TH! INFIJJENCE OF SEVERAL FACTORS
ON THE ACTION OF PENICILLIN AGAINST
SOME MEMBERS OF THE ENTEROBACTERIACEAE

Thai: for the Dogm of Ph. D.
MICHIGAN STATE UNIVERSITY
Dale Emil Bord?

5 ' 1955

 

 

This is to certify that the

thesis entitled

The Influence of Several Factors
on the Action of Penicillin Agains

Some i-fenifiers of the Enterohacteriacede.
presented In]

Dale Pordt

has been accepted towards fulfillment
of the requirements for

__Eh_11._ degree in W10 gy

\v l\:\vl l .:\ .\ . x x‘ 1K"\ \\ \

Iajor \professor

 

 

TEE INFLUHCE OF SEVERAL FACTORS ON THE ACTION
OF PENICILLIN AGAINST SOME MEMBERS OF
THE ENTEROBACTERIACEAE

3:
Belle mil Bordt

AN ABSTRACT

Submitted to the School of Graduate Studies of Michigan
Stete University or‘Agriculture end Applied Science
in partial fulfillment of the requirements
for the degree or
DOCTOR OF PHILOSOPHY

Depertnent or Iiercbiclcgy and Public Health
1955 '

Anya-Wed __ bWMmmA/Qg

1 w

 

3. Dale mu Bordt

AN ABSTRACT

Yericus teeters were studied to determine their in-
fluence on penicillin ection against selected members of
m W.

”The medium used was the synthetic medium of Young,
legs end Pent: (1944) Iith lactose as the sole carbon source.
In this indium soo units of penicillin per ml were found to
be bectericidel to W 2.9—1.6 Penicillin was most
,eetive‘ly beeterioidel when exposure took. place after ebout
six hours et 3'? 0, which corresponds to the length of lag
of L 933.1 in this medium.

Dilution see not an effective means for reduction of
penicillin there recovery of previously exposed organisms was
desired. hnicillinase was found to destroy penicillin
effectively and to allow recovery of previously exposed cells.
A euch higher concentration of the enzyme ves required for
recovery of cells exposed to penicillin then nee necessery to
inactivate the same amount of penicillin under identical con-
ditions of time and temperature.

W W was found to survive exposure to 200
units of penicillin per ml, provided a sufficient preincubation

tee enployed before exposure. This was evidently due to the

2 Dale m1 Bordt

fact that penicillin is active only against growing cells.
The addition of approximately one percent sterile fecal
material resulted in a bactericidal action of penicillin
08010“ E; m. ‘l'his was presumably due to the fact
that the fecal material contributed growth substances to the
nediun, and thus established the penicillin effect.

Sodium arsenite in proper concentration was shown to be
becterioetatic against members of the genus W, but
not against other gjggobgcteziaceae. Sodium arsenite in a
0.003 percent concentration in this synthetic medium pro-
tected certain selmonellee from penicillin activity even in
the presence of one percent fecal material.

The presence of two percent tryptose and 0.02 percent
cysteine in the plating medium materially enhanced the re-
covery of fl... w from exposure to penicillin.

m addition of 0.5 percent lactose and 0.005 percent
triphenyltetragsoliun chloride to the plating medium resulted
in colonies of diagnostic significance. Lactose non-ferment-
ing organisms reduced the tetrazolium salt and formed dark
red colonies, while those producing acid from lactose in
general failed to reduce the salt and remained colorless or
only faint pink.

A bisphenol , 2 , 2 'methylenebisoe-chloro-0~is opropylphenol ,
added to a level of 0.001 percent in the plating medium

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5 Dale Emil Bordt

effectively inhibited certain gram positive cocci which sur-
vived penicillin exposure.

A series of human fecal specimens was examined to deter-
mine whether this penicillin enrichment technique could be
used for the isolation of salmonellae from.feces. The results
of this limited series of examinations compare favorably with
those obtained with several media presently used for routine
diagnostic work.

Among the limitations of the method is the fact that
certain organisms other than the salmonellae do survive ex-
posure to penicillin and, when present in large numbers, pro-
duce overcrowded plates making isolation of typical colonies
difficult. Organisms involved in this type of interference are
(l) pseudo-chads, (2) nutritionally deficient coliforms, (3)
lactose non~fermenting or slow fermenting coliforms. Other.
limitations are the inability to recover quantitatively the
salmonellae, and the relatively small inoculum.necessary.

The method, with some modifications, could also be very
convenient for isolation of naturally occurring biochemically

deficient forms of bacteria.

loung, E.G., R.W. Begg and E.I. Pants. Inorganic nutrient

requirements of‘ggghgzighigwpgli. Arch. Biochem. 5:121-
130. 1944.

THE INFLUENCE OF SEVERAL FACTORS ON THE ACTION
Oi" PENICILLIN AGAIIS! SOME MEMBERS OF
THE ENTEROBAOTERIACEAE

By
Dale mi Bordt

A THESIS

Submitted to the School of Graduate Studies of Inchigen
State university of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of lflcrobiclogy and Public Health
1966

AOKNOWLEDGMENTB

The author wishes to express his appreciation for the
able guidance and advice given to him throughout his entire
graduate career by Dr. LL. Mallmann, Professor of Bacter-
iclogy and Public Health.

Sincere thanks to Ir. HJ. Cope and Miss Margaret
Murphy of the Michigan Department of Health at Lansing for
linking available the fecal specimens used in this study.

Thanks also to Pr. William Ferguson and 1m. William
Geldwell of the Salmonella Typing Station of the Michigan
Department of Health for generously agreeing to type sero-
logically the salmonellae isolated in this investigation.

In.

TABLE OF OONTH‘ITS

INTEODUCTION .00...COOOQOQO0......OQOCOOOOCOOOOO00....0..
PURPOSE OF THE STUDY .o...’...............................
THEORETICAL CONSIDERATIONS ..............................

DETERMNATION OF TIE EFFECTS OF '
SEVERAL FACTORS 0N PENICILLIN ACTION eeeeeeeeeeeeeeeee.

DEVELOPMENT OF A DIFFERE‘ITIAL PLATING MEDIUM FOR
DISTINGUISHING THE SALMONELLAE FROM COLIFORM BACTERIA"

use or TEE PENICILLIN micm'r TECHNIQUE FOR THE

I$OLATION OF SALMONELLA! FROM NATURALLY INFECTED
m PEGAL SPECIMENS .0.0...OOOOOOOOOOOOOOOOOOOOOOO0..

SMARY eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

p

15

'72

86
97

CONCLUSIONS eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 101
“TRIALS USED eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 1-03
LITmTURE CITED deeeeeeeeeeeeoeeeeeeeeeeeeeeeeeeeeeeeeee 104

eeoeel'ee'oeesee00ca..-.-eoo '

eoeedoeceeeeeedeelc '

...‘....ge"’ . »_ .

eeeeeeeeeeeoewevooeeeevoeseesee.

e.assesses-sosees-eetseeeese ' -'

Ole-sewersono-Onovfieofloo'- -

LIST 01' TABLES

The effect of varying the lactose concen-
tration on the initial growth of L 39;;
in‘IBPIn.d1nnoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

The residual bactericidal effect of pani-
eillin on Q‘ W after reduction of
' P.310illin concentration by dilution..ooo.....

The potency of Bacte-penase Concentrate

assayed against penicillin G potassium........ -.

Determination of the amount of penase
necessary for quantitative recovery of
% P209 which has been exposed to
0 uni s of penicillin per ml in 0.85
DOPOGIO 3‘11n.eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee

The amount cf penase required for a quan-
titative recovery of s o from 10
minutes exposure to 355' un ts o peni-
cillin per ml in 0.85 percent saline..........

The bactericidal effect of 800 units of
penicillin per ml on 003.1 in IE? me-
dium with 0.25 percen lactose at 37 0........

The efficiency of hill of 393,; by 300

units of penicillin per as influenced

by length of pre-incubati on and penicillin
exposure time in YBP medium containing

0.35 PQIOODt lao‘oseeeeeeeeeeeeeeeeeeeeeeeeeee

The influence of pre-incubation time on the
hill of fizggl; by 300 units of pmicillin
per ml in medium containing 0.25 per0

cent IQOOOICeeeeeeeeeeeeeeeeeeeeeseeeeeeeeeeee

nae effect of exposure time on the kill of

L, .9211. by 800 units of penicillin per ml
after a six hour pre-incubation in IBP me-
dium containing 0.85 percent lactose..........

Page

17

21

87

29

51

33

35

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10 The recovery of §_., 2%20f3! from exposure
to 800 units of pen ci in per ml at 3'7 0

in YEP medium containing 0.25 percent lac-

tOCCQeeeeeeeeeeeeeeeeeeeseseeseeeeeeseeseeeeee 40

ll Recovery of §_._ gallery? from exposure to
300 units Of P31110111 I after a prelimi-

nary 18-hour incubation in 0.85 percent
Bfilin. It 57 Cseeeeeeeeeeeeseeeeeeeeeeeeeeeeee ‘2

12 The effect of one parent sterile faces
on the survival of L 2233.923? after ex-
posure to 200 units of penici in per ml
1‘»YEP3-‘diumeesseeeeeeseeeeeeeeeeeessssoseese 45

13 The selectively inhibitory preperty of
sodium arsenate and sodium arsenite on

f; sell "mtg. new in untou-
a as

otOlO bro OOOOOOOOeeeeeeeeeeeeeeeeeeeeeee 48

la The effect of various concentrations of sodium
arsenite on viability and growth of L 2211,
in tryptose-lactose broth at 37 0............. 50

lit The effect of various concentrations of
sodium arsenite on viability and growth

of ESQ-$2M in tryptcse-laotose broth

.33? OOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 51
15 The effect of 0.01 percent sodium srsenite

on the growth of several members of the

We 1n tryptose-laotosc
30thesseseeeeeseeeeeeeeeseeeeeeeeeeeeeeseeeee 53
la The effect of 0.01 percent sodium arsenite

on penicillin action against _§,_ {#133133

and 1., 2934. in tryptose-lactose oth......... 55
17 The effect of various concentrations of

.scdium arsenite on growth and survival of

fig. W in YEP medium plus 0.25 par- -
OOBt 1‘°t°.eeOeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee 57

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18 The influence of 0.002 percent sodium ar-
senite on recovery of §,. pullogum from
exposure to 200 units of penicillin per
ml in IBP medium with 0.25 percent lac-
toss and approximatelylm percent sterile

rm.OOOOOOOOQOOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOO 60

19 The effect of two concentrations of sodium
arsenite on the growth of g... coli in YEP ’
medium plus 0.25 percent lactose.............. 63

80 The effect of varying penase concentrations
and incubation times on recovery of §_,_ 231-
from exposure to 1,000 units of penio
c n contained in five ml of YBP medium..... 64

21 The influence of the nature of the plating
medium on the recovery of S pugcgum '
from penicillin exposure...................... 69

23 The recovery of several Salmonella species
from exposure to penicim medium..... 71

23 The effect of tryptose concentration in the

plating medium on recovery of S “13%;," cm

from exposure to penicillin in'YB dium um..... 76
24 The effect of cysteine and sodium thic-

glyccllate on the recovery of §_,. W
from.pon101111n exposure...................... 80

35 The development of W in the
' presence of sodium auryl sulfate and

3975‘3eeeseeeeseeeeeeeeeeeeeeseeseeeeeeeeeeese 8‘

86 Results of the examination of fecal speci-
mens for salmonellae by the penicillin en-
richment technique and by four currently
OIfllOYOQ diagnostic media..................... 89

8'7 mm: reactions of ccliform strains which
dc not grow in YBP medium plus lactose
and which survived penicillin exposure in
thifl ‘Ddiumeeeeeeseeeeseeeeeeeeeeseeeeeeeeesso 95

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INTRODUCTION

flaming (1929) made the historic observation that a
filtrate of a figpicilligg culture showed a remrkable se-
lective action against the gram positive bacteria in general,
and a considerably lesser action against most gram negative
organisms. He was the first to take practical advantage
of this fact by using penicillin in an agar medium to facil-
itate the isolation of W W ( a gram negative
organism) from nasal washings which also contained. an abun-
dance of gram positive microorganisms.

lucLaan (1937) also applied this same principle with
marked success for the isolation of Hemophilgs pggtussis
from whooping cough cases.

Pissi (1945) reported the use of penicillin in a me-
dium for the cultivation of Trjpanosoma org; . The peni-
cillin is nontoxic to the protozoan while inhibiting the
growth of bacteria.

Burnet, Stone, and Anderson (1946) made use of a com-
bination of sulfonamide and penicillin for the isolation
of the virus of influenza B in chick embryos during an
epidemic of influenza in Australia. The penicillin and
sulfcnamide were effective in inhibiting growth of bacteria

in the chick amnion, and sufficiently nontoxic to the
embryo and the virus to allow invasion by and development
of the virus to take place.-

Hawking (1946) used ten units of penicillin per ml in
a tissue culture of chicken macrophage reticulc-endcthelial
sells for the isolation and growth of nasmodigg gelictggp.

Lacy (1946) utilized the selective action of penicillin
for the isolation of Egggella apogtgg from.milk.

llcrin and Turcctte (1946) used penicillin for the bio-
logical “purification" of vaccine emulsions. These authors
point out that 50 units of penicillin per ml of vaccine pre-
vented bacterial infection by vaccination without adversely
affecting the immunological potency of the vaccinia virus.

Dishes (1947) reported the use of penicillin for the
isolation of pleuropneumcnia-like organisms frombpathc-
logical specimens.

Bowitt and Barnett (1948), working with poliomyelitis
virus, report that the use of 1,000 units of penicillin and
20 mg of streptomycin per md.of fecal suspension.made intra-
abdcmdnal inoculation of monkeys possible without preventing
paralysis. "

Framer, flouhannes and chotzkie (1950) developed a
method for the quantitative recovery of mycobasteria from
sewage. They used Dubcs' medium plus five units of peni-

cillin and 50 units of grisein per ml. They also pre-
heated the sewage sample at 50 0 for 60 minutes. This_
treatment was reported to suppress sv-oe percent of sewage
organisms with.no loss of‘lyggpgggggigg,tubgggglgsis.

