain of S. pullorum with which they were
working would not grow in any of the synthetic media used by them.
simple synthetic medium containing glucose,

A

salts, and asparagine, com­

posed by Gray and Tatum 11944), supported the growth of two strains of
S. pullorum with which Lederberg (1947) worked when he added the amino
acids leucine and cystine for the one strain; and leucine, cystine, and
methionine for the other strain.

Tn an extensive study of the

tional requirements of 3. yjllorum by Johnson and Rettger

nutri­

(1943), no

4
attempt was made to devise a simple synthetic medium,

T5iey reported

that only 2 of the 45 strains studied required a vitamin supplement;
that 8 of these strains grew without glucose; that 3 would not grow
in the test media used; and that there were definite amino aoid re ­
quirements, but that these varied with strains*

Of Interest is the

fact that none of the 45 strains studied by these men required tryp­
tophane, and that leucine and aspartic acid were taken, on the whole,
as the most Important amino acids for 8 of 1 0 strains for which the
amino acid requirements were more thoroughly investigated*

Beard and

Snow (1936; were able to culture a strain of S. pullorum on Sahyun's
medium fflZ (Sahyun, Beard, Schultz, Snow, and Cross,

1936), when a

supplement of 2 0 amino acids, asparagine, and creatine were added.
Koser*s medium (Koser, 1923) with the addition of the salts of lactic,
succinic, fumaric, and citric acids would not support the growth of
three strains of S. pullorum, according to Hajna (1935)*
and Miller

When Weldin

(1932) investigated the utilization of citric acid and sodium

citrate by a number of strains of S. pullorum in Koser*s synthetic
medium, and in Simmons agar medium (Simmons, 1926), they found that
the majority of the strains would grow, but that growth was variable*
The foregoing literature covers the research that has been done
in relation to part one of the problem; namely, culture cycle and syn­
thetic media.

Literature pertinent to part two (factors that may or

may not affect the antibacterial action of neomycin} follows.
Neomycin was first described by Wakaman and Lechevalier (1949)
who isolated it from Streptomyoes fradiae.

Although neomycin has been

extracted from the same genus of actinomycetes as streptomycin,

5
streptothricin, aureomycin, Chloromycetin, and other less well-known
antibiotics, it has been defined chemically and biologically a s a diff­
erent antibiotic*

Neomycin is a generic term for a "neomycin complex"

of antibiotics from which Neomycin A has been isolated, according to
Peck, Hoffhine, Jr., Gale, and Polkers (1949).
Waksman and lechevalier (1949), Waksman, Lechevalier, and Harris
(1949), and Swart, Waksman, and Hutchinson (1949) characterized neomycin
chemically as a basic compound; soluble in water, slightly soluble in
methanol, insoluble in other organic solvents; thermostable in neutral
solution and at pH 2.0; and negative to Sagaguchi test for arginine.
A large variety of organisms have been tested in vitro and in vivo
vjith neomycin by Hobby, Lenert, and Dougherty (1949) and Felsenfeld,
Volini, Ishihara, Bachman, and Young (1950).
specifically mentioned S. pullorum.
(1949)

Neither of these papers

Waksman, f'rankel, and Graessle

and Waksman and Lechevalier (1949) pointed out that neomycin was

far more effective than streptomycin in suppressing the infection of
chick embryos with S. pullorum.
Neomycin is now being produced in a homogeneous state, and, as a
result, experimentation with human administration is beginning.

Duncan,

Glancey, Walgamot, and Beidleman (1951) concluded that neomycin is an
effective agent in the treatment of human infections, particularly those
of the urinary tract, but Waisbren and Spink (1950b) found that neomycin
was not always successful in eradicating Pseudomonas aeruginosa from the
urinary tract.

Oral administration of neomycin to people who were subse­

quently subjected to operations on the colon has indicated its value in

the prevention of post-operative peritonitis or fecal fistulae,

6
according to Path, Fromm, Wise, and Hsiang (1950).
In a study of the bactericidal activity of neomycin against
Escherichia coli. Waisbren and Spink (1950a) reported that 20 jig per
ml. is sufficient to kill in 2 hours all the organisms of a 2 x 1 0 ^
inoculum from an 18 hour culture suspended in saline.

Waksman,

Frankel, and Graessle (1949) showed that 20 units of neomycin, which
is rather crude and has an assay of 30-100 units per mg., incubated
with a heavy suspension of S. pullorum was completely bactericidal in
3£ hours.
Using E. coli and beta streptococcus, Worth, Chandler, and Bliss
(1950)

studied the effect of size of inoculum on the action of neomycin,

and found that the bactericidal concentrations of neomycin are affected
by this factor.

They concluded that the necessary increase in bacteri­

cidal concentration of neomycin with Increase in inoculum is of the
same order of magnitude as that for aureomycin, Chloromycetin, and
penicillin.
The antibacterial activity of neomycin is favored b y a mild alka­
line reaction of the medium.

The best activity occurs at pH 7.0 - 8.0

according to Waksman, Lechevalier, and Harris (1949).
Cysteine had no marked effect upon the activity of neomycin in
the experimental procedure used b y Waksman, Lechevalier, and Harris
(1949).

On the other hand, they reported that the presence of glucose

in the test medium reduces the potency of the antibiotic by favoring
either acid production or growth of the test organism.

Likewise, the

potency is reduced by oleic acid (Waksman, Katz, and Lechevalier, 1950).

7
Although a review of the work concerning the development of re­
sistant strains, synergisms, and toxicity is beyond the scope of this
study, it is Included because this antibiotic is relatively new and
these are subjects of importance to those interested in antibiosis*
Waisbren and Spink (1950a), Waksman, Katz, and Lechevalier (1950),
Demerec and Demerec

(1950), and Waksman and Lechevalier (1949) all came

to the same general conclusion - that developnent of resistance to neo­
mycin occurs slowly*

It resembles the "penicillin" rather than the

"streptomycin" type of drug resistance*
is a step-wise process of development.

That is, resistance to neomycin
It is of interest that strepto­

mycin-resistant, streptomycin-sensitive and streptomycin-dependent
strains are all sensitive to neomycin; whereas neomycin-resistant strains
are slightly more resistant to streptomycin than the original sensitive
strain (Demerec and Demerec, 1950).

These authors also pointed out that

strains resistant to Chloromycetin are sensitive to neomycin, and visa
versa*

Hobby, Lenert, and Dougherty (1949), and V/aksman, Lechevalier,

and Harris (1949) noted that neomycin is active against streptomycinsensitive and streptomycin—resistant tubercle bacilli.

Neomycin has

been used successfully by Duncan, Clancy, Walgamot, and Beidleman (1951)
in the treatment of human infections where organisms were completely or
moderately resistant to penicillin, aureomycin, chloramphenicol, and
streptomycin*
A possible synergistic action of neomycin and streptomycin in the
treatment of ten-day-old chick embryos infected with S* pullorum was
substantiated by Waksman, Frankel, and Graessle (1949).

Having treated

8
the infected chick embryos with different concentrations o f streptomycin
only, neomycin only, as well as with a mixture of
mycin, they found that 1 0 0 jig of

streptomycin and neo-

streptomycin had no protec tire effect;

25 units of neomycin gave 30

per cent protection; 50 units of neomycin

gave 70 per cent protection;

and a combination of 50 units of strepto­

mycin and 25 units of neomycin raised the protective effect of the
latter to 60 per cent, thus pointing to the potential synergistic action
of the two antibiotics.
The original work with crude neomycin by Waksman and Lechevalier
(1949) led these men to believe that it has either no toxicity or
limited toxicity for animals.

This was verified by Rake (1949), Hobby,

Lenert, and Dougherty (1949), Waksman, Frankel, and Graessle (1949),
Spencer, Payne, and Schultz (1950), Path, Fromm, Wise, and Hsiang
(1950), and Felsenfeld, Volini, Ishihara, Bachman, and Young (1950);
but Waisbren and Spink (1950b) noticed definite nephrotoxic and oto­
toxic reactions of human beings treated w ith neomycin.

MATERIALS AND METHODS
Cultures.

Three of the 5 strains of S. pullorum used were iso­

lated in 1950 from poultry by the Poultry Pathologist, Department of
Bacteriology and Public Health, Michigan State College; number 12 came
from a 2-month-old white leghorn, number 13 came from a 4-week-old
turkey, and number 17 came from an adult turkey.

It should be pointed

out that number 17 has produced only acid in dextrose and mannitol
since the original Isolation; nevertheless, it has been confirmed as
being S. pullorum at the Coordinating Bacteriology Section, Michigan
Department of Health Laboratory, Lansing.

Strains number 11 and 29

(original number 296X) were kindly supplied by R. Gwatkin.*

It is in­

teresting to note that number 29 is known as a variant which has the
same biochemical reactions in the routine tests as a regular strain but
a different antigenic structure.

For further information about these

strains the reader is referred to Gwatkin (1945) and Younie (1941).

Culture m e d i a .

All the media were used at pH 7.0 and sterilized

by autoelaving for 20 minutes at 15 pounds pressure except where other­
wise specified.
Nutrient broth was composed of Difco beef extract, 3 gm.; Difco
peptone, 5 gn.; and distilled water, 1 liter.

^Address:
Division of Animal Pathology, Science Service, Dominion
Department of Agriculture, Animal Diseases Research Institute, Hull,
Quebec, Canada.

10
Tryptos«-water was made up of Difco tryptose, 10 gm.; and distilled
water, 1 liter*
The synthetic medium desired was to he as simple as possible but of
such a composition that 2*5 ml* would support visible growth after 24
hours incubation at 37°C. w h e n an inoculum of approximately twenty-five
thousand organisms was used.
The glucose-asparagine-mineral base of the synthetic medium was of
the same composition and concentration as the one utilized by Qray and
Tatum (1944).

Part of the mineral base was a trace element solution r e ­

ported by Horowitz and Beadle (1943).
It was necessary to determine what specific amino acids to use;
therefore, a synthetic medium was made with the glucose-mineral base
(note that asparagine was omitted) and 17 amino acids.
page 89, for their source).

(See Appendix,

The amino acids were: DL-alpha alanine, L( + )

arginine monohydrochloride, DL-aspartic acid, L(-) cystine, glycine, L(+)
histidine monohydrochloride, L(-) leucine, L(+) lysine monohydrochloride,
DL-methionine, DL-phenylalanine, L(-) proline, L(+) glutamic acid, DLserine, DL-threonine, DL-tryptophane, L(-) tyrosine, and DL-valine.

They

were used in the concentrations stated by Johnson and Rettger (1943).
Threonine, w h ich was not used by these workers, wa s added to give a
i/4,000 concentration.
strains.
medium.

This medium supported luxuriant growth of all

One amino acid at a time was then omitted from this synthetic
Each medium thus composed was inoculated with approximately

twenty— five thousand organisms, incubated at 37°C. for 24 hours, and ob­
served for reduction in visible growth or no growth.

If either of these

conditions occurred, the amino acid omitted was considered to be of prob­
able nutritional value.

The effect on growth was then noted wh e n the

organisms were grown in the glucose-mineral base with these acids added

11
singly and in different combinations.
Whether vitamins and other organic nutrients would hasten the growth
of S. pullorum was then tested.

A medium composed o f cystine, methionine,

leucine, glucose, asparagine, and minerals in the concentrations stated
above, was prepared.
ly.

All the strains would grow in this medium, but slow­

Four different mixtures of vitamins and other organic nutrients were

employed initially for screening purposes.

The four different mixtures

screened, and the quantity of each substance used per liter of medium were
(1) thiamine hydrochloride, 1.0 mg.; riboflavin, 0.5 mg.; pyridoxine hydro
chloride, 0.5 mg.; calcium pantothenate, 2.0 mg.;

(2) para-eminobenzoic

acid, 0.5 mg.; nicotinamide, 2.0 mg.; i-inositol, 4.0 mg.; pimelic acid,
4.0 mg.;

(3) choline hydrochloride, 2.0 mg.; nucleic acid, 5.0 mg.; folic

acid, 0.106 jig; biotin 5.0 jig; (4) all the vitamins and other organic
nutrients of the other three mixtures.
given in the Appendix, page 89.

The source of these substances is

The substances of any mixture which had

appeared to accelerate the growth were then tested individually.
The final composition of the synthetic medium was: NH 4 CI, 5.0 gm.;
m ^ N 0 3 , 1.0 gm.; N a g S O ^

2.0

gm.; l'gS0 4 . 7 H 2 O, 0 . 1 gm.; CaClg, trace;

K 2 HPO 4 , 3 . 0 ®n.; K H 2 P O 4 , 1 . 0 ©n.; trace element solution, 1 ml.;

(salts

of boron, 0 . 0 1 mg.; molybdenum, 0 . 0 2 mg.; iron, 0 . 2 mg.; copper, 0 . 1 mg.;
manganese, 0 . 0 2 mg.; zinc, 2 . 0 mg.; and distilled water, 1 liter) glucose,
10.0 g m . ; asparagine,

1.5 ©ft.; L( — ) cystine,

60 mg.; DL—valine, 78 mg.;

L ( + ) arginine monohydrochloride, 43 mg.; L(+) histidine monohydrochloride,
39

mg.; thiamine hydrochloride, 1 . 0 mg.; calcium pantothenate, 2 . 0 mg.;

and distilled water,

1

liter.

This medium was prepared as follows.

The inorganic substances

12
were added to 750 ml. of distilled water and autoclaved.

Each of the

organic components then was added aseptlcally from the stock solutions
described below to give the proper concentrations.

The pH was adjusted

to 7.0 with 0.2N NaOH and the volume brought up to one liter with ster­
ile distilled water.
One and one-half grams of asparagine was dissolved in 30 ml. of
0.2N HC1; sixty mg. of cystine was added to 10 ml. of 0.2N HC1.