Iheae papers are cited to give examples of the wide
use of penicillin in the isolation or biological "puri-
fication' of selected organisms. They are by_no means
the only cases reported of the use of penicillin for this
purpose. Penicillin and other antibiotics are used rou-
tinely in virus laboratories as a practical means of pre-
venting bacterial infection of laboratory animals or chick
embryos by bacterial contaminants in virus preparations.

In all of the mentioned procedures advantage is taken
of the natural ability of certain organisms (such as viruses,
protozoa and most gram negative bacteria) to resist the
lethal action of a given concentration of penicillin. Under
the same conditions, contamunents are killed or inhibited
due to their natural penicillin sensitivity.

Hobby, lbyer, and Ohaffes (l942), studing the mechanism
of action of penicillin on pneumocoeci, homelytic strep?
tocoeoi and staphylococci, concluded that penicillin is
bactericidal under certain conditions. They found the rate
.of killing by penicillin to vary with different organisms,
and also that penicillin appears to be effective only when

(I

 

active multiplication of the exposed organisms takes place.

roster and Iilker (1945), in a study of penicillin
effects 03.W W mm, found that
survival from.penicillin exposure was not due primarily to
highly resistant fipersisters". 0n the contrary, cultures
from.surviving cells were found to have survival curves
identical with the parent culture. They conclude that peni-
cillin depends for its activity on the logarithmic prolong-
ation of generation time. This suggests also that the sur-
vival was due to some special metabolic state of the cell
at the time of exposure.

Bigger (19dd) lent additional support to the hypothesis
that only growing, or at least actively metabolizing cells
are susceptible to the lethal action of penicillin. He
found that by using a dilute solution of boric acid as a
bacteriostat fcr‘lgnggggus, the action of penicillin could
be inhibited.

lobby and Dawson (1944e,b) worked along similar lines.
they showed that an increase in growth rate of hemolytic
streptococci caused an increased rate of kill by penicillin.
conditions which decreased growth rate also decreased the
rate at which penicillin acted. In addition, these workers
observed that sulfadiasine, which inhibits growth, also,
inhibits penicillin action. It was also found that at d 0,

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penicillin action, as well as growth, was stopped or
greatly retarded.

Lee, Ioley, and Epstein (1944), working with Staphylo-
. m 9.9.33.9. m, came to the following conclusions:

.1. on. rate of kill by penicillin is greater in a
medium.where normally the growth rate is greater.

2. Physiologically young organisms are killed quicker
than old ones. This was believed to be due to the shorter
lag.involved.

.Ihsse workers also_showed that greatly increasing the
concentration of penicillin did not appreciably shorten the
time required to kill all the organisms in a suspension.
They showed a linear relationship during logarithmic growth
between the logarithm.of the viable population found in the
control tubes and the logarithm.of the number of viable cr-
’ ganisms in the presence of 1.5 units per ml of penicillin.
(Their final conclusion also is that actual growth of the
organism.seems to be necessary for penicillin to kill.

Rants and Kirby (1944), in their, in 2.1.22.9. studies on
the action of penicillin on staphylococci, observed that,
in order to establish the penicillin effect, the organisms
apparently must divide. They also confirm.the observation
_by roster and lilker (1943) that the bacteria remaining

viable after exposure to penicillin are in general as sen-

sitive to penicillin action as the parent strain. fhese
workers interpret this to mean that survival is not due to
an artificially induced penicillin resistance, but it is
due merely to the fact that the surviving organisms were
not in a metabolic state suitable for ditision to take place.

Schwartnman (1944), in studying the affect of peni-
cillin on Esghggigahig mi, showed that methionine enhances
(the action of penicillin; and he also states that substances
inhibitory to growth interfere with penicillin action.

chain and Duthie (1945) came to the conclusion that
penicillin acts as a bactericstatic or bactericidal agent
.depending on concentration._ These effects_occur only under
,conditions that will allow for bacterial growth in the absence
of penicillin. In a medium where growth is nutritionally
impossible, penicillin is ineffective. They also observed
that bacteriostatio concentrations of helvolic acid inter-
fare with the action of penicillin on.lL_gQ:§ng,

0n the other hand, Traffers (1946) found that inhibi-
tcry agents like iodcaoetic acid,sodium.azide, gentian
violet and.merthiolatc potentiate the inhibitory action of
penicillin. He postulates this potentiating effect to be
due to these agents affecting a part or all of a common
enzyme.syatem.

Dufrenouy, Strait, and Pratt (1947) studied the effect

of penicillin on 5,, col}, flgteus W, gt serene and
Egagllgg,§ngjlllg, is a result of these studies they came

to the conclusion that penicillin affects both gram.negative
and grem.positive organisms in essentially the some manner.
the threshold concentration necessary for lethal action
appears to be the only major difference between the two
groups.

Krampits and‘werkman (1947) showed that penicillin had
no deleterious effect on the metabolism of glucose by 3,, am,
“and also that it had no affection the enzymes concerned with
the intermediate products of carbohydrate breakdown. In a
synthetic medium, in the absence of penicillin, a gradual
acceleration of oxygen uptake would occur after about five
hours. In the presence of penicillin this acceleration of
oxygen uptake did not occur. Control experiments showed the
same accelerated uptake to occur with cells in a buffer sol-
ntion without carbohydrate substrate. The substances being
oxidised were evidently constitutents of the cells. During
the course of rapid endogenous activity, pentose disappeared.
The presence of penicillin inhibits this disappearance. these
(workers_ere of the opinion that the source of pentcse is
the ribose of ribose nucleic acid. .rhsy conclude that in
some manner, penicillin interferes withf”t"he dissimilation

of ribose nucleic acid and consequently with its assimilation

 

 

 

 

 

'4

 

 

during growth.

lagle and lhsselman (1949) made the observation.that
beta hemolytic streptococci, when exposed to penicillin in
e mediun.favorable for growth, did not resume growth.immed-
,iately after addition of the penicillin destroying enzyme
penase, but the organisms showed a slow recovery time. This
time of recovery was directly related to the time of ex-
posure._ They also noted that large doses. up to 10,000
times necessary for inhibition, did not further accelerate
bacterial death. On the contrary, in some cases such large
doses even retarded lethal action. These workers also ob-
served that when cells were exposed to penicillin in a me-
diam.unfavcrable for growth and were then placed in‘a favor»
able medium. they did not require the recovery period neces-
sary fcr cells which had been exposed in a favorable me-
dium. Their conclusions, drawn about the action of penicillin,
are as follows: "The Bactericidal action of penicillin may
thus depend on the continuation, in a favorable medium, of
certain.metabolic activities in the cells in which the pro-
cesses which normally lead to growth and cell division have
been interrupted by penicillin." This is in agreement with
the conclusions of Krampits and lerkman (1947) previously
cited. It was also noted that cells thus altered by peni-

cillin exposure in a medium favorable for growth are able

ms

-
.
I‘
s
.e.
. .
.
5s .. A
..
s r
o
t
e .
L
l
.w A
.
.
. e
o
O
.
. e
e .
t
‘e
..

 

to recover from this exposure only in.a msdium.favorable
for growth.

stis_(l948). and Lederberg and Zinder (1948) simul-
taneously reported the possibility of using penicillin for
the isolation of biochemically deficient mutants of bac-
teria. These were the first two deliberate and practical
applications of the previously discussed phenomenon that
penicillin kills bacteria only when in a medium.favorable
for growth.

The technique of Davis (1948), using gh,gg;;, was in
general the following: A suspension.of ghpggli,was ir-
radiated with ultra-violet light to induce mutation. The
cells were then incubated in a complete mediun.to allow the
surviving ones to multiply. The sells were then washed
with.saline and placed in a minimal medium in the absence
of tryptophane. After that they were exposed to 300 units
of penicillin.per:sl for 84 hours. The cells were removed
and plated on a complete medium. A high proportion of the
colonies which then developed were found to be made up_of
sells requiring tryptophane for growth. These mutants,
when exposed to penicillin in a complete medium, show no
increased natural resistance to the antibiotic. It is
apparent, then, that the surviving cells resisted the peni-

cillin exposure because they were exposed in a meter

lO

bclically inactive state. This inactive state of the cells
was due to the inability of the deficient mmdium.to support
their growth.

aincc these original observations, many other workers
have also used this approach for the isolation of bacterial

mutants.

11

.PUIPOSI or THE STUDY

ihe apparent success of the 'penicillin enrichment tech-
nique? prompted the present investigation. It was considered
worthewhile to investigate the possibility of using this
unique action of penicillin for the selection of bacteria frml
maxed populations containing closely related types.

l_classical example of this type of situation_involves
the isolation of members of the genus lemgggl g from.fecal
material containing large numbers of other members of the
Inlgzghsgfigzlgggsgl Iith this object in mind, the influence
of several factors on the penicillin susceptibility of coli-
form.bacteria and various members of the genus figlmonellg
was chosen as the primary object of this study.

the main portion of this work will be concerned with
the investigation of the effects of various conditions in a
medium.which.modify the action of penicillin on representse
tive members of the coliform.group and the genus §glmonella.

The two organisms chosen for the initial portion of the
study were;

(1) A typical strain of !h_ggli freshly isolated from
human.feces.

(2) s strain of Salmgnella pullggug freshly isolated

 

13

free chicken feces.

fih,pgglogum.was chosen to represent the genusIfigi;
plgngllg,because it is one of the least resistant of the
salmonellae to the action of penicillin. Thomas and
Levine (1945) shoved that as little as 10 units per ml
inhibited growth in beef extract broth. Also, §_‘ pullorum
is considered not to be highly pathogenic for man, which
minimises the danger of accidental infection while counting

colonies on plates, etc. in the laboratory.

13

IEEORETIGAL.CONSIDERATIONS

decording to the studies on the mode of action of penie
cillin previously cited, the following conditions must be
met in order to obtain the desired selective hill of the
colifcrns: ‘

1. Conditions must be formulated which are favorable
for the growth of the coliforms. Ibo presence of peni‘
.cillin in sufficient concentration should then be bacteri-
cidal to this group.

2. These conditions must at the same time be such
that members of the genus leggngllg, which may be present,
will not be able to initiate growth; but will, nevertheless,
remain viable in a metabolically inactive state.

In order to attain such conditions, physiological dire .
ferences between the two groups of organisms were considered.
the outstanding difference appears to be the ability of the
coliforns to utilise lactose as a source of carbon in an
otherwise inorganic medium, and the inability of members of
the genus W to do so. ..

On this basis, then, a synthetic medium with lactose as

the sole carbon source was considered adequate to fulfill

I.

14

the necessary requirements. A medium.0f this type is that
described by Ybung, Begg, and Pentz (1944).

In an intensive study of the nutrient requirements at
linggli, these authors found the optimum.inorganic require-
ments to be the following:

seal ............................... 0.5%
(N34)330‘ .......................... 0.5%
_rrzrt, ............................. 0.2%
legato. ............................ 0.2%
In ................................._0.515per ml
1‘6 ................................. 0.56Vrer 1.111

In the present study the magnesium.was supplied in the
form or1ugsc¢ovnao (0.005 grams per liter) and the iron in
the form.opreSd‘-7820 (0.00278 grams per liter). The pH of
this inorganic base medium.after autoclaving isde 0.5. The
anthers found that this base medium plus 3.0 percent glycerol
supported the growth of g‘flggli as well as did nutrient broth,
except that a longer lag period was observed.

this inorganic base medium.was dispensed in 50 ml amounts
in 125 ml flasks and sterilized by eutoclaving for 10 minutes
at 121 G. Lactose, as the sole carbon source, was added asep-

tioally from.a simdlarly autoclaved solution prior to in-

oculation to give the desired final concentration,

I I Q. o .
s - ‘1
e ‘ s
e~ceseeeetaeoeeesreooe
v6.0..‘0‘zteabee. '\- "
.o‘ee-«eueeeeeee.oee '.'
osteeoeDOOOsOOOOOc-O ..

15

hummus-Ion or; ran more or 33mm
' reasons on PENICILLIN ACTION

the first consideration in this study was to determine
the conditions necessary for the optimum lethal effect of
penicillin on.§h_gg;;,in the synthetic mediun.of Young, Begg,
and Pants (1944). for convenience this msdium.will here-
after be rarer-red to as mar medium.

from.the evidence obtained by the work of the several
authors quoted previously, conditions optimum.for nest rapid
initial growth of §h_gg;i should also be optimum for peni-
cillin action against this organism. Ebbby, Mbyer and Ghaffee
(19e2); nigger (1944); Hobby and Dawson (1944); Lee, Foley,
and Epstein (1944); Rants and Kirby (1944); Schwartsman (1944);
Chain and Duthie (1945); Dufrenouy, strait and Pratt (1947); '
Irampits and workman (1947); lagle and mseelman (1949). '

EIEBRIMENT I.
netermination of the Optimum.concentration of Lactose
in HP “than for the minimum Lag of 5,, 29.1.10
_.§:ocggurez fifty n1 quantities of YBP'medium.containing
,verying concentrations of lactose were prepared and seeded

with 0.5 ml of a 1-200,000 dilution of a 24shour brain heart

-.-

16

infusion broth culture of gh,ggli, Duplicate onerml quan-
tities were plated in brain heart infusion agar at hourly
intervals. After a 24-hour incubation period, the colonies
were counted and recorded as in Table 1. The incubation
temperature was 37 0.

W3, As can be seen in Table l, the optimum con-
centration of lactose for shortest lag of 3h,ggli,is from
0.25 to 0.5 percent.

these results also show that the length of leg at the
optimum.lactose concentration is approximately five to six
hours.

Before an attempt was made to demonstrate the bacteri-
cidal action of penicillin against A 39],; in this medium,
a consideration of the concentration of penicillin which
must be used was necessary.

homes and Levine (1945) studied the in m effects
of_penieillin on various intestinal bacteria and found that
the order of sensitivity was as follows: ,Salmonella,
W. 2121231.. W. Woh 0 is. and Lergbacter.
lbst lfighgnighig_strains required from.50 to 100 units per
ml for complete inhibition in beef extract broth, while
1,, W and L, 832529.21. usually required over 100 units
,per ml for_inhibition. On this basis then, a concentration

of 200 units per ml was chosen to insure kill of even the

an.
' I.
C
v
,.
e

17

TABLE 1

The Effect of Varying the Lactose
Concentration on the Initial
Growth of L 0011 in YBP lediml

A

_L

 

w—

VV. vvr

 

 

fasten;w ’ _ fl?" . H

Concentration (f) 0.1 0.25 0.5 1.0 2.5

Es .m'aftei? ‘ A # “ f

seeding

0 17,000* 18,000 17,500 f 18,100 +_ 16,000
1 17,400 17,800 18,000 .17,300 17,400
2 €17,200, 718.300 ,17.200 18.900 18,000
3 18,000 20,100 20,300 20,500 17,800
,4 31,000 _46,000 44,000 32,000 21,400
5 63,000 84,000 ,78,000 48,000 24,300
a 74,000 123,000 119,000 38,000

77,000

 

W‘W" i.

* average count per ml

_'

he. I!

Q

8

18

.Iore resistant coliform strains. The use of such a relatively
high concentration of penicillin is further justified by

the work of Iagle and Mhsselman (1949), who showed that

large doses of penicillin, even up to 10,000 times that
necessary for inhibition, did not accelerate bacterial death.

The next logical step in this investigation was to dee
termine the bactericidal activity of penicillin against 15‘
m; in YBP medium.

Before this could be determined, however, some means had
to be devised to reduce the residual penicillin concentration
below the threshold value for the organism prior to plating.

‘ fwo theoretically possible methods to accomplish this
presented themselves:

(1) the penicillin could be diluted beyond the three:
hold concentration before plating.

(8) The penicillin could be destroyed by incubation in
the presence of a sufficient amount of a penicillinvdestroyirg
332339!

apicer and Blitz (1948), working with_§t;eptocgccus xgzi:'
333;, showed that when'penicillin was removed by dilution,

no colonies developed upon plating the cells in an agar me-
dium. than the penicillin was removed by sufficient penis
cillinase, however, the exposed organisms regained their

viability and developed colonies in agar poured plates. The

19

reason for the cells failure to regain viability after peni-
cillin removal by dilution is indicated by the work of less
and Johnson (1949). These authors, using radioactive penis
cillin, showed a specific uptake of penicillin by suscepe
tible cells independent of the extracellular concentration.
this penicillin was not removable by dilution, and could
be rcsponsible.for the residual effect after dilution of the
antibiotic in the suspending medium beyond the threshold
level for the organism.in question.

s-It-was desired to determine whether this residual effect
is also ‘Operativc in the ease of §_,. mm. To determine
this the following was done:

EXPERIMENT II.
The Effect of Removal of Penicillin by Dilution on the
Recovery of i W.

_ ..2:29.911m8 s 1-100,000 dilution of a 24-hour culture
are, W was prepared in 0.85 percent saline. Peni-
cillin was added to a level of 200 units per ml. After 10
minutes this suspension was diluted 1-100 in saline and
plated in duplicate in 10 ml of brain heart infusion agar.
this gave a final concentration of 0.8 units per.ml in the
agar plate, which is far below the threshold concentration
of about 10 units per ml for §_, w (Levine 1945).

After 84-hours incubation, no colonies developed, as can be

20

seen from.!able 2. The control showed no loss of viability
in the saline during this short exposure, nor did 0.2 units
of penicillin per ml in the agar medium prove inhibitory to
cells not previously exposed to the higher concentration of
[the antibiotic. On the basis of this evidence it is apparent
that dilution could not be the method to use for the removal
of penicillin for recovery of §,.pu;;grg§u These findings
are also in agreement with those of Spioer and Blits (1948).

.The other alternative, as indicated by these same aue
thors, involves the enzymatic inactivation of penicillin by
the enzyme penicillinase.

Abraham.and Chain (1940) were the first to report the
existence of a bacterial enzyme capable of destroying penis
cillin.. They showed that various bacterial extracts and
culture filtrates destroyed penicillin with varying degrees
of effectiveness.

Hobby, Meyer and Ghaffee (1942) also confirmed the pro-
duction of a penicillinase by a strain ofwgpwggli.

Lawrence (1943), in an attempt to devise a sterility
test for penicillin preparations, showed that takadiastase
and clarsse were effective in destroying penicillin. Later,
however, Lawrence (1944) reported that the active penicillin
destroying substances in clarase were certain filterable

substances of bacterial origin. These bacteria were identified

21

TABLE 3

The Residual Bactericidal Effect of Penicillin on
§h_ c after Reduction of
Penicillin Concentration by Dilution

 

 

Control - count per.ml
before penicillin exposure. 9,600

A.—

v“—

Oount after 10 min. exposure to

800 units of penicillin per ml.

Penicillin reduced to 0.2 units

per ml by diluting 1-100 and plating 0
one ml in 10 ml of brain heart

infusion agar.

“A
fivw -w-w '7 WV 7w. —v—v 71

Control - count per ml. No initial

exposure to penicillin, but plated

in brain heart infusion agar ccn- 10,500
taining 0.2 units of penicillin

per ml.

22

by him.ss belonging to the figsillus cereus type which were
present as contaminants in some olarase preparations.

loodruff and Foster (1945) made a rather intensive
study of a bacterial penicillinase preparation.. These workers
found that penicillinase is destroyed rapidly by temperatures
above 50 0, but that it is quite stable at 4 0. In addition,
they found that the preparation was stable over a remarkably
wide pH range of from.3.0 to 11.0, with an optimum.activity
of from.6.5 to 8.0. It was also found that penicillinase
activity was maximum.at 37 0, and that sarcontaining comp
pounds such as sodium.thioglycollate and cysteine enhanced
its activity. These authors also found that penicillinase
activity is not limited to bacteria, but that certain yeasts,
aetinomycetes and other fungi show it to varying degrees.

Beets-penase concentrate, a product of the Difco Come
pany of Detroit, Michigan was used here. lccording to the
manufacturers, one ml of penase concentrate is capable of
inactivating 500,000 units of penicillin in two hours when
incubated together in 15 ml of fluid thioglycollate mwdium
or brain heart infusion broth at room.tempereture.

Different lots of penicillin and penase are apt to vary
slightly in potency. Therefore, it was considered necessary
to assay the potency of this particular lot of penase with

the lot of penicillin used in order to express the penase

 

 

 

II

 

potency in terms of the penicillin used.

EPEBIMENT III.

The Assay of Bacto-penase Concentrate Carried Out
according to the Procedure Outlined in the Difco manual
(ninth edition), 1955. '
- ,zzggggugg; Dilutions of Bacto-penass Concentrate of
1-2, 1-5, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, and 1-10 were pre-
pared in sterile distilled water. One ml of each dilution
was added to a series of test tubes containing 15 ml of Bacte-
rluid Thicglycollats medium. One ml of a solution of peni-
cillin containing 50,000 units per ml was added to each tube
and allowed to remain at room.temperature for two hours.
lash tube was then inoculated with one ml of a 1-1.000 di-
lution of a 24-hour broth culture of !k_pyogengs Egggpgurggg
P809, and incubated at 37 0 for 24 hours. The highest_
dilution permitting growth in this period was detarmdned, and
this dilution of haste-penase Concentrate was multiplied by
50,000 to determine its potency in terms of the number of
units of penicillin inactivated by one ml of the enzyme pre-
paration. The results of this assay are given in Table 3. ,
‘ Eiscgssiog: from these results it is evident that 300,000
units of penicillin can be inactivated by one ml of penase
in two hours_st room temperature. The potency than is 350,000
”units" per m1.

24

TABLE 3

The Potency or Bacto-penase concentrate
Aesayed Against Penicillin 0 Potassium

*— v w “7 “WV fi—

‘__— *7 v- m

lube lumber Bacto-penese Turbidity after

 

dilution . 24 hours
1‘ 1-2 }
2 1-3 }
3 1-4 I
4 1-5 /
5 1-0 }
a 1-7 I
7 1-3 .
8 1-9 -
9 1-10 -

 

W W ’1' W W ‘7...

' lash tube contained 50,000 units or penicillin per :1.

35

It must be recognized that in the assay of penase, the
susceptible bacteria were added to the assay tubes after peni-
cillin inactivation. In actual sterility-testing procedures,
or in procedures designed to recover bacteria after penicillin
exposure, a very different situation exists. That is, the
bacterial cells are in contact with penicillin before the addi-
tion of penase, and remain so during the entire inactivation
procedure.

'It was considered important, therefore, to determine
whether the concentration of penase adequate for inactivation
of a given amount of penicillin will also be adequate to al-
1cw for recovery of susceptible cells which had been pre-

viously exposed to the penicillin for a short time.

EXPIRIIINT IV.

Deterndnation of the amount of Penase necessary for
Quantitative Recovery of 1h_gggggg P209, Exposed to zoo units
of Penicillin.per ml in 0.85 Percent Saline.

W: A 84-hour culture, of 3,, m P209 was
diluted 1-10.000 in saline and 200 units of penicillin per
ml were added. One-ml quantities were removed after 10 min-
utes and placed in nine ml of saline, brain heart infusion
broth and fluid thioglycollatc medium, containing various
amounts of penase, After two hours at room.tempereture one-

ml quantities were plated in brain heart infusion agar and

86

incubated for 84 hours at 37 0. The results are shown in
Table d.

W: These results show that a much higher con-
centration of penase is necessary for the recovery of bac-
teria from penicillin exposure than is necessary for the
more inactivation of penicillin the same length of time.

It is evident from these data that treatment with 200 in-
activating units of penase for two hours is insufficient
for recovery of ]h_au eus, exposed to 800 units of peni-

. cillin in a medium.unfavorab1e for growth. A concentration
as high as 35,000 inactivating units for four hours is
necessary to cause quantitative recovery in the three media
tested. brain heart infusion broth appears to be the most
efficient medium.for penicillin inactivation. ‘However, since
the counts after five hours inactivation in fluid thiogly-
collate medium.and brain heart infusion broth exceeded the
eount in the control tube, it is evident that comm recovery
and multiplication occurred.

The next experiment was designed to determine the con-
centration of penase necessary for quantitative recovery of
1, 223-339—133 from a short exposure to 200 units of penicillin

per ml in.a medium.unfavorable for growth.

27

Ha Hog cameo owsuoes *

 

0mm own cum mason 0 on naaaaos
idem omiaonpsoo
com o 0 com o o can 0 o e
an o o own 0 o m o o m
m o o n o o a o to o
mason ma

cad» soapspfipossH

 

ooo.mm cow.m oom coo.mm oo«.m com ooo.mm oom.m com ononom no noon:
endows cusaaoomaw moons codename osaasm sodps>apowna
loans chasm ounce nanom smm.o nod sons salons

 

 

onaanm snooeom mm.o on as non naaaaofimwm no moans
com on vomomnm moon was moana.momm unease .2 ho unopooom
opapsuapsssd you anoomoooz season we pssoa4 on» we soapssasuopon

q Hum<B

28

EXPERIMENT'V.

The Determination.of the Amount of Penase Required for
a Quantitative Recovery of §_,. w from 10 Minutes Ix-
posure to 200 Units of Penicillin per md in 0.85 Percent
Saline.

oce e: A 24-hour brain heart infusion broth cul-
ture of §_,, W was diluted 1-10 million in 0.85 percent
saline, and penicillin was added to a level of 200 units per
m1. rive ml quantities were removed after 10 mdnntcs and
penase was added to give the final concentrations shown in
Table 5. After a two-hour incubation at 37 0, one m1 quan-
tities were plated in brain heart infusion agar. lbodruff
and Foster (1945) showed 37 0 to be the optimum temperature
for penase activity, and on that basis it was adopted here.
The results are shown in Table 5.

W8 in examination of these data reveal tint
in order to inactivate the 1,000 units of penicillin con-
tained in the five ml of saline sufficiently in two hours
at 3'7 0 to allow for quantitative recovery of g, M, '
at least 35,000 inactivating units of penase were necessary.
This is a ratio of 35 units of penase to one unit of peni-
cillin. This proportion of penase to penicillin was used

in further experiments for determining conditions optimum
for kill of L 3234.

29

IABLE 5

lbs Amount of Penase Required for a Quantitative
Recovery of.§L pullorgm from.lxp0sure
to 200 Units of Penicillin per
ml in 0.85 Percent Saline

 

 

rabe* units of Count
penase added per ml
1 3,500 0
a 8,750 0
a 17,500 0
a 26,250 as
5 35,000 10‘
a 70,000 139
Control (no
penicillin) ---~-- 149

 

w-v

W

‘—

* Itch tube contained five ml for a total of 1,000 units
of penicillin per tube.

av

30

‘IIPIRIMENT‘YI.

Determination of the Optimum Time of Incubation of g;
19;; in the Medium Plus 0.25 Percent Lactose for the Maximum
Kill by 200 Units of Penicillin per ml.

Ezgceduzgz Five-tenths ml of a l-200,000 dilution of
a 24-hour brain heart infusion broth culture of £5,22li,was
seeded into 50 ml of YBP medium.plus 0.25 percent lactose.
Two hundred units of penicillin per ml were added and incu-
bated at 37 0. it two hour intervals, five-ml euantities
were removed and incubated for two hours at 37 0 in the pre-
sence of 55,000 inactivating units of penase. At the end of
two hours, duplicate one-ml quantities were plated in brain
heart infusion agar. The colonies were counted after 24 hours
at 37 0. The results are given in Table c.

.nigggggiggt These results show that an exposure of 13
hours to 200 units of penicillin per md.in IBP‘msdium.with
0.25 percent lactose will result in a 99.7 percent reduction
in viability of 5,, gig.

Beverel workers, Hobby, Mbyer and Ohaffee (1942); Bigger
(1944); Hobby and Dawson (1944); Lee, Foley and-Epstein (1944);
Rants'and Kirby (1044), i... found that penicillin is most
rapidly lethal for bacteria when the cells are in an actively
growing state. On this basis, then, the most advantageous
timn for the addition of penicillin should be at the end of

31.