They

were filter sterilized with a frittered-glass bacteriological filter.
A solution of 87 mg. of leucine in 10 ml. of 0.2N HOI was made and autoclaved for 20 minutes at 12 pounds pressure.
dissolved in 2 0 ml. of distilled water.

Ten grams of glucose was

Valine, arginine, and histidine,

in the amounts given above, were dissolved separately in 1 0 ml. of dis­
tilled water.

Stock solutions of thiamine hydrochloride, 0.2 mg. per ml.,

and calcium pantothenate, 0.4 mg. per ml., were made separately in dis­
tilled water.

Glucose, valine, arginine, histidine, thiamine hydro­

chloride, and calcium pantothenate were autoclaved for 2 0 minutes at 1 2
pounds pressure.

Growth studies.

The stock cultures were maintained on tryptose

agar slants in the refrigerator at 4 °C. and transferred to fresh slants
once a month.

At least 4 days before the culture cycle was determined,

individual tubes containing approximately 1 0 ml. of the medium being
studied, i.e., nutrient broth, 1 per cent tryptose-water, or the syn­
thetic medium, were inoculated with organisms from the stock cultures;
and a transfer was made to fresh medium every 24 hours.

At the beginning

of the growth curve determination, or zero time, a 250 ml. Erlenmeyer

13
flask containing 100 ml. of medium at the incubation temperature, 37°C.,
was inoculated with approximately ten thousand organisms that were taken
from a 24 hour culture.

The actual size of the inoculum was determined

with duplicate pour-plates.

The inoculated medium was incubated at 37°C.;

at 3 hour intervals a sample was taken, diluted properly, if necessary,
in physiological saline dilution blanks; and pour— plates were made in
duplicate.

This procedure was repeated until the organisms reached the

stationary phase of growth.

At each 3 hour interval, except when the

sample was actually being taken, the cultures were maintained at a
constant temperature of 37°C. Before a sample was taken from the culture
medium, it was mixed by alternating clockwise and counter-clockwise circu­
lar motions for one minute.
through a two foot arc.

The dilution bottles were shaken 25 times

With the aid of a Quebec Colony Counter, colonies

on the dilution plates were counted approximately 48 hours after being
made.
Another procedure that was tried with 1 per cent tryptose-water and
nutrient broth was to inoculate several tubes containing exactly 1 0 ml.
of medium at zero time.

Each tube was inoculated with approximately one

thousand organisms from the same 24 hour culture which had been previously
transferred three times.

At each 3 hour interval a different one of these

tubes was used to determine the number of organisms present.

The rest of

the procedure was the same as stated above.

Neomycin.*

A solution containing 1 mg. of the salt of neomycin,

*Neomycin used in this study was supplied through the courtesy of
The Upjohn Co., Kalamazoo, Michigan.

14
neomycin sulfate, per ml. of solvent was prepared fresh each time that
it was used.

The solvent was either the medium in which the tests were

made or sterile physiological saline.

The results wi t h neomycin are

reported in micrograms in this study.

It had been assayed by the Ba­

cillus subtllis plate method and the Klebsiella pneumoniae turbidlmetric methods and found to contain 200 Waksman units per milligram.
Paper chromatography, using several solvent systems, had showed that
this material is homogeneous.

Sensitivity to neomycin.

The sensitivity of S. pullorum to neo­

mycin was determined by the tube dilution method.

Five-tenths ml. of

a 1:100 dilution of a 24 hour culture was added to 10 X 75 mm. rimless
culture tubes containing 0.5 ml. of two-fold serial dilutions of neo­
mycin.

The solvent for the antibiotic, the diluent for the bacterial

suspension, and the diluent for the serial dilutions were the same, and
were varied with the medium in which the organisms had been growing.
That is, if the sensitivity of the organisms grown in 1 per cent tryptose-water was being checked, then 1 per cent tryptose-water was used
as the solvent for the antibiotic, and as the diluent for both the bac­
terial suspension and the serial dilution.

A control tube contained

0.5 ml. of the test medium and 0.5 ml. of the 1:100 dilution of the 24
hour culture.

The tubes were incubated at 37°C. and observed macro-

scopically at 24 hour intervals for 72 hours and, again, after one week
to see whether growth had occurred.

Factors that may or may not Influence antibacterial action of neo­
mycin,

The cultures for all of the tests that are described under this

15
heading were grown in 1 per cent tryptose-water#
In order to study the effect of pH on the activity of neomycin, 5
lots of 1 per cent tryptose-water with different pH values, pH 5, 6 , 7,
8

, and 9, were prepared by adding IN NaOH or IN HG1 to the medium.

The

pH values were limited to a range of pH 5 to 9 because it had been shown
by Chang, Kuan-how and Stafseth (1950) that S. pullorum would not grow
at pH values beyond these extremes#
made with a Beckman pH meter#

The measurement of the pH value was

The same procedure as stated in the de­

scription of the sensitivity tests was followed with these media#
The effect of some of the constituents of the different media and
sodium chloride was determined with two sets of serial dilutions of neo­
mycin.

One set was made with 1 per cent tryptose-water to which had been

added a quantity of a constituent of a medium to {rive the same final con­
centration as that of this particular constituent in its regular medium.
7/hen sodium chloride was tested, a quantity of it sufficient to give a
final concentration of 0.85 per cent was added to 1 per cent tryptosewater#

The other set of the serial dilutions of neomycin was made with

1 per cent tryptose-water itself.

This was the control set.

In addi­

tion, a control tube, as stated in the description of the sensitivity
test, was included#

An inoculum of 0.5 ml, of a 1:100 dilution of a

24 hour culture was added to each tube#

The tubes were incubated at

27°C• and observed macroscopically at 24 hour intervals for 72 hours
and, again, after one week to see whether growth had occurred.
In the experiments to test the effect of temperature,

size of in­

oculum, and growth phase on the antibacterial activity of neomycin, the
size of the sample taken from the experimental tubes to determine the

16
number of organisms present was limited to 0.1 ml. because of the lack
of knowledge of a neutralizer for neomycin.

It was assumed that 15 to

20 ml. of tryptose agar, used to make a pour-plate, would dilute the
neomycin removed with 0.1 ml. of a sample to a point
cidal concentration.
at the same time.

below its bacteri­

To check this assumption two samples were taken

One was plated out directly and the other one was

washed and centrifuged three times with saline before plating.

It was

found that when plates made with a direct inoculum were free of growth
so were those made with the washed inoculum.

Also, at the same time that

a sample was taken for a pour-plate, another one was taken and inoculated
into 20 ml. of 1 per cent tryptose-water.

It was found that when plates

made with a direct inoculum were free of growth so were the tubes of 1
per cent tryptose-water, except on rare occasions.
To test whether temperature would influence the action of neomycin,
undiluted 12 hour cultures were divided into 6 equal portions of 10 ml.
each.

To each of 3 samples, 0.1 ml. of a solution of neomycin was

added, giving a final concentration of 0.5 pg. per ml.

To each of the

remaining 3, which were the control tubes, 0.1 ml. of 1 per cent tryp—
tose—water was added.
and 37°C.

One tube from each set was incubated at 4°, 20°,

It should be noted that before neomycin or 1 per cent tryp­

tose-water was added to the cultures they were either cooled or heated
to the temperature at which they were to be incubated.

The number of

organisms per ml. was determined at the beginning and at the end of 18
hours* incubation by a colony count of pour-plates.
To observe the effect of the size of inoculum, 6 different sizes
of inocula with 3 different concentrations of neomycin were employed.

17
Ons and 0*1 ml* quantities or an undiluted 24 hour culture; and 1
0.1 ml. quantities of a 1-100, and 1-10,000 dilution of a 24 hour cul­
ture mere used for the inoculum.

The final concentration of organisms

per ml. of medium was of a range of approximately 3.5 X 107 to 3.5 X
10^ with a 10—fold increment.
this manner.

Four sets of 6 tubes were prepared in

Neosiycin was added to three sets of 6 tubes in the amount

of 0.5, 5.0, and 50.0 jxg respectively, giving a final concentration of
0.0495, 0.495, and 4.95 ^ig. per ml., and the cultures were incubated at
37°C.

For the control, a set of 6 tubes without neomycin were incubated

simultaneously.

The number of organisms per ml. at 0, 30, 60, 120, and

240 minutes was ascertained by colony counts in pour-plates.
To find out whether the action of neomycin was affected by the
phase of growth, organisms were tested in the logarithmic and station­
ary phases.

Nine tubes, each containing 9.9 ml. of 1 per cent tryptose-

water, were inoculated with approximately a thousand organisms from a
24 hour culture and incubated at 37°C.
each strain.

This procedure was repeated for

After 12 hours (logarithmic phase), the number of organ­

isms in 5 of the 9 tubes was determined by taking a sample from each
one and making pour—plates.

Then, to 2 of the 5 tubes enough neomycin

was added to give a final concentration of 1.09 pg. per ml., and to the
other 2 a quantity of it was added to give a final concentration of 3.2
pg. per ml.

No neomycin was added to tube 5, which was the control.

These 5 tubes were incubated for 18 hours and then the number of organ­
isms per ml. was determined again.

After 30 hours (stationary phase),

the number of organisms in the other 4 tubes of the set of 9, plus tube

18
5 which was the control, was determined, and neomycin added as above.
These 5 tubes were incubated for 18 hours and then the number of organ­
isms per ml. was determined again.

All plates were counted approximately

48 hours after Inoculation and incubation.

The data were handled by

computing the percentage of organisms that was killed with different
concentrations of neomycin in each phase of growth.

RESULTS AND DISCUSSION

Growth studies*

The results of the growth studies that are de­

scribed first were accomplished by taking samples from the same culture
of 1 per cent tryptose-water or nutrient broth for a period of at least
24 hours and, in the case of the synthetic medium, for a period of 60
hours*
Washing the inoculum and adapting organisms to a culture medium
are two of several things which can influence the bacterial culture
cycle.

Chesney (1916) made the interesting observation that an inocu­

lum of washed cells showed a greater lag than one of unwashed cells*
Adapting bacteria to enhance reactions, normally but slowly performed,
or to grow in media previously insufficient to support growth has been
reported by many workers (Knight, 1936; Dubos, 1940; and Rahn, 1938).
Sraham-Smith (1920) demonstrated that the initial stationary phase and
lag phase were shorter when the inoculum was from bacteria which had
been frequently subcultured than from those in which few previous sub­
cultures had been made*
In this study 5 strains of S. pullorum were subcultured for at
least 4 days in the culture medium being tested prior to the growth
determination.

Strain 11 was subcultured for at least 6 days when

grown in the synthetic medium and each subculture was inoculated with
two loopfuls rather than one.

The inoculum was not washed but simply

diluted with physiological saline to give the desired quantity of organ­
isms*

This procedure was used in order to abolish the initial

20
stationary phase and to
The results of the
V,

reduce the lag phase time.
growth determinations are given in tables I, III,

(pages 22, 25, 28) and in figures 1, 2, 3 (pages 23, 26, 29).

All

strains, except 11, had a lag phase of approximately 3 hours when they
were grown in 1 per cent tryptose-water.
phase for 3 to 6 hours.

Strain 11 remained in this

A lag phase of 3 hours occurred for all strains

when they were grown in nutrient broth.

Three of the 5 strains had a

noticeable lag phase when grown in the synthetic medium.

Strains 13 and

29 were in this phase for about 6 hours, whereas strain 12 was in it for
about 12 hours.

The absence of a lag phase with the other two strains

was probably due to the fact that they were inoculated with organisms
which were in the logarithmic phase.

Barber (1908) showed that when a

transfer was made from a culture in the logarithmic phase to the same
medium and under the same conditions, the new culture multiplied at
once at a logarithmic rate.
The pattern of the
5

logarithmic phase was not consistent among all

strains when grown in the same medium.

Strains 12, 13, and 17, grown

in 1 per cent tryptose-water, were in the logarithmic phase of growth
from about the third hour to the eighteenth hour of incubation or for a
period of 15 hours.

These strains had an average generation time of 52

to 53 minutes which was shorter than that of strains 11 and 29.

Table

II (p. 24) gives the reproductive rate of all strains growing in 1 per
cent tryptose-water.

Strain 11 was dividing at a maximum rate from about

the sixth hour to the twenty-fourth hour of incubation or for a period of
18 hours.

It had an average generation time of 66 minutes.

Strain 29

was in this phase from the third to the twenty-fourth hour of incubation
or for a period of 21 hours.

This extended logarithmic phase was

accompanied by a long generation tine,

it had an average generation

time of 74 minutes which was 21 to 22 minutes longer than those of
strains 12, 13, and 17.
In nutrient broth strains 12, 13, 17, and 29 were in the logar­
ithmic phase from the third hour to the fifteenth hour of incubation
or for a period of 12 hours.

The average generation time for these

strains was 42 to 45 minutes.

Strain 11 was In this phase from the

third to the eighteenth hour of incubation or for a period of 15 hours
It had an average generation time of 53 minutes.

Table IV (page 27)

gives the reproductive rate of all strains growing in nutrient broth.
The length of the logarithmic phase varied with each strain when
all strains were grown in the synthetic medium.

Strains 11 and 17

were in this phase from the time of inoculation to about the fortyeighth and sixtieth hours respectively.

Strain 11 had an average gen­

eration time of 138 minutes; strain 17 had an average generation time
of 160 minutes which was the longest one of all the strains.

Strains

12 and 29 had the shortest over-all logarithmic phase, which was 30
hours.

Strain 12 was in this phase from the twelfth to the forty-

second hour of incubation, and it had an average generation time of
114 minutes.

Strain 29 was in the logarithmic phase fran the sixth to

the thirty-sixth hour of incubation, and it had an average generation
time of 107 minutes which was the shortest one of all the strains,
strain 13 was in this phase from the sixth hour to the forty-eighth
hour of incubation or for a period of 42 hours.
generation time of 133 minutes.

It had an average

Table VI (page 30) gives the repro­

ductive rate of all strains growing in the synthetic medium.

TABLE I
LEAN POPULATION AT 3 HOUR INTERVALS OF 5 STRAINS OF S. HJLLORUK
GRO'.7N IN 1 PER CENT TRYPTCSE-VtATER

Hours

0
3
6
9
12
15
18
21
24

Strains
11

12

13

17

29

65
203
760
6,297
32,113
238,666
2,866,666
12,055,000
58,433,333

105
275
3,255
35,580
481,000
5,686,666
48,766,666
*

115
347
4,333
41,183
469,333
6,346,666
60,266,666

124
394
5,560
52,866
658,000
7,103,333
73,366,666
*

159,500,000

199,166,666

270,000,000

91
256
1,270
5,983
24,966
130,000
1,043,000
3,930,000
62,666,666

Figures are based on the average of the populations of 3 different growth determinations
and represent organisms per ml. The populations of the 3 individual growth determinations are
given in the Appendix, Tables XIII, XIV, and XV,
*No count made at this time.

23

Figure 1
Growth curves of 5 Strains of S. pullorum
grown in 1 per cent tryptose-water
on the data in table I)

8
6

4

2

0

Log

cells

per ml

0

3

Strain 13
6 9 12 15 18 21 24

8

8

6

6

4

4

2

2

0

0

3

Strain 12
6 9 12 15 18 21 24

0

8

8

6

6

4

4

2

2

0
0

3

3traln 11
6 9 12 15 18 21 24

atraia 17_________

0
3

6

9 12

0 3

6

9

15 18 21 24

0

Hours

12 15 18 21 24

TABLE II
REPRODUCTIVE RATE AT 3 HOUR INTERVALS OF 5 STRAINS OF S. PULLORUM
GROW; IK 1 PER CENT TRYPTCSE-VJATER

Hours

Strains
11
n

0
3
6
9
12
15
18
24

—

1.62
1.88
3.03
2.34
2.87
3.56
4.32

12
2

n

n
—

•

111.1
95,7
59.4
76.1
62.7
50.6
83.3

1.37
3.54
3.42
3.40
3.54
3.07
1.67

17

13

131.4
50.8
52.6
52.9
50.8
58.6
215.6

1.06
3.62
3.23
3.49
3.73
3.23
1.71

8
•

—

169.8
49.7
55.7
51.6
48.3
55.7
210.5

n
*

29
g

'

1.67
3.79
3.23
3.38
3.41
3.34
1.88

«.

107.8
47.5
55.7
53.2
52.9
53.9
191.5

n
•

1.48
2.31
2.22
2.06
2.36
2.98
5.87

g
—

121.6
77.9
81.1
87.4
76.3
60.4
61.3

These figures were computed from the data given in Table I.
n ■ number of generations occurring in time (t) in minutes.
g = generation time in minutes, or time for one complete cellular division to take place,
n and g were computed on the basis of the following two formulae:
n * 3.3 log10 b
F

g* _______ t_____
3.3 log10 b

5

where b * number of bacteria at the end of a given time (t), and B = number of bacteria at
the beginning of a given time, taken as t = 0.

TABLE III
LEAK POPULATION AT 3 HOUR INTERVALS OF 5 STRAINS OF S. PULLORW.
GROTZN IN NUTRIENT BROTH

Strains

Hours
11
0
3
6
9
12
15
18
21
24

76
166
1,141
17,100
162,000
2,323,000
25,516,666
138,833,333
205,500,000

12
62
202
1,811
53,400
940,000
22,466,666
56,333,333
148,666,666
231,500,000

13
50
212
4,585
138,500
3,971,666
39,550,000
150,333,333
213,500,000
223,000,000

17
61
281
5,383
55,800
2,458,333
27,625,000
102,333,333
186,000,000
232,666,666

29
105
468
6,258
114,167
3,015,000
78,833,333
172,000,000
203,000,000
270,333,333

Figures are based on the average of the populations of 3 different growth determinations
and represent organisms per ml. The populations of the 3 individual growth determinations are
given in the Appendix, Tables XVII, XVIII, and XIX.

Figure 2
Growth curves of 5 strains of _3. pullor»pp
grown in nutrient broth
(These graphs are baaed on the data in table III)

8
6

4

2
0
3

Strain 13
6 9 12 15 18 21 24

8

8

6

6

4

4

2

O
c^

0
3

Strain 12
6 9 12 15 18 21 24

0

8

8

6

6

4

4

2

2

0
3

Strain 11
6 9 12 15 18 21 24

0

Hours

Strain 17

Strain 29

26

TABLE IV
REPRODUCTIVE RATE AT 3 HOUR INTERVALS OF 5 STRAINS OF S. FULLORUM
GROAN IN NUTRIENT BROTH

Hour 8
11

n
0
3
6
9
12
15
18
21
24

1.13
2.77
3.87
3.23
3.81
3.42
2.42
0.58

12

n
159.3
65.0
46.5
55.7
47.2
52.6
74.4
310.3

1.71
3.15
4.85
4.11
4.55
1.31
1.37
0.67

Strains_________________
17
29

13

g
105.3
57.1
37.1
43.8
39.5
137.4
131.4
268.7

n

S

2.06
87.4
40.9
4.40
36.9
4.88
4.81
37.4
3.30
54.5
1.91
94.2
0.48 375.0
0.06 3000.0

5_____ £ _
2.19
4.23
3.36
5.43
3.46
1.88
0.84
0.38

82.2
42.6
53.6
33.1
52.0
95.7
214.3
473.7

S_____ £
2.12
3.72
4.16
4.69
4.67
1.13
0.26
0.36

84.9
48.4
43.3
38.4
38.5
159.3
666.6
500.0

These figures were computed from the data given in Table III.
n * number of generations occurring in time in minutes.
g * generation time in minutes, or time for one complete cellular division to take place,
"n" and "g" were computed on the basis of the same 2 formulae given below Table II.

to
o

TABUS V
MEAN POPULATION AT 6 HOUR INTERVALS OF 5 STRAINS OF S. FULLORUM
GROWN IN A SYNTHETIC MEDIUM

Strains

Hours
11
0
6
12
18
24
30
36
42
48
54
60

42
301
2,063
8,353
44,033
255,916
2,089,750
16,153,250
114,283,333
341,500,000
612,500,000

12
130
488
2,768
19,700
178,800
2,957,416
53,666,666
286,291,666
969,166,666
930,000,000
809,000,000

13
126
512
3,266
17,200
119,692
983,333
8,720,833
44,933,333
347,333,333
905,666,666
887,250,000

17
106
446
2,092
6,858
44,050
221,908
1,004,166
9,180,000
47,745,833
349,666,666
1,232,500,000

29
126
538
6,075
47,725
467,083
5,065,000
75,166,666
439,000,000
1,544,333,333
932,500,000
977,250,000

Figures are based on the average of the populations of 3 different growth determinations
and represent organisms per ml* The populations of the 3 individual growth determinations are
given in the Appendix, Tables XXI, XXII, and XXIII.

29

Figure 3
Growth curves of 5 strains of 3. pullorum
grown in a synthetic medium
(These graphs are based on the data in table V)

8

6

4

2
0

”3

3traln 13______________
12 18 24 30 36 42 <*8 54 60

8
6
4

4

2

2

0

6

Strain 12
12 18 24 30 36 42 48 54 60

8

8

6

6

4

4

2

2

0

Strain 11

0

Strain 17
6 12 18 24 30 36 42 48

0

Strain 29
6 12 18 24 30 36 42 48 54 60

0

0

6 12 18 24 30 36 42 48 54 60

Hours

TABLE VI
REPRODUCTIVE RATE AT 6 HOUR INTERVALS OF 5 STRAINS OF S. FULLOR!!,;
GRO'.TN IN A SYNTHETIC 1/EDItfc

Hours
11
n
0
6
12
18
24
30
36
42
48
54
60

2.83
2.77
1.99
2.39
2.52
3.02
2.93
2.81
1.57
0.84

Strains
13

12
«

127.2
130.0
180.9
150.6
142.9
119.2
122.9
128.1
229.3
428.6

n

1.91
2.49
2.81
3.16
4.02
4.15
2.39
1.75
*
*

g

188.5
144.6
128.1
113.9
89.6
86.7
150.6
205.7
*
*

n

2.02
2.66
2.39
2.79
3.02
3.13
2.36
2.93
1.37
¥

17
g

178.2
135.3
150.6
129.0
119.2
115.0
152.5
122.9
262.8
*

n

2.06
2.22
1.71
2.66
2.31
2.16
3.16
2.36
2.85
1.80

29
g

174.8
162.2
210.5
135.3
155.8
166.7
113.9
152.5
126.3
200.0

n

2.09
3.48
2.96
3.27
3.41
3.86
2.52
1.80
*
*

£

172.2
103.4
121.6
110.1
105.6
93.3
142.9
200.0
*
*

These figures were computed from the data given in Table V.
n = number of generations occurring in time in minutes.
g * generation time in minutes, or time for one complete cellular division to take place,
"n" and ’’g" were computed on the basis of the same 2 formulae given below Table II.
*Organisms decreasing in number.

k

31
In 1 per cent tryptose-water a negative acceleration phase of
approximately 3 hours followed the logarithmic phase*

In nutrient

broth there was a negative acceleration phase of approximately 3 hours
for all

strains except number 12 which was in this phase for about 6

hours*

This phase varied in duration from 0 to 12 hourB for these

strains in the synthetic medium.
29

Strain 17 had none; strains 11 and

had a 12 hour one, from the forty-eighth to sixtieth hour and from

the thirty-sixth to the forty-eighth hour of incubation respectively;
strains 12 and 13 had a 6 hour one, from the forty— second to the fortyeighth hour and from the forty—eighth to the fifty-fourth hour of incu­
bation respectively*
The stationary phase was entered after 27 hours by strains 11 and
29, and after 21 hours by 12, 13, and 17 when they were grown in 1 per
cent tryptose-water.

When cultured in nutrient broth, strains 13, 17,

and 29 entered it in 18 hours, and strains
hours*

11 and 12 reached it in 21

In the synthetic medium, 11 and 17 attained the stationary phase

in 60 hours,

strains 12 and 29 arrived at this phase in 48 hours, and 13

reached it in 54 hours*

The maximum number of viable organisms in the

stationary phase was about the same for all strains when they were grown
in either 1 per cent tryptose-water or nutrient broth for 36 hours, but
it was a good deal higher when they were grown in the synthetic medium
for 60 hours.

See tables XVI, XX, XXI, XXII, and XXIII in the appendix.

These results do not support the assumption of Bail

(1929) who studied

the problem of maximum population and decided that for each individual
bacterial species there was a maximum population which could not be sur­
passed no matter what factors were controlled.

Korinek (1939) found

32
that the maximum population was directly proportional to the concen­
tration of food.
In summarizing the material on growth presented thus far it should
be pointed out that there was no initial stationary phase evidenced when
a sample was taken as late as 3 hours after inoculation.

When grown in

1 per cent tryptose-water, all the strains, except 11, were in the lag
phase for a period of 3 hours.
hours.

Strain 11 was in this phase for about 6

All the strains were in the lag phase for 3 hours when grown in

nutrient broth.

Two of the 5 strains had no lag phase when grown in the

synthetic medium and the others had one of 6 to 12 hours, depending upon
the strain.

When cultured in nutrient broth, all these strains of S.

pullorum had the shortest logarithmic phase and most of them were in it
for about the same duration of time; in 1 per cent tryptose-water this
phase was somewhat more variable in duration from strain to strain and
all strains were in it longer than when cultured in nutrient broth;

in

the synthetic medium the reproductive rate was slow and thus extended
this phase considerably over that in the other two media.

The negative

acceleration phase was about the same length for most of these strains
whether they were growing in 1 per cent tryptose-water or in nutrient
broth but varied from 0 to 12 hours when they were cultured in the syn­
thetic medium.

The stationary phase was reached in 18 to 21 hours by

the nutrient broth cultures,

in 21 to 27 hours by the 1 per cent tryp­

tose-water cultures, and in 48 to 60 hours by the synthetic medium
cultures.

The maximum number of viable organisms in the stationary

phase was higher in the synthetic medium than in either 1 per cent tryp­
tose-water or nutrient broth.

The maximum number of viable organisms

33
in the stationary phase was about the sane in the latter two media.
7/hen growth determinations were made in 1 per cent tryptose-^water
or ntitrient broth using a different tube (tube method) at each 3 hour
interval, a pattern almost identical with that given by use of a single
source culture was found with nutrient broth.

Throe of the strains (13,

17, and 29) showed little, if any, negative acceleration phase when grown
in 1 per cent tryptose-water and, as a result, reached the stationary
phase 3 hours sooner by this method than when each pour-plate was made
from the same culture for a 24 hour period.

Also,

strains 11 and 12 had

a three-hour-shorter logarithmic period by the tube method.

However, by

this method the negative acceleration phase was 6 hours for strain 11;
hence it reached the stationary phase in 27 hours,
method.

just as with the other

Strain 12 had the usual 3 hour negative acceleration phase and

attained the stationary phase in 18 hours.

It must be kept in mind that

this method was used only once in order to determine whether the two
methods were at all comparable and, since the results were so much alike,
the procedure was not repeated.

These results are given in the appendix,

tables XVI and XX.

Synthetic m e d i u m .

A short discussion of the development of the syn­

thetic medium is included because the survey of the amino acid and vita­
min requirements of S. pullorum was of sufficient scope to be of value to
anyone interested in the nutrition of this organism.