TABLES

the Bactericidal Effect of 200 Units of Penicillin
per ml on L, 0913 in YBP Radium with
0.85 Percent Lactose at 3'7 0

Wy- w V

b __

'_ V— r— 7"“- v w

Hours exposed Surviving cells per ml

—v—-— ._v -v—-——

10,800
4,700
1,990

700
380
300
ll _180
13 109

coconut-o

 

 

A
. l 0 '-
. - , - s u . V
< ' e . s - < , e e
e
. l / , , . . . . s
e e . s o - e i l s , s v

 

32

the lag period of growth or in the early phase of logarithmdc
growth. An experiment was next performed to determine whe-
ther or not this is actually the case with‘gi coli.

IIPIRIMENT VII.

Determination of the Influence of Pre-incubation Time
on Penicillin Action Against 5, 391.3,.

229239323; Identical flasks containing 50 ml of YEP
medium.with 0.25 percent lactose were seeded with 0.5 ml of
a l-20,000 dilution of a 24-hour brain heart infusion broth
culture cf‘l“ggli. These flasks were incubated at 57 0 and
200 units of penicillin per ml were added to consecutive
flasks at two-hour intervals. it nine hours from.the time
of seeding of the flasks, five-ml quantities were removed
from each, and 35,000 inactivating units of penase were added.
after two hours at 57 0, one ml quantities were plated in
duplicate in brain heart infusion agar.

an examination of the results given in Table 7 shows
that a six-hour pro-incubation and a three-hour penicillin
exposure is more effective for the kill of gh,gg;; than is
a nine-hour penicillin exposure without a pro-incubation.
This is in agreement with the findings of the authors cited
above. Referral to Table 1 shows that after six hours in-
eubation in YBP medium plus 0.85 percent lactose, .24 2.9.1.1
is in an actively dividing state. This fact is apparently

33

TABLE 7

The Efficiency of Kill of L. gel; by 800 Units of Peni-
cillin per ml as Influenced by Length of
Pre-incubation and Penicillin Exposure

Time in YEP medium.00ntaining

0.25 Percent Lactose

 

w—r—Yr

'—

 

Length of Penicillin Numbers of
pre-incubation exposure time cells surviving
period, in hours in hours

0 9 838

g 7 185

a 5 177

6 3 310

8 l 265
Control

0 0 84,000

responsible for the more rapid kill by penicillin added at
this point, than when it is added before the apparent growth
phase has begun. From.the above findings it becomes evident
that an optimum pro-incubation time for maximum kill of g,

3211 can be established.

WWT VIII.

Determination of the Optimum Pro-incubation of g, 93;;
in YBP ledium roi- Maximum Kill by Penicillin. '

Eggcedurez‘ The pre-incubation time here was varied and
the penicillin exposure time was kept constant. fifty-ml
quantities of YEP medium.in 125 m1 flasks with 0.25 percent
lactose were seeded with 0.5 ml of a 1-2,000 dilution of a
24-hour brain heart infusion broth culture cf‘gh,gg;i, These
flasks were incubated at 57 0 and at hourly intervals peni-
cillin was added to give a final concentration of 200 units
per ml. an arbitrary three hour penicillin exposure period
was then employed. At the end of the penicillin exposure,
five-ml quantities were removed from each flask and placed
in test tubes in the presence of 55,000 inactivating units
of penase. lfter two hours at 57 0, duplicate one-ml quan-
tities were plated in brain heart infusion agar. The colonies
were counted after 24 hours at 37 a.

W: Prom the results of this experiment given
in Thble 8, it is evident that e pre-incubation of six hours

35

EABLE 8

Inc Influence of Presincubation Time on the Kill
eff§s by 200 Uhits of Penicillin per ml in
p , lbd um.Oontaining 0.25 Percent Lactose

w V w *—
V-v—v V T T
w—.— W __

 

‘nours pro-incubation Count per ml after
at 37 a 3-hr penicillin exposure
7 3 min"
4 < mm
a 1.350
6 910
7 1570
8 1980

Control - no pro-incubation and no penicillin exposure -
-84sJ-60 POI Ill

W fiv
_r~ v.

* euro - too numerous to count.

 

before the addition of penicillin is optimum.for maxinnn.kill
of 5., 39;; in IBP medium plus 0.25 percent lactose.

‘ These results further confirm the hypothesis that peni-
eillin action is greatest when the exposed cells are in an
actively growing state. Table 1 shows that after six hours
incubation in IBP:medium.plus 0.25 percent lactose without
penicillin, £L,gg;i,is past its lag phase and is in an ac-
tively dividing state.

Having established the optimum.pre~incubation time be~
fore penicillin exposure for maximum.kill of §L_33li, the
next step was the determination of the optimnn.penicillin

exposure time after pro-incubation for the optimum kill of

lhbgflilfi

EXPERIMENT IX.

Determination of the lffeet of Exposure Time on the £111
of; 93;; by 200 Units of Penicillin per Ill after a Six-
nour Pre-inenbstion.

Ezgggggggj 5 24-hour brain heart infusion broth cul-
ture of 1h,2gliywas diluted l-2,000 and 0.5 ml was added to
50 ml of IBP'nsdium.with 0.25 percent lactose. After a pre-
incubation time of six hours at 37 c, 200 units of penicillin
per ml were added. At hourly intervals thereafter, five—ml
quantities were removed and 55,000 "units” of penase added.

moi- two hours at 37 c, duplicate one-ml’quantities were

fi“

3'7

plated in brain heart infusion agar. Counts after 24 hours
at 37 0 are recorded in Table 9.

Qisgussioni From.this set of data it is evident that
a pro-incubation time of six hours, followed by a penicillin
exposure time of five hours, results in s 99.3 percent reduc-
tion of lhwggli in YBP medium. talso an exposure as short
as one hour results in a 95.7 percent less in viability.
This also indicates that pre-incubation time, and not ex-
posurc time is of most critical importance.

The next logical step in the investigation was to deter-
mine whether §_,, pullogyg would survive these conditions Just
established for optimum.kill of E. coli.

EXPERIMENT.X.

The Determination of the Recovery of a m from
lxposure to 200 Uhits of Penicillin per ml in YBP medium.

.Ezgggggggj A 24-hour brain heart infusion broth cul-
ture‘ offi,’ W was diluted 1-100,000. Five-tenths III
of this dilution was added to 50 ml of YEP medium plus 9.25
percent lactose. The flask was incubated at 37 0 for six
hours, at which time 200 units of penicillin per ma were
added. After an additional four-hour incubation, five ml
were removed and placed with 35,000 "units” of penase for
two hours at 37 0. Duplicate one-ml quantities were then
plated in brain heart infusion agar and incubated for 24

38

7331.39

The meet of Exposure Time on the Kill 01’ L 93;;
by 200 Units or Penicillin per ml after a
81; Hour Presincubaticn in YEP Medium
Containing 0.25 Percent Lactose

 

"V

 

Hours or exposure Bacteria per Percent loss
to 800 units per ml ml surviving in viability

0 25,000 "-9-

1 1,060 . 95.7

a 590 97,7

3 350 98.5

a 238 99.0

5. 181 99.3

 

w—v ‘rvw—u-r— w. .7

p... O

-‘

39

hours at 37 0. The resulting counts are recorded in Table
10.

niscussicg; The results show that under these conditions
_S_,, M can survive the effects of penicillin exposure
to the extent of about 70 percent.

Thus, under identical conditions of exposure, gh_gg;;,
which is normally considered very resistant to penicillin,
is killed to the extent of more than 99 percent while the
more sensitive'§‘,pgllogum.survives to the extent of about
70 percent.

’ The reason that §_,_ pulloggg was not quantitatively re-
covered is not clear. The control tube (Table 10) shows
that the medium.itself was not sufficiently toxic to account
for the decrease in numbers. A possible explanation is that
the six-hour pre-incubation period was not sufficient to
render all the cells in an inactive metabolic state.

’ In order to determine whether or not this is the cast,

the following was done:

EXPERIMENTZII.

Determination of the neccvary of g‘ pullcrum after a
12-Hour Presineubaticn in 0.85 Percent Saline and an Ex-
posure to 800 units of Penicillin per ml.

mm A zeehour culture of _8_,. pullogg was die
luted 1-100,000 and 0.5 ml was added to 50 ml of 0.85 per-

.I
y .‘
a l .-
| /
. .
. e. . v u.
_ . 0
.
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a
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i ‘I
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w v
s.
.5
.

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.
a .
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v .
I l
s \.
n ‘
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i e
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to
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er, . n
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.r
v .. .-
O
. I 4.
. o I
.
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.c .
.
. r In A
. O
l e
u
.

£0

ILBLE 10
The Recovery of llo from.Exposure to 200

units of Penicil in per ml at 57 0 in YBP Medium
Containing 0.25 Percent Lactose

 

 

 

EIposure tine Penicillin Control - no
an, i a es *1, openicillin
0 hrs. 250’ 230
4 hrs. 162 209

 

* average count per ml

 

41

cent saline. The flask was incubated for 12 hours at 57 c
to rid the cells of stored nutrients. Two hundred units of
penicillin per ml were then added, and at hourly intervals
fiveeml samples were removed and placed in test tubes con-
taining 35,000 ”units” of penase. These tubes were then
incubated at 3700 fortwo hours, at the end of which time
duplicate one-ml quantities were plated in brain heart in-
fusion agar and incubated at 37 0 for 24 hours. The results
are given in table 11.

cu o : These findings show that an exposure as
long as eight hours to 200 units of penicillin per ml will
eause no significant decrease in numbers of £33, w when
in a ”resting" condition in saline. The1most logical expla-
nation fcr the results observed in Table 10 would then be
as follows; The conditions established for optimum.kill of
1‘93}; will not allow for simultaneous quantitative str-
vival of §_,_ w due to the fact that some of the cells
are still able to metabolize sufficiently to establish the
penicillin effect. This metabolism may well be due to nuts-
rients stored in the cell from.the previous rich medium.in
which they were grown.

One possible way to obtain a greater survival of §_,. p3};

lgggn'would be to pre-incubate for a longer period of time

before adding penicillin to allow the cells to assume an ins

Ill-Ills'iIIIlIIlrllvt.

 

42

TABLE 11

Recovery of §_,_ W from Exposure to 200 Units
of Penicillin after a Preliminary 12-Hour
Incubation in 0.85 Percent Saline at 57 0

r V T' “——

r—w rm W v '— w

 

Penicillin exposure Count per ml after
in hours penicillin inactivation
0 208
1 821
2 206
4 219
6 818
8 204

43

active state. However, this would not be feasible in a
procedure designed to obtain selective kill of the coliforms
in.a mixed population. as was seen in Table l, a pre~
incubation of more than six hours allows §h_ggli,to multiply.
This increase_in numbers of gh_g§;;,would tend to offset the
advantage of the pro-incubation for in. pullogg and would
decrease the efficiency of the selective process. See also
Table 8.

s simular difficulty was anticipated when attempts to
obtain selective killwof the coliforms from.fecal material
were to be made. The fecal material would very likely con-
tribute growth substances to the medium.which would enable
the salmonellae to initiate growth also, and, hence, be killed.

In order to determine whether or not this anticipated
difficulty is a real one, the following experiment was per-

formed:

EXPERILENT XII.

Determination whether the Survival of §_,_ W; to
Penicillin Exposure in YBP fledium.Iould be adversely Af-
fected in the Presence of fecal NIterial.

W: ,1 24-hour culture of §,, W was die
luted 1.100.000 in 0.85 percent saline. Five-tenths ml of
this dilution was added to 50 ml of YBP medium.with 0.25

percent lactose and approximately 0.5 grams of sterilized

O
»
.
e
.. 7‘
.
,.
. v
e
.-
o-
. .
.

,—..

 

human fecal material. After a six-hour pro-incubation period
at 37 O, 200 units of penicillin per ml were added. The flask
was further incubated for four hours at 37 C, after which time
five ml was removed and mixed with 35,000 "units" of penase.
After two more hours at 37 0, duplicate oneeml portions were
plated in brain heart infusion agar and incubated for 24 hours.
nisgussign: The results given in Table 12 reveal that
under these conditions _s_,_ pgllgrg does not survive exposure
to 800 units of penicillin per ml. However, the control tube
containing no penicillin shows that no appreciable multipli-
cation of fi‘upgllgggg takes place under these conditions.

The interpretation given these results is that the fecal
material contributes substances to the medium.which enable
§,, mm to metabolize sufficiently to establish the penio
cillin effect, but which do not allow for appreciable mul-
tiplioation in the time interval used.

A possible means to obviate this difficulty is suggested
by the studies previously cited. Bigger (1944). Hebby and
Dawson (1944), Schwartzman (1944), Chain and mm. (194.5),
These authors all noted that certain bacteriostatic snb~
stances render bacteria resistant to penicillin by virtue
of the fact that they inhibit growth.

For this phenomenon to be of use here, a bacteriostat

would have to be employed which would selectively inhibit,

45

TABLE 12
The Effect of One Percent Sterile Feces on the Survival

of g. pgllorum after Exposure to see Units of
Penicillin per ml in YB? Medium

"

 

 

 

Count before Count after
exposure , exposure
YEP medium plus '
0.25; lactose 126 120
E? medium plus
0.25% lactose plus
1% sterile feces 158

 

46

but not kill the salmonellae while allowing the coliforms
to 310'.

inoculloch (1945) stated that ”Small amounts of arsenous
acid are toxic to many pathogenic organisms, such as £h.£l:.
299.2,. L m and many streptococci, while the closely
related L, £14,, L. m, and many staphylococci actually
are stimulated by such amounts."

Dubos (l947) indicated that it is now considered that
the arsenicals, whether tervalent or quinquevalent, are first
oxidized or reduced to their corresponding arsenoxides, which
in turn react with some reduced sulfhydryl groups of the cell.
He pointed out that the simultaneous injection of an arsenical
and a compound containing an SH group slows the rate of dis-
appearance of trypanosomes from.an infected animal. This
lends support to the idea that arsenicals are active due to
their ability to block SH groups of enzyme systems. This
also suggests that arsenic may be bacteriostatic rather than
bactericidal. If indeed this is the case, then an arsenic
compound could be of use here as a selective bacteriostat.

Two arsenic compounds were tested to determine whether
a selective bacteriostasis of the salmonellae could be

achieved.

as5’

’ .
o
- .
\
U . - .
‘ .
n
A '5
r
’ I
I a to
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.

q
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0 a
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s U
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t! .
‘ ‘ . s
0
I
.
I
b
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5 .

 

 

4'7

EXPERIMENT XIII.

Determination of the Selectively Inhibitory Property of
Sodium lrsenate and Sodium Arsenite ong‘,’ pullorum and $4.
w.

W; Ten-ml quantities of tryptose-lactose broth,
(Derby and Hallmann 1939), containing various concentrations
of sodium arsenate and sOdium arsenite were prepared. One
set of tubes containing each of the two compounds was seeded
with 0.1 ml of a l-lOOO dilution of a 24-hour culture of _E_,_
323.1. A similar set of tubes was seeded with 0.1 ml of a
l-lOOO dilution of a 24-hour culture of §,. W

Growth, as determined by visual turbidity, was recorded
after 24 hours. These results are given in Table 13.

Was he can be seen from this table, L221}.
grows dwell in much higher concentrations of both chemicals
than does L W. However, it is seen that sodium ar-
senite inhibits §_,, W over a much wider range than
does sodium arsenate. For this reason the effect of sodium

arsenite on L 93;; and g“ pullogum was further, investigated.

EXPERIMENT XIV.

Determination of the Influence of Various Concentrations
of Sodium Arsenite on Viability of s, W and Growth of
L 993.; in Tryptose-lectose Broth.

W; Fifty-ml quantities of tryptose-lactose

 

 

TABLE 13

The Selectively Inhibitory Property of Sodium
Arsenate and Sodium lrsenite on E coli and
§_,_ pullozg in Tryptose-lactose “Broth

 

 

A;
7‘ ‘r—v—f

Organism E. coli S. pullorum

 

firc'ent of so-
dium arsenate

0.06 3/* -
0.05 4/ -
0.04 4/ -
0.03 4} -
p 0.08 4} -
0.01 4} ' 1}
Control
0 a; 4}

 

fircent fir so-
dium arsenite

0.00 ,3} . -
0.05 4f -
0.03 4f -
0.01 4} -
0.008 4/ -
0.000' 4f -
0.004 4} -
control
0 4/ 4}

 

or. degree of turbidity compared 700‘ control at 4F

49

broth were prepared containing various concentrations of
sodium.arsenite. Five-tenths ml of a 1-600,000 dilution
of a 24-hour culture of £h.22$$ was seeded into each flash
of one series. In a similar manner a l-l00,000 dilution of
a 84-hour culture of §,,pgllgzgg,was seeded into another
series. At hourly intervals one-md.quantities were plated
in duplicate in brain heart infusion agar. After 34 hours
incubation at 37 C, the colonies were counted and recorded
as in Table 14a and 14b.

fligcgggion: From.these results it is seen that in
trypt0se-lactose broth a concentration of sodium.arsenite
as high as 0.01 percent has no inhibitory effect on the
growth of gh,gg;i. Also, a concentration as low as 0.006
percent is bacteriostatic for §,_ REE-.9233.

At this point the effect of 0.01 percent sodium.ar-
senite on several other enteric organisms was also deter-

mined.

EXPERIMENT XV.

The Effect of 0.01 Percent Sodium Arsenite on the
Growth of Several members of the Eagerobggteziggegg in
Tryptcse-lactose Broth.

fizggggggg; Ten-ml quantities of tryptose-lactose
broth containing 0.01 percent sodium arsenite were prepared

and sterilized by autoclaving for ten minutes at 121 0.

e.

50

TABLE 14.

The Effect of Various Concentrations of Sodium Arsenite
on Viability and Growth of £1. 00;; in hyptose-
lactose Broth at 37 c .

 

__'_i

Incubation time
in hours 0 2 4

 

307101 arsenite
eoneentrati on (33 )

0 28* 118 2.000
0.0004 21 125 3,900
0.0000 21 120 2,400
0.0000 28 125 3,000
0.001 25 115 3,700
0.002 31 128 3,400
0.000 28 121 2,700
0.000 27 118 2,400
0.008 24 122 2,000
0.01 20 132 2,800

 

" average count per ml

51

TABLE 14b

The Effect of Various Concentrations of Sodium Arsenite
on Viability and Growth of h u orum in
Iryptoee-lactose Broth at 3 G

 

v j

Incubation time .
in hours 0 2 4 6

 

iodine areeni te
concentration (i)

o 123* 300 0, 200 mm
0.0004 107 152 300 950
0.0006 117 141 147 160
0.0008 120 110 132 140
0.001 107 110 107 105
0.002 124 147 125 120
0.004 130 120 122 105
0.000 113 137 124 103
0.008 112 117 120 109
0.01 107 120 125 111

"’ average count per ml

52

These tubes were seeded with 0.1 m1 of 1-100 dilutions of
80-hour brain heart infusion broth cultures of several mem-
bers of the thgbactgriaceae. Growth, as evidenced by
visual turbidity, was determined after 24 and 48 hours and
recorded in hble 15.

pissugsiog: It is evident from this table that all the

members of the genus Salmonella tested were inhibited.

However, 22W: Alkaligenes fec lis, Pseudomonas

gegnginosg, L coli, A, agrogenes and the gigellas did show
turbidity within 24 hours.’

In the light of these results, and the hypothesis that
penicillin affects only growing cells, it seemed advisable
to determine whether a selective kill of L 00 1 and simul-
taneous survival or L w could be obtained by use of
penicillin in this tryptose-lactose medium containing 0.01

percent sodium arsenite.

EXPERIMENT XVI.
Determination of the Effect of 0.01 Percent Sodium
“senite on Penicillin Action against §,_ pullorum and g,-
821.}. in Tryptcse-lactose Broth. _ .
W: fifty-ml quantities of tryptcse-laetose
broth with 0.01 percent sodium arsenite were pepared. One
of these flasks was seeded with 0.5 ml of a 1-100,000 di-
. luticn of a brain heart infusion broth culture of g; pgllorun.

'e O'

\O

53

IIBLE 15
The Irrect of 0.01 Percent Sodium.lrsenite on the Growth

of Several numbers or the Engeropactggiageae
in Tryptosc-lactose Broth

r fifi vi

 

 

 

Organiam{ 24 hours ,‘ .48.hours,_
Proteus vulgaris ’ ‘ f* }
Alkaligeaes fecalis f ‘f

.Peeudomcnas aerugincsa } f
E. coli 0.3. f f
I. 0011 #83 ,l ,3
Aerobacter aerogenes f f
Shigella ambigua } f
Bhigella alkaleseens { ./
Salmonella typhimuriwn -** -
Sal. enttritldls - -
Sal. typhosa #1 - -
Bel. paratyphi B p- -
Sal. typhosa #2 - -
Sal. typhosa #3 - -
Bel. pullorum ~ ? -
:* { s_growth as evidenced by visual turbidity

a no growth

I,

A..-

.e

. e

4

§ 0
- e

“1

r

\b

-.

54

The other was seeded with 0.5 ml of a l-lOOO dilution of a
similar culture of _E_,, 321;. Control flasks containing no
sodium arsenite were also prepared. After a one hour pre-
_ incubation, .200 units of penicillin per ml were added. After
four more, hours at. 37 0, five ml amounts were removed and
placed in test tubes containing 35,000 'units' of penase.
these tubes were incubated for two hours at 07 0 and plated
in duplicate, in brain heart infusion agar. _ After 24 hours
at 37 o, the plates were counted. Ihe results are given
in Table 10.

, ‘ _Mu_s_s_i_gg; As can be seen here, the viability of 5,,
293:1. was reduced about 99.8 percent by this penicillin ex-
posure. ‘l'he recovery of L, pullogg was about 20 percent.
however, 1,, pullorum in tryptose-lactose broth without so-
dium arsenite showed no recovery at all. This is evidence
for the fact that a ,baeteriostatic concentration of sodium
arsenite does inhibit penicillin action to some extent.
However, under. the conditions of this experiment, a quan-
titative recovery. of g, W from exposure to .800 units
of penicillin per ml was not attained.

, {the inhibition of, penicillin action by sodium arsenite
must be considered as a secondary effect. The primary effect
appears to be that of bactericstssis. . 'this hypothesis is
substantiated by the fact that 0.01 Ptrcent sodium arsenite

sJ

55

TABLE 16

The lrfeet of 0.01 Percent Sodium.lrsenite on
Penicillin Action against §,_ W and
.Jh,ggli in Tryptose-lactoae Broth

w a. v ‘7

‘L

 

8e pullorum ,, no 0911
hetero peni-
cillin exposure 115‘ 19,200
”tar penit-
cillin exposure 81 38
, control - no
sodium arsenite 0 p 34

 

fifi ——— v—fiv

" average count per ml

tall.

0‘.

'. ’

.

no-9»

56

does not inhibit the action of penicillin against 35,, 993.1,
but does inhibit the penicillin effect against §_,_ W
to some extent. It is significant that this sodium arsenite
concentration is bacteriostatic for _S_,_ m but not
for I. .9311.

...... It ,is a more difficult utter to demonstrate the bee-
teriostatickeffect of sodium arsenite on g, mllorum in YEP
medium containing lactose. The obvious reason for this is
that the medium itself will not support the growth of the
organism. However. the highest ccncentrati on of sodium
arsenite which will not cause a kill of h mallow in YEP

mdiun with 0.25 percent lactose was next determined.

MIMI XVII.

Determination of the Effect of Various Concentrations
of Sodium Arsenite on Survival of g, m in YBP medium
with 0.25 meant Lactose.

. W: fifty-ml quantities of YBP medium were pre-
pared with various concentrations of sodium arsenit‘c. After
autoclaving, 0.35 percent lactose was added to each flask.
the flasks were than seeded with 0.0 ml at a 1400.000 di-
lution of a 24-hour culture of g, M and incubated at
3'7 0. After hourly intervals, one ml quantities were plated
in duplicate in brain heart infusion agar. Counts obtained

after 24 hours at 37 C are recorded in Table 17.

 

57

um 17

the Effect of'Various Concentrations of Sodium
Arsenite on Growth and Survival of §,_ 23;;
low in IBP Medium plus 0.25 Percent Lactose

 

_—_.—._’ V‘— w

Sodium arsenite

 

concentration (f) 0.001 0.002 0.004 ‘ 0.000
Mme) ’ - 1
0 430* 430 450 42”
5 430 420 320 51
10 440 480 890 ' 18

*4

"' count per ml
** due to inhibition, not low inoculum

 

58

mgcgsaiog: As is seen from Table 17, a concentration
of sodium arsenite as high as,0.002 percent causes no
appreciable decrease of g, pullorum after as long as 11 hours
at 57 C. This concentration proved to be bacteriostatie
even in tryptose-lactose broth, Table 14, and on this basis,
it was assumed to be bacteriostatie in TBP medium also.

It was next determined whether 0.002 percent sodium
arsenite in In? medium containing 0.25 percent lactose would
protect a W from exposure to 200 units of penicillin
per ml in the presence of approximately one percent fecal

”tul‘le

EXPERIMENT XVIII.

Detrmination of the Effect of 0.008 Percent Sodium
Arsenite on the Recovery of §_,_ W from Exposure to
Penicillin in YEP Hedium Containing One Percent Sterile
feces.

M: A 50 ml quantity of YBP medium containing
0.25 percent lactose was prepared. A similar quantity of
YBP medium was prepared but 0.002 percent sodium arsenite
and approximately one percent sterile human fecal material
were added. Each flask was seeded with 0.5 ml of a 1-1oo,000
dilution of a 24-hour brain heart infusion culture of _S_..
“112m. After a six-hour incubation at 57 C, lo-ml por-

tions were removed from each flask to serve as controls on

 

 

.
.
.
.
.
ow-
.

59

the effect of the medium.itself on the organism. Penicillin
was then added to the original flasks to give a level at}
200 units per ml. After an additional four-hour incubation,
five ml amounts were removed and.mixed with 55,000 ”units"
of penase. Two hours later, duplicate one-ml quantities were
plated in brain heart infusion agar pour plates. The results
are given in Table 18.
niggussiog: ' It can be seen here that in IBP’medium
with 0.25 percent lactose, 0.002 percent sodium arsenite
and one percent fecal material, g‘,ppllorum.can survive
penicillin exposure to the extent of about 85 percent. Under
identical conditions, except for the absence of sodium.ar-
senite, no survival oecurred. This also confirms the finding
in.lxpsriment XVI that sodium.arsenite in bacteriostatie
concentrations does tend to inhibit the action of penicillin.
It now became necessary to determine whether this con-
eentration of sodium.arsenite will also have an inhibitory
effect on the growth of 5,, 92.1; in YBP medium plus 0.25 per-
cent lactose, and a consequent effect on the penicillin
action against this organism. For this determination the

following was done:

60

TABLE 18

The Influence of 0.002 Percent Sodium.Arsenite on Recovery
ef,§‘,pgllorum.from.Exposure to 200 units of Penicillin
per ml in IBP lledium with 0.25 Percent Lactose and
Approximately 1.0 Percent Sterile races

4‘

 

0.002$ Sodium no Sodium 1r-
srsenite and senite plus
1.0% feces 1.0% feces
Control - no
penicillin 155* 162
after penicillin
exposure 128 0

 

' average count per ml

 

 

61

EXPERIMENT.XIX.

Determination of the Effect of Two Coneentrstions of
Sodiun Arsenite on the Growth of ‘13,, 3213,, in YBP Medium.

223335331; Fiftyeml quantities of IBP’nedium were
prepared containing 0.001 and 0.002 percent sodium.arsenite.
Each.flask.was seeded with 0.5 ml of a 1-200,000 dilution
of a 24-hour brain heart infusion broth culture of,§,_col .
it hourly intervals, appropriate dilutions were made and
plated in brain heart infusion agar. The resulting counts
‘nade after 24 hours at 37 c are given in Table 19.

W: By comparing these eounts with those of
the control flask, it becomes evident that 0.001 and 0.008
percent sodium.arsenite in I!P*nediun.caused no appreciable
inhibition of growth of 3,. 9.21.1.0 Since penicillin action
has been shown to be positively correlated with Growth,
[these eoneentrations of arsenite should also show no inhi-
bition of penicillin action against gh,ggl;. This will be
further confirmed in subsequent experiments.