On the basis of the

statements of Lederberg (1947) and Gray and Tatum (1944), which have been
cited in the review of literature, it was assumed in the beginning of
this study that a synthetic medium such as they used would be suitable

34
for the strains of S. puljorum being studied*

Furthermore,

it was de­

cided arbitrarily that the synthetic medium should be one in which all
strains would grow sufficiently to change it macroscopically in 24 hours
when an inoculum of about twenty—five thousand organisms was added to
2*5 ml, of it and in which subcultures would grow similarly when pre­
pared in the same manner.

Growth, in this discussion, is considered

synonymous with visible growth.
One of the media used by Lederberg (1947) was composed of glucose,
asparagine, cystine, leucine, methionine,

salts, and trace elements.

The concentrations of these substances have been given in the chapter
on materials and methods.

These concentrations were used throughout

this investigation unless otherwise mentioned.
grew in this medium in 24 hours.
of growth,

None of the strains

In 48 hours strain 12 gave evidence

'llhen either cystine, or leucine and cystine were omitted

from this medium,

there was no growth in 48 hours.

By plating out these

media after 48 hours incubation, it was found that the organisms in the
medium with all three amino acids were increasing in number, but slowly.
In the following discussion the above medium containing all three
amino acids is designated by the term "base."

A vitamin mixture of

thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, and calci­
um pantothenate added to the base accelerated the growth of strains 12,
13, and 29 so that there was a macroscopic change in 24 hours.

By the

end of 48 hours growth had occurred in the tube containing strain 17
but not in the one containing strain 11.

Strain 12 grew in a mixture

of para-eminobenzoic acid, nicotinamide, i-inositol, and pimelic acid,

35
plus the base, but the growth did nob become evident until it had been
incubated for 48 hours.

This is not an acceleration as it grew this

well in the base alone.

Strains 29 and 17 grew in 48 hours in a mixture

of choline hydrochloride, nucleic acid, folic acid, and biotin, plus the
base, but they grew only slightly - the latter strain doing this on the
third subculture.

Strains 12, 13, and 29 grew in 24 hours in a mixture

of all the vitamins and other organic nutrients plus the base.
17 grew in it in 48 hours;

Strain

strain 11 did not grow in it in 48 hours.

From this set of data it was concluded that more than just a supplement
of the vitamins and other organic nutrients tested is needed to stimulate
more rapid growth of strain 11, and that each vitamin of the mixture co n ­
taining thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, and
calcium pantothenate should be tested by itself.
The following results are based on experimentation with the vitamins
just mentioned, each tested separately with the base.

No acceleration of

growth occurred in tubes in which either riboflavin or pyridoxine hydro­
chloride was added to the base.

Strains 13 and 17 grew in 48 hours when

thiamine hydrochloride was added to the base.

Strain 29 responded simi­

larly with added calcium pantothenate.
In summary, these experiments showed that a vitamin mixture of thia­
mine hydrochloride, riboflavin, pyridoxine hydrochloride, and calcium
pantothenate accelerated the growth of strain 12 but that they did not
accelerate growth of strain 12 when added individually to the base;
a supplement of thiamine hydrochloride accelerated strains 13 and 17;
a supplement of calcium pantothenate accelerated strain 29; neither
the base itself nor any of the vitamin and other organic nutrient

mixtures tested accelerated strain 11; and, finally, none of these
media were satisfactory for all 5 strains according to the criteria
set down at the outset of this discussion.
Experiments to determine what amino acids were actually essential
for these strains were tried by the method outlined in the chapter on
materials and methods.

The medium used w as composed of glucose, salts,

trace elements, and 17 amino acids.

Essentially, the procedure was to

omit one amino acid at a time from the entire mixture and observe
whether the organisms would grow in the medium remaining.

7/hen a strain

failed to grow 2 out of 3 times in 24 hours in the absence of an amino
acid, that amino acid was then designated as essential; when a strain
failed to grow at a maximum rate 2 out of 3 times in 24 hours in the
absence of an amino acid, that amino acid was then designated as ac ­
cessory.

These designations were designed for this study only.

They

were merely used in order to have some means of classifying the data
accumulated from three different experiments with all 17 amino acids.
Leucine, histidine, valine, arginine, and proline were essential to at
least one or more strains; histidine, valine, cystine, serine, tryp­
tophane, phenylalanine, and glutamic acid were accessory to at least
one or more strains.
amino acids.

Strain 11 seemed to need the largest number of

Leucine, histidine, valine, arginine, and proline were

essential and cystine, serine, and glutamic acid were accessory.
Strain 29 needed the fewest amino acids.
histidine was accessory.
Cystine

Leucine was essential and

Arginine was essential to strains 12 and 13.

serine were accessory to the former strain; valine and

cystine were accessory to the latter strain.

Leucine only was

37
essential to strain 17; valine, cystine, and serine were accessory*
Only the absence of leucine curtailed growth of all strains for over
24 hours*

Without leucine no growth occurred even after 2 weeks in­

cubation*

Xt was concluded that leucine is an essential amino acid

for these strains when this word is used in its narrowest sense of
meaning*

Johnson and Rettger (1943) concluded that leucine was one

of the most important amino acids for most of the strains of S. pullorum with which they worked.
Since leucine seemed to be the key amino acid, an experiment was
tried in which leucine, in 3, 6, and 9-fold greater concentrations than
that ordinarily used, was added to glucose, salts, and trace elements*
No growth occurred in any of these concentrations during a two week
period of observation*

Also, a 10—fold increase in concentration of

leucine was tried with glucose, thiamine hydrochloride, calc1van panto­
thenate, salts, trace elements, and ascorbic acid (at the rate of 0,62
mg,/l00 ml* of medium).

No growth occurred in 48 hours.

Leucine, histidine, arginine, valine,

serice, and cystine were

tried in different combinations with glucose,
The combinations tried were:

salts, and trace elements.

leucine* leucine and histidine; leucine,

histidine, and cystine; leucine, histidine, cystine, and arginine;
leucine, cystine, histidine, arginine, and valine; leucine, histidine,
cystine,

serine, and arginine;

and arginine.

leucine, arginine, and cystine;

leucine

It should be noted that proline was not included among

this group of amino acids because it was specific for strain 11 only.
Cystine and serine were tried in preference to proline, although not
essential, because their absence consistently resulted in reduced

38
growth of the majority of strains.

All strains grew well in 48 hours

when arginine, cystine, histidine, leucine, and valine were combined,
except 11, which required a larger inoculum to get a similar growth
response.

When the concentrations of arginine, cystine, histidine,

and valine were doubled and leucine was quadrupled, growth occurred in
24 hours in all strains after the third subculture but growth was very
slight for strains 11 and 13.
From the results of the experiments with the amino acids the follow­
ing summarizing statements may be made.

Leucine was essential for all

strains and the usage of essential here means that without leucine they
will not grow.

None of the strains grew in a medium containing glucose,

salts, and trace elements with leucine added in 3 different amounts.
None of the strains grew in 48 hours in a medium in which leucine, in
10-fold greater concentration than that ordinarily used, was added to
glucose, thiamine hydrochloride, calcium pantothenate, salts, trace
elements, and ascorbic acid.

Good growth occurred in 48 hours in a

medium composed of arginine, cystine, histidine,

leucine, valine, glu­

cose, salts, and trace elements except for strain 11 which responded
similarly only when a larger inoculum was used.

Doubling the concen­

tration of all these amino acids except leucine, which was increased
4-fold, gave a medium in which all strains grew in 24 hours after at
least 3 subcultures.
Finally, either asparagine or one of the combinations (thiamine
hydrochloride and calcium pantothenate; asparagine and thiamine hydro­
chloride; asparagine and calcium pantothenate; asparagine, thiamine

39
hydrochloride, and calcium pantothenate) was added separately to the
medium containing glucose, salts, trace elements, arginine, cystine,
histidine, leucine, and valine to determine whether it would enhance
growth.

The concentrations of the amino acids were those stated at the

outset of the discussion on the development of the synthetic medium.
All strains grew in 24 hours in a medium containing the combination of
asparagine, thiamine hydrochloride, and calcium pantothenate; however,
strain 11 did not grow consistently in 24 hours.

This medium also gave

best results when subcultures were made from it. Finding that this medium
gave better results than any other combination tested was expected since
it had already been determined that strains 13 and 17 grev; better with
the addition of thiamine hydrochloride and that strain 29 grew better
with the addition of calcium pantothenate.

None of the strains grew

consistently in 24 hours in any of the other combinations of media.
The synthetic medium that was used for the growth studies and the
work with neomycin was composed of arginine, cystine, histidine,

leucine,

valine, thiamine hydrochloride, calcium pantothenate, glucose, salts,
asparagine, and trace elements.

This medium was chosen over the one

with these amino acids alone in higher concentrations because growth
occurred upon initial inoculation in 24 hours,

subculturing was neces­

sary to attain similar results with the amino acids alone in higher con­
centrations and even then strains 11 and 13 grew only slightly in 24
hours.
bensltivity to neomycin.

As is shown in tables VII and VTII

(pages

41 and 43), about the same concentration of neomycin was sufficient to
stop the growth of all the strains when they were grown and tested in
either 1 per cent tryptose-water or nutrient broth.

However, when the

40
bactericidal concentration of neomycin necessary in 1 per cent tryp­
tose-water and nutrient broth was compared wi t h that necessary in the
synthetic medium,

shown in table IX (page 43), it was revealed that

from 41 to 321 times more neomycin was needed to kill the organisms
when they were grown and tested in the synthetic medium.

The latter

fact was probably due to the interference of neomycin action by the in­
organic salts of the synthetic medium.

This point will be discussed

further under the heading of the effect of the constituents of the
medium and sodium chloride on the antibacterial action of neomycin.

In

only one case w h e n they were grown and tested in either 1 per cent tryp­
tose-water or nutrient broth was there growth after 72 hours.

This was

exactly diametrical to the results with the synthetic medium in which
all except one strain grew after 72 hours in a tube with a higher con­
centration of neomycin.

Although there was growth after 24 hours in

tubes with a higher concentration of neomycin, this growth usually did
not occur in a tube that contained greater than a 4—fold increase in
concentration of the antibiotic.

And, since only very small concen­

trations of neomycin were necessary to kill these strains of S. pullorum
this difference in concentrations of neomycin which stopped growth for
24 hours and that which inhibited growth for a week ma y be considered
negligible.

It was concluded from these observations that neomycin is

bactericidal in concentrations only slightly higher than that in which
it is bacteriostatic.

This finding is in agreement with those reported

by '//aisbren and Spink (1950a), V«aksman, Frankel, and Graessle (1949),
Waksman, Lechevalier, and Harris
(1949).

(1949), and v'/aksman and Lechevalier

41
TABLE VII
SENSITIVITY OF S. FULLQRUM TO NEOMYCIN AT VARIOUS pH VALUES

Strain
1.25
11
12
13
17
29
11
12
13
17
29

11
12
13
17
29

72*
48
72
48
24

0.625
72
48
72
48
24
24

—
-

—

48
24

0.312
72
48
72
48
24
24
48
48
24
24

jog./ml. Neomycin
0.156 0.078 0.039

mm

—

0.009

Control

72
48
72
48
24

pH 5.0
72
48
72
48
24

72
48
72
48
24

72
48
72
48
24

72
48
72
48
24

48
48
72
48
24

24
24
24
24
24

pH 6.0
24
24
24
24
24

24
24
24
24
24

24
24
24
24
24

24
24
24
24
24

24
24
24
24
24

24
48
48
24
24

24
48
24
24
24

24
24
24
24
24

24
24
24
24
24

96

24
96
48
48
48

24
24
24
24
24

48
24
24
24
24

48
24
24
24
24

72
—

0.019

—

—

—

—

—

—

—

—

—

-

-

-

-

pH 7.0
24
168
48
—

48
pH 8.0

11
12
13
17
29

—
_

-

_

—

—

—

-

—

_

—

—

-

•

_

—

—

-

48
120

-

-

-

-

-

-

pH 9.0
11
12
13
17
29

—
—

-

—

—

—

-

—

-

_

—

-

-

—

—

96
168
120
48
144

96
48
48
24
24

* Indicates the number of hours before growth occurred.
** Indicates no growth after incubation for 1 week.

42

Figure 4

Effect of pH oa the antibacterial aotivity of neomycin
(These graphs are based on data in table VII)
8traln 13
pH
pH
pH
pH

019

078

6.0
7.0
8.0
9.0

312

1.25
0

2

4

6

8
^Strain 17

a
£
o
<u
a

019

019

078

078

312

312

So-t
E

^

1.25

1-25

Strain 11

Strain 29

019

019

078

078

312

312

1.25

- • - — ft

1.25

Base line
(showing
growth)
Period of incubation in days

• -

—

ft

43
TABLE VIII
SENSITIVITY O F S. FULLORm! TO NEOMYCIN V7HEN GRC7/N AID TESTED
IN NUTRIENT BROTH

S t r a i n ________________________ tig./ml. neomyoin
1.25
0.625
0.312
0.078
0.156
11
12
13
17
29

-

—

-

-

—

_

72

-

—

-

—

-

—

-

72

-

-

-

-

72**
48
72
48
48

0.039
48
24
48
48
48

Control
24
24
24
24
24

’’‘Indicates no growth after incubation for 1 week.
**Indioates the number of hours before growth occurred.

TABLE IX
SENSITIVITY OF S. FULLORm. TO NEOMYCIN ’THEN GROVJN AND TESTED
IN A SYNTHETIC MEDIUM

Strain
11
12
13
17
29

________________________ jig./ml. neomyoin
3.12
25.0
6.25
50.0
12.5
_

168**

—

—

—

—

—

—

—

-

-

168
168
168

168
72
48
72
48

24
24
24
24
24

1.56

Control

24
24
24
24
24

24
24
24
24
24

’’’Indicates no growth after incubation for 1 week.
’’’’’’Indicates the number of hours before growth occurred.

44
To test whether there w a s a n y difference in the sensitivity
of the organisms themselves w h e n grown in different media, they were
grown in the synthetic medium, then washed and centrifuged three times
with physiological saline, and tested with different concentrations of
neomycin in both the synthetic medium and 1 per cent tryptose-water.
A greater concentration of neomycin was found necessary to kill an
organism which had been grown and tested in the synthetic medium than
when it had been grown in the synthetic medium and tested in 1 per
cent tryptose-water.

Moreover, about the same concentration of neo­

mycin was necessary to kill all organisms when they were tested in 1
per cent tryptose-water whether they had been grown previously in the
synthetic medium or in 1 per cent tryptose-water.

Although experimen­

tation along this line was limited to one strain of S. pullorum, the
tentative conclusion may be drawn that the sensitivity of an organism
to neomycin is governed by the medium in which it is tested rather
than by that in which it is grown.

Therefore, w h e n the sensitivity

of an organism to a particular antibiotic is reported, the medium in
which the tests were carried out should be an integral part of the
report.
The effect of pH on the antibacterial activity of neomycin.

The

data in table VTI (page 41) and figure 4 (page 42) show that a pH of
8,0 definitely favored the action of neomycin over that of the other
pH values at which the experiments were run.
of neomycin was destroyed.
mycin at this pH.

At pH 5.0 the activity

The organisms grew in all dilutions of neo­

7/hen the bactericidal titers of neomycin at pH values

45
6.0, 7.0, and 9.0 were compared with the bactericidal titer at pH 8.0
it is seen that 16 to 64 times more neomycin was needed at pH 6.0, 4
to 16 times more neomycin was needed at pH 7.0, and 2 to 4 times more
neomycin was needed at pH 9.0 than at pH 8.0.

The general conclusion

ma y be drawn that neomycin activity is favored by an alkaline medium.
Similar results have been reported by Waksman, Lechevalier, and Harris
(1949) and VJaksman and Lechevalier (1949).

The most likely explanation

for this phenomenon is that the basic property of neomycin, which prob­
ably is essential to its activity, is neutralized by acid or neomycin
becomes more dissociated in an alkaline medium.

The effect of temperature on neomycin activity.
table X

As is shown in

(page 45), neomycin at 4°C. and 20°C. was not quite a s effect­

ive as it was at 37°C.

At 4°C., 96.5 per cent to 99.9

organisms were killed; at 20°C.,

per cent of the

99.9 per cent of the organisms were

killed; at 37°C., 99.9 per cent of the organisms were killed - less than
10 organisms survived.

On the basis of these results, when temperature

alone was considered as

a factor which might influence

the action of neo

myein,

that the action of neomycin is

notaffected sig­

it was concluded

nificantly by lowering the temperature from 37°G. to 4°C.

These results

seem to indicate that neomycin is somewhat unique wit h respect to the
effect of temperature, as a closely related antibiotic, streptomycin,
has been shown to be practically useless at 5°C. and to have a marked
reduction in its activity at 20°G.
Chaffee

(Garrod, 1948).

Hobby, Meyer, and

(1942) investigated the effect of temperature on penicillin

activity and reported results w h i c h are similar to those given above
for streptomycin.

table x

EFFECT CF TEMPEEh TTIRE CL EEQLYCIN ACTIVITY

Incubation
Temperature

Organisms per ml. after 18 hours incubation
20°C.

4°C.
Control

4°C.
20°C.
37°C.

6.9 X 104
6.9 X 104
6.2 X 104

6.5 X 104

12
12
12

4°C.
20°C.
37°C.

2.9 X 106
2.9 X 106
4.8 X 106

3.1 X 106

13
13
13

4°C.
20°C.
37°C.

3.1 X 106
3.1 X 106
3.3 X 106

3.4 X 106

17
17
17

4°C.
20°C.
37°C,

1.7 X 106
1.7 X 106
3.1 X 106

1.7 X 106

29
29
29

8.8 X 104
8.8 X 104
2.0 X 104

7.1 X 104

20°C.
37°C.

•

11
11
11

tp»
o
o

Strain

Organisms
per ml. at
0 hours

0.5 jig./ml.
neomycin

Control

37°C.
Control

0.5 jig./ml.
neomycin

0.5 jig./ml.
neomycin

2.3 X 103
7.4 X 105

2 X 101
1.6 X 108

<10

2.3 X 108

<10

2.2 X 108

<10

2.7 X 108

<10

2.6 X 10b

<10

1.1 X 103
4.5 X 107

5 X 101

1.3 X 103
5.4 X 107

5 X 101

1.4 X 103
4.8 X 107

5 X 101

1.0 X 103
5.2 X 105

<10

Data for the part of the experiment at 37°C. came from another experiment.

47
It should be noted that the number of organisms in the control
tube remained about the same after 18 hours Incubation at 4°C.

The

fact that neomycin killed 96*5 per cent to 99.9 per cent of the organ­
isms at this temperature then indicates that it was very effective when
they were multiplying and metabolizing at a very slow rate.

There was

only a slight increase in the total population in the control tube
after 18 hours incubation at 20°C.

Nevertheless, 99.9 per cent of the

organisms were killed by neomycin, which again indicates the antibac­
terial activity of neomycin was not restricted to organisms that were
multiplying rapidly.

The organisms in the control tube incubated at

37°C. continued to divide rapidly and were in the stationary phase at
the end of the 18 hour incubation period.

The fact that neomycin

killed 99.9 per cent (less than 10 organisms survived) then Indicates
that it was very effective when organisms were multiplying rapidly.
These results were further substantiated in another set of experiments
which will be discussed next tinder the heading of the effect of growth
phase on neomycin activity.
Effect of growth phase on neomycin activity.

The antibacterial

action of neomycin did not seem to be affected significantly by the
particular phases of growth tested.

This is revealed in table XI

(page 48) which shows that, with either concentration of neomycin,
almost as large a per cent of organisms was killed when they were in
the stationary phase as when they were in the logarithmic phase.

The

slight difference in the per cent killed in the 2 phases might have
have been due to some factor in the old culture which interfered
slightly with the action of neomycin.

4«

TABLE Al
EFFECTIVENESS 0? NEOMYCIN AGAINST 3. FTTLLCRUM IN THE LOGARITHMIC
AND STATIONARY PHASES OF GR07/TH

Strain

Growth
phase

Jig./ml.
neomycin

11
11

Log.
Log.

1.0
3,2

31,650
24,750

410
<10

99.9
99.9

11
11

Sta.
Sta.

1.0
3.2

240,000,000
259,000,000

2,910,000
620,000

98.7
99.7

12
12

Log.
Log.

1.0
3.2

3,230,000
3,150,000

410
<10

99.9
99.9

12
12

Sta.
Sta.

1.0
3.2

276,000,000
232,000,000

16,075,000
1,047,500

94.2
99.6

13
13

Log.
Log.

1.0
3.2

4,445,000
3,470,000

410
410

99.9
99.9

13
13

Sta.
Sta.

1.0
3.2

267,000,000
277,500,000

30,700,000
1,710,000

88.5
99.4

17*
17

Log.
Log.

1.0
3.2

1,845,000
1,910,000

< 10
<10

99.9
99.9

17
17

Sta.
Sta.

1.0
3.2

282,000,000
273,000,000

1,295,000
139,500

99.5
• 99.9

29
29

Log.
Log.

1.0
3.2

25,900
34,000

<10
<10

99.9
99.9

29
29

Sta.
Sta.

1.0
3.2

370,500,000
247,500,000

8,200,000
1,507,000

97.8
99.4

Organisms
per ml. before
adding neomycin

Per cent
Organisms
per ml. remaining
of
Organ i sr.:s
after 18 hours
killed
incubation with
neomycin

Log. - logarithmic; Sta. - stationary.
Controls with each strain were run simultaneously wit h the above
experiment.
They followed the normal growth pattern.
*Data for strain 17 added from another experiment.

49
The significant thing about the results of this set of experi­
ments and those results gathered with the experiments on the effect
of temperature was that neomycin acted just as lethal when the organ­
isms were not multiplying rapidly as when they were multiplying rapid­
ly.

On the other hand, Hobby, l*eyer, and Chaffee (1942) reported that

penicillin appeared to be effective only when active multiplication
was taking place; Lenert and Hobby (1947) stated that streptomycin
more constantly and completely inhibited the growth of 6 hour cultures
than 16 hour ones.

Chang, Kuan-how and Stafseth (1950) reported that

the older the culture of S. pullorum the higher the bactericidal titer
of streptomycin.
Effect of size of inoculum on the action of neomycin.

The results

of this set of experiments are given in figures 5, 6, 7, 8, and 9 (pages
50, 51, 52, 53, and 54).

The first part of this discussion pertains to

those results gathered when various quantities of organisms were incu­
bated at 37°C. for 240 minutes with a quantity of neomycin large enough
to give a final concentration of 0.495 )ig./ml.

It was concluded from

previous experiments, see results of sensitivity tests in 1 per cent
tryptose-water at pH 7.0, table VTI,(page 41) that this quantity of neo­
mycin would render a culture sterile in a week or less.
stated conditions,

Under the above

the size of inoculum, within rather broad limits, did

not seem to affect the action of neomycin significantly.

That is, with

all strains the inoculum could be varied from 1,000 to 1,000,000 organ­
isms without influencing the action of neomycin.

The time that it took

this concentration of neomycin to reduce the number of organisms to be­
low 10 per ml. was definitely influenced b y the size of the inoculum.
As the inoculum was increased in size so was the time lengthened for
neomycin to reduce the number of organisms below 10 per ml.

Figure 5
Effect of size of Inoculum on action of neomycin (Strain 11)
(These graphs are bused on data in appendix, table XXIV)

Control
0.0495 jig./ml
0.495 ;ig./ml.
*.95 n6*/ml.

C>

CM

in

CD

CM

to o>

CM

Log. cells per ml.

tO

vO

CM

1

8
6

4

2
0 o

u —o

o
eo

CD

Minutes

>H

CM

Figure 6
Effect of size of inooulun on action of neomyoin (Strain 12)
(These gruj)hs are baaed on data in appendix, table X3QV)

1*
2.
3.
4.

o

o

to

o

o

at

o
02

o

Control
0.0*95 pg./inl.
0.495 jig./ml.
4.95 jjg./ml.

o

9

8

6

4

2

CT>

0o

02
CO

1

02

CM

CM

8

6
4

2

O

o>

0 o

o
CM

Minutes

CM

52

Figure 7

Effect of size of Inoculum on action of neomycin (Strain 13)
(These graphs

ire based on data in appendix,

8

8

1.
2.
3.
4.

6

t ble XXIV)

Control
0.0*95 /lg./ml.
0.495 ^if’./ml.
4.95 jif/./ml.

4

2

0

Log. cells

>—I

»—(

i—4

CVi

9

CVI

8

8

6

6

4

4

2

2

0

o

o o
to ID

o

ct>

o
w

o
in

O

GO
iH

O

<-H
CVJ

0

Q

o

o

o

o

o

n

io

o

( ji

w

o

o
io
r-H

o

o

Cvi

8

8

6

6

4

4

2

2

0

o

cd

s 9
CNJ CV2

0
o o
00 r-t
f-H r-H W CVJ

o

Minutes

n

ID

o

<71

W

ID

o

CO

S

Cvi

3

CVI

Figure 8

53

Effect of size of inoculum on action of neomycin
(Those graphs are based on data in appendix,

8

(Strain IV)

t a b l e

XXIV)

Control
0.0495 /ig. / m l .
0. 495 ^ig./ml.
4. 95 /ig./ml.

6
4

2

0
O

O

to

O

(O

O
m

O
N

O

O

m

r-4

O

Q

CO
H
-J
«H CVJ CO

H

rH

iH CV CV

Log. cella per ml

e

6
4

2

0

O

O O
to to

O

a*

O

O

o

cv? in cp

2

CO

o

3

o

CO

a

8
6

4

2
0

o

o

CO

o

to

o

CT»

o

CVJ
—t

o
in

r->

O O Q
CD

r-*

«—»

CV

o
CM

Minute8

o

CO

O
»o, O O O
CT>

to

lO

O

CO
-^

O Q

r—< V*
CV CO

Figure 9

^

Ef fect of size of inoculum on action of neomycin
(These granhs are based on data

8

8

6

6

4

4

2

2

0

o

o

rO

o

o

'~ o

ct»

o

to

o

iO

o

(C

o o
•—
l

a

O

6

6

O

ro «o

O

O

e> c

r-1

O

o
H

O

c;

O

Q

CJ

C\i

4

2

o

0

o o
to o

o
;D

8

8

6

6

4

4

2

2

0

O

♦

2

0

9on + rol
0. 0^95 /»(•*./jnl
0. 495 jift. /ml,
4.95
/r I •

cv?

8

C

taVie XIOLV)

0
7'

8

4

in appendix,

(Strain 29)

o
•o oto

o o
CO H
—I CV)

0

O

O

to

O

VC

O

O

O

C,

O

1C

O O Q
CO
i—I t
t
'
ta cj

O
O
Q
CC
r-<

H

Minutes

Of

*V
CV

55
With a 10-fold increase in the concentration of neomycin, 4,95
jig./ml., all 6 of the different sized inocula tested were reduced to
less than 10 organisms per ml. by the end of 2 to 4 hours; inocula of
ten thousand organisms or less were reduced to less than 10 organisms
per ml. by the end of 30 minutes.

Table XXIV in the appendix shows

that there was considerable reduction in the number of organisms before
the original sample for a pour-plate could be removed from the tubes
containing 4.95 jig./ml. of neomycin.

That is, the action of neomycin

was so rapid that the original (0 time) colony count of those pourplates containing neomycin was considerably less than that of the con­
trol plate.