The experiments done thus far have established the
cptinum.conditions of time and mediun.for kill of g‘,ccl
by penicillin, and simultaneous survival of g‘, lo um.
the next step deals with determining the cptinun.penase
eoncentration and time to use for maximum recovery of g,
pullorun.frcm.exposure to penicillin in YBP’mediun.under

the conditions established above.

 

in

 

"

.04 o

 

68

TIBLE 19

The Effect of Two Concentrations of Scdium.Arsenite

on the Growth of‘g‘yggli in YBP Medimm

plus 0.25 Percent Lactose

 

 

 

Sodiun.arsenite
concentration (i) 0 0.001 0.002
‘"’ rifle (hours)
‘ 0 15,800* 10,200 15,900
1 17,300 15,700 16,700
a 10,200 15,800 15,000
3 19,400 21,100 20,000
4 41,000 42,000 30,000
5 04,000 91,000 90,000
0 123,000 131,000 127,000

 

v——V——f

’ average count per ll

a1

.0

65

mum II.

Determination of the 0ptimum.Penase Concentration and
Incubation Time for Maximum Recovery of g, mllorum from
,Exposure to 200 units of Penicillin per ml in IBP.nedium.

Exaggggzg: Fifty ml of YBP msdium.containing 0.002
percent sodium.arsenite, 0.25 percent lactose and one per-
cent sterile fecal material was prepared. This was seeded
with 0.5 ml of a 1-100,000 dilution of a 24-hour culture of
g, m and incubated at 3'7 0 for six hours. 'fwc huh-
dred units of penicillin per ml were then added. After four
hours, five-ml quantities were removed and mixed in test
tubes with various amounts of penase. After hourly intervals,
duplicate one-m1 quantities were plated in brain heart in-
fusion agar. The colonies which developed were counted after
34 hours at 07 C. These results are found in Table 20.

W: It is clearly seen from these data that
under these conditions 3,500 and 17,500 ”units“ of penase
are inadequate for recovery of §_,, W from the 1,000
units of penicillin contained in the five ml of medium. Alt
is further evident that 35,000 “units" of penase did permit
a recovery of about 80 percent.‘ The percent of recovery was
not appreciably influenced by as much as 70,000 “units" of
penase. It is also evident here that a penase exposure time

of more than one hour at 37 C does not further enhance the

‘l

64

TABLI 20

The lrfect of varying Penase Concentrations and Incubation
Times on Recovery of g, Whom Exposure to 1,000
units of Penicillin Contained in live ml of YEP ibdimm

W7 f v

’1 rw —'

Hours of penase . ‘
exposure 1 2 3

 

Wpenase “ fi . ’—
3,000 , 0* o 0
17,300 0 . 0 * 0
33,000 121 ' 110 110

70,000 ‘110 94 112

Control - no
penicillin 149

_‘
'r'

" average count per ll

.0

65

recovery of a, W Thus, a one hour incubation at

37 C~in the presence of 35,000 'unita' of penase is considered
adequate for recovery of §_,, w from 1,000 units of peni-
cillin contained in five m1 of IBP'medium.with 0.25 percent
lactose and one percent sterile fecal material. The reason
for the failure to attain a quantitative recovery of the
organisms is to be sought elsewhere.

There are several possible explanations for failure to
attain a quantitative recovery. These possibilities are
considered below:

1. The fairly long period of bacteriostasis necessary
here may have been lethal to some of the organisms. However,
the results in Thble 17 show that this is not responsible for
the reduction in.numbers. Here it is shown that a 10-hour
incubation period at 37 C in Ilemedium, containing 0.002
pereent sodium.arsenite and 0.25 percent lactose, does not
cause a significant decrease in numbers of §,_ W

2. Another possibility is that not all the cells are
in a completely static state, and therefore, do not resist
the penicillin effect. This is a rather difficult point to
determine with certainty. However, the results of Table 17
show that g, Wdces not multiply in YEP medium with
0.002 pereent sodium.arsenite. This is evidence for, but

not proof of, the completely static state of the organism.

 

(a

66

It is possible that some of the organisms are able, under
these conditions, to metabolize sufficiently to become
irreversibly injured by penicillin without actually under-
going multiplication.

3. The third possibility is that the plating medium
used may not provide optimum conditions for recovery from
penicillin exposure. This third possibility was considered
worthy of investigation in the light of the findings of
Iainwright and mllaney (1934). These authors showed that
the growth of 3... all exposed to penicillin in a synthetic
medium was profoundly influenced by the availability of the
carbon.source in a synthetic medima.

Curran and Trans (1937) also showed that the composition
of media used for recovery of bacterial spores and vegeta-
tive cells previously exposed to lethal agents greatly in-
fluenced the viable count. They conclude that organisms
which survive killing factors are more fastidious in their
food requirements than the same culture before exposure.

An attempt was now made to determine whether the com?
position of the plating medium actually does influence the

amount of recovery after penicillin exposure.

67

mm XII.

Determination of the Influence of the Final Plating
lbdium on Recovery of g. M from Penicillin Exposure.

Mm: rive-tenths ml of a l~100,000 dilutions of a
uvhcur culture of k W was added to 50 ml of YBP me-
dium containing 0.25 percent lactose and 0.002 percent sodium
arsenite. After a six-hour pro-incubation st 37 0, 200 units
of penicillin per ml were added. four hours later a five-ml.
quantity was removed and nixed with 35,000 "units" of penase.
After one hour at 57 0, one-ml quantities were plated in
duplicate in each of two media. These media were plain nu.
trient agar and brain heart infusion agar. As can be seen
from the results in table 21, the nature of the medium has
a very marked effect on recovery. Drain heart infusion agar
again gave approximately an Bl percent recovery, while nu-
trient agar gave no recovery at all. control plates show
that when not previously exposed to penicillin, essentially
the same number of g. M eolonies develop. on both media,
although it was noted that the colonies formed on brain heart.
infusion agar are somewhat larger than those on nutrient agar.

The fact that a greater recovery is obtained when using
brain heart infusion agar rather than plain nutrient agar
indicates that after penicillin exposure in a static state,
am is still viable. However, the percent of via-

s4

68

bility is a function of the plating medium used for evalua-
tion of this viability.

Brain heart infusion agar is generally considered a
very rich medium; and, by comparison, plain nutrient agar
may be considered relatively poor. However, the difference
in survival of §_,. W after penicillin exposure, when
plated in these two media, must be due to a factor or factors
other than this general “richness”. hidence for this is
the fact that equal numbers of organisms develop on each me-
dium when k W is plated without a previous penicillin
exposure (See table 21 - controls).

At this point in the investigation it was desired to
determine to what extent other W species would sur-
vive this penicillin enrichment technique.

mmnmnr ml.

Detamination of the Survival of Several W
strains from Exposure to Penicillin in tap Medium.

W3 Twenty-four hour brain heart infusion broth
cultures of five W species were diluted l~l,000,000
in saline. fen-ml quantities of BP medium with 0.008 per-
cent sodium arsenite, 0.25 percent lactose and 1.0 percent
sterile fecal material were seeded with one ml of the above
dilutions. After a six-hour incubation at 3'7 0, five-ml

quantities were removed and 800 units of penicillin per ml

 

v1.1.1: .

69

15mg].

‘lhe Influence of the Nature of the Plating Mhdium on the
Recovery of §_._ m from Penicillin Exposure

v' fiv a v

 

 

Plating medium lutrient agar IBreln heart
infusion agar

Before exposure 14.5"‘ 149

After exposure 0 131

v — Y— v—r V ‘—

’ average count per ml

‘c‘

~. 0
P-Q
e

I s .
O . . C
t a a .l
.- e Q

.0».

. .

#0..

‘ u

v

70

Iere added. The remainder from each tube was held until the
end of the experiment and plated as a control on the toxicity
of the medium. After four more hours at 37 0, 55,000 "units"
of penase were added and mixed thoroughly by shaking. -0ne
hour later, duplicate one ml quantities were plated in brain
heart infusion agar. The results are given in Table 22.
W: Rom these results, it is evident that
these five W strains also survive penicillin ex-
posure in for medium but to varying degrees; gm,
mammals. mnmwmam minimum . our-
. viral of from so to so percent, while s, 223.22.23.11; survived
only about le percent. It is not clear why g... ggggitidis
did not survive bettc. ‘l'he control tubes show that this
sharp reduction was not due to the toxicity of the medium.
Table 15 also shows that sodium arsenite is beateristatie
to the organism. A repeated determimtion with the same

organism showed essentially the same result.

A

D!
V.‘
e
4
I

HE mom passe awakens *

 

 

on me nasananooco .m
mam «mm oneness .2
omfl mam mammompmfiomo .m
as omfi Fwd m annahcncn .m
mam sfimm ananznahdao .m
huwoflxop Shaves mammogxo chomcm Emficwmao

no Honpcoo

 

4 mm» mm cHHHHowccm or madmoexm Scam

mamwgosfiwm Hmpoomm mo hnmroomm axe

N N miHmdfi

'72

nmnormr 01A nurmmrm memo MEDIUM
roe DISTINGUISHING THE mum: ’
mom oouroan mama * ‘ '

In this investigation under certain derinite conditions
of incubation in the proper medium, E. coli was selectively
killed by penicillin. under these same conditions several
gulggngllg’epeciea were shown to survive at least to the
extent of about 80 percent.

In order to determine thether this selectivity would
apply to other colifornl and salmonellae in naturally in-
fected fecal material, it became evident that a good, none
toxic differential medium.ehich.eould distinguish the eel-
nonellee tron the colitorns must be employed.

It was desired to be able to plate relatively large
volunes or IE? mediun.in which the organisms had been ex-
posed to yenicillin. this would enhance the statistical
probability of recovering the salmonellae, especially ehere
they eceur in relatively small numbers. 0n the basis or
these considerations then, a nediumwwhich could be used as
a pour plate medium.uould be the one or choice.

,A relatively nontoxic nadiun.such as MhoGonkey agar is

unsuitable because only surface colonies are differentiated.

 

73

Bis-nth sulfite agar is satisfactory as a poured plate
medium. This was considered unsatisfactcn-y for use here be-
cause of the presence of the dye, brilliant green. A medium
containing no substances inhibitory for the gram negative or-
ganism was desired in order that all the salmonellae cur-
viving penicillin exposure would have the maximum opportunity
to initiate growth and form colonies.

Lederberg (1948) employed 0.005 percent triphenyltetra-
zoliun chloride in nutrient agar plus one percent, lactose as
a medium for. the detection of fermentative variants of 334
an. fleobservcd tint the biological reduction of the tetra-
solium salt to the highly colored, insoluble formazan is
probably pH dependent. ‘i'he parent type of 35, .321}, which is
capable of producing acid from lactose in the medium, does
notreduce the tetrazclium. Therefore. colonies of the
parent type which develop remain colorless. 0n the other
hand, occasional variants which do not produce acid from
lactose do reduce the tetrasolium salt and take on a very
intense red color. This is also advantageous in that the
color remains confined to the colony and does not obscure the
true nature of nearby colonies by diffusion throughout the
nediua. as often occurs 'with ordinary pH indicators.

Lederberg (1948) also suggested the possible value or
tetrenolium in this respect in a differential mdium roi-

9‘

74

the eel-onellee.

various tetrazolium.sa1ts are being used in the field
of bacteriology for diagnostic purposes. Chapman (1951)
incorporated triphenyltetrazolium.chloride in his tergitol-

7 nedium:for the differentiation of various types of coli-
forms. Reinhold, Swarn.and Hussong (1953) also describe the
use of a tetrazolium.salt in a medium for the isolation and
enumeration of the enterococoi.

Preliminary work using nutrient agar with 0.5 percent
lactose and 0.005 percent triphenyltetrazolium.chloride re-
vealed that in. W produced rather small, vary intensely
red colonies in poured plates as well as on streak plates.
with the same medium. L. 293.1, produced colorless or very
faint pink colonies, somewhat larger in size. It thus
appeared that tetrazolium.could be used as an indicator to
differentiate lactose fermenters from lactose non-fermenters.

However. the results of ExperimenthII show that nutrient
agar is not a satisfactory medium for recovery of §_,. M
after penicillin exposure. Therefore.soms other nadium.must
be used as a base to insure adequate recovery. It see also
shown.in Experiment III that brain heart infusion agar enabled
a relatively high recovery rate of gh,pgllorum. '

One main difference between brain heart infusion agar

and nutrient agar is that a higher concentration of peptone

75

is present in the former.

The following experiment was run in order to determine
whether this higher peptone concentration‘could be at least
partially responsible for the enhanced recovery of §_‘ 22$:
m from penicillin exposure. Bacto-tryptose was the

peptone used.

mm XXIII.

i'he Iffeet of Varying the 'rryptose Concentration in the
Plating Medium on the Recovery org. W from Penicillin
Exposure in Y8? Medium.

m: l so-mi quantity of YBP medium with 0.25
percent lactose and 0.002 percent sodium arsenite was seeded
with 0.5 ml of a l-lO0,000 dilution of a 24-hour brain heart
infusion broth culture of §_,. m. After six hours at
37 0, 300 units of penicillin per ml were added. ' lfter a
further four-hour incubation, a lO-ml quantity eas removed
to a test tube containing 70,000 ”units” of penase. after
one hour at 37 o, one-m1 amounts eere plated in duplicates
in media containing 0.5 percent sodium chloride, 1.5 percent
agar and various concentrations of tryptose. After fee-hours
incubation at 37 0, the colonies were counted.‘ The results
are shosn in Table 23. y .

mm: It can be seen from these results that the-
amcunt of peptonc in the form of bactc-tryptcae in the platiig

O ‘ x
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.
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76

TABLE 83
the Effect of Tryptoee Concentration in the

Plating Medium on Recovery of W
from kpoeure to Penicillin in d an

M k _.‘A_.
.7 v , fl. 1 w vwr ll fl?
; .4

w ————r w

Tryptose concentration ‘ Count per ml “ta 5

 

 

f W w Y W w exposure
0.55 o
l.0$ 42
1.53‘ 70
2.05 93
2.5% so

control-Brain heart infusion agar 160

Control-no penicillin exposure 190

A

77

medium does have a pronounced effect on the recovery of g‘
m from exposure to penicillin in YBP medium. 5 two
percent concentration of tryptose appears to be optimum.
Lower concentrations tested, appreciably decreased the re-
covery. while a higher concentration did not increase re-
covery to any extent. The recovery of §_,, w using
this optimum concentration of tryptostwas approximately
50 percent. In comparing this result with that obtained
using brain heart infusion agar as the plating medium, it
is as evident that some surviving cells still failed to
develop in 2.0 percent tryptose agar.