Garrod (1948) reported that streptomycin acts more rapidly

at higher concentrations but that the action of penicillin is not ac­
celerated by an increased concentration of it above a minimum level.
This same phenomenon was demonstrated earlier with penicillin by Hobby,
Meyer, and Chaffee (1942).
A concentration of 0.0495 jig. of neomycin per ml. was effective in
4 hours for all strains only when the smallest inoculum, one thousand
organisms, was used.

It failed to reduce the growth of any of the

strains to below 10 organisms per ml. when an inoculum of one million
organisms or more was used.

However, this concentration of neomycin

did kill over 99 per cent of the organisms of each different sized in­
oculum in 4 hours.

Chang, Kuan-how and Stafseth (1950) reported that

the smaller the size of inoculum of S. pullorum, the higher the bacteri
cidal titer of streptomycin.

Lenert and Hobby (1947) found that with

many strains the number of organisms in relation to the concentration
of streptomycin so greatly influences its effect that size of inoculum

56
is probably a limiting factor in the usefulness of streptomycin* These
same authors state that the number of units of penicillin necessary
for the inhibition of a culture is altered only slightly by a large
variation in the number of organisms present.

Garrod (1948) reported

that a small inoculum is destroyed rapidly by streptomycin, whereas a
proportion of a large one always survives*
In summary, with all strains the inoculum could be varied from
one thousand to one million organisms without influencing the action
of 0*495 pg. of neomycin per ml* significantly when there was an incu­
bation period of 4 hours.

An increased concentration of neomycin, 4.95

p g . / m 1*, was effective in 2 to 4 hours against all 6 of the different
inocula tested.

A decreased concentration of neomycin, 0.0495 p g . / m l . ,

reduced the number of organisms to below 10 per ml. only in the tube
with the smallest inoculum but did kill over 99 per cent of the organ­
isms of each different inoculum in 4 hours.
the 5 strains of S. pullorum used,

In these experiments wi t h

it w a s possible to change the size

of the inoculum one hundred thousand-fold with 3 different concentra­
tions of neomycin, 4*95, 0.495, 0.0495 p g . / m l . , without influencing
significantly its action.

Using S. coli and a beta streptococcus,

Worth, Chandler, and Bliss (1950) studied the effect of the size of
inoculum on the action of neomycin and concluded that the necessary
increase in bactericidal concentration of neomycin w i t h increase in
inoculum is of the same

o r d e r

Chloromycetin, and penicillin.
rapid

in

of

m a g n i t u d e
The

r a t e

as

t h a t

for aureomycin,

of action of neomycin was most

the highest concentration used; it decreased noticeably with

each 10-fold decrease in concentration.

This condition held for all

57
inocula.

Waisbren and Spink (1950a) reported that 20 jig. of neomycin

per ml. killed all of the organisms of a 2 X 10® inoculum of E. coli
in 2 hours.

.Vaksman, Frankel, and 3-raessle (1949) have shown that 20

units of neomycin, assay 30 to 100 units per mg.,

incubated with a

heavy suspension of S. pullorum were completely bactericidal in 3g
hours.

Effect of some of the constituents of two culture media and
sodium chloride on the antibacterial activity of neomycin.
pertaining to the effect of arginine, cystine, histidine,

The data
leucine, and

valine on the antibacterial activity of neomycin is presented in table
TIT

(pai-e 61).

Repetition of experiments to test the effect of these

acids on neomycin activity did not consistently give the sane results.
That is, although the amount of neomycin necessary to kill strain 11,
when leucine was added, was 2-fold greater than that in the control
tube according to this particular data,

it was found in another experi­

ment that the same concentration of neomycin was lethal in both the
tube to which leucine had been added and in the control tube.

Also, i:

the presence of different amino acids, the bactericidal titer of neo­
mycin varied in different experiments with other strains.
tentatively concluded,

It may be

therefore, that no one particular amino acid or

group of amino acids, in the concentrations tested, affects markedly
the action of neomycin.

The fact that the bactericidal concentration

of neomycin varied as much as 2 to 4-fold when the same experiments
were repeated, although all conditions were maintained constant as
much as possible,

is probably not unique for this antibiotic or this

58
set of experiments*

West, Doll, and Edwards (1945) tested the sensi­

tivity of Salmonella cultures to streptomycin and reported that its
bactericidal titer varied when the same experiments were repeated.
Chang, Kuan-how (1949) reported similar difficulties when he tested
the sensitivity of S. pullorum to streptomycin.
Table XII

(page 61) gives the results of an experiment in which

asparagine, calcium pantothenate, and thiamine hydrochloride were
added individually to 1 per cent tryptose-water, and the effect on
the action of neomycin was noted.

The bactericidal titer of neomycin

often varied from 2 to 4-fold with the same strain in the same medium
when the same experiment was repeated.

As with amino acids, there

seems to be no distinct influence on the action of neomycin by these
substances.
7/hen glucose was added to 1 per cent tryptose-water, a 2-fold in­
crease in concentration of neomycin was needed to kill strains 11, 13,
17, and 29; and a 4-fold increase was needed to kill strain 12.
results are given in table XII.
this experiment was repeated.

These

Similar results were obtained when
Also, all strains were tested in the

synthetic medium with the concentration of glucose increased 4 and 8fold.

A 2 to 4-fold increase in concentration of neomycin was neces­

sary to kill all strains in both media.

These results then indicate

that glucose interferes slightly with the activity of neomycin. Waksman,
Lechevalier, and Harris (1949) reported that the presence of glucose
in a test medium reduces the potency of neomycin by favoring either
acid production or growth of the test organisms.
Referring to table XII again, it is seen that a 2 to 4-fold

59
increase in the concentration of neomycin was necessary to kill each
strain w h e n either peptone or beef extract w a s added to 1 per cent
tryptose-water.
repeated*

Similar data were obtained whe n this experiment was

On the other hand, the data from the sensitivity tests

(see tables VTI and VIIT, pages 41 and 43) show that about the same
concentration of neomycin w a s bactericidal in either 1 per cent tryp—
tose-water or nutrient broth.

On the basis of these results it was

concluded that either peptone or beef extract in 1 per cent tryptosewater interfere slightly with the action of neomycin but that peptone
and beef extract together in nutrient broth do not interfere.
A 10 to 20-fold increased concentration of neomycin was necessary
to kill these strains when a quantity of sodium chloride sufficient to
give a final concentration of 0.85 per cent was added to 1 per cent
tryptose-water.
table XII.

This effect was noted consistently and is given in

Thus, there seems to be no doubt that sodium chloride in­

terferes with the action cf neomycin.

As was pointed out in the d i s ­

cussion of the sensitivity tests in the synthetic medium, a greater
concentration of neomycin was necessary to kill the different strains
in it than when they were tested in either 1 per cent tryptose-water
or nutrient broth.

This might be due to the interference of tie salts

of the synthetic medium for it had as part of its composition a mineral
base of different salts.
In summary, arginine, cystine, histidine,

leucine, valine, calcium

pantothenate, asparagine, and thiamine hydrochloride,

in the concen­

trations tested, did not seem to influence significantly the action
of neomycin;

glucose, peptone, and beef extract, in the concentrations

tested, seemed to interfere slightly with the action of neomycin;
sodium chloride, in the concentration tested, definitely affected
the action of neomycin.

61
TABLE XII
THE EFFECT OF SOL1E OF THE CONSTITUENTS OF 2 CULTURE MEDIA AID
SODIUlu CHLORIDE ON THE ANTIBACTERIAL ACTIVITY' OF NEOKXCIN

Strain
0.312

11
12
13
17
29

*

—

-

0.156

-

Ug./ml. neomycin
0.078
0.039
0.019
Arginine
48* *
24
48

-

-

—

-

-

-

-

-

11
12
13
17
29

-

-

-

-

-

-

-

—

-

—

—

-

-

-

11
12
13
17
29

-

—

—

—

—

—

—

—

—

-

-

-

11
12
13
17
29

—

72

—

—

—

—

—

_

—

-

-

-

11
12
13
17
29

_

—

_

—

-

•

—

—

_

—

—

-

-

168

48

48
48
Cystine
24
168
-

48

48
-

48
48
72

168
48

72

Histidine
24
48
—

48
48
Leuc ine
24
48
-

48
48
Valine
24
48
-

48
48

0.009

Control

24
24
48
24
24

24
24
24
24
24

24
24
24
24
24

24
48
48
24
24

24
24
24
24
24

24
24
24
24
24

24
48
48
24
24

24
24
24
24
24

24
24
24
24
24

24
48
48
24
24

24
24
24
24
24

24
24
24
24
24

24
24
48
24
24

24
24
24
24
24

24
24
24
24
24

24
24
48
24
24

24
24
24
24
24

24
24
24
24
24

Control
11
12
13
17
29

_
_

48

—

—

_

_

-

_

—

-

72

72
48

24
-

48
48

’’'Indicates no growth after incubation for 1 week.
’’"’‘Indicates the number of hours before growth occurred.

62

TABLE XII (continued)
THE EFFECT CF SOLE OF THE CCNSTITUEirES OF 2 CULTURE MEDIA AND
SODIUM CHLORIDE OK THE ANTIBACTERIAL ACTIVITY OF NEOMYCIN

Strain

11
12
13
17
29
11
12
13
17
29

_ _ _____________________ pg»/ml, neomycin
1.25
0.625
0.312
0.156
0.078

-

-

-

-

—

-

-

-

158

-

—

24
24
24
24
24

24
24
24
24
24

Beef Extract
72
24
48
24
24
24
24
24
24
24

24
24
24
24
24

24
24
24
24
24

Ug./nl. neomycin
0.0781
0.019
0.039

0.009

-

72
72
72
72
-

-

-

-

—

—

—

Strain
0.312

0.156

Control

24
24
24
24
24

mm

-

Peptone
72
24
24
48
48

0.039

Control

Control
11
12
13
17
29

_

_

—

—

—

—

—

—

—

—

158
72
48
48
48

48
24
24
24
24

24
24
24
24
24

24
24
24
24
24

24
24
24
24
24

63
TABLE XII (continued)
THE EFFECT OF SOME OF THE CONSTITUENTS OF 2 CULTURE kRDIA AND
SODITlt CHLORIDE ON TEE ANTIBACTERIAL ACTIVITY OF NBOIYCIN

jig./ml. neomycin

Strain
0.312

0.156

0.078

0.039

0 .019

0.009

Control

Glucose
11
12
13
17
29
11
12
13
17
29

-

72
72
24
168
24
48

24
24
24
24
24

24
24
24
24
24

24
24
24
24
24

24
24
24
24
24

24
24
24
24
24

Asparagine
24
48
48
168
24

24
24
48
48
24

24
24
24
48
24

24
24
24
48
24

24
24
24
24
24

24
24
24
24
24

-

-

-

-

24
168
168

—

-

-

-

-

11
12
13

—

17

—

158

29

-

mm

—

-

24

Calcium Pantothenate
168
72
24
48
24
168
24
24
72
24
24
72
46
Thiamine Hydrochloride

11
12
13
17
2?

-

-

—

—

—

—

-

72

^ 4
XX

168
48
-

48

24

24

24

168

4?
24
48

48

24
24

24

24

24

24

24

24
48
48
48
24

24
24

24
24

24

24

24
24

24

48
—

72

48

48

Control
48
48
168

12

—

—

-

13

-

-

168

17
29

—

—

48

48

_

—

72

48

24

24

64
TABLE XII (continued)
THE EFFECT OF SOLS OF THE CONSTITUENTS OF 2 CULTURE MEDIA AID
SODIUM CHLORIDE ON THE ANTIBACTJRIAL ACTIVITY OF NEOMYCIN

Strain
12.5

6.25

ixg./xnl. neomycin
3.12
1.56
0.78

0.39

Control

Sodium Chloride
11
12
13
17
29

24

24
24
24
48
24

24
24
24
24
24

jig ./ml. neomycin
0.078
0.039
0.019

0.009

-

—

-

-

-

-

-

-

-

-

-

-

—

—

-

Strain
0.312

0.156

—

24

24
24
24
24
24

Control

Control
11
12
13
17
29

..

—

48

—

-

-

—

-

—

—

—

—

168
48
72

48
48
168
48
48

24
48
48
48
24

24
24
24
24
24

24
24
24
24
24

Sm'KARY AND CONCLUSIONS

The five strains of S. pullorum studied g r e w most rapidly in
nutrient broth;

they grew more rapidly in 1 per cent tryptose-water

than in the synthetic medium.
There was no evidence of an initial stationary phase for any
of the strains when they were grown in the three different culture
media.
The lag phase was of about the same duration for all strains,
except 11, when they were grown in 1 per cent tryptose-water.

It

was the same for all strains when they were grown in nutrient broth.
Two of the 5 strains had no lag phase when grown in the synthetic
medium, and the others had one of 6 to 12 hours, varying with the
strain.
The average generation time during the logarithmic phase was
longer when these strains were grown in 1 per cent tryptose-water
than when they were grown in nutrient broth.

As a result, they were

in this phase longer when cultured in 1 per cent tryptose-water than
when cultured in nutrient broth.

The average generation time during

this phase was the longest when they were grown in the synthetic
medium, and thus extended considerably this phase.
The negative acceleration phase was of about the same length
for most of these strains whether they were growing in 1 per cent
tryptose-water or in nutrient broth, but varied from 0 to 12 hours
when they were cultured in the synthetic medium.

66
The m aximum number of viable organisms in the stationary phase
was about the same for all strains whe n they were grown in either 1
per cent tryptose-water or nutrient broth for 36 hours, but it was
a good deal higher when they were grown in the synthetic medium for
50 hours.
Comparable growth curves fo r organisms grown in the same m e d i u m ,
either 1 per cent tryptose-water or nutrient broth, were established
by two different procedures.
The synthetic medium developed for these strains of S. pullorum
was composed of arginine, cystine, histidine, leucine, valine, thiamine
hydrochloride, calcium pantothenate, glucose, salts, asparagine, and
trace elements.
Leucine was found essential for all strains.
About the same concentration of neomycin wa s sufficient to stop
the growth of these strains when they were grown and tested in either
1 per cent tryptose-water or nutrient broth.

Forty-one to 321 tim.es

more neomycin was needed to kill the organisms when they were grown and
tested in the synthetic medium than when they were grown and tested in
either 1 per cent tryptose-water or nutrient broth.
Neomycin was bactericidal in concentrations only slightly higher
than that in which it was bacteriostatic.
The sensitivity of an organism to neomycin was governed by the
medium in which it was tested rather than by that in which it was
grown.
The action of neomycin was favored by an alkaline medium.

67
The antibacterial activity of neomycin was not affected sig­
nificantly by lowering the temperature from 37°C. to 4°C»
Neomycin was just as lethal to these organisms when they were
in the stationary phase or dividing very slowly as when they were in
the logarithmic phase or dividing at a maximum rate.
With the 5 strains of S. pullorum used, it was possible to change
the size of the inoculum one hundred thousand-fold with 3 different
concentrations of neomycin, 4.95, 0.495, and 0.0495 jig./n1., without
influencing significantly its action.
The rate of action of neomycin was most rapid in the highest con­
centration used;
concentration.

it decreased noticeably with each 10-fold decrease in
This condition was true for the different sized inocula.

Arginine, cystine, histidine, leucine, valine, asparagine, calcium
pantothenate, and thiamine hydrochloride,

in the concentrations tested,

did not influence significantly the action of neomycin; glucose, peptone,
and beef extract, in the concentrations tested, seemed to interfere
slightly with the action of neomycin;

sodium chloride, in the concen­

tration tested, definitely affected the action of neomycin.

LITERATURE CITED
Bail, 0, Ergebnisse experimenteller Populationsforschung.
Z.
Immunitats und Exper. Therapie, 60: 1-22, 1929.
Cited by
J. R. Porter, Bacterial Chemistry and Physiology. New York:
John Wiley & Sons.
1946. Pp. 1073.
Barber, k. A.
The rate of multiplication of Bacillus coli at
different temperatures.
J. Infectious Diseases, 5: 379-400,
1908.
Beard, P. J. and Snow, J. E. Antigenic characteristics of related
organisms after cultivation on synthetic medium.
J. Infectious
Diseases, 59: 40-42, 1936.
Chang, Kuan-how.
Studies on the bacteriostatic and bactericidal
action of streptomycin and sulfadiazine on Salmonella pullorum.
Unpublished Doctor*s dissertation. East Tensing: Michigan State
College. 1949. Pp. 48.
_______ , and Stafseth, E. J. Influence of various factors on the
bacteriostatic and bactericidal action of streptomycin on
Salmonella pullorum. Poultry Sci., 29: 130-138, 1950.
Chesney, A. k.
The latent period in the growth of bacteria.
Exp. Led., 24: 387-418, 1916.

J.

Davis, P., and Solowey, k.
The utilization of same organic compounds
by one strain each of Salmonella anaturn. S. oranienburg, and S.
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Demerc, k., and Demerc, R.
Bacterial resistance to neomycin and
Chloromycetin.
Bact. Proc. for 1950, 101.
Dubos, R. J.
The adaptive production of enzymes by bacteria.
Hera*, 4: 1-16, 1940.

Bact.

Duncan, C. C., Clancy, C. F., Walgamot, J. R., and Beidleman, E.
Neomycin: Results of clinical use in 10 cases.
J. Air.. I'ed.
Assoc., 145: 75-80, 1951.
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J. Lab. Clir. led.,
35: 428-433, 1950.

69
Gerrod, L. P. Bactericidal action of streptomycin.
4547: 382-386, 1948.

Brit. Med. J.,

Graham-Smith, G. S.
The behavior of bacteria in fluid cultures as
indicated by daily estimates of the numbers of living organisms.
J. Hyg., 19: 133-204, 1920.
Gray, C. H., and Tatum, B. L. X-ray induced growth factor require­
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Proc. Nat. Acad. Sci., 30: 404-410, 1944.
Gwatkin, R.
Studies in pullorum disease IV.
The effect of bacterio­
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Comp. Med. Vet. Sci., 9: 43-45, 1945.
~
Hajna, A. A.
Decomposition of salts of organic acids by bacteria of
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Hobby, G. L . , Meyer, K., and Chaffee, E.
Observations on the mecha­
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_______ , Lenert, T. F . , and Dougherty, N.
The evaluation of neomycin
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775-781, 1949.
Horowitz, N. H., and Beadle, G. W. A microbiological method for the
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The nutrition of Salmonella.
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Arch. Biochem., 13: 287-

70
Lenert, T. P. and Hobby, G. L. Observations on the action of
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Streptomyces antibiotics. XXIII.
Isolation of neomycin A.
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On the nature of adaptive enzymes.

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Rake, G.
The streptomycins and neomycin in murine
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The animal disease situation in the United States.
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The isolation and
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Twenty-five years of poultry practice.
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J. Am. Vet.

Waisbren, B. A. and Spink, W. W.
Comparative action of aureomycin,
Chloromycetin, neomycin, Q, 19, and polymyxin 3 against gram nega­
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Proc. Soc. Exp. Biol. Med., 74: 35-40, 1950a.
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. A clinical appraisal of neomycin.
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Ann. Internal

Waksman, S. A., Frankel, J., and Graessle, 0 . The in vivo activity of
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J. Bact., 52: 229-237, 1949.

71
Wakaman, S. A., and Lechevalier, H. A. Neomycin, a new antibiotic
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tuberculosis organisms.
Science, 109: 305-307, 1949.
_______ , ________ , and Harris, D. A. Neomycin: production and anti­
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J. Clin. Invest., 28: 934-939, 1949.
_______ , Katz, E . , and Lechevalier, H. A. Antimicrobial properties
of neomycin.
Bact. Proc. 1950, 94-95.
Tfeldin, J. C., and filler, A. R. Utilization of citric acid and
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West, M. G . , Doll, E. R., and Edwards, P. R.
Inhibition of Salmonella
cultures by streptomycin. Proc. Soc., Exp. Biol, lied., 60:
363-364, 1945.
Worth, P. T., Chandler, C. A., and Bliss, E. A.
The antibacterial
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Younie, A. R.
Fowl infection like pullorum disease.
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Can. J. Comp.

ATPETTDIX

TABLE XIII
POPULATION AT 3 HOUR INTERVALS OF 5 STRAINS OF S. PULLORUM
GROWN IN 1 PER CENT TRYPTOSE-WATER*”
Experiment ft 17
Hours

0
3
6
9
12
15
18
21
24

Strains
11

12

13

17

29

68
95
750
3,000
14,000
160,000
1,430,000
**

60
172
1,490
24,300
330,000
3,500,000
28,100,000
**

99
241
2,950
21,000
400,000
4,100,000
35,000,000
**

65
276
2,400
40,000
680,000
2,350,000
44,000,000
**

111
236
1,600
6,900
47,000
160,000
1,440,000
**

68,000,000

138,000,000

186,000,000

278,000,000

102,000,000

Figures represent organisms per milliliter.
*Data derived from an experiment by the single source culture method.
**No count made at this time interval.

TABLE XIV
POPULATION AT 3 HOUR INTERVALS OF 5 STRAINS OF S. PULLORUK
GROV.N IN 1 PER CENT TRYPTOSE-V/ATER*”
Experiirient

Strains

Hours
11
0
3
6
9
12
15
18
21
24
27
30
33
36

20

28
29
264
1,755
7,640
171,000
1,170,000
4,810,000
36,000,000
106,500,000
219,000,000
269,000,000
304,500,000

12

13

17

162
419
4,180
32,900
313,000
3,360,000
27,200,000
**

76
179
1,750
16,250
158,000
1,140,000
19,800,000
**

226
571
8,140
32,500
174,000
1,460,000
13,100,000
**

132,500,000

143,500,000

159,000,000

29
32
102
501
5,000
14,100
101,000
1,030,000
6,400,000
64,000,000
215,500,000
223,000,000
270,000,000
281,500,000

Figures represent organisms per milliliter*
Data for strains 11 and 29 have been added from a separate experiment with them*
♦Data derived from an experiment by the single source culture method.
**No count made at this time.

TABLE XV
POPULATION AT 3 HOUR INTERVALS OF 5 STRAINS OF S. PULLQRUd
GROM IN 1 PER CENT TRYPTOSE-VZATER*”
Experiment # 21
Hours

0
3
6
9
12
15
18
21
24

Strains
11

12

98
482
1,267
14,135
74,700
385,000
5,000,000
19,300,000
71,000,000

92
310
4,095
49,550
800,000
10,200,000
91,000,000
**

171
690
8,300
86,300
850,000
13,800,000
126,000,000
**

81
369
6,140
86,100
1,120,000
17,500,000
163,000,000
**

208,000,000

268,000,000

373,000,000

13

29

17

130
429
1,710
6,050
13,800
99,000
660,000
1,460,000
22,100,000

Figures represent organisms per milliliter.
Data for strains 11 and 29 hare been added from a separate experiment with them,
♦Data derired frcm an experiment by the single source culture method.
♦♦No count made at this time.

TABLE X7I
POPULATION AT 3 HOUR INTERVALS OF 5 STRAINS OF S. PULLORUM
GROWN IN 1 PER CENT TRYPTQSE-WATER*"
Experiment jf 46

Hours

Strains
11

0
3
6
9
12
15
18
21
24
27
30
33
36

175
260
380
2,100
15,000
830,000
3,000,000
30,000,000
110,000,000
220,000,000
230,000,000
227,000,000
**

12
165
440
10,900
121,000
1,190,000
30,300,000
113,000,000
214,000,000
201,000,000
175,000,000
200,000,000
210,000,000
231,000,000

13
223
530
14,400
88,000
1,220,000
22,300,000
178,000,000
174,000,000
185,000,000
190,000,000
220,000,000
256,000,000
230,000,000

Figures represent organisms per milliliter.
* Data derived from an experiment by the tube method.
**Failed to inoculate the tube.

17
129
420
8,300
102,000
990,000
15,400,000
147,000,000
220,000,000
208,000,000
248,000,000
302,000,000
340,000,000
320,000,000

29
115
350
1,830
13,000
42,000
210,000
2,300,000
18,000,000
141,000,000
223,000,000
261,000,000
280,000,000
250,000,000

TABLE XVII
POPULATION AT 3 HOUR INTERVALS OF 5 S m i N S OF S. PULLORUM
GROWN IN NUTRIENT BROTH*
Experiment f 9

Hours
11
0
3
6
9
12
15
18
21
24

101
208
1,337
16,400
161,000
2,860,000
25,350,000
132,000,000
208,000,000

12
40
50
280
60,000
310,000
3,800,000
21,000,000
138,000,000
265,000,000

Strains
13
42
135
1,180
62,000
1,060,000
7,600,000
116,000,000
232,000,000
182,000,000

17
39
120
1,100
43,000
430,000
7,100,000
26,000,000
168,000,000
223,000,000

Figures represent organisms per milliliter.
Data for strains 11 and 29 added from another experiment.
*Data derived from an experiment by the single source culture method.

29
95
619
9,025
210,000
6,105,000
57,500,000
113,000,000
213,500,000
254,000,000

TABLE XVIII
POPULATION AT 3 HOUR INTERVALS OF 5 STRAINS OF S. FULLORTi:
GROViI; IN NUTRIENT BROTH*
Experiment # 13
Hours

Strains
11

0
3
6
9
IE
15
18
21
24

91
258
1,437
17,900
165,000
2,060,000
24,250,000
142,000,000
212,500,000

12
97
465
2,542
77,200
1,940,000
56,800,000
109,000,000
186,000,000
226,500,000

13

17

29

27
157
3,800
136,000
5,100,000
**

37
207
4,000
86,000
2,640,000
**

164,000,000
**

119,000,000
**

281,000,000

216,000,000

110
474
5,090
99,500
1,470,000
91,000,000
207,000,000
190,500,000
280,000,000

Figures represent organisms per milliliter.
Data for strains 13 and 17 added from another experiment.
*Data derived from an experiment by the single source culture method.
**No count made at this time.

TABLE m
POPULATION AT 3 HOUR INTERVALS OF 5 SPRAINS OF S. PULLORUK
GROWN IN NUTRIENT BROTH*
Experiment # 11

Hours

Strains
11

0
3
6
9
12
15
18
21
24

35
33
650
17,000
160,000
2,050,000
26,950,000
142,500,000
196,000,000

12
50
92
2,610
23,000
570,000
6,800,000
39,000,000
122,000,000
203,000,000

13
81
345
8,775
217,500
5,755,000
71,500,000
171,000,000
195,000,000
206,000,000

17
108
517
11,050
384,000
4,305,000
48,150,000
162,000,000
204,000,000
259,000,000

Figures represent organisms per milliliter.
Data for strain 29 added from another experiment.
♦Data derived from an experiment by the single source culture method.

29
105
310
4,652
133,000
1,470,000
88,000,000
196,000,000
205,000,000
277,000,000

TABLE XX
POPULATION AT 3 HOUR INTERVALS OF 5 STRAINS OF S. PULLORUM
GROM IN NUTRIENT BROTH*
Experiment if 45

Hours
11
0
3
6
9
12
15
18
21
24
27
30
33
36

152
290
2,450
20,000
193,000
1,520,000
30,800,000
116,000,000
109,000,000
120,000,000
142,000,000
110,000,000
100,or0,000

12
73
142
5,500
157,000
3,300,000
20,000,000
37,000,000
164,000,000
159,000,000
167,000,000
140,000,000
250,000,000
**

Strains
13
121
340
9,700
408,000
5,200,000
87,000,000
123,000,000
164,000,0o0
193,000,000
165,000,000
150,000,000
240,000,000
340,000,000

Figures represent organisms per milliliter.
*Data derived from an experiment by the tube method.
*vFailed to inoculate the tube.