Ioodruff and Foster (1945), while studying bacterial
penicillinase, made the observation that cysteine is sap-
able of inactivating penicillin. Also other 83 containing
compounds were found to enhance penicillinase activity.

It was next deemed advisable to determine whether the
addition of such 33 containing substances to the plating me-
dium would increase the rate of recovery of §._ w from

exposure to penicillin.

EXPERIMENT XXIV.

Determination of the Effect of cysteine and Sodium Thio.
glycollate on the Recovery of g. M from Penicillin a:-
poem.

0 ed a A 60-ml qusntity of YBP medium with 0.25

78

percent lactose and 0.003 percent sodium.arsenite was pre-
pared and seeded with 0.5 ml of a 1-100,000 dilution of a
24-hour culture of a W. After six hours at 37 G.
300 units of penicillin.per ml were added. .After a further
fourehour incubation at 37 0, a twenty-ml quantity was mixed
with 140,000 "units” of penase. After one hour, duplicate
one-ml quantities were phated in two percent tryptose agar
containing various concentrations of cysteine and sodium
thioglycollate. These results may be found in Thble 24.

Discussion: It is evident from.these data that the
addition of 0.02 percent cysteine to 2.0 percent tryptose
. agar increases the recovery of L m from penicillin
exposure to about 83 percent. This is comparable to the
recovery experienced using brain heart infusion agar. Any
further increase in cysteine concentration does not appear
to enhance recovery additionally.

Also, it can be m. that the addition of sodium thic-
glycollate does enhance the recovery to some extent, but
not to the extent experienced with cysteine.

from.the information gained in the previous experiments,
the following was formulated as a possible-differential me-
dium.for the detection of members of the genus §glgpnelia in

the presence of other fecal bacteria:

79

Iryptose ........................... 2.05
Sodium Chloride .................... 0.5‘
Cysteine ........................... 0.002%
friphenyltetrasolium chloride ...... 0.005%
Lactose ............................ 0.5%

Mixtures of l... gig, and pg, w plated in this me-
dium gave a very clear cut differentiation. The _S_,, 293.3213.
colonies were a very intensely red, while the L 23;; colonies
were a light pink and somewhat larger.

The next experiment was designed to determine the effect-
iveness of this tryptcse lactose tetrazolium agar in differen-
tiating g, M from other fecal organisms which survive
the penicillin exposure in m medium.

MW? m.

Beta-nineties of the Ability of fryptose Lactose Tetra-
solium Agar to Differentiate 3‘, m from Other Fecal 0r-
ganisms which Swvive Penicillin Exposure in YEP lbdium.

W: A 50-ml quantity of YBP medium with 0.003
percent sodium arsenite and 0.25 percent lactose was pre-
pared. Approximately 0.5 grams of fresh human feces and 0.5
ml of a 1400.000 dilution of a 24-hour culture of g“ Bill-92233
were added. This was incubated for six hours at 37 0. Peni-
cillin was added to a level of 200 units per ml, and further
incubated for four hours. A five-ml quantity was then mixed

5‘. .
esO'
bIse
e'. D

C I 6
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a U .

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I
.
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O

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.
v
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u
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v
0

80

IABLE 24

The lrfect of cysteine and Sodium.Thioglycollate~

on the Recovery of pgllggnl
from Penicillin fipos ure

 

concentration (f) ‘ Average count per mu

Cysteine
0 126
0.0085 140
0.005 138
0.01 148
0.08 186
0.03 ' l90

Sodium.thioglycollate
0 ' 186
0.01 148
0.025 ' 145
0.05 151
0.1 143

control - before
penicillin 230

—-—____

81

with 35,000 “units" of penase and incubated further for one
hour. Onednl quantities were then plated in tryptose lec-
tose tetrezoliun agar. After 24 hours, the plates were ex-
amined with the following result:

§L_pgl;gzgg colonies which developed were deeply dark
red. The relatively few ccliform.colonies (lactose fer-
:menting gran.negetive rods) varied frcn.neerly colorless
to pink. These were also considerably larger than the g.
m colonies. A fairly large proportion of the null
intensely red colonies proved to be gren.pcsitive cceci. end
cocci in short chains or pairs chsrecteristic of the entere-
cocci.

niscgsliog: This nediun.shcwed a sherp distinction
between a. W end the other gram negative fecal ferns
encountered in this specimen. However, the development of
the gren.pcsitive cocci made it apparent thet some neens of
eliminsting these forms wss necessary if the differential
value of triphenyl tetrasolium.chlcride wes to be used here.

Severel chemical substances mdght be added to this agar
to inhibit selectively the gren.pcsitive organisms. Among
these are the aniline dyes, various wetting agents, and cer-
tsin bis-phenols.

Litsky, Millmann and Iifield (1952) showed that in
sufficient concentrations all the basic dyes which they tested

82

showed some inhibition of the gram negative as well as a
marked inhibition of the gram positive organisms. Litsky,
Hellman, and finale (1953) worked with ethyl violet, which
is the least toxic of thesedees. These authors show that
the enterecocci are intermediate in sensitivity between the
gram positive and gram negative organism. The results of .
these studies indicate that a concentration of ethyl violet,
which is sufficient to inhibit the enteroeocci, could also
show considerable toxicity toward gram negative organisms.
since the enterococei are among the organisms which it is
desired to inhibit, these dyes were not considered further.
human and Darby (1941) showed that the anionic wetting
agent sodium lauryl sulfate in a concentration of 0.01 percent
is effective in inhibiting several gram positive organism in
tryptcse broth. This same concentration proved completely
nontoxic to 1., 29.11. the gram negative organism tested.

Bordt (1.951), working with the bisphenol 8,2' methylenebis-
e-chloro-c-isoprcpylphenol (x4643), duonstrated a marked
selective property of this compound also. a concentration
as low as 0.000001 percent inhibited the gowth of the gran
positive organisms tested, while the highest concentration
tested, 0.001 percent, showed no toxicity toward 1,, 293,1.

These two compounds were next considered as possible

agents to use for the elimination of the gram positive forms

which interfere with the diagnostic efficiency of tryptose

lactose tetrazolium agar.

EXPERIMENT xva. .

Determination of the Efficiency of 0.01 Percent Sodium
Lauryl Sulfate and 0.001 Percent 2,2'Methylenebis-4-ohloro-
6-iscprcpylphenol in Suppressing the Growth of Gran Positive
fecal Organisms lhich Survive Penicillin Exposure in YBP
medium.

m: Ten gram positive organisms isolated from
plates in Experiment XXIV were grown in brain heart infusion
broth. Five of these were morphologically typical of micro-
cocci, and five were large cocci in short chains or pairs
morphologically typical of the enterccccci. One drcp of a
1.10.000 dilution of a 24-hour culture of each of these or-
ganisms was plated in tryptose lactose tetrazolium agar con-
taining 0.01 percent sodium lauryl sulfate. Identical plates
were poured using tryptose lactose tetrazolium agar with
0.001 percent of 2,a'nethylenebis-e-ohlorc-eoiscprcpylphencl.
Duplicate plates of each medium, as well as tryptose lactose
tetrazclium agar base, were also seeded with one ml of a
14,000,000 dilution of a 24-hour culture of §_., W
these were controls on the toxicity of the two selective
agents for g. We The plates were examined after 24
hours at 37 0, and recorded in Table 25.

I
n
e
. V
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o
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-~.—.

IABLE 25

The Development of a

lo in the Presence

. of Sodium.Lauryl Sulfate and K-7643

 

 

Inhibitor Average count per ml
lone 264
Sodium lauryl sulfate 0.01% 259
1-7043 0.001f 266

 

85

luugmggiggj The results of this study show that sodium
lauryl sulfate was effective in completely inhibiting three
of the five micrococcus types, while two did grow and pro-
duce dark red colonies. All five of the enterococcus types
developed dark.red colonies.

The plates containing the bisphenol, however, showed
no growth of either type of gram positive organism.

An examination of Table 25 shows that neither sodium
lauryl sulfate nor 2,2'methylenebisod-chlorc-0~iscpropyl-
phenol exhibit an appreciable toxicity toward §,, W
as far as numbers of colonies developing is concerned.

0n the basis of these results then, it was decided to
use the following as a differential plating medium.for the
detection of salmonellae in subsequent studies:

fryptose .................................... 3.0%

Lactose ..................................... 0.5%

Sodium.chloride .........e................... 0.5f

Cysteine .................................... 0.025

Iriphenyltetrsaolium.chloride ............... 0.005!

2,2'lhthylenebis-4-chlorc-6~isopropylphenol . 0.0001?

85

piscgsgiogx The results of this study show that sodium
lauryl sulfate was effective in completely inhibiting three
of the five nicrococcus types, while two did grow and pro-
duce dark red colonies, All five of the enterococcus types
developed dark red colonies.

rhe plates containing the bisphenol, however, showed
no growth of either type of gram.pcsitive organism.

An examination of Table 25 shows that neither sodium
lauryl sulfate nor 2,8'nethylenebis-d-ohloro-d-isopropyl-
phenol exhibit an appreciable toxicity toward g, m
as far as numbers of colonies developing is concerned.

as the basis of these results then, it was decided to
use the following as a differential plating medium.for the
detection of salmonellae in subsequent studies:

fryptose .................................... 3.0%

Lactose ..................................... 0.5%

8odium.chloridc ............................. 0.5$

Cysteine .................................... 0.02‘

Iriphenyltetrasolium.chloride ............... 0.005%

2,8'nethylsnebis-4-chlorc-e-visopropylphenol . 0. 0001%

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86

USE OF THE PENICILLIN ENRICHMENT TECHNIQUE FOR
THE ISOLATION OF almonmln FROM NATURALLY
INFECTED HUMAN FECAL srncxms "

The final portion of this work was concerned with de-
ternining whether or not this "penicillin enrichment" pro-
sedure could be used for the isolation of manners of the
genus W from naturally infected human fecal mater-
ial.

W: The specimens used here were ob-
tsined from the diagnostic laboratory of the Inchigan Department
of Health, Lansing, Michigan. They consisted of fecal samples
from known carriers, suspected carriers, suspected active
eases of W infections, and from persons in contact
with carriers or active cases. The specimens were usually
obtained at weekly intervals, and varied in age from three
to twelve days after the date of receipt at the laboratory.

W23: Approximately two gm of the
specimen was suspended by shaking in ten ml of sterile physio-
logical saline in a large test tube. this suspension was
then freed of gross particles by slowly forcing a loosely
packed ectton plug down through the suspension to the bottom

Ow...

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86

use or m rmxcmm ENRICHMENT TECHNIQUE FOR
THE ISOLATION or SALMONELLAE FROM NATURALLY
‘ INFECTED HUMAN men. SPECIMENS ' ~

The final portion of this work was concerned with as.
termining whether or not this "penicillin enrichment” pro-
cedure could be used for the isolation of manners of the
genus W from naturally infected human fecal mater-
ial.

gggrce 2f gpgcigeg: The specimens used here were ob-
tained from the diagnostic laboratory of the Inchigan Department
of Health, Lansing, Michigan. they consisted of fecal samples
from known carriers, suspected carriers, suspected active
asses of W infections, and from persons in contact
with carriers or active cases. The specimens were usually
obtained at weekly intervals, and varied in age from three
to twelve days after the date of receipt at the laboratory.

WW: Approximately two gm of the
specimen was suspended by shaking in ten ml of sterile physio-
logical saline in a large test tube. This suspension was
then freed of gross particles by slowly forcing a loosely
peeked eotton plug down through the suspension to the bottom

87

of the tube with a sterile pipette. One-and 0.1 md-quantities
of the supernatant were then placed in five-ml quantities of
YEP medium containing 0.25 percent lactose and 0.008 Percent
sodium.arsenite. After thorough mixing, these tubes were
placed in a water bath at 37 c for six hours. Penicillin was
than.added to the level of 200 units per ml. After four hours
at the same temperature, 55,000 ”units" of penase were added
to each tube. One hour later, 0.1-and five-ml amounts were
plated in the differential tryptose, lactose tetrazolium
agar described on page 85. The plates were examuned after 24
and 48 hours at 37 0, and the very intensely red colonies
characteristic of the salmonellae were picked and inoculated
into tubes of Baetoexligler Iron Agar. Ihose tubes showing
an alkaline slant, and acid butt with or without gas or hy-
drogen sulfide were further studied. The following.msdia
were used for a partial identification of the organisms iso-
lated: lecto-Purple Broth base containing 0.5 percent con»
centrations of dextrose, lactose, maltose, and sucrose. Also
seeded were one percent tryptose solution for determination
of indole production.from tryptophanc, semisolid motility
test agar for determination of motility, Simmons' citrate
agar for citrate utilisation, and urea broth to detect the
organisms ability to hydrolyze urea. Each culture was also

gram-stained to check for purity and gm.reaction.

88

In order to establish some standard to indicate the
relative efficiency of this penicillin enrichment procedure,
the specimens were also examined by using four of the sev-
eral diagnostic media designed for salmonella isolation.

In this parallel procedure, tso ml of the original saline
suspension was placed in test tubes containing ten-ml quan-
tities of Bacto-Selenite broth, and incubated for 24 hours.
‘After this preliminary enrichment, 0.leml quantities were
spread on the surface of BactoenacConkey agar, Bacto~SS
agar, and fiesta-Bismuth Sulfite agar plates. Also five-ml
and one drop quantities of the original saline suspension
were plated in bismuth sulfite agar pour plates. After 84
and 48 hours, these plates were examined and suspected
salmonella colonies were picked and partially identified by
the procedure outlined above.

Pure cultures showing reactions typical of the genus
W were sent to the Salmonella Typing Station of
the Michigan Department of Health for positive identification.
The results obtained by the two methods of isolation are
given in Table 86.