17
179
320
11,400
226,000
4,000,000
70,000,000
108,000,000
212,000,000
210,000,000
187,000,000
179,000,000
300,000,000
250,000,000

29
105
175
6,400
114,000
1,590,000
46,000,000
138,000,000
127,000,000
189,000,000
141,000,000
121,000,000
130,000,000
230,000,000

TABLE XXI
POPULATION AT 6 HOUR INTERVALS OF 5 STRAINS OF S. HTLLORUiv.
®nv/N IN A SYNTHETIC LFDIUM*
non v Tr •'->
Hour 8

strains

11
0
6
12
18
24
30
36
42
48
54
60

65
492
3,610
17,000
61,000
270,250
1,708,000
15,549,750
103,350,000
320,500,000
**

12
168
650
3,520
22,000
176,000
2,642,250
36,000,000
130,875,000
937,500,000
**
**

13
167
520
3,210
13,300
106,500
680,000
5,812,500
27,600,000
93,000,000
777,000,000
***

17
148
720
2,980
8,200
50,000
332,125
1,137,500
9,040,000
29,737,500
487,000,000
1,635,000,000

Figures represent organisms per milliliter,
*Data derived from an experiment by the single source culture method,
**No count made at this time.

29
189
812
9,475
58,475
645,000
9,600,000
140,000,000
692,000,000
1,735,000,000
**
**

TABLE XXII
POPULATION AT 6 HOUR INTERVALS OF 5 STRAINS OF S. POLLORDK
GROVJN IN A SYNTHETIC LEDIM:*
Experiment § 78a
Hours

Strains
11

0
6
12
18
24
30
36
42
48
54
60

31
181
1,180
4,850
38,100
253,500
2,311,250
16,660,000
119,500,000
384,000,000
585,000,000

12
98
378
2,750
19,550
214,900
5,080,000
93,500,000
558,000,000
1,062,000,000
1,085,000,000
407,000,000

13
92
503
3,680
22,450
174,875
1,570,000
17,550,000
87,200,000
856,000,000
1,390,000,000
691,500,000

17
78
179
1,055
3,900
29,150
165,000
845,000
7,800,000
36,500,000
180,000,000
962,500,000

Figures represent organisms per milliliter.
*Data derived from an experiment by the single source culture method.

29
85
473
4,750
43,000
369,750
3,345,000
45,500,000
395,000,000
1,785,000,000
1,330,000,000
838,500,000

TABLE m i l
POPULATION AT 6 HOUR INTERVALS OF 5 S1RAINS OF S. PULLCRUM
GROWN IN A SYNTHETIC MEDIUM*
Experiment # 78b
Strains

Hours
11
0
6
12
18
24
30
36
42
48
54
60

30
230
1,400
3,210
33,000
244,000
2,250,000
16,150,000
120,000,000
320,000,000
640,000,000

12
123
437
2,035
17,550
145,500
1,150,000
31,500,000
170,000,000
908,000,000
775,000,000
1,211,000,000

13
118
512
2,910
15,850
77,700
700,000
2,800,000
20,000,000
93,000,000
550,000,000
1,083,000,000

17
93
440
2,240
8,475
53,000
168,600
1,030,000
10,700,000
77,000,000
382,000,000
1,100,000,000

Figures represent organisms per milliliter.
*Data derived from an experiment by the single source culture method.

29
103
329
4,000
41,700
386,500
2,250,000
40,000,000
230,000,000
1,113,000,000
535,000,000
1,116,000,000

84
TABLE XXIV
EFFECT OF SIZE OF INOCULUM OK ACTION OF NEOMYCIN
A. Strain 11

pg./ml.
Time neomycin

Tube
1

2

3

4

5

6

0*
0*
0
0
0

0.0495
0.0
0.495
4.95
0.0

11,000,000
13,800,000
22,000,000
10,400,000
22,000,000

1,210,000
1,010,000
2,510,000
910,000
2,340,000

78,000
110,000
254,000
133,000
196,000

12,300
10,800
15,800
4,300
25,800

1,120
1,450
1,690
1,000
2,020

110
70
250
40
220

30*
30*
30
30
30

0.0495
0.0
0.495
4.95
0.0

8,300,000
13,500,000
1,872,000
13,650
22,900,000

1,060,000
2,000,000
416,000
740
2,390,000

132,000
130,000
65,000
220
227,000

10,700
12,500
2,180
10
22,800

1,270
1,060
380
<10
1,850

60
80
20
<10
270

60*
60*
60
60
60

0.0495
0.0
0.495
4.95
0.0

4,300,000
19,400,000
107,250
110
19,900,000

310,000
1,970,000
19,500
<10
2,630,000

53,000
184,000
4,260
<10
210,000

6,000
13,700
20
<10
17,200

580
910
<10
<10
2,280

10
130
<10
<10
180

120*
120*
120
120
120

0.0495
0.0
0.495
4.95
0.0

767,000
21,000,000
7,800
<10
54,000,000

28,000
2,880,000
600
<10
7,100,000

7,400
630,000
80
<10
640,000

190
22,000
<10
<10
37,000

90
2,410
<10
<10
4,100

<10
170
<10
<10
390

240*
240*
240
240
240

0.0495
0.0
0.495
4.95
0.0

1,000
38,600
45,000,000 9,900,000
<10
180
<10
<10
87,000,000 21,200,000

250
2,300,000
<10
<10
2,560,000

<10
106,000
<10
<10
166,000

<10
11,800
<L0
<10
25,000

<10
720
<10
<10
1,290

Figures under time represent minutes; figures under tube repre­
sent organisms per nl.
*Data taken from another experiment.

85
TABU! XXIV (continued)

EFFECT OF SIZE OF INOCULUM OK ACTION OF NEOMYCIN
B. Strain 12

Time

jig ./ml.
neomycin

Tube
1

2

3

4

5

6

0
0
0
0*
0*

0.0495
4.95
0.0
0.495
0.0

22,100,000
5,600,000
21,100,000
19,266,666
20,933,333

2,960,000
1,290,000
2,440,000
2,455,000
2,436,666

242,000
88,000
222,000
195,833
206,333

13,800
2,300
15,100
16,083
17,766

2,260
450
2,280
1,132
1,930

270
70
240
120
245

30
30
30
30*
30*

0.0495
4.95
0.0
0.495
0.0

18,450,000
60
27,200,000
85,000
27,366,666

1,430,000
<10
2,760,000
11,491
3,143,333

148,000
<10
283,000
2,150
238,333

13,650
<10
23,000
95
18,633

2,390
<10
2,880
17
2,490

300
<10
260
<10
240

60
0.0495
60
4.95
60
0.0
60* 0.495
60’’' 0.0

1,105,000
120
26,700,000
225
26,333,333

260,000
<10
2,680,000
138
3,106,000

180,700
<10
237,000
50
285,000

5,910
<10
27,800
<10
25,766

1,240
<10
2,570
<10
3,530

50
<10
290
<10
205

120
120
120
120*
120*

0.0495
4.95
0.0
0.495
0.0

20,800
<10
71,000,000
82
63,500,000

6,500
<10
8,800,000
12
6,150,000

1,300
<10
910,000
<10
795,000

230
<10
59,000
<10
38,500

280
<10
8,000
<10
6,050

<10
<10
460
<10
400

240
240
240
240*
240*

10
740
0.0495
<10
<10
4.95
146,000,000 47,000,000
0.0
<10
<10
0.495
33,000,000
345,000,000
0.0

70
<10
5,020,000
<10
4,650,000

<10
<10
198,000
<10
219,500

<10
<L0
37,200
<10
31,500

<10
<10
2,560
<10
2,700

*Data taken from the average of 3 individual experiments.

86
TABLE XXIV (continued)
EFFECT OF SILE OF INOCULUM CN ACTION OF NEOMYCIN
C. Strain 13

Time

jxg./ml.
neomycin
1

2

Tube
3

4

5

6

0
0
0
0

0*0495
0.495
4*95
0.0

48,100,000
36,300,000
31,100,000
38,000,000

2,960,000
2,120,000
1,160,000
4,100,000

332,000
223,000
105,000
325,000

25,100
17,800
8,000
30,100

1,720
980
660
2,410

230
110
40
270

30
30
30
30

0.0495
0.495
4.95
0.0

17,200,000
931,600
730
31,500,000

2,910,000
3,800
20
4,920,000

327,000
880
30
382,000

13,500
50
<10
43,800

1,600
10
<10
2,900

60
<10
<10
150

60
60
60
60

0.0495
0.495
4.95
0.0

3,240,000
2,500
250
44,500,000

366,000
540
10
5,100,000

74,000
80
<10
427,000

3,700
<10
<10
42,800

550
<10
<10
2,400

60
<10
<10
170

120
120
120
120

0.0495
0.495
4.95
0.0

149,500
210
<10
87,000,000

77,000
50
<10
7,600,000

24,100
<10
<10
1,500,000

110
<10
<10
90,000

210
<10
<10
2,100

30
<10
<10
220

240
240
240
240

0.0495
0.495
4.95
0.0

4,740
1,920
20
20
<10
<10
92,000,000 14,900,000

1,640
<10
<10
2,360,000

10
<10
<10
502,000

<10
<10
<10
25,000

<10
<10
<10
210

TABLE XXIV (continued)
EFFECT OF SINE OF IKOCULUk ON ACTION OF NEOMYCIN
D. Strain 17

yg./ml.

Time

0

neomyc in _______________________________Tube______________
1
2
3
4
5

0
0

0.0495
0.495
4.95
0.0

34.600.000
34.900.000
26,000,000
34.800.000

3.620.000
3.760.000
2.400.000
3.520.000

342.000
364.000
301.000
355.000

28,700
17,400
12,200
28,500

4,140
2,840
1,000
3,800

30
30
30
30

0.0495
0.495
4.95
0.0

32.600.000
1,755,000
8,450
44.500.000

3.520.000
13,000
8,760
3.620.000

338.000
22,750
370
323.000

21,400
1,350
<10
32,600

2,720
90
<10
3,480

60
60
60
60

0.0495
0.495
4.95
0.0

17,000,000
33,150
200
45,200,000

1.300.000
3,240
1,530
4.180.000

227.000
1,350
<10
516.000

8,100
60
<10
32,000

1,880
<10
<10
3,880

120
120
120
120

0.0495
1,625,000
430
0.495
<10
4.95
109,000,000
0.0

150,000
<10
<10
8,500,000

61,600
30
<10
2,640,000

650
< 10
<10
67,000

390
<10
<1 0
8,600

240
240
240
240

7,540
16,900
0.0495
<10
160
0.495
<10
<10
4.95
261,000,000 46,800,000
0.0

3,940
<10
<10
5,490,000

60
<10
<10
359,000

160
<1 0
<10
39,500

0

88

TABLE XXIV (continued)
EFFECT OF SIZE OF IKOCULUK ON ACTION OF NEOI..YCIN
E. Strain 29

Time

pig./ml.
neomycin

Tube
1

2

3

4

5

6

0*
0*
0
0
0

0.0495
0.0
0.495
4.95
0.0

17,200,000
18,300,000
33,800,000
15,300,000
32,700,000

2,910,000
2,840,000
3,010,000
1,140,000
2,770,000

185,000
201,000
362,000
322,000
275,000

20,400
22,900
28,100
1,100
25,100

1,710
2,150
2,830
1,200
3,020

100
250
300
40
220

30*
30*
30
30
30

0.0495
0.0
0.495
4.95
0.0

18,600,000
22,800,000
4,797,000
6,630
31,900,000

2,380,000
2,480,000
565,500
1,180
2,930,000

215,000
198,000
80,000
130
256,000

18,900
20,300
3,620
370
30,000

1,920
2,440
940
<10
2,390

120
220
60
<10
180

60*
60*
60
60
60

0.0495
0.0
0.495
4.95
0.0

2,800,000
25,400,000
35,750
200
44,900,000

241,000
3,620,000
10,400
10
3,580,000

75,000
351,000
3,570
<10
321,000

4,000
28,600
160
<10
31,200

920
3,130
30
<10
3,430

40
240
<10
<10
390

120*
120*
120
120
120

0.0495
0.0
0.495
4.95
0.0

41,700
60,000,000
720
10
85,000,000

9,900
6,700,000
70
50
4,200,000

4,500
640,000
<10
<10
1,310,000

30
45,600
<10
<10
40,000

30
7,300
<10
<10
9,000

<10
400
<10
<10
430

240*
240*
240
240
240

900
3,700
0.0495
149,000,000 13,200,000
0.0
20
100
0.495
<10
<10
4.95
172,000,000 24,000,000
0.0

75
1,880,000
<10
<10
3,970,000

<10
147,000
<10
<10
198,000

<10
22,700
<10
<10
31,400

<10
1,080
<10
<10
1,790

*Data taken from another experiment.

SOURCE OF A?UNO ACIDS AND OTHER ORGANIC NUTRIENTS

Biotin was purchased from Hoffmann-La Roche Inc., Nutley, New
Jersey.
Pimelic acid was purchased from Eastman Kodak Co., Rochester,
New York.
Alpha alanine, aspartic acid, choline, cystine, folic acid, lysine,
glycine, histidine,

inositol, leucine, methionine, nicotinamide, nucleic

acid, pyridoxine, pantothenate, para-aminobenzoic acid, phenylalanine,
riboflavin, thiamine, tryptophane, tyrosine, and valine were purchased
from General Biochemicals, Inc., Laboratory Park, Chagrin Falls, Ohio.
Arginine, glutamic acid, proline, serine, and threonine were
purchased from Nutritional Biochemicals Corporation, Cleveland, Ohio.