W8 The results of this limited number of exam-
inations indicate that the efficiency of the penicillin en-
richment procedure compares quite favorably with that of the
combination of currently employed media. A total of 103

 

89

am 86

Results of the Examination of Fecal Specimens for
Salmonellae by the Penicillin Enrichment

Technique and by Four Currently
uployed Diagnostic Media

 

w

 

Specimen Current Penicillin Organism
number media techni que is elated
es f - _s_,, typhos;
59 f 2‘ § typhosg
57 - f §_,, txphosg
53 f f g... txphosg
as f f g, montivegec
M f f i gggesgee
36 f 2‘ a. mm:
m I I a. W
189 f f a, typhosa
“5 f / fie. when
405 f i gt granienbggg
731 f ;‘ §_,, tnhosa
all f I g, gngosa
335 1‘ - g, gentgvidec
3“ i‘ i‘ §_._ mppevidgg
868 I 1‘ §_‘ typhimurium
899 - f _S_,_ typhcga

\ e O
. _ "e
‘ e v v-OIV
. -. _ - t ‘6
. .
. l e
- . . . n
e -, . e
e D -
. . . .
e ,
‘ . e

.'

 

90

TABLE 26 (Continued)

 

 

Tv—wv—w—

 

Specimen Current Pe pl 01 11 in Organism
number media technique isolated
761 f f g. uphimurig
1153 f ,l g, tnhose
1168 f f _S_,, oranicnbgg
1889 h :3 - a. Me
1299 I / §_,_ tzphcsa
1314 f f _S_,_ typhosa
1315 f - _8_,_ 11229.99.
1331 f 1‘ §... txphoga
1470 f f §_,. worthington
1611 - r‘ §... mm;

 

U

..
..e.e.
I ..
, e
v a-

0

e
' ~
, I

90

TABLE 26 (Continued)

 

 

w

 

Specimen current Peni ci llin Organism
number media technique isolated
m 5‘ f a. W
1153 f f é; tnhosa
1168 f f g; cranienbgg
1889 ‘ f - g, mntgigeo
1299 I f §_,, typhcsa
1314 f f _3_., typhoga
1315 2‘ - in when
1331 f f g, typhoga
1470 f f g, worthington
1611 - f g, typhoga

 

91

specimens were examined. Irom.28 of these,1§glg9ggllg_species
were isolated by the penicillin enrichment method, while '25
were positive by the commonly employed media. As is seen
from.the tables, in three cases salmonellae were isolated by
the penicillin enrichment procedure but missed by the cur-
rently used media, while in four cases the penicillin tech-
nique failed, but the currently used media did not.

During the examination of these several specimens, it
was noted that in the majority of the cases the nonesalmo-
nella population of the fecal specimen was reduced sufficiently
to allow for a ready isolation of discrete colonies. However,
in a few cases the resulting plates were very heavily pop-
ulated, making differentiation and isolation of discrete
colonies impossible. Upon closer examination of these over-
crowded plates it was discovered that the organisms involved
were most often those of the genus Pseudomonas. These cols
onies were dark red, but where not too abundant, they could
be distinguished from.the salmonellae by the presence of a
greenish-white halo surrounding the colony.

Also in a few specimens the resulting plates were over-
crowded with light pink colonies characteristic of the coli-
fcrms. Isolates from.suoh colonies produced acid and gas
in lactose broth and gave other typical coliform.reactions.

The reason for their surviving the penicillin exposure in

98

large numbers was not immediately apparent. However, when
these organisms were placed in YEP medium containing 0.25
percent lactose, but no sodium arsenite, about 90 percent
of them failed to show growth after 24 hours at 3? c. A
number of such isolates are listed in Table 2‘? along with
their IMViO reactions. Evidently these are intermediate
colifcrm strains which do not possess the nutritional effi-
ciency characteristic of the tribe Bohezicheag. The sur-
vival of these types imadiately suggests the possible use
of a penicillin enrichment technique for the isolation of
naturally occurring biochemically deficient forms of bac-
teria in much the same manner that artifioally induced
mutant forms. are isolated.

Boepke, Libby and Small (1944) also reported the nat- ‘
ural occurrence of nutritionally deficient strains of L 391i.
mess workers investigated the added requirements of some of
these strains and found them to be one or the following:
methionine, thiamine, nicotinic acid or its amide, lysine,
oyatine, arginine, threcnine, or tryptophane.

Occasionally dark red colonies appeared on the plates
which were indistinguishable from those of the salmonellae
except for their slightly greater size. In Kligler's iron
agar these gave acid and abundant gas in the. butt, and an
alkaline slant. Further biochemical studies revealed that

 

0‘

 

93

TABLE 87

Mic Reactions of Golirorm Strains which do not Grow in
131’ ledium‘Plus Lactose and which Survived
Penicillin hpcsure in this Medium

 

 

—v —~—v— v ‘v

 

 

m 1‘: meme: mm mm“
1 AG 4-;
2 m 4-,:
3 as ”in:
4 so ~/--
5 AG ”2--
6 as -/-K
7 AG Mb}
9 ac -}--
10 AG I 4-}
u m uh)
is As -2..-
13 AG ~k-i
15 as -}-2
16 AG -/-}
1? Ac -3...
18 AG ~¥~f
N no -;-;
22 AG -}--

93

TABLE 87

mm Reactions of Caliform Strains which do not Grow in
RP lediumPlus Lactose and which Survived
Penicillin hposure in this Medium

 

 

Me: 24 hour reaction IMHO formula

 

 

V in lactose broth
1 AG -x-/
3 AG -}-x
s so -).}
4 m « -/--
5 so -).-
6 AG -x-}
v m 4...:
9 AG -}--
10 AG .4-)
u tG ~i-k
12. AG -}.-
13 AG ~2-i
w w 4-;
16 AG -/-}
1? Ac ”2...
l8 AG -{.,c
1° AG -;-;
22 AG -3-..

13'

94

IABLE 27 (Continued)

 

 

 

lumber 24 hour reaction mnc formula
in lactose broth__ . w A
33' + m - f 4
34. so - f - f ’
so AG - ,c - ,c
as so - ,c .. -
50 m - g - -
31 AG - ,1 - ,t
as is - f - -

as A6 -}--

 

95

in general these were very slow lactose fermenters or none
fermenters otherwise typical of members of the coliform.sroup.
These, according to Kauffmann (1951), are probably best
called slow lactose fermenting or nonfermenting coliforms.

It is postulated that these interfering types survived
exposure to penicillin because they were unable to grow in
the synthetic IBP medium. The members of the PIGEQOmOgaS
group probably survive due to their inability to utilize
lactose as a carbon source. this also may be true of the
lactose nonfermenting coliforms. The majority of the lac-
tose fermenting types which survive probably do so because
of some other nutritional deficiency which they possess.
‘3221331,species would be expected to interfere here also.
Bhwever. these were only very rarely encountered and caused
no serious difficulty in the procedure.

One cf the chief limitations to the penicillin enrich-
ment technique is the relatively small sample which can be
ordinarily examdnedu As can be seen from the procedure on
page 8?, the maximum.amount of fecal material plated in
any one plate is about 0.2 grams. This is in contrast to
about one gram.which can be plated in bismuth sulfite agar.

.another limitation, of course, is the fact that a bun-
dred percent survival of the salmonella is not obtained.

In most cases, 80-99 percent survival is experienced, but

‘\

96

with one organism it was as low as 15 percent. This is most
serious, of course, when dealing with specimens containing
relatively small numbers of salmonellae.

96

with one organism.it was as low as 13 percent. This is most
serious, of course, when dealing with specimens containing

relatively small numbers of salmonellae.

97

sums!

Various factors were investigated to determine their
influence on the action of penicillin against selected mem-
bers of the gtgcbacteriacgge. These factors were composi-
tion of the medium, pro-incubation time, penicillin exposure
time, methods“ of inactivation of penicillin prior to plating,
and composition of the plating medium.

Two hundred units of penicillin per ml was bactericidal
for 5,, 39],}, in a synthetic medium with lactose as a carbon
source. 1130 penicillin was found to be most actively bee-
tericidal when exposure took place after the lag phase of
growth had been passed and the organisms had begun to divide.

Dilution was not an effective means for reduction of
penicillin concentration where recovery of previously exposed
organisms was desired. Penicillinase was found to destroy
penicillin effectively and allow recovery of previously ex-
posed cells. However, a much higher concentration of the enqne
was required for recovery of cells exposed to penicillin than
was necessary to inactivate completely the same amount of peni-
cillin under identical conditions of time and temperature. s,
W when pre-incubated for a sufficient time in a medium
unfavorable for growth and then exposed to 800 units of peni-

98

cillin per ml, survived this exposure. This was presumably
due to the fact that penicillin is effective only against
growing cells.

Uhder identical conditions in YBP medium with lactose
as the sole carbon source, 200 units of penicillin was highly
bactericidal to g. 391,1, but showed very little action against
§_,_ m The addition of approximately one percent sterile
fecal material resulted in a bactericidal action against g,
m as well. This was presumably due to the fact that
the fecal mterial contributed substances to the medium which
were utilizable for growth by §_,_ w, and thus established
the penicillin effect.

Sodium arsenite in proper concentration was found to be
selectively bacteriostatie against members of the genus is};
m, but not auinst other W stulied.
Sodium arsenite in a concentration of 0.003 percent in YBP
medium protected members of the genus W from peni-
cillin exposure even in the presence of one percent fecal
material.

The presence of a two percent concentration of tryptcsc
as well as 0.08 percent cysteine in the plating mdium mat-
erially enhanced the recovery of _S_,. w from exposure
to penicillin.

' Also, the addition of 0.5 percent lactose and 0.005 per-

 

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99

cent triphenyltetrazclium.chloride to the plating medium
resulted in colonies of diagnostic significance. Lactose
nonofernenting organisms reduced the tetrazolium.salt and
developed intensely red colonies. Those organisms which
produced acid from lactose failed to reduce the salt or did
so only very slightly and gave rise to colorless or faint
pink colonies.

In early attempts to utilise this penicillin enrichment
technique for isolating salmonellae from.fecal material. it
was noted that certain gram positive bacteria also survived
the exposure and interfered with the efficiency of the dif-
ferential medium by forming colonies indistinguishable from
those of the salmonellae. The addition of 0.001 percent of
a bisphenol, 2,8'methylenebie—a-chlorc-G-isOpropylphenol,
to the plating medium successfully inhibited these forms.

A series of human fecal samples was examined to deter-
mine whether this penicillin enrichment technique could be
used for the isolation of salmonellae from feces. Compara-
tive results obtained indicate that the technique is work-
able. !he result of this limited series of examinations re-
veals a favorable comparison with several media presently
used in routine diagnostic procedures. Among the limitations
of the method is the fact that certain organisms other than

the salmonellae survive the penicillin exposure, and when

100

initially present in very large numbers, these result in over-
crowded plates which.makes isolation of typical colonies dif-
ficult or impossible. The organisms involved in this type of
interference are (l) Pseudompnas species, (2) nutritionally
deficient coliforms. (3) lactose non-fermenting or slow fer-
menting cclifarms. other limitations of the method are the
lack of quantitative recovery of the salmonellae and the
necessarily relatively small inoculum.used.

In addition to its use in isolating salmonellae from
fecal material, this penicillin enrichment technique with
modifications might also be used for the isolation of bio-

chemically deficient forms of bacteria from.nature.

 

 

101

CONCLUSIONS

1. 200 units of penicillin per ml was bactericidal for 35,
.qg;i in.a synthetic medium with lactose as a carbon source.

2. Penicillin was most actively bactericidal when exposure

of the cells took place after the lag phase of growth had been
passed and the organisms had begun to divide.

3. Dilution was not an effective means for reduction of peni-
cillin concentration where recovery of previously emposed cells
was desired.

4. A proper concentration of penicillinase did destroy peni-
cillin effectively and allowed subsequent recovery and growth
of cells previously exposed to the antibiotic.

5. g... M was found to survive exposure to 200 units of
penicillin per ml when in a synthetic medium.where growth was
nutritionally impossible.

6. Sodium.arsenite in a concentration of 0.002 percent was
found to be selectively bacteriostatie against various sal-
monellae. this bacteriostatic action of sodium.arsenite also
tended to protect salmonellae from.the action of penicillin.

7. Members of the genus §glmgpglla,were successfully isolated

from.infccted fecal material by this selective action of peni-

102

cillin against growing cells.

8. the addition of two percent tryptose and 0.02 percent
cysteine to the final plating medium.increased the percent
recovery of _s_. W from penicillin action.

9. The presence of 0.5 percent lactose and 0.005 percent
triphenyltetrasolium.chloride in the plating medium.resulted

in the development of dark red salmonella colonies and light
pink or colorless coliform.solonies.

lO. a concentration of 0.01 percent 2,2'methylenebis-4-chlorc-
Fiscprcpylphenol in the plating medium inhibited gram positive
fecal organisms which survived penicillin exposure.

11. This ”penicillin enrichment” technique compared favorably
in efficiency with media currently employed for isolation of

salmonellae from.fecal material.

103

mm ILLS USED

menium Sulfate (mass. C.P.
Beets Lactose ’ '

Dectc Penase Concentrate. Control
lumber 424.0“

Bactc Tryptcse
ferrous Sulfate reSOp'sz 0.P.
hgnesium Sulfate 15360407320 0.P.

2 , 8 'methylenebis-e-chlorc—c-is o-
prepylphencl

Penicillin 0 Potassium, Control
number 358827 .

Petals inn Phos pha ts monobas io
WPOQ 0e Po

Sodium arsenate dibasic leaflAsOp'Inzo
reagent

Sodium ”senite Rodeo; anal. reagent
Sodium Chloride ReOl O.P.

Sodium Phosphate dibesic Ream 0.P.
2 , :5 , 5 , Triphcnyl-zfi-Te trazoliun Chloride

Baker
Difco

Difco

Dif cc

Kerk

Eimer d: amend

Dow

30311 DB

Baker

Merl:
fillinekrcdt
Baker

Baker

Distillation
.Products

104

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‘ t
O
.
.‘ . .
_ .
-
.
I
I
‘-
. .
. 0
. . . .
. .
O
‘ C
‘ I

so

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136. 1944.

{£65331 USE Giéii’

 

 

   

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